Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Keiichi Nakayama Last modified date:2019.06.01

Professor / Department of Molecular and Cellular Biology / Medical Institute of Bioregulation


Papers
1. Keiichi Nakayama, Y. I. Shinkai, K. Okumura, H. Nakauchi, Isolation and characterization of the mouse CD8 β-chain (Ly-3) genes. Absenced of an intervening sequence between V- and J-like gene segments, Journal of Immunology, 142, 7, 2540-2546, 1989.01, We have isolated and determined the nucleotide sequence and genomic organization of the genes encoding Ly-3.1 and Ly-3.2. These genes span approximately 14 kb on chromosome 6 and consist of six exons and five introns. The exons correlate roughly with the putative functional domains, namely, a leader exon, a variable and joining region-like exon, a hinge region-like exon, a transmembrane exon, and two intracytoplasmic exons. There is no intervening sequence between V- and J-like gene segments, indicating that rearrangement is not necessary for the expression of the Ly-3 gene. In the 5'-flanking region there is no'TATA' box nor 'CAAT' box; however, three 'GC' boxes are located upstream of the ATG initiator codon. There are short stretches of sequence homologous to 5'-flanking sequences of the Ly-2 gene. In addition, the sequences CTCTGTGGCA at -748 exhibits homology to the enhancer core sequence of the human Ig H chain and TCR genes. Comparison of the nucleotide sequence corresponding to the extracellular portion between Ly-3.1 and Ly-3.2 revealed a single base difference which results in an amino acid substitution. Therefore it is likely that this amino acid difference is responsible for the previously defined Ly-3 allotypes..
2. Keiichi Nakayama, H. Nakauchi, An improved method to make sequential deletion mutants for DNA sequencing., Trends in genetics : TIG, 5, 10, 1989.01.
3. Keiichi Nakayama, Satoki Tokito, Ko Okumura, Hiromitsu Nakauchi, Structure and expression of the gene encoding CD8α chain (Leu-2/T8), Immunogenetics, 10.1007/BF02425282, 30, 5, 393-397, 1989.11.
4. Keiichi Nakayama, Satoki Tokito, Christian Jaulin, Christiane Delarbre, Philippe Kourilsky, Hiromitsu Nakauchi, Gabriel Gachelin, Comparative structure of two duplicated T1a class I genes (T10c and 37) of the murine H-2d MHC
Implications on the evolution of the T1a region, Journal of Immunology, 144, 6, 2400-2408, 1990.03, The class I Ag encoded in the Qa/T1a regions of the marine MHC are much less polymorphic, and usually have a more restricted tissue distribution than the classical histocompatibility class I Ag, encoded by genes located in the H-2K, D, and L loci. The isolation of a quasi-ubiquitously expressed, poorly polymorphic class I gene of the T1a region of the H-2d mouse MHC, namely gene 37 (or T18d), has been recently reported. We describe the nucleotide sequence of a closely related gene, T10c gene, the counterpart of the gene 37 in the large duplicated parts of T1a region of the BALB/c (H-2d) MHC. The T10c gene structure and sequence are very similar to those of gene 37, but T10c gene is most likely a pseudogene. In A/J mouse strain, there appears to be a single gene related to 37, which is also found expressed in a variety of tissues. We show that this gene is likely to be a chimeric one derived from T10c for its 3′ part, and from a gene closely related to gene 37 for its 5′ part, which potentially encodes for an unusual class I molecule composed of the first two domains. Finally, Southern blot analysis of a number of wild mice and related animals suggests that a gene closely related to the present T10c gene may be the ancestor of this subfamily of class I genes characterized by the presence of an unusual second domain..
5. Akira Ohara, Toshio Suda, Naomi Tokuyama, Junko Suda, Keiichi Nakayama, Yasusada Miura, Shin Ichi Nishikawa, Hiromitsu Nakauchi, Generation of b lymphocytes from a single hemopoietic progenitor cell in vitro, International Immunology, 10.1093/intimm/3.7.703, 3, 7, 703-709, 1991.01, The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were Individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed In 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells..
6. Keiichi Nakayama, Satoki Tokito, Christophe Pannetier, Hiromitsu Nakauchi, Gabriel Gachelin, MHC gene Q8/9d of the BALB/cJ mouse strain cannot encode a Qa-2,3 class I antigen, Immunogenetics, 10.1007/BF00230499, 33, 4, 225-234, 1991.04, We have determined the nucleotide sequence of Q8/9d gene of the BALB/c strain of mice, isolated from Steinmetz's cosmid library. As for all other class I genes of the Qa region, the Q8/9d gene spans approximately 4.7 kilobases (kb) and consists of seven exons and six introns. A seven bases deletion in exon 3 results in the occurrence of an early termination codon. Thus the Q8/9d gene cannot encode a normal class I protein. Comparison of the nucleotide sequence of the Q8/9d gene with that of other class I MHC genes revealed a stronger homology to Q7 and Q8 than to K, D, L, TL, and other Q genes. However, the gene cannot originate from a mere fusion between Q8 and Q9 genes except if the ancestor to putative Q8d was markedly different from the present Q8b gene. Using polymerase chain reaction (PCR) technology, we have confirmed the presence of a Q8/9 gene, identical to that present in cosmid 46.1, in the genome of BALB/cJ (Qa-2low). Finally, it has been reported that cDNA clone 94-A, which codes for a Qa-2 antigen, could derive from a transcript of gene Q8/9d. The nucleotide sequences of gene Q8/9d and of cDNA clone 94-A are distinctly different in their 5′ regions, in spite of an almost perfect matching in their 3′ regions. Thus, clone 94-A cannot derive from an mRNA transcribed from the Q8/9d gene..
7. Keiichi Nakayama, Akinori Sarai, Hiromitsu Nakauchi, Single amino acid substitutions determine mouse CD8 allotypes
epitope mapping of mouse CD8, Immunogenetics, 10.1007/BF01719243, 33, 3, 206-209, 1991.05.
8. Keiichi Nakayama, Y. Kawachi, S. Tokito, N. Minami, R. Yamamoto, T. Imai, G. Gachelin, H. Nakauchi, Recent duplication of the two human CD8 β-chain genes, Journal of Immunology, 148, 6, 1919-1927, 1992.01, Isolation of cDNA clones encoding the β-chain of the human T cell surface glycoprotein CD8 revealed the presence of five distinct forms of cDNA resulting from alternative splicing. In the process of analysis of the gene organization, we found that there exist two recently duplicated genes for CD8β. These genes, designated CD8β1 and β2, consist of nine and seven exons, respectively. The organization of CD8β1 and β2 genes is almost identical except in their 3'-ends. There are nine nucleotide differences between the coding regions of the CD8β1 and β2 genes in spite of the extremely high similarity of these genes which extends over the entire genes including introns. Pulse field gel analysis demonstrated that CD8β1 and β2 genes are located more than 1.5 Mb apart. It was found that the CD8β1 gene is approximately 25 kb upstream from the CD8α gene in the same transcriptional orientation on chromosome 2. Although both CD8β1 and β2 genes appear functional from the nucleotide sequence, the five distinct forms of CD8β cDNAs and corresponding mRNAs found in thymus, PBL, and leukemic cell line HPB-ALL are all derived by alternative splicing from CD8β1 transcripts..
9. Keiichi Nakayama, Dennis Y. Loh, No requirement for p56lck in the antigen-stimulated clonal deletion of thymocytes, Science, 10.1126/science.1621101, 257, 5066, 94-96, 1992.01, Activation of protein-tyrosine kinases (PTKs) is required for signal transduction during T cell activation, although the pathway used during thymic selection is unknown. An in vitro system was established in which T cell receptor transgenic thymocytes underwent clonal deletion in response to peptide antigen. The effects of two PTK-specific inhibitors, herbimycin A and genistein, on the clonal deletion of immature thymocytes and the activation of mature thymocytes were examined. Clonal deletion occurred while T cell activation was inhibited and when no p56lck activity was evident. Thus, p56 lck is not required for the antigen-stimulated step of clonal deletion of immature thymocytes, and negative selection proceeds via a distinct pathway..
10. K. Iwabuchi, Keiichi Nakayama, R. L. McCoy, F. Wang, T. Nishimura, S. Habu, K. M. Murphy, D. Y. Loh, Cellular and peptide requirements for in vitro clonal deletion of immature thymocytes, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.89.19.9000, 89, 19, 9000-9004, 1992.10, Thymocytes from DO10 T-cell-receptor transgenic mice undergo apoptosis, or programmed cell death, when chicken ovalbumin-(323-339) peptide is administered in vivo. Using DO10 mice thymocytes, we have now developed a simple in vitro model system that recapitulates the in vivo clonal-deletion process. When transgenic thymocytes were cocultured with fibroblasts, B cells, or thymic nurse cell lines (all bearing I-A(d)) in the presence of chicken ovalbumin-(323-339), deletion of the transgenic TCR+CD4+CD8+ thymocytes was seen within 8-20 hr. Thymocytes designed to bear I-A(d) on their surface could mediate the deletion themselves. Thus, thymocyte clonal deletion entirely depends on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the nature of the peptide antigen- presenting cells. Furthermore, thymic nurse cell line TNC-R3.1 could cause deletion, strongly suggesting that some thymic epithelial/stromal components are potentially capable of participating in negative selection. In all cases examined, little deletion could be induced at a peptide concentration <10 nM, thus defining the minimum amount of peptide antigen required for negative selection. The peptide-dependent in vitro negative-selection system will allow further dissection of the molecular and cellular processes involved in clonal deletion due to apoptosis in the thymus..
11. Yoichi Shinkai, Shigeo Koyasu, Keiichi Nakayama, Kenneth M. Murphy, Dennis Y. Loh, Ellis L. Reinherz, Frederick W. Alt, Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes, Science, 259, 5096, 822-825, 1993.01, Introduction of TCRα transgene, TCRβ transgene, or both into RAG-2-/- mice differentially rescues T cell development. RAG-2 -/- mice have small numbers of TCR-CD4-CD8 - (double negative, DN) thymocytes that express CD3γδ ∈ and ζ proteins intracellularly. Introduction of a TCRβ transgene, but not a TCRα transgene, into the RAG-2-/- back-ground restored normal numbers of thymocytes. These cells were CD4 +CD8+ (double positive, DP) and expressed small amounts of surface TCRβ chain dimers in association with CD3γδ∈ but not ζ. RAG-2-/- mice that expressed α and β TCR transgenes developed both DP and single positive thymocytes. Thus, the TCRβ subunit, possibly in association with a novel CD3 complex, participates in the DN to the DP transition..
12. Keiichi Nakayama, Keiko Nakayama, Izumi Negishi, Keisuke Kuida, Yoichi Shinkai, Marjorie C. Louie, Larry E. Fields, Phillip J. Lucas, Valerie Stewart, Frederick W. Alt, Dennis Y. Loh, Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice, Science, 10.1126/science.8372353, 261, 5128, 1584-1588, 1993.01, The bcl-2 proto-oncogene can prevent the death of many cell types. Mice were generated that were chimeric for the homozygous inactivation of bcl-2. Lymphocytes without Bcl-2 differentiated into phenotypically mature cells. However, in vitro, the mature T cells that lacked Bcl-2 had shorter life-spans and increased sensitivity to glucocorticoids and γ-irradiation. In contrast, stimulation of CD3 inhibited the death of these cells. T and B cells with no Bcl-2 disappeared from the bone marrow, thymus, and periphery by 4 weeks of age. Thus, Bcl-2 was dispensable for lymphocyte maturation, but was required for a stable immune system after birth..
13. Keiichi Nakayama, Rie Yamamoto, Shunsuke Ishil, Hiromitsu Nakauchl, Binding of c-Myb to the core sequence of the CD4 promoter, International Immunology, 10.1093/intimm/5.8.817, 5, 8, 817-824, 1993.08, We identified a regulatory region of the mouse CD4 promoter by both in vivoand in vitro analysis. The results of transient transfection assays indicated that the dominant transcription activating element within the CD4 promoter is located at -82 to -42. Elimination of this element, by linear deletion or specific mutation, significantly reduced transcriptional activity from this promoter. DNase I footprinting and gel mobility shift assays confirmed that the region -90 to -64 acts as the binding site of a specific nuclear factor, designated NF-CD4. In this region, an 11 bp core motif (CAACAACTGGG; -82 to -72) was found to be sufficient for the binding and transcriptional activation of the NF-CD4. This motif contains a consensus sequence for binding of c-Myb and proteins with helix-loop-helix structures. Indeed, bacterially-synthesized c-Myb specifically binds to this motif for NF-CD4. Furthermore, binding of NF-CD4 to the promoter region was specifically inhibited by the addition of anti-Myb antibodies. The evidence strongly suggests that c-Myb binds, In a sequence-specific fashion, to the core region of the CD4 promoter defined by functional assays and that this proto-oncogene product appears to play a role in the complex regulation of CD4 gene expression during T cell development..
14. Keiichi Nakayama, Hiromitsu Nakauchi, Cyclosporin a inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation, International Immunology, 10.1093/intimm/5.4.419, 5, 4, 419-426, 1993.12, Cyclosporin A (CsA) is a powerful immunosuppressive drug widely used in transplantation medicine. A major effect of CsA is inhibition of the differentiation of immature double-positive (DP) CD4+CD8+ thymocytes into mature single-positive (SP) CD4+CD8- or CD4-CD8+thymocytes. The mechanisms underlying the changes in CD4/CD8 expression during normal differentiation of thymocytes and the way CsA interferes with this differentiation process are still unknown. Here we show that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) causes a decrease of both CD4 and CD8 expression at the cell surface level and at the mRNA level in a CD4+CD8+ T cell line and in freshly isolated thymocytes. A PKC inhibitor, staurosporin, interferes with the differentiation from DP to SP in fetal thymus organ culture system. These data suggest that the alternation of CD4/CD8 expression from DP to SP is dependent on PKC activation. CsA blocks this decrease of CD4/CD8 expression by PMA in vitro. Moreover, this PMA effect is also blocked by treatment with cycloheximlde. These results suggest that the reduction of CD4/CD8 expression requires de novo synthesis of a protein(s) induced in response to a signal conveyed by activated PKC. CsA may block the transition from DP to SP by inhibition of CD4/CD8 down-regulation induced by PKC activation..
15. Keiichi Nakayama, Keiko Nakayama, Izumi Negishi, Keisuke Kuida, Marjorie C. Louie, Osami Kanagawa, Hiromitsu Nakauchi, Dennis Y. Loh, Requirement for CD8 β chain in positive selection of CD8-lineage T cells, Science, 10.1126/science.8108731, 263, 5150, 1131-1133, 1994.01, CD8 is either an αα homodimer or an αβ heterodimer, although most peripheral CD8-lineage T cells express only the CD8αβ heterodimer. The physiological function of CD8β was elucidated with mice that were chimeric for the homozygous disruption of the CD8β gene. The CD8β-/- T cells developed normally to CD4+CD8 + stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8β gene into CD8β-deficient T cells. Thus, CD8β is necessary for the maturation of CD8+ T cells..
16. Keiko Nakayama, Keiichi Nakayama, Izumi Negishi, Keisuke Kuida, Hirofumi Sawa, Dennis Y. Loh, Targeted disruption of Bcl-2αβ in mice
Occurrence of gray hair, polycystic kidney disease, and lymphocytopenia, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.91.9.3700, 91, 9, 3700-3704, 1994.04, Mice carrying ablated coding regions of the bcl-2α and bcl-2β transcripts have been made. bcl-2(-/-) mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+ T cells disappeared most quickly followed by CD4+ T cells, whereas B cells were least affected. bcl- 2(-/-) lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice..
17. P. J. Lucas, I. Negishi, Keiichi Nakayama, L. E. Fields, D. Y. Loh, Naive CD28-deficient T cells can initiate but not sustain an in vitro antigen-specific immune response, Journal of Immunology, 154, 11, 5757-5768, 1995, Naive T cells require an Ag-specific signal, as well as a costimulatory signal to mount a primary Ag-specific response. Because of their low precursor frequency, it has been difficult to study costimulatory requirements of these Ag-specific T cells. We have generated a CD28-deficient mouse that has been bred to a TCR transgenic (Tg) mouse to better study the function of CD28 during CD4+ T cell responses to Ag. In the absence of CD28, naive TCR Tg T cells responded vigorously to peptide, but responded poorly to mitogen activation. Comparison of activation-induced cell-surface molecules, including CD25, CD44, CD69, and CD71, showed no significant differences between CD28+ and CD28- TCR Tg T cells during the first 24 to 48 h after Ag stimulation. Despite relatively normal surface phenotype and normal proliferative response to Ag, CD28- T cells produced little IL-2, had a decreased sensitivity to lower Ag concentrations, and were unable to maintain their proliferative response. These results suggest that naive T cells are able to utilize other costimulatory signals to initiate a primary Ag-specific response, but require CD28 for optimal, sustained proliferation..
18. Noboru Motoyama, Fanping Wang, Kevin A. Roth, Hirofumi Sawa, Keiichi Nakayama, Keiko Nakayama, Izumi Negishi, Satoru Senju, Qing Zhang, Satoshi Fujii, Dennis Y. Loh, Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice, Science, 10.1126/science.7878471, 267, 5203, 1506-1510, 1995.01, Bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems..
19. Anita J. Merritt, Christopher S. Potten, Alastair J M Watson, Dennis Y. Loh, Keiichi Nakayama, Keiko Nakayama, John A. Hickman, Differential expression of bcl-2 in intestinal epithelia
Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia, Journal of Cell Science, 108, 6, 2261-2271, 1995.06, The cell-positional incidence of both spontaneous and damage-induced apoptosis of epithelial cells was assessed in longitudinal sections of the crypts of small intestine and colon of BDF1 mice. This was compared, using immunohistochemistry, with the pattern of expression of bcl-2, a suppressor of apoptosis. In the small intestine, apoptosis was maximal around cell position 4 from the base of the crypt; this closely corresponds to the position considered to contain the stem cells. In the colon, however, apoptosis was not confined to the area considered to harbour the stem cells (position 1 and 2). Instead, apoptosis was attenuated and distributed along the length of the crypt. Some cells at the base of murine colonic crypts expressed bcl-2 protein, whereas bcl-2 was absent in the crypts of the small intestine. Most pertinently, bcl-2 was absent from small intestinal crypt cells at positions 4-5 (the stem cell region). The importance of the expression of bcl-2 to the attenuation of apoptosis in stem cells was confirmed by analysis of the levels of both spontaneous and induced apoptosis in homozygously bcl-2 null C57BL/6 mice: in colonic crypts the level of spontaneous apoptosis rose significantly, and selectively at the base of the crypt, in comparison with crypts from wild-type animals. In contrast, there was no rise in spontaneous apoptosis in the small intestinal crypts from the bcl-2 null animals. Analysis of sections of human colon and small intestine also showed that expression of bcl-2 was confined to the base of the colonic crypt. The attenuation of apoptosis by bcl-2 in the region of the stem cells of the colonic crypts may dispose these to neoplastic transformation. Indeed, analysis of human carcinomas revealed expression of bcl-2, which in some samples was reciprocal with the expression of p53..
20. Izumi Negishi, Noboru Motoyama, Keiichi Nakayama, Keiko Nakayama, Satoru Senju, Shigetsugu Hatakeyama, Qing Zhang, Andrew C. Chan, Dennis Y. Loh, Essential role for ZAP-70 in both positive and negative selection of thymocytes, Nature, 10.1038/376435a0, 376, 6539, 435-438, 1995.08, DURING thymic development, T cells that can recognize foreign antigen in association with self major histocompatibility complex (MHC) are selected for survival (positive selection) and autoreac-tive T cells are eliminated (negative selection). Both of these selective events are mediated by interaction between the T-cell receptor (TCR) and the peptide-MHC complex1. But the signalling pathways that lead to cell survival or to cell death are still unclear. ZAP-70 is a protein tyrosine kinase (PTK) that is associated with the TCR signalling subunits (CD3 and ζ) and is expressed in T cells and natural killer cells2. It has been shown that ZAP-70 plays a crucial role in T-cell activation2-5 and development6-8. Here we show that mice lacking ZAP-70 had neither CD4 nor CDS single-positive T cells, but human ZAP-70 reconstituted both CD4 and CDS single-positive populations. Moreover, ZAP−/− thymocytes were not deleted by peptide antigens. Natural killer cell function was intact in the absence of ZAP-70. These data suggest that ZAP-70 is a central signalling molecule during thymic selection for CD4 and CD8 lineage..
21. Keiichi Nakayama, Keiko Nakayama, Lynn B. Dustin, Dennis Y. Loh, T-B cell interaction inhibits spontaneous apoptosis of mature lymphocytes in Bcl-2-deficient mice, Journal of Experimental Medicine, 10.1084/jem.182.4.1101, 182, 4, 1101-1110, 1995.10, Bcl-2 expression is tightly regulated during lymphocyte development. Mature lymphocytes in Bcl-2-deficient mice show accelerated spontaneous apoptosis in vivo and in vitro. Stimulation ofBcl-2-deficient lymphocytes by anti-CD3 antibody inhibited the spontaneous apoptosis not only in T cells but also in B cells. The rescue of B cells was dependent on the presence of T cells, mainly through CD40L and interleukin (IL)-4. Furthermore, we generated Bcl-2--deficient mice transgenic for a T cell receptor or an immunoglobulin, both specific for chicken ovalbumin, to test for antigen-specific T-B cell interaction in the inhibition of the spontaneous apoptosis. The initial T cell activation by antigenic peptides presented by B cells suppressed apoptosis in T cells. Subsequently, T cells expressed CD40L and released ILs, leading to the protection of B cells from spontaneous apoptosis. These results suggest that the antiapoptotic signaling via CD40 or IL-4 may be largely independent of Bcl-2. Engagement of the Ig alone was not sufficient for the inhibition of B cell apoptosis. Thus, the physiological role of Bcl-2 in mature lymphocytes may be to protect cells from spontaneous apoptosis and to extend their lifespans to increase the opportunity for T cells and B cells to interact with each other and specific antigens in secondary lymphoid tissues. Bcl-2, however, appears to be dispensable for survival once mature lymphocytes are activated by antigen-specific T-B cell collaboration..
22. Satoru Senju, Izumi Negishi, Noboru Motoyama, Fanping Wang, Keiichi Nakayama, Keiko Nakayama, Philip J. Lucas, Shigetsugu Hatakeyama, Qing Zhang, Shin Yonehara, Dennis Y. Loh, Functional significance of the Fas molecule in naive lymphocytes, International Immunology, 10.1093/intimm/8.3.423, 8, 3, 423-431, 1996.03, The Fas molecule mediates apoptotic signal in many cell types. Mouse mutations (Ipr, Ipr(cg), gld), which impair the function of Fas, cause spontaneous autoimmune disease. We generated Fas-deficient (Fas(-/-)) mice by homologous recombination. In embryonic stem cells Fas(-/-) mice developed Ipr-like disease, confirming that the abnormality of Fas is causal in the Ipr phenotype. We also made Fas(-/-) chimeric mice composed of a mixture of Fas(+/+) and Fas(-/-) cells. The chimeric mice also showed the Ipr phenotype. In Fas(-/-) chimeric mice, the Fas-deficient population expanded progressively among mature T and a lymphocytes. The expansion of Fas-deficient lymphocytes occurred at the naive, pre-primed, lymphocyte stage. These results suggest that the Fas molecule functions not only after antigenic stimulation, as previously hypothesized, but also at the naive lymphocyte stage..
23. Keiko Nakayama, Noriko Ishida, Michiko Shirane, Akira Inomata, Tomoaki Inoue, Nobuyuki Shishido, Ikuo Horii, Dennis Y. Loh, Keiichi Nakayama, Mice Lacking p27Kip1 Display Increased Body Size, Multiple Organ Hyperplasia, Retinal Dysplasia, and Pituitary Tumors, Cell, 10.1016/S0092-8674(00)81237-4, 85, 5, 707-720, 1996.05, Mice lacking p27Kip1 have been created by gene targeting in embryonic stem cells. These mice are larger than the control animals, with thymus, pituitary, and adrenal glands and gonadal organs exhibiting striking enlargement. CDK2 activity is elevated about 10-fold in p27-/- thymocytes. Development of ovarian follicles seems to be impaired, resulting in female sterility. Similarto mice with the Rb mutation, the p27-/- mice often develop pituitary tumors spontaneously. The retinas of the mutant mice show a disturbed organization of the normal cellular layer pattern. These findings indicate that p27Kip1 acts to regulate the growth of a variety of cells. Unexpectedly, the cell cycle arrest mediated by TGFβ, rapamycin, or contact inhibition remained intact in p27-/- cells, suggesting that p27Kip1 is not required in these pathways..
24. Michio Nagata, Hiromitsu Nakauchi, Keiichi Nakayama, Keiko Nakayama, Dennis Loh, Teruo Watanabe, Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl-2-deficient mice, American Journal of Pathology, 148, 5, 1601-1611, 1996.05, Renal development in bcl-2-deficient mice was monitored to examine the temporal and spatial function of this gene during nephrogenesis in vivo. Extensive apoptosis occurred during abnormal nephrogenesis in bcl-2-deficient mice. In embryos and newborn mice, the sequence of morphological events was monitored by morphology in conjunction with morphometry, and bcl-2 -/-, bcl- 2 +/-, and bcl-2 +/+ mice were compared. In bcl-2 -/- mice, initial induction of nephrons was detected by embryonic day 13 (E-13) as normal. Then, apoptotic cells became five times more frequent at E-13 to E-16 with a significant reduction (1/5) in nephron number at E-17 to E-19 in bcl-2 -/- mice compared with bcl-2 +/+ mice. No morphological difference was evident between bcl-2 +/- mice and bcl-2 +/+ mice by morphometry. Apoptotic cells were found mainly among the mesenchyme and less frequently in tubuli. Little apoptosis among ureteric buds was noted. In bcl-2 -/- mice at E-17 to E-19, inactive branching and insufficient convolution of ureteric buds were accompanied by fulminant apoptosis in the mesenchyme. Neonatal bcl-2 -/- mice lacked the nephrogenic zone, exhibiting renal hypoplasia. Thus, bcl-2 seems to inhibit apoptosis in renal stem cells during the induction of nephrons in vivo..
25. Keiichi Nakayama, K. Takahashi, L. D. Schultz, K. Miyakawa, K. Tomita, Erratum
Abnormal development and differentiation of macrophages and dendritic cells in viable motheaten mutant mice deficient in haematopoietic cell phosphatase (International Journal of Experimental Pathology (1997) 78 (245-257)), International Journal of Experimental Pathology, 78, 5, 1997.01.
26. Yumi Matsuzaki, Keiichi Nakayama, Keiko Nakayama, Takashi Tomita, Miu Isoda, Dennis Y. Loh, Hiromitsu Nakauchi, Role of bcl-2 in the development of lymphoid cells from the hematopoietic stem cell, Blood, 89, 3, 853-862, 1997.02, To investigate the role of bcl-2 in lymphohematopoiesis, a long-term bone marrow reconstitution system was established. Transplantation of 1,000 c-Kit+ Sca-1+ and lineage markers negative cells from bcl-2-/- mouse bone marrow resulted in long-term reconstitution of nonlymphoid cells. However, T cells were totally absent and B-lymphocyte development was severely impaired at a very early stage of differentiation in the chimeric mouse. On the other hand, transplantation of day 14 fetal liver cells from bcl-2-/- mice resulted in generation of both T and B cells in the recipient, albeit transiently. These data suggest that bcl-2 plays a critical role in the development of lymphoid progenitor cells from the hematopoietic stem cell (HSC), but is not essential for the development of nonlymphoid cells and the self-renewal of HSC. In addition, lymphopoiesis from fetal liver HSC appears to be less dependent on bcl-2 than adult bone marrow HSC..
27. Keiichi Nakayama, Keiko Nakayama, Cip/Kip cyclin-dependent kinase inhibitors
Brakes of the cell cycle engine during development, BioEssays, 10.1002/(SICI)1521-1878(199812)20:12<1020::AID-BIES8>3.3.CO;2-4, 20, 12, 1020-1029, 1998.01, Precise control of cell-cycle progression is believed to be critical for normal development, while oncogenesis may be a direct result of its disturbance. Cell-cycle progression is regulated predominantly by a series of serine/threonine kinases, the cyclin-dependent kinases (CDKs). The activities of the CDKs are controlled by a variety of mechanisms, and a group of molecules that inhibit CDK activity, CDK inhibitors (CKIs), has recently become the focus of interest, particularly in the fields of development and tumorigenesis. To date, seven CKIs have been identified in mammals and categorized into two families, the Cip/Kip and Ink4 families. The Cip/Kip family is well conserved phylogenetically, suggesting that it is biologically important. Despite the structural and biochemical similarities among the Cip/Kip members, the phenotypes of knockout mice of each Cip/Kip member are surprisingly different, which suggests that the Cip/Kip CKIs have a variety of physiological functions. In this review, the biological roles of Cip/Kip CKIs in development and tumor suppression are discussed..
28. Michio Nagata, Keiichi Nakayama, Yoshio Terada, Sachi Hoshi, Teruo Watanabe, Cell cycle regulation and differentiation in the human podocyte lineage, American Journal of Pathology, 10.1016/S0002-9440(10)65739-2, 153, 5, 1511-1520, 1998.01, Mature podocytes are regarded as growth-arrested cells with characteristic phenotypic features that underlie their function. To determine the relationship between cell cycle regulation and differentiation, the spatiotemporal expression of cyclin A, cyclin B1, cyclin D1, the cyclin- dependent kinase inhibitors (CKIs) p27 and p57, and markers of differentiating podocytes in developing human kidneys was investigated by immunohistochemistry. In S-shaped body stage, Ki-67, a cell proliferation marker that labels the G1/S/G2/M phase, was expressed in the majority (more than 80%) of presumptive podocytes, along with cyclin A (~20% of the Ki-67- positive cells) and cyclin B1 (less than 5% of Ki-67-positive cells) expression. Among these cells, cyclin D1 and CKIs were markedly down- regulated. At the capillary-loop stage, by contrast, CKIs and cyclin D1 were intensely positive in podocytes, whereas no Ki-67, cyclin B1, or cyclin A expression was seen. Moreover, double-immunolabeling and serial-section analysis provided evidence that CKIs and markers specific for differentiating podocytes, namely PHM-5 (podocalyxin-like protein in humans), synaptopodin (a foot process-related protein), and C3b receptor, were co-expressed at the capillary-loop stage. Podocytes were the only cells within the glomeruli that expressed CKIs at immunohistochemically detectable levels. Furthermore, bcl- 2 (an apoptosis inhibitory protein) showed a reciprocal expression pattern to that of CKI. These results suggest that 1) the cell cycle of podocytes is regulated by cyclin and CKIs, 2) CKIs may act to arrest the cell cycle in podocytes at the capillary-loop stage, and 3) the specific cell cycle system in podocytes may be closely correlated with their terminal differentiation in humans..
29. Shigetsugu Hatakeyama, Azumi Hamasaki, Izumi Negishi, Dennis Y. Loh, Fujiro Sendo, Keiko Nakayama, Keiichi Nakayama, Multiple gene duplication and expression of mouse bcl-2-related genes, A1, International Immunology, 10.1093/intimm/10.5.631, 10, 5, 631-637, 1998.05, Here we report the genomic cloning and characterization of the murine A1 genes, which belong to the bcl-2 gene family. Southern analysis indicated the existence of at least four A1 genes in the murine genome and four different A1 genes, designated A1-a, -b, -c and -d, were cloned from the murine genomic library. The A1-a, -b and -d genes consisted of two exons, whereas the A1-c gene contained 1 bp insertion in the coding region which may result in an aberrant and truncated protein by frame-shift. With the exception of A1-c, the coding regions among A1 genes are highly conserved at > 97% at the nucleotide level and at > 96% at the amino acid level. A1-a, -b and -d genes appeared to be expressed specifically in organs containing many neutrophils. In neutrophils, A1-a, -b and -d transcripts were detected at a comparable level. Our data suggest that the multiple A1 genes in mice were generated by gene duplication and each of them may function as antiapoptotic molecules in neutrophils..
30. Tomoaki Tanaka, Ichiro Tatsuno, Yoshihiko Noguchi, Daigaku Uchida, Toru Oeda, Shuh Narumiya, Tatsuji Yasuda, Hideaki Higashi, Masatoshi Kitagawa, Keiichi Nakayama, Yasushi Saito, Aizan Hirai, Activation of cyclin-dependent kinase 2 (Cdk2) in growth-stimulated rat astrocytes
Geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E gene expression, Journal of Biological Chemistry, 10.1074/jbc.273.41.26772, 273, 41, 26772-26778, 1998.10, The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G1/S transition. The expression of p27(kip1), cyclin D1, and the activities of Cdk4 and Cdk- activating kinase (CAK), composed of Cdk7 and cyclin H, were not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl- CoA (HMG-CoA) reductase mRNA at mid-G1 phase. Moreover, an inhibitor of HMG- CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G1 arrest in the astrocytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G1/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G1/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G1/S transition in rat astrocytes..
31. Azumi Hamasaki, Fujiro Sendo, Keiko Nakayama, Noriko Ishida, Izumi Negishi, Keiichi Nakayama, Shigetsugu Hatakeyama, Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene, Journal of Experimental Medicine, 10.1084/jem.188.11.1985, 188, 11, 1985-1992, 1998.12, To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (Al-a(-/-) mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a(-/-) mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a(+/-) mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a(-/-) and A1-a(+/-) animals. On the other hand, the extent of tumor necrosis factor α-induced acceleration of neutrophil apoptosis did not differ among A1-a(-/-), A1- a(+/-), and wild-type mice. The descending order oral mRNA expression was wild-type, A1-a(+/-), and A1-a(-/-). Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis..
32. S. Hatakeyama, M. Kitagawa, K. Nakayama, Michiko Shirane, Masaki Matsumoto, K. Hattori, H. Higashi, H. Nakano, K. Okumura, K. Onoe, R. A. Good, Keiichi Nakayama, Erratum
Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul1/F-box protein FWD1 (Proceedings of the National Academy of Sciences of the United States America (March 30, 1999) 96:7 (3859- 3863)), Proceedings of the National Academy of Sciences of the United States of America, 96, 11, 1999.01.
33. Shigetsugu Hatakeyama, Masatoshi Kitagawa, Keiko Nakayama, Michiko Shirane, Masaki Matsumoto, Kimihiko Hattori, Hideaki Higashi, Hiroyasu Nakano, Ko Okumura, Kazunori Onoé, Robert A. Good, Keiichi Nakayama, Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.96.7.3859, 96, 7, 3859-3863, 1999.03, Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF- κB through control of IκB protein stability..
34. Michiko Shirane, Yumiko Harumiya, Noriko Ishida, Aizan Hirai, Chikara Miyamoto, Shigetsugu Hatakeyama, Keiichi Nakayama, Masatoshi Kitagawa, Down-regulation of p27(Kip1) by two mechanisms, ubiquitin-mediated degradation and proteolytic processing, Journal of Biological Chemistry, 10.1074/jbc.274.20.13886, 274, 20, 13886-18893, 1999.05, The intracellular level of p27(Kip1), a cyclin-dependent kinase (CDK) inhibitory protein, is rapidly reduced at the G1/S transition phase when the cell cycle pause ceases. In this study, we demonstrated that two post- translational mechanisms were involved in p27(Kip1) breakdown: degradation via the ubiquitin (Ub)-proteasome pathway and proteolytic processing that rapidly eliminates the cyclin-binding domain. We confirmed that p27(Kip1) was ubiquitinated in vitro as well as in vivo. The p27(Kip1)-ubiquitination activity was higher at the G1/S boundary than during the G0/G1 phase, and p27(Kip1) ubiquitination was reduced significantly when the lysine residues at positions 134, 153, and 165 were replaced by arginine, suggesting that these lysine residues are the targets for Ub conjugation. In parallel with its Ub-dependent degradation, p27(Kip1) was processed rapidly at its N terminus, reducing its molecular mass from 27 to 22 kDa, by a ubiquitination- independent but adenosine triphosphate (ATP)-dependent mechanism with higher activity during the S than the G0/G1 phase. This 22-kDa intermediate had no cyclin-binding domain at its N terminus and virtually no CDK2 kinase inhibitory activity. These results suggest that p27(Kip1) is eliminated by two independent mechanisms, ubiquitin-mediated degradation and ubiquitin- independent processing, during progression from the G1 to S phase..
35. Masatoshi Kitagawa, Shigetsugu Hatakeyama, Michiko Shirane, Masaki Matsumoto, Noriko Ishida, Kimihiko Hattori, Ikuo Nakamichi, Akira Kikuchi, Keiichi Nakayama, Keiko Nakayama, An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of β-catenin, EMBO Journal, 10.1093/emboj/18.9.2401, 18, 9, 2401-2410, 1999.05, β-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of β-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. β-catenin levels are regulated by the ubiquitin-dependent proteolysis system and β-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3β (GSK-3β)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/βTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with β-catenin, Axin, GSK-3β and APC. Mutations at the signal-induced phosphorylation site of β-catenin inhibited its association with FWD1, FWD1 facilitated ubiquitination and promoted degradation of β-catenin, resulting in reduced cytoplasmic β-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized β-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCF(FWD1))-ubiquitin ligase complex is involved in β-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated β-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of β-catenin in response to external signals. SCF(FWD1) may be critical for tumor development and suppression through regulation of β-catenin protein stability..
36. Koko Urase, Takashi Momoi, Eriko Fujita, Kyoko Isahara, Yasuo Uchiyama, Akinori Tokunaga, Keiichi Nakayama, Noboru Motoyama, Bcl-xL is a negative regulator of caspase-3 activation in immature neurons during development, Developmental Brain Research, 10.1016/S0165-3806(99)00076-0, 116, 1, 69-78, 1999.08, Caspases and Bcl-xL, the mammalian homologues of the Caenorhabditis elegans (C. elegans) ced-3 and ced-9 genes, respectively, regulate apoptosis of various cells. Caspase-3 is processed into an active form (p20 or p17 and p12) during apoptosis. We investigated the relation between caspase-3 and Bcl-xL during development by examining activation of caspase-3 and apoptotic cells in Bcl-x-deficient (bcl-x(-/-)) mice at embryonic (E) day 11.5. We used a double-staining technique with a cleavage site-directed antibody against caspase-3 (anti-p20/17) and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Bcl-xL-deficiency increased both numbers of p20/17-positive and -negative apoptotic cells in dorsal root ganglia (DRG); the numbers of p20/17-positive apoptotic cells in the caudal parts of the ventral hindbrain and ventral spinal cord; and the numbers of p20/17-negative apoptotic cells in the dorsal midbrain, dorsal hindbrain, and dorsal spinal cord. Thus, Bcl-xL blocks the caspase-3-dependent apoptotic pathway in the restricted regions of the nervous system during development. Furthermore, these observations suggest that Bcl-xL protects against activation of the caspase-3-independent apoptotic pathway. Other caspases or apoptotic mechanisms may also be activated in the nervous systems of bcl-x(-/-) mice. Copyright (C) 1999 Elsevier Science B.V..
37. Yumi Watanabe, Takeshi Watanabe, Masatoshi Kitagawa, Yoichi Taya, Keiichi Nakayama, Noboru Motoyama, pRb phosphorylation is regulated differentially by cyclin-dependent kinase (Cdk) 2 and Cdk4 in retinoic acid-induced neuronal differentiation of P19 cells, Brain Research, 10.1016/S0006-8993(99)01844-2, 842, 2, 342-350, 1999.09, The retinoblastoma protein (pRb) is a key regulator of cell growth, differentiation and survival. pRb(-/-) mice show abnormal neuronal cell death in the developing brain. The function of pRb is regulated by its phosphorylation state. In this study, the phosphorylation of pRb during retinoic acid (RA)-induced neuronal differentiation of P19 cells was examined using site-specific antibodies against pRb phosphorylated at Ser601, Ser605 and Ser773. Although pRb was hyperphosphorylated in undifferentiated P19 cells, Ser601 and Ser773 were not phosphorylated. Upon exposure to RA, however, these two sites became strongly phosphorylated. Cdk4 kinase activity was almost undetectable in undifferentiated P19 cells, but was strongly activated on exposure to RA. In contrast, Cdk2 kinase activity and the phosphorylation of Ser605 were observed in undifferentiated cells as well as in RA-treated cells. These observations suggest that Cdk2 and Cdk4 may phosphorylate different sites of pRb in vivo and that the two sites of pRb examined here are newly phosphorylated during RA-induced neuronal differentiation in P19 cells..
38. Kimihiko Hattori, Shigetsugu Hatakeyama, Michiko Shirane, Masaki Matsumoto, Keiichi Nakayama, Molecular dissection of the interactions among IκBα, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of IκBα, Journal of Biological Chemistry, 10.1074/jbc.274.42.29641, 274, 42, 29641-29647, 1999.10, The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus β-TrCP) functions as the ubiquitin ligase for IκBα. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IκBα. The structural requirements for the interactions of FWD1 with IκBα and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IκBα abolished the binding of IκBα to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IκBα. The IκBα mutant D31E still exhibited binding to FWD1 and underwent ubiquitination. These results suggest that, in addition to site- specific phosphorylation at Set32 and Set36, an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IκBα. Deletion analysis of Skp1 revealed that residues 61-143 of this protein are required for binding to FWD1. On the other hand, the highly conserved residues Pro149, Ile160, and Leu164 in the F-box domain of FWD1 were dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IκBα, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex..
39. Michiko Shirane, Shigetsugu Hatakeyama, Kimihiko Hattori, Keiko Nakayama, Keiichi Nakayama, Common pathway for the ubiquitination of IκBα, Iκbβ, and IκBε mediated by the F-box protein FWD1, Journal of Biological Chemistry, 10.1074/jbc.274.40.28169, 274, 40, 28169-28174, 1999.10, FWD1 (the mouse homolog of Drosophila Slimb and Xenopus βTrCP, a member of the F-box- and WD40 repeat-containing family of proteins, and a component of the SCF ubiquitin ligase complex) was recently shown to interact with IκBα and thereby to promote its ubiquitination and degradation. This protein has now been shown also to bind to IκBβ and IκBε as well as to induce their ubiquitination and proteolysis. FWD1 was shown to recognize the conserved DSGψXS motif (where ψ represents the hydrophobic residue) present in the NH2-terminal regions of these three IκB proteins only when the component serine residues are phosphorylated. However, in contrast to IκBα and IκBβ, the recognition site in IκBε for FWD1 is not restricted to the DSGψXS motif; FWD1 also interacts with other sites in the NH2-terminal region of IκBε. Substitution of the critical setine residues in the NH2- terminal regions of IκBα, IκBβ, and IκBε with alanines also markedly reduced the extent of FWD1-mediated ubiquitination of these proteins and increased their stability. These data indicate that the three IκB proteins, despite their substantial structural and functional differences, all undergo ubiquitination mediated by the SCF(FWD1) complex. FWD1 may thus play an important role in NF-κB signal transduction through regulation of the stability of multiple IκB proteins..
40. Masatoku Miura, Shigetsugu Hatakeyama, Kimihiko Hattori, Keiichi Nakayama, Structure and expression of the gene encoding mouse F-box protein, Fwd2, Genomics, 10.1006/geno.1999.5965, 62, 1, 50-58, 1999.11, A novel class of ubiquitin ligases, termed the SCF complex, consists of invariable components, Skp1 and Cullin, and variable components called F-box proteins, which have a primary role in determining substrate specificity. We have isolated a cDNA encoding the mouse F-box protein Fwd2 (also known as MD6) as a possible constituent of an SCF-type ubiquitin ligase. Fwd2 cDNA contains 1890 bp with a 1362-bp open reading frame and encodes an approximately 51.5-kDa protein. Fwd2 is expressed predominantly in liver and, to a lesser extent, in the testis, lung, heart, and skeletal muscle. Immunofluorescence staining for Fwd2 protein shows a pattern with the cytoplasm. A coimmunoprecipitation assay has revealed the in vivo interaction between Skp1 and Fwd2 through the F-box domain. Fwd2 also interacts with Cul1 through Skp1, suggesting that Skp1, Cul1, and the F-box protein Fwd2 form an SCF complex (SCF(Fwd2)). We have also isolated and determined the nucleotide sequence and genomic organization of the gene that encodes mouse Fwd2. This gene spans approximately 17 kb and consists of six exons and five introns. Our results suggest that Fwd2 is an F-box protein that constitutes an SCF ubiquitin ligase complex and that it plays a critical role in the ubiquitin- dependent degradation of proteins expressed in the liver..
41. K. Nakayama, Keiichi Nakayama, Ubiquitin-dependent proteolysis by SCF complex, Seikagaku. The Journal of Japanese Biochemical Society, 72, 2, 113-117, 2000.
42. Kazuya Shimoda, Koji Kato, Kenichi Aoki, Tadashi Matsuda, Akitomo Miyamoto, Masafumi Shibamori, Masakatsu Yamashita, Akihiko Numata, Ken Takase, Shinji Kobayashi, Shouichirou Shibata, Yoshinobu Asano, Hisashi Gondo, Kazuo Sekiguchi, Keiko Nakayama, Toshinori Nakayama, Takashi Okamura, Seiichi Okamura, Yoshiyuki Niho, Keiichi Nakayama, Tyk2 plays a restricted role in IFNα signaling, although it is required for IL-12-mediated T cell function, Immunity, 10.1016/S1074-7613(00)00055-8, 13, 4, 561-571, 2000.01, Janus kinases (Jaks) play an important role in signal transduction via cytokine receptors. Tyk2 is a Janus kinase, and we developed tyk2-deficient mice to study the requirement for tyk2 in vivo. Tyk2-deficient mice show no overt developmental abnormalities; however, they display a lack of responsiveness to a small amount of IFNα, although a high concentration of IFNα can fully transduce its signal even in the absence of tyk2. Furthermore, IL-12-induced T cell function is defective in these mice. In contrast, these mice respond normally to IL-6 and IL-10, both of which activate tyk2 in vitro. These observations demonstrate that tyk2 plays only a restricted role in mediating IFNα-dependent signaling while being required in mediating IL-12-dependent biological responses..
43. Katsuhiko Takahashi, Keiichi Nakayama, Keiko Nakayama, Mice lacking a CDK inhibitor, p57(Kip2), exhibit skeletal abnormalities and growth retardation, Journal of biochemistry, 10.1093/oxfordjournals.jbchem.a022586, 127, 1, 73-83, 2000.01, p57(Kip2), one of the cyclin-dependent kinase (CDK) inhibitors, has been suggested to be a tumor suppressor candidate. To elucidate its biological roles in mouse development and tumorigenesis, we created p57(Kip2)-deficient mice. The p57(Kip2)-deficient mice exhibited a cleft palate and defective bone formation resulting in severe dyspnea. Most of the p57(Kip2)-deficient mice died within 24 h after birth, while about 10% of them survived beyond the weaning period. All of the surviving mice showed severe growth retardation. The males showed immaturity of the testes, prostate and seminal vesicles, and the females showed vaginal atresia, immaturity of the uterus, and an increased number of atretic follicles. Although Yan et al. and Zhang et al. have already reported p57(Kip2)-deficient mice, they could not investigate the phenotypes of the surviving p57(Kip2)-deficient mice. Also, most of the symptoms of Beckwith-Wiedemann syndrome could not be reproduced in the mutant mice. Embryonic fibroblasts prepared from p57(Kip2)-deficient mice showed no differences in the proliferation rate and saturation density, suggesting that G1 arrest is largely independent of p57(Kip2) function. Our results suggest that p57(Kip2) plays a critical role in development, but do not support the hypothesis that the p57(Kip2) gene is a tumor-suppressor gene or is responsible for Beckwith-Wiedemann syndrome..
44. A. Yamanaka, S. Hatakeyama, K. I. Kominami, M. Kitagawa, Masaki Matsumoto, Keiichi Nakayama, Cell cycle-dependent expression of mammalian E2-C regulated by the anaphase-promoting complex/cyclosome, Molecular biology of the cell, 10.1091/mbc.11.8.2821, 11, 8, 2821-2831, 2000.01, Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity..
45. Yulchiro Tojima, Atsushi Fujimoto, Mireille Delhase, Yi Chen, Shigetsugu Hatekeyama, Keiichi Nakayama, Yoko Kaneko, Yuji Nimura, Noboru Motoyama, Kyoji Ikeda, Michael Karin, Makoto Nakanishi, NAK is an IκB kinase-activating kinase, Nature, 10.1038/35008109, 404, 6779, 778-782, 2000.04, Phosphorylation of IκB by the IκB kinase (IKK) complex is a critical step leading to IκB degradation and activation of transcription factor NF- κB. The IKK complex contains two catalytic subunits, IKKα and IKKβ, the latter being indispensable for NFKB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKβ activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-κB- activating kinase), that can activate IKK through direct phosphorylation. NAK induces IκB degradation and NF-κB activity through IKKβ. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NFKB by protein kinase C-ε (PKCε). Thus, NAK is an IKK kinase that may mediate IKK and NF-κB activation in response to growth factors that stimulate PKCε activity..
46. Keiko Nakayama, Hiroyasu Nagahama, Yohji A. Minamishima, Masaki Matsumoto, Ikuo Nakamichi, Kyoko Kitagawa, Michiko Shirane, Ryosuke Tsunematsu, Tadasuke Tsukiyama, Noriko Ishida, Masatoshi Kitagawa, Keiichi Nakayama, Shigetsugu Hatakeyama, Targeted disruption of Skp2 results in accumulation of cyclin E and p27 (Kip1), polyploidy and centrosome overduplication, EMBO Journal, 19, 9, 2069-2081, 2000.05, The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G2 phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCF(Skp2) ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators..
47. Hiroyuki Takai, Kaoru Tominaga, Noboru Motoyama, Yohji A. Minamishima, Hiroyasu Nagahama, Tadasuke Tsukiyama, Kyoji Ikeda, Keiko Nakayama, Makoto Nakanishi, Keiichi Nakayama, Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice, Genes and Development, 14, 12, 1439-1447, 2000.06, The recent discovery of checkpoint kinases has suggested the conservation of checkpoint mechanisms between yeast and mammals. In yeast, the protein kinase Chk1 is thought to mediate signaling associated with the DNA damage checkpoint of the cell cycle. However, the function of Chk1 in mammals has remained unknown. Targeted disruption of Chk1 in mice showed that Chk1(-/-) embryos exhibit gross morphologic abnormalities in nuclei as early as the blastocyst stage. In culture, Chk1(-/-) blastocysts showed a severe defect in outgrowth of the inner cell mass and died of apoptosis. DNA replication block and DNA damage failed to arrest the cell cycle before initiation of mitosis in Chk1(-/-) embryos. These results may indicate that Chk1 is indispensable for cell proliferation and survival through maintaining the G2 checkpoint in mammals..
48. Kyoko Kitagawa, Toshihiro Kawamoto, Naoki Kunugita, Tadasuke Tsukiyama, Kohji Okamoto, Akira Yoshida, Keiko Nakayama, Keiichi Nakayama, Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse, FEBS Letters, 10.1016/S0014-5793(00)01710-5, 476, 3, 306-311, 2000.07, A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype. Copyright (C) 2000 Federation of European Biochemical Societies..
49. Noriko Ishida, M. Kitagawa, S. Hatakeyama, Keiichi Nakayama, Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability, Journal of Biological Chemistry, 10.1074/jbc.M001144200, 275, 33, 25146-25154, 2000.08, The association of the p27(kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser10 accounted for ≃70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was ≃25- and 75-fold greater than that at Ser178 and Thr187, respectively. The phosphorylation of p27(kip1) was markedly reduced when the positions of Ser10 and Pro11 were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser10. The extent of Ser10 phosphorylation was markedly increased in cells in the G0-G1 phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser10 was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(kip1) in which Ser10 was replaced with glutamic acid in order to mimic the effect of Ser10 phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser10 is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr187, contributes to regulation of p27(Kip1) stability..
50. H. Nagahama, S. Hatakeyama, K. Nakayama, M. Nagata, K. Tomita, Keiichi Nakayama, Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development, Anatomy and Embryology, 10.1007/s004290000146, 203, 2, 77-87, 2001, The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development..
51. Keiichi Nakayama, Shigetsugu Hatakeyama, Keiko Nakayama, Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2001.4627, 282, 4, 853-860, 2001.01, The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCFSkp2. The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed..
52. Hidetoshi Ikeda, Takashi Yoshimoto, Naoki Shida, Ichiro Miyoshi, Keiko Nakayama, Keiichi Nakayama, Masanobu Oshima, Makoto M. Taketo, Morphologic and molecular analysis of estrogen-induced pituitary tumorigenesis in targeted disruption of transforming growth factor-β receptor type II and/or p27 mice, Endocrine, 10.1385/ENDO:16:1:55, 16, 1, 55-65, 2001.01, The critical genes and products involved in estrogeninduced tumorigenesis of the pituitary gland were investigated in heterozygous transforming growth factor-β (TGF-β) receptor type II and p27 knockout mouse models. Tgfbr2(+/-), p27(+/-);Tgfbr2(+/-), and p27(+/-) mice and C57BL/6J wild-type mice received sc implantation of estrogen or placebo pellets for 16 or 25 wk, after which the mice were sacrificed and their pituitary glands removed for examination. The bromodeoxyuridine labeling indexes in tissue from both the anterior and intermediate pituitary lobes from p27 (+/-) and Tgfbr2(+/-);p27(+/-) mice were significantly higher than those from wild-type and Tgfbr2(+/-) mice after treatment with estrogen for 16 wk. Pituitary tumorigenesis was significantly accelerated in Tgfbr2(+/-), p27(+/-), and Tgfbr2(+/-);p27(+/-) mice compared with wild-type mice after treatment with estrogen for 16 wk. Pituitary tumorigenesis was not accelerated in Tgfbr2(+/-);p27(+/-) mice compared with Tgfbr2(+/-) or p27(+/-) mice. Expression of TGF-β receptor type II mRNA was lower in the pituitary gland of Tgfbr2(+/-) mice than in wild-type mice before estrogen treatment and was significantly reduced after treatment. Pituitary tumorigenesis is accelerated in mice with severe TGF-β resistance, and greatly accelerated in mice with TGF-β resistance combined with decreased p27 expression compared with wild-type mice. Both the TGF-β receptor type II gene and p27 gene and their products are involved in estrogen-induced tumorigenesis..
53. Naoko Doira, Takashi Kanematsu, Miho Matsuda, Hiroshi Takeuchi, Hito O. Nakano, Yushi Ito, Keiko Nakayama, Keiichi Nakayama, Masato Hirata, Hyperinsulinemia in PRIP-1 gene deleted mice, Biomedical Research, 10.2220/biomedres.22.157, 22, 3, 157-165, 2001.01, The protein p130, named from its molecular size, was originally identified as an inositol 1, 4,5-trisphosphate binding protein similar to phospholipase C (PLC)-δ1, but lacking any PLC activity. It was recently renamed PLC-related catalytically inactive protein-1 (PRIP-1). In the present study, PRIP-1 gene-targeted mice were analyzed for glycogen metabolism based on the previous finding that PRIP-1 interacts with protein phosphatase-1 (26). Compared to the control mice, mutant mice exhibited lower phosphorylase activity and higher levels of glycogen in the liver, which appeared to be consistent with the inhibition of protein phosphatase-1 by PRIP-1. These observation could also be attributed to the increased levels of plasma insulin. Hyperinsulinemia, observed even in the young mice, was progressive with aging, and was accompanied by the accumulation of fat tissues, increased body weight and decreased sensitivity to insulin in the mutant mice at the age of 12 months. These results suggest that PRIP-1 is a molecule involved in the control of plasma insulin..
54. T. Tsukiyama, Noriko Ishida, Michiko Shirane, Y. A. Minamishima, S. Hatakeyama, M. Kitagawa, K. Nakayama, Keiichi Nakayama, Down-regulation of p27Kip1 expression is required for development and function of T cells, Journal of Immunology, 10.4049/jimmunol.166.1.304, 166, 1, 304-312, 2001.01, The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27Kip1 is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4-CD8- thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27Kip1 expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4+CD8+, CD4+CD8-, and CD4-CD8+ stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27Kip1 expression. The development of thymocytes in the transgenic strain in which p27Kip1 is most abundant (p27-Tghigh mice) appeared to be blocked at the CD4-CD8-CD25+CD44low stage. Peripheral T cells from p27-Tghigh mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tghigh mice. These results suggest that down-regulation of p27Kip1 expression is required for the development, proliferation, and immunoresponsiveness of T cells..
55. Hirofumi Morishita, Tomoko Makishima, Chie Kaneko, Yun Shain Lee, Neil Segil, Katsuhiko Takahashi, Akio Kuraoka, Takashi Nakagawa, Junichi Nabekura, Keiko Nakayama, Keiichi Nakayama, Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2001.4939, 284, 1, 142-149, 2001.01, Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3-/- mice was significantly elevated compared to that of caspase-3+/+ mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3-/- mice at 15 days of age. Caspase-3-/- mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3-/- mice may represent a model of this human condition,.
56. Shintaro Kamizono, Toshikatsu Hanada, Hideo Yasukawa, Shigeru Minoguchi, Reiko Kato, Mayu Minoguchi, Kimihiko Hattori, Shigetsugu Hatakeyama, Masayoshi Yada, Sumiyo Morita, Toshio Kitamura, Hirohisa Kato, Keiichi Nakayama, Akihiko Yoshimura, The SOCS Box of SOCS-1 Accelerates Ubiquitin-dependent Proteolysis of TEL-JAK2, Journal of Biological Chemistry, 10.1074/jbc.M010074200, 276, 16, 12530-12538, 2001.04, Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases..
57. Hiroshi Ageta, Akihiko Kato, Shigetsugu Hatakeyama, Keiichi Nakayama, Yasushi Isojima, Hiroyuki Sugiyama, Regulation of the Level of Vesl-1S/Homer-1a Proteins by Ubiquitin-Proteasome Proteolytic Systems, Journal of Biological Chemistry, 10.1074/jbc.M011097200, 276, 19, 15893-15897, 2001.05, The vesl-1S/homer-1a gene is up-regulated during seizure and long term potentiation. Other members of the Vesl family, Vesl-1L, -2, and -3, are constitutively expressed in the brain. We examined the regulatory mechanisms governing the expression level of Vesl-1S protein, either an exogenously introduced one in COS7 or human embryonic kidney 293T cells or an endogenous one in rat brain neurons in cultures. In both cases, application of proteasome inhibitors increased the amount of Vesl-1S protein but not that of Vesl-1L, -2, or -3 protein. Deletion analyses revealed that the C-terminal 11-amino acid region was responsible for the proteolysis of Vesl-1S by proteasomes. Application of proteasome inhibitors promoted ubiquitination of Vesl-1S protein but not that of the Vesl-1S deletion mutant, which evaded proteasome-mediated degradation. These results indicate that ubiquitin-proteasome systems are involved in the regulation of the expression level of Vesl-1S protein..
58. Shigetsugu Hatakeyama, Masayoshi Yada, Masaki Matsumoto, Noriko Ishida, Keiichi Nakayama, U Box Proteins as a New Family of Ubiquitin-Protein Ligases, Journal of Biological Chemistry, 10.1074/jbc.M102755200, 276, 35, 33111-33120, 2001.08, The U box is a domain of ∼70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity..
59. Shun Ichiro Maruyama, Shigetsugu Hatakeyama, Keiko Nakayama, Noriko Ishida, Koichi Kawakami, Keiichi Nakayama, Characterization of a mouse gene (Fbxw6) that encodes a homologue of Caenorhabditis elegans SEL-10, Genomics, 10.1006/geno.2001.6658, 78, 3, 214-222, 2001.09, The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box-WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immuno-precipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently..
60. R. E. Kiernan, S. Emiliani, K. Nakayama, A. Castro, J. C. Labbé, T. Lorca, Keiichi Nakayama, M. Benkirane, Interaction between cyclin T1 and SCFSKP2 targets CDK9 for ubiquitination and degradation by the proteasome, Molecular and Cellular Biology, 10.1128/MCB.21.23.7956-7970.2001, 21, 23, 7956-7970, 2001.11, CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCFSKP2 was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45SKP2. CDK9 accumulated in p45SKP2-/- cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCFSKP2 is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9..
61. Zou Xiang, Ahmed A. Ahmed, Christine Möller, Keiichi Nakayama, Shigetsugu Hatakeyama, Gunnar Nilsson, Essential role of the prosurvival bcl-2 homologue A1 in mast cell survival after allergic activation, Journal of Experimental Medicine, 10.1084/jem.194.11.1561, 194, 11, 1561-1569, 2001.12, Mast cells reside in tissues, where upon activation through the high-affinity-IgE-receptor (FcΕRI) they degranulate and orchestrate the allergic reaction. Mast cells survive this activation and can thus be reactivated. In this study we demonstrate that this process depends on the prosurvival gene Al. Activation of mast cells through FcΕRI resulted in degranulation, strong induction of A1 mRNA and protein, and cell survival. In contrast, Al-deficient mast cells released granule mediators similar to the wild-type control, but the cells did not survive an allergic activation. Furthermore, A1-/- mice that had been sensitized and provocated with allergen exhibited a lower number of mast cell compared with littermate controls. The induction of A1 was dependent on calcium, as EDTA prevented A1 expression. The calcium ionophore, ionomycin, induced A1 expression and mast cell survival, whereas compound 48/80, a well-known mast cell secretagogue, did not. This study uncovers the importance of A1 for mast cell survival in allergic reactions, and it proposes A1 as a potential target for the treatment of allergic diseases..
62. Taichi Hara, Takumi Kamura, Keiko Nakayama, Kiyotaka Oshikawa, Shigetsugu Hatakeyama, Keiichi Nakayama, Degradation of p27Kip1 at the G0-G1 Transition Mediated by a Skp2-independent Ubiquitination Pathway, Journal of Biological Chemistry, 10.1074/jbc.M107274200, 276, 52, 48937-48943, 2001.12, Targeting of the cyclin-dependent kinase inhibitor p27Kip1 for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27Kip1 at the G0-G1 transition of the cell cycle has now been shown to proceed normally in Skp2-/- lymphocytes, whereas p27 Kip1 proteolysis during S-G2 phases is impaired in these Skp2-deficient cells. Degradation of p27Kip1 at the G 0-G1 transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2 -/- lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27Kip1 accumulation during S-G2 phases of the first cell cycle. Polyubiquitination of p27 Kip1 in the nucleus is dependent on Skp2 and phosphorylation of p27Kip1 on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2-/- cells, even with a threonine 187 → alanine mutant of p27Kip1 as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27Kip1 degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G0 to G 1..
63. Yohji A. Minamishima, Keiko Nakayama, Keiichi Nakayama, Recovery of liver mass without proliferation of hepatocytes after partial hepatectomy in Skp2-deficient mice, Cancer Research, 62, 4, 995-999, 2002, The abundance of p27Kip1, an inhibitor of cell proliferation, is determined by Skp2-dependent proteolysis, the deregulation of which is associated with cancer progression. Lack of Skp2 results in p27Kip1 accumulation as well as enlargement and polyploidy of hepatocytes. The role of Skp2 in cell growth and proliferation was investigated in Skp2-deficient mice subjected to partial hepatectomy. Skp2-/- mice exhibited restoration of liver mass without cell proliferation; rather, hepatocytes increased in size, an effect that was accompanied by increased polyploidy and p27Kip1 accumulation. Lack of Skp2 thus impairs hepatocyte proliferation, which is compensated for by cellular enlargement, during liver regeneration..
64. Shinsuke Tomari, Hiroyasu Nagahama, Yujing Shu, Sachi Hoshi, Keiko Nakayama, Keiichi Nakayama, Michio Nagata, Glomerular differentiation in p27 and p57 double-mutant metanephroi, Anatomy and Embryology, 10.1007/s00429-002-0274-5, 206, 1-2, 31-36, 2002, The cell cycle inhibitors p27 and p57 are initially concurrently expressed at a critical point of glomerulogenesis and podocyte differentiation. The present study generated mice lacking both p27 and p57, in order to investigate the synergistic roles of these molecules in glomerular differentiation. It appeared that p27 and p57 double-mutant mice died between E16.5 and E18.5, before glomerular differentiation can take place. We harvested E13.5 metanephroi to advance the glomerulogenesis in a metanephric organ culture. Metanephroi of double-mutant and wild-type mice showed no great difference in size and shape at harvest or after 6 days in culture. Histology and morphometry revealed that average glomerular size in metanephroi from double-mutant mice was significantly larger than those in any other mutants. Larger glomeruli in double-mutant metanephroi are composed of an increased number of podocytes. The glomeruli in the double-mutant metanephroi expressed synaptopodin and WT-1 with the same pattern and intensity as those found in wild type. In addition, electron micrography showed the presence of foot processes and slit membrane in podocytes in double mutant. Western blot analysis of metanephroi after 6 days in culture showed an up-regulation of p21 protein in p27 mutant and double-mutant, but not in p57 mutant metanephroi. These findings suggest that p27 and p57 are synergistically involved, in part, to determine the number, but not the differentiation, in podocytes..
65. Chiho Ikebe, Keiichi Nakayama, Chiho Ikebe, Kin ichiro Kominami, Kei Ichi Nakayama, Kin ichiro Kominami, Kei Ichi Nakayama, Takashi Toda, Isolation and characterization of a novel F-box protein Pof10 in fission yeast, Biochemical and Biophysical Research Communications, 10.1006/bbrc.2002.6344, 290, 5, 1399-1407, 2002.01, The SCF complex is a type of ubiquitin-protein ligase (E3) that consists of invariable components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Using a yeast two-hybrid system, we isolated six proteins that interact with Schizosaccharomyces pombe Skp1. Among them, Pof10 is a novel F-box protein consisting of 662 amino acids, harboring the F-box domain required for the binding to Skp1 and followed by four WD40 repeats. Overexpression of Pof10 in fission yeast resulted in loss of viability with marked morphological changes that are similar to those in pop1 mutant yeast. Coexpression of Skp1 with Pof10 prevented the lethality, suggesting that the lethality from Pof10 overexpression results from the sequestration of Skp1 from other F-box proteins including Pop1. Whereas most F-box proteins show rapid turnover, Pof10 has a remarkably long half-life in vivo and has been shown to be localized predominantly in cytoplasm. These results suggest that the stable F-box protein Pof10 might target abundant cytoplasmic proteins for degradation in fission yeast..
66. Yuzuru Imai, Mariko Soda, Shigetsugu Hatakeyama, Takumi Akagi, Tsutomu Hashikawa, Keiichi Nakayama, Ryosuke Takahashi, CHIP is associated with Parkin, a gene responsible for familial Parkinson's Disease, and enhances its ubiquitin ligase activity, Molecular Cell, 10.1016/S1097-2765(02)00583-X, 10, 1, 55-67, 2002.01, Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity..
67. Luis F. García-Fernández, Alejandro Losada, Victoria Alcaide, Álvarez Alberto M., Ana Cuadrado, Laura González, Keiko Nakayama, Keiichi Nakayama, José María Fernández-Sousa, Alberto Muñoz, José María Sánchez-Puelles, Aplidin™ induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C δ, Oncogene, 10.1038/sj.onc.1205972, 21, 49, 7533-7544, 2002.01, Aplidin™ a new antitumoural drug presently in phase II clinical trials, has shown both in vitro and in vivo activity against human cancer cells. Aplidin™ effectively inhibits cell viability by triggering a canonical apoptotic program resulting in alterations in cell morphology, caspase activation, and chromatin fragmentation. Pro-apoptotic concentrations of Aplidin™ induce early oxidative stress, which results in a rapid and persistent activation of both JNK and p38 MAPK and a biphasic activation of ERK. Inhibition of JNK and p38 MAPK blocks the apoptotic program induced by Aplidin™, demonstrating its central role in the integration of the cellular stress induced by the drug. JNK and p38 MAPK activation results in downstream cytochrome c release and activation of caspases -9 and -3 and PARP cleavage, demonstrating the mediation of the mitochondrial apoptotic pathway in this process. We also demonstrate that protein kinase C delta (PKC-δ) mediates the cytotoxic effect of Aplidin™ and that it is concomitantly processed and activated late in the apoptotic process by a caspase mediated mechanism. Remarkably, cells deficient in PKC-δ show enhanced survival upon drug treatment as compared to its wild type counterpart. PKC-δ thus appears as an important component necessary for full caspase cascade activation and execution of apoptosis, which most probably initiates a positive feedback loop further amplifying the apoptotic process..
68. Atsushi Yamanaka, Masayoshi Yada, Hiroyuki Imaki, Makoto Koga, Yasumi Ohshima, Keiichi Nakayama, Multiple Skp1-related proteins in Caenorhabditis elegans
Diverse patterns of interaction with Cullins and F-box proteins, Current Biology, 10.1016/S0960-9822(02)00657-7, 12, 4, 267-275, 2002.02, Background: The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. Results: Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities. Conclusions: The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans..
69. Kazuhiko Yoshida, Keiko Nakayama, Hiroyasu Nagahama, Takayuki Harada, Chikako Harada, Junko Imaki, Akira Matsuda, Kazuyuki Yamamoto, Miyuki Ito, Shigeaki Ohno, Keiichi Nakayama, Involvement of p27KIP1 degradation by SKp2 in the regulation of proliferation in response to wounding of corneal epithelium, Investigative Ophthalmology and Visual Science, 43, 2, 364-370, 2002.02, PURPOSE. To examine the expression of the p27KIP1 in the normal and epithelial-scraped cornea and whether degradation of p27KIP1 by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium. METHODS. C57B16, p27KIP1-/-, Skp2-/-, and Skp2-/-/p27KIP1-/- double-knockout mice were examined. Normal and epithelial-scraped corneas were analyzed by immunocytochemistry using anti-p27KIP1 antibody. Cells in the S phase of DNA synthesis were analyzed by immunocytochemistry using anti-bromodeoxyuridine (BrdU) antibody. RESULTS. The p27KIP1 was expressed in basal cells of the central and peripheral region of the cornea and limbus. This expression was not detected 24 hours after the epithelial scraping, when there were many cells in the S phase of DNA synthesis in the corneal epithelium. There were no obvious differences in the thickness and anti-BrdU staining in the corneal epithelium of p27KIP1-/- mice from that of control animals. Twenty-four hours after epithelial scraping in the Skp2-/- mice, the corneal epithelium was thinner than in wild-type mice and had many p27KIP1-positive cells and few BrdU-positive cells. In contrast, 24 hours after epithelial scraping in the Skp2-/- /p27KIP1-/- double-knockout mice, the corneal epithelium was as thick as in wild-type mice and had many BrdU-positive cells. CONCLUSIONS. These results suggest that degradation of p27KIP1 by Skp2 is involved in the regulation of cell proliferation in response to wounding of the corneal epithelium..
70. Takashi Kanematsu, Il Sung Jang, Taku Yamaguchi, Hiroyasu Nagahama, Kenji Yoshimura, Kiyoshi Hidaka, Miho Matsuda, Hiroshi Takeuchi, Yoshio Misumi, Keiko Nakayama, Tsuneyuki Yamamoto, Norio Akaike, Masato Hirata, Keiichi Nakayama, Role of the PLC-related, catalytically inactive protein p130 in GABAA receptor function, EMBO Journal, 10.1093/emboj/21.5.1004, 21, 5, 1004-1011, 2002.03, The protein p130 was isolated from rat brain as an inositol 1, 4, 5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ-aminobutyric acid (GABA). Yeast two-hybrid screening identified GABARAP (GABAA receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl-current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit..
71. Kazuya Shimoda, Hiroko Tsutsui, Kenichi Aoki, Koji Kato, Tadashi Matsuda, Akihiko Numata, Ken Takase, Tetsuya Yamamoto, Hideyuki Nukina, Tomoaki Hoshino, Yoshinobu Asano, Hisashi Gondo, Takashi Okamura, Seiichi Okamura, Keiichi Nakayama, Kenji Nakanishi, Yoshiyuki Niho, Mine Harada, Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice, Blood, 10.1182/blood.V99.6.2094, 99, 6, 2094-2099, 2002.03, Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-γ (IFN-γ) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-KB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-γ production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-γ was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the crosstalk between Jak-Stat and mitogen-activated protein kinase pathways in cytokine signaling..
72. Ken Koguchi, Yuji Nakatsuji, Keiichi Nakayama, Saburo Sakoda, Modulation of astrocyte proliferation by cyclin-dependent kinase inhibitor p27Kip1, GLIA, 10.1002/glia.10017, 37, 2, 93-104, 2002.03, We previously demonstrated that type 1 astrocytes exhibited homotypic cell contact-dependent inhibition of proliferation with increased expression of cyclin-dependent kinase inhibitor p27Kip1. Here, we investigated the functional role of p27 in contact-dependent inhibition of astrocytes and reactive gliosis in vitro and in vivo. An increase in the number of proliferating cells was detected in high-density cultures of astrocytes derived from mice carrying a targeted deletion in the p27 gene compared to astrocytes from wild-type mice. Overexpression of p27 by adenovirus vectors inhibited astrocyte proliferation, which was accompanied by downregulation of cyclin A. In a gliosis model in vitro, a transient decrease in the p27 level and an increase in the proliferation rate were observed. Astrocyte proliferation following cortical injury lasted longer in p27-deficient mice than in wild-type mice. Forced expression of p27 in both in vitro and in vivo models of gliosis effectively suppressed astrocyte proliferation. In summary, we demonstrated that p27 contributed to the cell contact-dependent inhibition of astrocyte proliferation and to the cessation of proliferation in reactive astrocytosis. p27 may be used to modulate reactive astrocytosis..
73. Noriko Ishida, Taichi Hara, Takumi Kamura, Minoru Yoshida, Keiko Nakayama, Keiichi Nakayama, Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export, Journal of Biological Chemistry, 10.1074/jbc.C100762200, 277, 17, 14355-14358, 2002.04, Phosphorylation of the cyclin-dependent kinase inhibitor p27Kip1 has been thought to regulate its stability. Ser10 is the major phosphorylation site of p27Kip1, and phosphorylation of this residue affects protein stability. Phosphorylation of p27Kip1 on Ser10 has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27Kip1 protein was translocated from the nucleus to the cytoplasm at the G0-G1 transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27Kip at the G0-G1 transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser10 with Ala (S10A) markedly reduced the extent of p27Kip1 export, whereas substitution of Ser10 with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27Kip1 on Ser10 is required for its binding to CRM1 and for its subsequent nuclear export..
74. Akitomo Miyamoto, Keiko Nakayama, Hiroyuki Imaki, Sachiko Hirose, Yi Jiang, Masaaki Abe, Tadasuke Tsukiyama, Hiroyasu Nagahama, Shigeo Ohno, Shigetsugu Hatakeyama, Keiichi Nakayama, Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cδ, Nature, 10.1038/416865a, 416, 6883, 865-869, 2002.04, Protein kinase C (PKC), which comprises 11 closely related isoforms, has been implicated in a wide variety of cellular processes, such as growth, differentiation, secretion, apoptosis and tumour development1-4. Among the PKC isotypes, PKC-δ is unique in that its overexpression results in inhibition of cell growth5-11. Here we show that mice that lack PKC-δ exhibit expansion of the B-lymphocyte population with the formation of numerous germinal centres in the absence of stimulation. The rate of proliferation in response to stimulation was greater for B cells from PKC-δ-deficient mice than for those from wild-type mice. Adoptive transfer experiments suggested that the hyperproliferation phenotype is B-cell autonomous. Production of interleukin-6 was markedly increased in B cells of PKC-δ-null mice as a result of an increase in the DNA-binding activity of NF-IL6. Furthermore, the PKC-δ-deficient mice contain circulating autoreactive antibodies and display immune-complex-type glomerulonephritis, as well as lymphocyte infiltration in many organs. These results suggest that PKC-δ has an indispensable function in negative regulation of B-cell proliferation, and is particularly important for the establishment of B-cell tolerance..
75. Takaaki Masuda, Hiroshi Inoue, Hideto Sonoda, Shinji Mine, Yasuji Yoshikawa, Keiko Nakayama, Keiichi Nakayama, Masaki Mori, Clinical and biological significance of S-phase kinase-associated protein 2 (Skp2) gene expression in gastric carcinoma
Modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis, Cancer Research, 62, 13, 3819-3825, 2002.07, Reduced expression level of p27, a cyclin-dependent kinase inhibitor, is associated with high aggressiveness and poor prognosis of various malignant tumors, including gastric carcinoma. S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of substrate-recognition subunits of Skp1-Cullin-F-box ubiquitin-protein ligase complexes, is necessary for p27 ubiquitination and degradation. In the present study, we examined the clinical and biological significance of Skp2 expression in human gastric carcinoma and the relationship between the expression of Skp2 and p27. Northern blot analysis showed that Skp2 mRNA was overexpressed in carcinoma tissues (P < 0.05), and the high Skp2 expression group showed significantly poorer prognosis in 98 patients with gastric carcinoma (P < 0.05). Immunohistochemical analysis showed that Skp2 protein was expressed predominantly in carcinoma cells. We also found an inverse correlation between the expression of Skp2 mRNA and p27 protein in vivo (P < 0.01). To analyze the biological behavior of Skp2, we established stably Skp2-transfected gastric carcinoma cell lines. Western blot analysis showed that Skp2-transfected cells expressed lower levels of p27 protein than the control cells. Skp2-transfected cells showed significantly higher levels of growth rate (P < 0.05), percentage of bromode-oxyuridine-positive cells after serum starvation (P < 0.01), resistance to apoptosis induction by actinomycin D treatment (P < 0.05), and invasion potential (P < 0.01) than the control cells. These findings indicate that Skp2 expression can modulate the malignant phenotype of gastric carcinoma, possibly via p27 proteolysis. Skp2 can play an important role in gastric carcinoma progression and would be a novel target for the treatment of gastric carcinoma as well as a strong prognostic marker..
76. Ikuo Nakamichi, Shigetsugu Hatakeyama, Keiichi Nakayama, Formation of Mallory body-like inclusions and cell death induced by deregulated expression of keratin 18, Molecular biology of the cell, 10.1091/mbc.01-10-0510, 13, 10, 3441-3451, 2002.10, Mallory bodies (MBs) are cytoplasmic inclusions that contain keratin 8 (K8) and K18 and are present in hepatocytes of individuals with alcoholic liver disease, nonalcoholic steatohepatitis, or benign or malignant hepatocellular neoplasia. Mice fed long term with griseofulvin are an animal model of MB formation. However, the lack of a cellular model has impeded understanding of the molecular mechanism of this process. Culture of HepG2 cells with griseofulvin has now been shown to induce both the formation of intracellular aggregates containing K18 as well as an increase in the abundance of K18 mRNA. Overexpression of K18 in HepG2, HeLa, or COS-7 cells also induced the formation of intracellular aggregates that stained with antibodies to ubiquitin and with rhodamine B (characteristics of MBs formed in vivo), eventually leading to cell death. The MB-like aggregates were deposited around centrosomes and disrupted the microtubular array. Coexpression of K8 with K18 restored the normal fibrous pattern of keratin distribution and reduced the toxicity of K18. In contrast, an NH2-terminal deletion mutant of K8 promoted the formation of intracellular aggregates even in the absence of K18 overexpression. Deregulated expression of K18, or an imbalance between K8 and K18, may thus be an important determinant of MB formation, which compromises the function of centrosomes and the microtubule network and leads to cell death..
77. Toyohi Isse, Tsunehiro Oyama, Kyoko Kitagawa, Koji Matsuno, Akiko Matsumoto, Akira Yoshida, Keiko Nakayama, Keiichi Nakayama, Toshihiro Kawamoto, Diminished alcohol preference in transgenic mice lacking aldehyde dehydrogenase activity, Pharmacogenetics, 10.1097/00008571-200211000-00006, 12, 8, 621-626, 2002.11, Aldehyde dehydrogenase (ALDH) 2 plays a major role in the detoxification of aldehyde and is known to be responsible for alcohol preference. A diminished enzyme activity due to mutation of the Aldh2 gene is associated with high alcohol sensitivity and a low alcohol tolerance in humans. The genomic background distinguishing an alcohol preference and avoidance in various inbred mouse strains is not clear. We created Aldh2-negative mice by transgenic knockout of the Aldh2 gene into the high alcohol preference C57BL/6 background. The Aldh2 gene targeting (Aldh-/-) mice exhibited an alcohol avoidance characteristic. After free-choice ethanol and water drinking, brain and liver acetaldehyde concentrations of Aldh2-/- mice were almost equal to those of wild-type (Aldh2+/+) mice although the Aldh2-/- mice drank less ethanol than the Aldh2+/+ mice. This result indicates that a direct effect of the Aldh2 genotype plays an important role on alcohol preference and acetaldehyde concentration in the brain is correlated with alcohol avoidance. This highlights the potential benefits of alcoholism and alcohol-related disease research in the animal model of ALDH2 alleles..
78. Kazuya Shimoda, Kenjiro Kamezaki, Akihiko Numata, Kenichi Aoki, Tadashi Matsuda, Kenji Oritani, Sadafumi Tamiya, Koji Kato, Ken Takase, Rie Imamura, Tetsuya Yamamoto, Toshihiro Miyamoto, Koji Nagafuji, Hisashi Gondo, Seiho Nagafuchi, Keiichi Nakayama, Mine Harada, Cutting edge
Tyk2 is required for the induction and nuclear translocation of Daxx which regulates IFN-α-induced suppression of B lymphocyte formation, Journal of Immunology, 169, 9, 4707-4711, 2002.11, IFN-α inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-α inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-α-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-α signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis..
79. Kazuhiko Yoshida, Takayuki Harada, Chikako Harada, Satoru Kase, Hiromi Ikeda, Masaharu Sakai, Shinzo Nishi, Junko Imaki, Keiko Nakayama, Hiroyasu Nagahama, Keiichi Nakayama, Shigeaki Ohno, Proliferative regulation in the cornea and lens, Nippon Ganka Gakkai zasshi, 107, 11, 678-686, 2003.01, PURPOSE: To examine the mechanism of regulation of proliferation in epithelial scraped cornea and the developing lens. METHOD AND RESULTS: C57B16 mouse, p27 (KIP 1)-/- mice, Skp2-/- mice and Skp2-/-/p27 (KIP 1)-/- double knockout mice were examined by immunocytochemistry using anti-p27 (KIP1) antibody, and cells in the "S" phase of DNA synthesis were analyzed by immunocytochemistry using anti-BrdU antibody. The p 27 (KIP 1) was expressed in basal cells of the central and peripheral region of the cornea and limbus. This expression was not detected 24 hr after epithelial scraping, when there were many cells in the "S" phase of DNA synthesis in the corneal epithelium. There was no obvious difference in the thickness and anti-BrdU staining in the corneal epithelium of p 27(KIP 1)-/- mice from that of controls. 24 hr after the epithelial scraping in the Skp 2-/- mice, the corneal epithelium was thinner than in wild-type mice and had many p 27(KIP 1) positive cells and few BrdU positive cells. In contrast, 24 hr after the epithelial scraping in the Skp 2-/-/p 27(KIP 1)-/- double knockout mice, the corneal epithelium was as thick as in wild-type mice and had many BrdU positive cells. CONCLUSIONS: These results suggest that degradation of p27(KIP1) by Skp 2 is involved in the regulation of proliferation in response to wounding of the corneal epithelium. To examine the involvement of the c-maf gene in the proliferation of the lens cells, eyes of the E13 and E18 stages of wild-type and c-maf-/- mice were analyzed by BrdU incorporation assay, TUNEL assay, and immunocytochemistry using an anti-P 27 (KIP 1) and an anti-P 57 (KIP 2) antibody. In the E 13 and E 18 c-maf mutant lens, BrdU-positive cells were detected at the posterior region of the lens. Cell-cycle inhibitor P 27 (KIP 1) and P 57 (KIP 2) were expressed in the equatorial and posterior region of the lens of both wild-type and c-maf-/- lenses. These results suggest that the expression of c-maf is required for differentiation and cell cycle arrest of lens cells independent of p 27 (KIP 1) and p 57 (KIP 2)..
80. Kenichi Aoki, Kazuya Shimoda, Kenji Oritani, Tadashi Matsuda, Kenjiro Kamezaki, Ryuta Muromoto, Akihiko Numata, Sadafumi Tamiya, Takashi Haro, Fumihiko Ishikawa, Ken Takase, Tetsuya Yamamoto, Taro Yumioka, Toshihiro Miyamoto, Koji Nagafuji, Hisashi Gondo, Seiho Nagafuchi, Keiichi Nakayama, Mine Harada, Limitin, an interferon-like cytokine, transduces inhibitory signals on B-cell growth through activation of Tyk2, but not Stat1, followed by induction and nuclear translocation of Daxx, Experimental Hematology, 10.1016/j.exphem.2003.08.011, 31, 12, 1317-1322, 2003.01, Objective. Limitin, an interferon-like cytokine, suppresses B lymphopoiesis through ligation of the interferon-α/β (IFN-α/β) receptor. The aim of this study was to examine the intracellular signal transduction pathways activated by limitin. Materials and Methods. The effects of limitin on cell growth, the activation of Jak kinase and Stat proteins, and the induction of interferon regulatory factor-1 (IRF-1) and Daxx were examined using the mouse pre-B-cell line 18.81, wild-type, and Tyk2-deficient mouse bone marrow cells. In addition, the change of localization of the Daxx protein after limitin treatment in wild-type and Tyk2-deficient mice was examined. Results. Limitin phosphorylates Tyk2, Jak1, Stat1, and Stat2 and rapidly induces IRF-1 mRNA production. Phosphorylation of Stat1 by limitin is partially dependent on Tyk2. Suppression of B-cell growth by limitin, however, is severely impaired in the absence of Tyk2, whereas it is unaffected by the absence of Stat1. Limitin also induces the expression and nuclear translocation of Daxx, which is essential for IFN-α-induced inhibition of B-lymphocyte development. The absence of Tyk2 abrogates this induction of Daxx expression and nuclear translocation. Conclusions. Limitin suppresses B-cell growth through activation of Tyk2, resulting in the up-regulation and nuclear translocation of Daxx. This limitin-mediated signaling pathway does not require Stat1..
81. Yohei Seto, Hiroshi Nakajima, Akira Suto, Kazuya Shimoda, Yasushi Saito, Keiichi Nakayama, Itsuo Iwamoto, Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice, Journal of Immunology, 10.4049/jimmunol.170.2.1077, 170, 2, 1077-1083, 2003.01, Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-γ and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-αβ, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2-/-) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2-/- mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2-/- mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4+ T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2-/- mice. Adoptive transfer experiments revealed that CD4+ T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2-/- mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2-/- mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2-/- mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways..
82. Chie Kaneko, Shigetsugu Hatakeyama, Masaki Matsumoto, Masayoshi Yada, Keiko Nakayama, Keiichi Nakayama, Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(02)02834-6, 300, 2, 297-304, 2003.01, UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (∼5700bp) Ube4b cDNA was isolated and the corresponding gene spans 100kb, comprising 27 exons. Luciferase reporter gene analysis of the 5 flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway..
83. Shigetsugu Hatakeyama, Keiichi Nakayama, U-box proteins as a new family of ubiquitin ligases, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(03)00245-6, 302, 4, 635-645, 2003.03, Ubiquitin-protein ligases (E3s) determine the substrate specificity of ubiquitylation and, until recently, had been classified into two families, the HECT and RING-finger families. The U-box is a domain of ∼70 amino acids that is present in proteins from yeast to humans. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. We recently showed that mammalian U-box proteins, in conjunction with an E1 and an E2, mediate polyubiquitylation in the absence of a HECT type or RING-finger type E3. U-box proteins have thus been defined as a third family of E3s. We here review recent progress in the characterization of U-box proteins and of their role in the quality control system that underlies the cellular stress response to the intracellular accumulation of abnormal proteins..
84. Koichi Kitamura, Keiko Mizuno, Akiko Etoh, Yoshiko Akita, Akitomo Miyamoto, Keiichi Nakayama, Shigeo Ohno, The second phase activation of protein kinase C δ at late G1 is required for DNA synthesis in serum-induced cell cycle progression, Genes to Cells, 10.1046/j.1365-2443.2003.00635.x, 8, 4, 311-324, 2003.04, Background: Cell lines that stably over-express protein kinase C (PKC) δ frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKCδ has a negative effect on cell proliferation. However, the mode of PKCδ activation, the cell cycle stage requiring PKCactivity, and the exact role of PKCδ at that stage remains unknown. Results: Here we show that the treatment of quiescent fibroblasts with serum activates PKCδ at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKCδ. Importantly, an inhibitor of PKCδ, rottlerin, suppresses the biphasic activation of PKCδ, and suppression of the second phase of PKCδ activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKCδ mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKCδ plays a positive role in cell cycle progression. While the over-expression of PKCδ enhances serum-induced DNA synthesis, this was not observed for PKCE. Similar experiments using a series of PKCδ/E chimeras showed that the carboxyl-terminal 51 amino acids of PKCδ are responsible for the stimulatory effect. On the other hand, the over-expression of PKCδ suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors. Conclusions: We conclude that PKCδ plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase..
85. Kiyotaka Oshikawa, Masaki Matsumoto, Masayoshi Yada, Takumi Kamura, Shigetsugu Hatakeyama, Keiichi Nakayama, Preferential interaction of TIP120A with Cul1 that is not modified by NEDD8 and not associated with Skp1, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(03)00501-1, 303, 4, 1209-1216, 2003.04, The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cul1 in the nucleus and that this interaction is mediated by a central region of Cul1 distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cul1 that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase..
86. You Wei Zhang, Keiko Nakayama, Keiichi Nakayama, Ikuo Morita, A novel route for connexin 43 to inhibit cell proliferation
Negative regulation of S-phase kinase-associated protein (Skp 2), Cancer Research, 63, 7, 1623-1630, 2003.04, Accumulated evidence suggests that connexin43 (Cx43) serves as a tumor-suppressing gene. We have previously shownA. B. that Cx43 suppressed the G1-S phase cell cycle transition via increasing the level of p27 (Zhang, Y. W., et al., Oncogene, 20: 4138-4149, 2001). Here we report that Cx43 inhibited expression of Skp2, the human F-box protein that regulates p27 ubiquitination. This reduction was attributed to an increased degradation of Skp2. The Cx43 antisense oligonucleotide blocked this inhibitory effect of Cx43 on Skp2 expression and led to p27 down-regulation. In contrast, the antisense oligonucleotide of Skp2 induced a further increase in the level of p27. However, ectopic expression of Skp2 reversed the Cx43-induced Skp2 reduction, p27 accumulation, and cell proliferation inhibition. Cx43 increased p27 expression only in the SKP2 +/+ mouse embryo fibroblasts (MEFs), but not in the SKP2 -/- MEFs, indicating that Skp2 plays a critical role in the Cx43-induced p27 up-regulation. We also show that both Skp2 and p27 are required for Cx43 to inhibit cell proliferation, in that Cx43 hardly inhibited cell proliferation of the SKP2 -/- and p27 -/- MEFs, whereas it clearly did both in the SKP2 +/- and in the p27 +/- MEFs. Our findings suggest a new route for Cx43 to inhibit tumor growth by linking it with the key cell cycle regulators..
87. Natalie Von Der Lehr, Sara Johansson, Siqin Wu, Fuad Bahram, Alina Castell, Cihan Cetinkaya, Per Hydbring, Ingrid Weidung, Keiko Nakayama, Keiichi Nakayama, Ola Söderberg, Tom K. Kerppola, Lars Gunnar Larsson, The F-box protein Skp2 participates in c-Myc proteosomal degradation and acts as a cofactor for c-Myc-regulated transcription, Molecular Cell, 10.1016/S1097-2765(03)00193-X, 11, 5, 1189-1200, 2003.05, The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S phase transition of the cell cycle in normal lymphocytes. Surprisingly, Skp2 enhances c-Myc-induced S phase transition and activates c-Myc target genes in a Myc-dependent manner. Further, Myc-induced transcription was shown to be Skp2 dependent, suggesting interdependence between c-Myc and Skp2 in activation of transcription. Moreover, Myc-dependent association of Skp2, ubiquitylated proteins, and subunits of the proteasome to a c-Myc target promoter was demonstrated in vivo. The results suggest that Skp2 is a transcriptional cofactor for c-Myc and indicates a close relationship between transcription activation and transcription factor ubiquitination..
88. Shigetsugu Hatakeyama, Keiichi Nakayama, Ubiquitylation as a quality control system for intracellular proteins, Journal of Biochemistry, 10.1093/jb/mvg106, 134, 1, 1-8, 2003.07, Quality control of intracellular proteins is essential for cellular homeostasis. Molecular chaperones recognize and contribute to the refolding of misfolded or unfolded proteins, whereas the ubiquitin-proteasome system mediates the degradation of such abnormal proteins. Ubiquitin-protein ligases (E3s) determine the substrate specificity for ubiquitylation and have been classified into HECT and RING-finger families. More recently, however, U-box proteins, which contain a domain (the U box) of about 70 amino acids that is conserved from yeast to humans, have been identified as a new type of E3. The prototype U-box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor (E4) that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and an E3 to catalyze the formation of a ubiquitin chain on artificial substrates. Yeast Ufd2 is functionally implicated in cell survival under stressful conditions. This review addresses recent progress in characterization of the role of E3 enzymes, especially that of U-box proteins, in quality control of intracellular proteins..
89. Keiko Nakayama, Shigetsugu Hatakeyama, Shun Ichiro Maruyama, Akira Kikuchi, Kazunori Onoé, Robert A. Good, Keiichi Nakayama, Impaired degradation of inhibitory subunit of NF-κB (IκB) and β-catenin as a result of targeted disruption of the β-TrCP1 gene, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1133216100, 100, 15, 8752-8757, 2003.07, β-TrCP1 (also known as Fbw1a or FWD1) is the F-box protein component of an Skp1/Cul1/F-box(SCF)-type ubiquitin ligase complex. Although biochemical studies have suggested that β-TrCP1 targets inhibitory subunit of NF-κB (IκB) proteins and β-catenin for ubiquitylation, the physiological role of β-TrCP1 in mammals has remained unclear. We have now generated mice deficient in β-TrCP1 and shown that the degradation of IκBα and IκBβ is reproducibly, but not completely, impaired in the cells of these animals. The nuclear translocation and DNA-binding activity of NF-κB as well as the ability of this transcription factor to activate a luciferase reporter gene were also inhibited in β-TrCP1-/- cells compared with those apparent in wild-type cells. The subcellular localization of β-catenin was altered markedly in β-TrCP1-/- cells. Furthermore, the rate of proliferation was reduced and both cell size and the percentage of polyploid cells were increased in embryonic fibroblasts derived from β-TrCP1-/- mice pared with the corresponding wild-type cells. These results suggest that β-TrCP1 contributes to, but is not absolutely required for, the degradation of IκB and β-catenin and the consequent regulation of the NF-κB and Wnt signaling pathways, respectively. In addition, they implicate β-TrCP1 in the maintenance of ploidy during cell-cycle progression..
90. Hiroyuki Imaki, Keiko Nakayama, Sophie Delehouzee, Hiroshi Handa, Masatoshi Kitagawa, Takumi Kamura, Keiichi Nakayama, Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein, Cancer Research, 63, 15, 4607-4613, 2003.08, Skp2 is the F-box protein component of an SCF-type ubiquitin ligase that interacts specifically with p27Kip1 and thereby promotes its ubiquitylation and degradation. The abundance of Skp2 mRNA oscillates in a cell cycle-dependent manner, being maximal in S and G2 phases. The regulation of Skp2 transcription was investigated by cloning the promoter region of the mouse gene and determination of its activity in a luciferase reporter assay. Deletion analysis identified a minimal ∼0.3-kb promoter region with marked transcriptional activity and a 105-bp essential sequence within this region. Electrophoretic mobility shift assays indicated the presence in nuclear extracts of proteins that bind to this sequence. Site-directed mutagenesis revealed that the core binding motif, CACTTCCG, which is similar to that of GA-binding protein (GABP), is essential for Skp2 transcription. "Supershift" analysis indicated that the protein-probe complexes detected by electrophoretic mobility shift assays contain GABP. Endogenous GABP bound to Skp2 promoter element in a cell cycle-dependent manner. Furthermore, overexpression of GABPβ increased Skp2 promoter activity, and suppression of GABPα or GABPβ by a small interfering RNA resulted in the reduction of Skp2 promoter activity. These data suggest that the cell cycle-dependent binding of GABP to the Skp2 promoter plays an important role in the regulation of Skp2 expression and cell cycle progression from G1 to S phase..
91. Takumi Kamura, Taichi Hara, Shuhei Kotoshiba, Masayoshi Yada, Noriko Ishida, Hiroyuki Imaki, Shigetsugu Hatakeyama, Keiko Nakayama, Keiichi Nakayama, Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1831009100, 100, 18, 10231-10236, 2003.09, The abundance of the cyclin-dependent kinase (CDK) inhibitor p57 Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57 Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57 Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1..
92. James S. Foster, Romaine I. Fernando, Noriko Ishida, Keiichi Nakayama, Jay Wimalasena, Estrogens Down-regulate p27Kip1 in Breast Cancer Cells through Skp2 and through Nuclear Export Mediated by the ERK Pathway, Journal of Biological Chemistry, 10.1074/jbc.M302830200, 278, 42, 41355-41366, 2003.10, The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer, p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17β-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16 Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 → Ala; S10A) interfering with CRM1/ p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1 caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway..
93. Tong Shin Chang, Myung Jin Kim, Kanghyun Ryoo, Jihyun Park, Soo Jung Eom, Jaekyung Shim, Keiichi Nakayama, Keiko Nakayama, Motowo Tomita, Katsuhiko Takahashi, Min Jae Lee, Eui Ju Choi, p57KIP2 Modulates Stress-activated Signaling by Inhibiting c-Jun NH2-terminal Kinase/Stress-activated Protein Kinase, Journal of Biological Chemistry, 10.1074/jbc.M309421200, 278, 48, 48092-48098, 2003.11, p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK..
94. Keiichi Nakayama, Mechanisms to control degradation of polyglutamine-containing protein, Clinical Neurology, 43, 11, 906-908, 2003.11, Machado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is neurodegenerative disease which is caused by polyglutamine expansion in a responsible gene product, MJD1/Ataxin3.MJD1 has now been shown to undergo ubiquitylation and degradation by proteasome-dependent pathway. MJD1 with expanded polyglutamine tract was more resistant to degradation than normal MJD1. We established an in vitro system of ubiquitylation of MJD1, thereby biochemically purified activity to mediate polyubiquitylation of MJD1 from rabbit reticulocyte lysate. An AAA-family ATPase VCP was isolated from the active fraction, and found to binds to MJD1. Furthermore, UFD2a, a mammalian ubiquitin-chain assembly factor (E4), associated with VCP and induced polyubiquitylation of MJD1. UFD2a markedly promoted ubiquitylation and degradation of MJD1 with expanded polyglutamine tract, resulting in the clearance of MJD1 protein. In contrast, dominant-negative mutant UFD2a reduced the degradation rate of MJD1, leading to the formation of intracellular aggregation. In Drosophila model, overexpression of UFD2a significantly suppressed the neurodegeneration induced by expression of MJD1 with expanded polyglutamine tract. These findings suggest that E4 is a rate-limiting factor of degradation of pathologic polyglutamine-containing proteins, and may give a potential tool for gene therapy to control the clinical conditions of MJD..
95. Koji Kato, Kenjiro Kamezaki, Kazuya Shimoda, Akihiko Numata, Takashi Haro, Kenichi Aoki, Fumihiko Ishikawa, Ken Takase, hiroshi ariyama, Tadashi Matsuda, Toshihiro Miyamoto, Koji Nagafuji, Hisashi Gondo, Keiichi Nakayama, Mine Harada, Intracellular signal transduction of interferon on the suppression of haematopoietic progenitor cell growth, British Journal of Haematology, 10.1046/j.1365-2141.2003.04650.x, 123, 3, 528-535, 2003.11, Interferon (IFN)-α and IFN-γ suppress the growth of haematopoietic progenitor cells. IFN-α activates Janus kinase-1 (Jak1) and Tyrosine kinase-2 (Tyk2), followed by the phosphorylation of the signal transducers and activators of transcription, Stat1 and Stat2. IFN-γ activates Jak1 and Jak2, followed by the activation of Stat1. Activated Stats bind the promoter regions of IFN-inducible genes. We evaluated the role of Tyk2 and Stat1 in the IFN-mediated inhibition of haematopoietic progenitor cell growth. While IFN-α (1000 U/ml) suppressed the number of granulocyte-macrophage colony-forming units (CFU-GM) or erythroid burst-forming units (BFU-E) from wild-type mouse bone marrow cells, this suppression was partially inhibited by a deficiency in Tyk2 and completely inhibited by a deficiency in Stat1. High levels of IFN-α (10 000 U/ml) suppressed the CFU-GM or BFU-E obtained from Stat1-deficient mice, but did not suppress this growth in cells from Tyk2-deficient mice, Stat1 was phosphorylated by IFN-α in Tyk2-deficient cells, although the level of phosphorylation was weaker than that observed in wild type mice. Thus, the inhibitory signal on haematopoietic progenitor cells mediated by IFN-α may be transduced by two signalling pathways, one regulated by Tyk2 and the other dependent on Stat1. IFN-γ also suppressed the number of CFU-GM or BFU-E, and this pathway was mediated by IFN-γ in a Stat1-dependent manner, independently of Tyk2..
96. Etsuo Susaki, Keiichi Nakayama, Deregulated Proteolysis of the Cell Cycle Regulator p27Kip1 and Cancers, Biotherapy, 17, 6, 546-555, 2003.11, Reduced expression of p27, a member of CDK inhibitors, is observed in a variety of human cancers, and many studies have indicated that downregulation of the p27 expression correlates with poor prognosis. p27 acts as a negative regulator of the cell cycle, and its degradation by the ubiquitin-proteasome pathway is a critical event for the cell cycle progression to S phase. The degradation of p27 is regulated by phosphorylations prior to ubiquitination that is mediated by the SCFSkp2 ubiquitin ligase complex. The low levels of p27 protein in many cancer cells seem attributable to enhanced proteolysis. In fact, increased levels of Skp2 protein are found in many tumors, and the overexpression of Skp2 may also be related to the high grade of cancers. Previous reports have suggested that p27 is clinically useful as an independent prognostic marker for cancer patients, and trials of new treatments targeting p27 have already started. p27 is also implicated to play a role in other cell functions, such as cell differentiation, apoptosis, and cell-cell adhesion. Thus, a variety of mechanisms seem to underlie the deregulation of p27 in cancer cells, which remain to be elucidated..
97. Takayuki Tsukuba, Kuniaki Okamoto, Yoshiko Okamoto, Michiyo Yanagawa, Keiko Kohmura, Yoshiyuki Yasuda, Uchi Hiroshi, Takeshi Nakahara, Masutaka Furue, Keiko Nakayama, Tomoko Kadowaki, Kenji Yamamoto, Keiichi Nakayama, Association of Cathepsin E Deficiency with Development of Atopic Dermatitis, Journal of Biochemistry, 10.1093/jb/mvg216, 134, 6, 893-902, 2003.12, Atopic dermatitis (AD) is a pruritic inflammatory skin diseases associated with a family history of atropy. Here we show that mice lacking the endolysosomal aspartic proteinase cathepsin E spontaneously develop skin lesions similar to those of humans with AD when reared under conventional conditions but not under specific pathogen-free conditions. These mice showed the increase in the ratio of CD4+/CD8+ T cells, the strong polarization of naïve T cells to T helper 2 cells, and the systemic accumulation of IL-18 and IL-1β accompanied by a marked increase in IL-4, IL-5, and IgE. The relative rates of degradation of IL-18 and IL-1β were significantly lower in cathepsin E-deficient mice than wild-type mice. These results strongly suggest that the development of AD in cathepsin E-deficient mice is initiated by systemic accumulation of IL-18 and IL-1β, mainly due to their reduced turnover rates. In addition, the reduced expression of cathepsin E was also observed in erythrocytes of both humans with AD and the AD mouse model NC/Nga. Cathepsin E deficiency might thus be responsible for the induction of AD in humans and mice..
98. Yumiko Mori, Koichi Hirose, Kotaro Suzuki, Hiroshi Nakajima, Yohei Seto, Kei Ikeda, Kazuya Shimoda, Keiichi Nakayama, Yasushi Saito, Itsuo Iwamoto, Tyk2 is essential for IFN-α-induced gene expression in mast cells, International Archives of Allergy and Immunology, 10.1159/000077789, 134, SUPPL. 1, 25-29, 2004, Mast cells are recognized not only as the major effector cells of type I hypersensitivity reactions but also as an important player of innate immune response against bacterial infection. Type I IFNs are also involved in the response against bacterial infection. However, the role of type I IFNs and their associated Janus kinase Tyk2 in mast cell functions remains to be determined. In this study, we addressed this issue using Tyk2-deficient (Tyk2-/-) bone marrow-derived mast cells (BMMCs). When BMMCs from wild-type (WT) mice were stimulated with IFN-α, they expressed mRNA for IFN-γ-inducible protein 10 (IP-10) and monocyte chemoattractant protein-5 (MCP-5). Interestingly, IFN-α-induced expression of IP-10 and MCP-5 was severely decreased in Tyk2-/- BMMCs. In addition, IFN-α-induced Stat1 phosphorylation was decreased in Tyk2-/- BMMCs. On the other hand, IFN-α-induced Stat1 phosphorylation and IP-10 and MCP-5 expression were normal in Tyk2-/- fibroblasts. These results indicate that IFN-α induces the expression of TNF-α and the chemokines IP-10 and MCP-5 in mast cells and that Tyk2 plays a nonredundant role in IFN-α signaling in mast cells..
99. Florian Blaschke, Olli Leppanen, Yasunori Takata, Evren Caglayan, Joey Liu, Michael C. Fishbein, Kai Kappert, Keiichi Nakayama, Alan R. Collins, Eckart Fleck, Willa A. Hsueh, Ronald E. Law, Dennis Bruemmer, Liver X receptor agonists suppress vascular smooth muscle cell proliferation and inhibit neointima formation in balloon-injured rat carotid arteries., Circulation research, 95, 12, 2004.01, The liver X receptors alpha and beta (LXRalpha and LXRbeta) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1-->S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1-->S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase-associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1-->S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand-mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org..
100. Michiko Shirane, Keiichi Nakayama, Immunophilin FKBP38, an inherent inhibitor of calcineurin, targets Bcl-2 to mitochondria and inhibits apoptosis, Nippon rinsho. Japanese journal of clinical medicine, 62, 2, 405-412, 2004.01, Various apoptotic stimuli induce mitochondrial dysfunction. Bcl-2 and Bcl-xL antagonize apoptosis by blocking the release of caspase activators such as cytochrome c from mitochondria. We demonstrated that FKBP38, a member of the immunophilin family, interacts and targets these anti-apoptotic proteins Bcl-2 and Bcl-xL, thereby assisting them in their pro-survival role. FKBP38 is specifically localized on mitochondria, at which FKBP38 is colocalized with Bcl-2 and Bcl-xL. Expression of exogenous FKBP38 promotes mitochondrial targeting of Bcl-2 and Bcl-xL, while dominant-negative FKBP38 or siRNA of FKBP38 disturbs their localization. On the other hand, unlike FKBP12, FKBP38 inhibits serine/threonine phosphatase calcineurin in an FK506-independent manner. Overexpression of FKBP38 inhibits apoptosis, while expression of dominant-negative FKBP38 or depletion of endogenous FKBP38 increases the sensitivity for apoptosis. Thus, FKBP38 has unique features among members of the immunophilin family..
101. Daisuke Uchida, Shigetsugu Hatakeyama, Akemi Matsushima, Hongwei Han, Satoshi Ishido, Hak Hotta, Jun Kudoh, Nobuyoshi Shimizu, Vassilis Doucas, Keiichi Nakayama, Noriyuki Kuroda, Mitsuru Matsumoto, AIRE Functions as an E3 Ubiquitin Ligase, Journal of Experimental Medicine, 10.1084/jem.20031291, 199, 2, 167-172, 2004.01, Autoimmune regulator (AIRE) gene mutation is responsible for the development of autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy, an organ-specific autoimmune disease with monogenic autosomal recessive inheritance. AIRE is predominantly expressed in medullary epithelial cells of the thymus and is considered to play important roles in the establishment of self-tolerance. AIRE contains two plant homeodomain (PHD) domains, and the novel role of PHD as an E3 ubiquitin (Ub) ligase has just emerged. Here we show that the first PHD (PHD1) of AIRE mediates E3 ligase activity. The significance of this finding was underscored by the fact that disease-causing missense mutations in the PHD1 (C311Y and P326Q) abolished its E3 ligase activity. These results add a novel enzymatic function for AIRE and suggest an indispensable role of the Ub proteasome pathway in the establishment of self-tolerance, in which AIRE is involved..
102. Masaki Matsumoto, Masayoshi Yada, Shigetsugu Hatakeyama, Hiroshi Ishimoto, Teiichi Tanimura, Shoji Tsuji, Akira Kakizuka, Masatoshi Kitagawa, Keiichi Nakayama, Molecular clearance of ataxin-3 is regulated by a mammalian E4, EMBO Journal, 10.1038/sj.emboj.7600081, 23, 3, 659-669, 2004.02, Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3..
103. Ohtsuka Takao, Hoon Ryu, Yohji A. Minamishima, Salvador Macip, Junji Sagara, Keiichi Nakayama, Stuart A. Aaronson, Sam W. Lee, ASC is a Bax adaptor and regulates the p53-Bax mitochondrial apoptosis pathway, Nature Cell Biology, 10.1038/ncb1087, 6, 2, 121-128, 2004.02, The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein-protein interaction domain1-5. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53-Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53-Bax network..
104. Ryosuke Tsunematsu, Keiko Nakayama, Yuichi Oike, Masaaki Nishiyama, Noriko Ishida, Shigetsugu Hatakeyama, Yasumasa Bessho, Ryoichiro Kageyama, Toshio Suda, Keiichi Nakayama, Mouse Fbw7/Sel-10/Cdc4 Is Required for Notch Degradation during Vascular Development, Journal of Biological Chemistry, 10.1074/jbc.M312337200, 279, 10, 9417-9423, 2004.03, Mammalian Fbw7 (also known as Sel-10, hCdc4, or hAgo) is the F-box protein component of an SCF (Skp1-Cul1-F-box protein-Rbx1)-type ubiquitin ligase, and the mouse Fbw7 is expressed prominently in the endothelial cell lineage of embryos. We generated mice deficient in Fbw7 and found that the embryos died in utero at embryonic day 10.5-11.5, manifesting marked abnormalities in vascular development. Vascular remodeling was impaired in the brain and yolk sac, and the major trunk veins were not formed. In vitro para-aortic splanchnopleural explant cultures from Fbw7-/- embryos also manifested an impairment of vascular network formation. Notch4, which is the product of the proto-oncogene Int3 and an endothelial cell-specific mammalian isoform of Notch, accumulated in Fbw7-/- embryos, resulting in an increased expression of Hey1, which encodes a transcriptional repressor that acts downstream of Notch signaling and is implicated in vascular development. Expression of Notch1, -2, or -3 or of cyclin E was unaffected in Fbw 7-/- embryos. Mammalian Fbw7 thus appears to play an indispensable role in negative regulation of the Notch4-Hey1 pathway and is required for vascular development..
105. Kwang Jin Oh, Anna Kalinina, Jing Wang, Keiko Nakayama, Keiichi Nakayama, Srilata Bagchi, The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase, Journal of Virology, 10.1128/JVI.78.10.5338-5346.2004, 78, 10, 5338-5346, 2004.05, Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2-/- mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells..
106. Keiko Nakayama, Hiroyasu Nagahama, Yohji A. Minamishima, Satoshi Miyake, Noriko Ishida, Shigetsugu Hatakeyama, Masatoshi Kitagawa, Shun ichiro Iemura, Tohru Natsume, Keiichi Nakayama, Skp2-Mediated degradation of p27 regulates progression into mitosis, Developmental Cell, 10.1016/S1534-5807(04)00131-5, 6, 5, 661-672, 2004.05, Although Skp2 has been thought to mediate the degradation of p27 at the G1-S transition, Skp2 -/- cells exhibit accumulation of p27 in S-G2 phase with overreplication. We demonstrate that Skp2 -/- p27 -/- mice do not exhibit the overreplication phenotype, suggesting that p27 accumulation is required for its development. Hepatocytes of Skp2 -/- mice entered the endoduplication cycle after mitogenic stimulation, whereas this phenotype was not apparent in Skp2 -/- p27 -/- mice. Cdc2-associated kinase activity was lower in Skp2 -/- cells than in wild-type cells, and a reduction in Cdc2 activity was sufficient to induce overreplication. The lack of p27 degradation in G2 phase in Skp2 -/- cells may thus result in suppression of Cdc2 activity and consequent inhibition of entry into M phase. These data suggest that p27 proteolysis is necessary for the activation of not only Cdk2 but also Cdc2, and that Skp2 contributes to regulation of G2-M progression by mediating the degradation of p27..
107. Masayoshi Yada, Shigetsugu Hatakeyama, Takumi Kamura, Masaaki Nishiyama, Ryosuke Tsunematsu, Hiroyuki Imaki, Noriko Ishida, Fumihiko Okumura, Keiko Nakayama, Keiichi Nakayama, Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7, EMBO Journal, 10.1038/sj.emboj.7600217, 23, 10, 2116-2125, 2004.05, The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7-1- embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively..
108. Kazuhiko Yoshida, Keiko Nakayama, Satoru Kase, Hiroyasu Nagahama, Takayuki Harada, Hiromi Ikeda, Chikako Harada, Junko Imaki, Kazuhiro Ohgami, Kenji Shiratori, Shigeaki Ohno, Shinzo Nishi, Keiichi Nakayama, Involvement of p27(KIP1) in proliferation of the retinal pigment epithelium and ciliary body, Anatomy and Embryology, 10.1007/s00429-004-0382-5, 208, 2, 145-150, 2004.05, According to observations in various cell lines, elimination of the cyclin-dependent kinase inhibitor p27(KIP1) during the late G1 phase of the cell cycle is required for progression to the S phase. Eyes from C57BL/6 mice at embryonic days 13, 14, and 18, and at 4 weeks of age, were analyzed by a bromodeoxyuridine cell proliferation assay and by immunocytochemistry using anti-p27(KIP1) antibody. On embryonic days 14 and 18, p27(KIP1) was detected in the ciliary body. This protein also was detected in the nuclei of the many cells of the retinal pigment epithelium on embryonic day 18, and was present in all such cells at 4 weeks of age. When p27(KIP1)-/- knockout and control mice were injected with bromodeoxyuridine between postnatal days 7 and 10 and analyzed on day 11, positive cells were abundant in the retinal pigment epithelium and the ciliary body of p27(KIP1)-/- mice, whereas few cells were positive in control mice. By fluorescent nuclear staining in whole mounts of retinal pigment epithelium at 12 weeks of age, more nuclei were present in p27(KIP1)-/- than in the wild-type mice. These results suggest that p27(KIP1) was involved in regulation of proliferation in the RPE and the ciliary body..
109. Kazuhiko Yoshida, Satoru Kase, Keiko Nakayama, Hiroyasu Nagahama, Takayuki Harada, Hiromi Ikeda, Chikako Harada, Junko Imaki, Kazuhiro Ohgami, Kenji Shiratori, Shigeaki Ohno, Keiichi Nakayama, Distribution of p27(KIP1), cylin D1, and proliferating cell nuclear antigen after retinal detachment, Graefe's Archive for Clinical and Experimental Ophthalmology, 10.1007/s00417-004-0861-7, 242, 5, 437-441, 2004.05, Purpose: To examine the expression of the p27(KIP1), cyclin D1, and proliferating cell nuclear antigen (PCNA) in the retina and retinal pigment epithelium (RPE) after retinal detachment. Methods: Normal eyes and eyes at 2 or 4 days after retinal detachment with the C57B16 mouse were analyzed by immunocytochemistry using anti-p27(KIP1), anti-cyclin D1, and anti-proliferating cell nuclear antigen (PCNA) antibodies as well as anti-glutamate synthetase (GS) antibody. Results: fhe p27(KIP1) positive nuclei were distributed in the inner nuclear layer (INL) and the RPE of the normal mice eye. In the INL, p27(KIP1) was detected in the middle sublayer, where the nuclei of glutamate synthetase positive Müller cells were situated. In contrast, cyclin D1 was not detected either in the retina or in the RPE. At 2 and 4 days after the retinal detachment, RPE cells under the detached retina were negative for p27(KIP1) and positive for cyclin D1 and PCNA. In the INL of the detached retina, p27(KIP1) was detected after 2 days, but was not detected after 4 days. In contrast, PCNA was not detected in the INL after 2 days, but was detected after 4 days. Cyclin D1 was detected in the middle sublayer of the INL at both 2 and 4 days after the retinal detachment. Conclusion: These results suggested that degradation of p27(KIP1) and expression of cyclin D1 was involved in the proliferation of the Müller cells as well as RPE cells after retinal detachment..
110. Shigetsugu Hatakeyama, Masaki Matsumoto, Masayoshi Yada, Keiichi Nakayama, Interaction of U-box-type ubiquitin-protein ligases (E3s) with molecular chaperones, Genes to Cells, 10.1111/j.1356-9597.2004.00742.x, 9, 6, 533-548, 2004.06, Members of the U-box family of proteins constitute a class of ubiquitin-protein ligases (E3s) distinct from the HECT-type and RING finger-containing E3 families. Two representative mammalian U-box proteins, UFD2a and CHIP, interact with the molecular chaperones VCP and either Hsp90 or Hsc70, respectively, and are implicated in the degradation of damaged proteins. We have now investigated the roles of mammalian U-box proteins by performing a comprehensive screen for molecules that interact with these proteins in the yeast two-hybrid system. All mammalian U-box proteins tested were found to interact with molecular chaperones or cochaperones, including Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP. These observations suggest that the function of U box-type E3s is to mediate the degradation of unfolded or misfolded proteins in conjunction with molecular chaperones as receptors that recognize such abnormal proteins..
111. Kazuhiko Yoshida, Satoru Kase, Keiko Nakayama, Hiroyasu Nagahama, Takayuki Harada, Hiromi Ikeda, Chikako Harada, Junko Imaki, Kazuhiro Ohgami, Kenji Shiratori, Iliyana Bozhidarova Ilieva, Shigeaki Ohno, Shinzo Nishi, Keiichi Nakayama, Involvement of p27KIP1 in the proliferation of the developing corneal endothelium, Investigative Ophthalmology and Visual Science, 10.1167/iovs.03-1238, 45, 7, 2163-2167, 2004.07, PURPOSE. To examine the involvement of p27KIP1 in the regulation of the proliferation of the developing corneal endothelium. METHODS. Central and peripheral corneas in C57Bl6 mice at postnatal day (P)1, P11, and 12 weeks after birth were analyzed by immunocytochemistry with anti-p27KIP1, -p57KIP2, and -proliferating cell nuclear antigen (PCNA) antibodies. Nuclear staining was performed with 4′,6′-diamino-2-phenylindole (DAPI) in wholemounts of corneal endothelium of the center and peripheral cornea in wild-type and p27KIP1 knockout (-/-) mice at 12 weeks of age. p27KIP1-/- and control mice were injected with bromodeoxyuridine (BrdU) once on P7, twice per day on P8 and P9, and once on P10 and then were analyzed by a BrdU cell-proliferation assay on P11. RESULTS. On P1, p27 KIP1 immunoreactivity was detected in a small number of corneal endothelial cells, and many endothelial cells expressed PCNA. At P11 and 12 weeks after birth, p27KIP1 immunoreactivity was detected in many corneal endothelial cells. PCNA-positive cells in the endothelium were rare on P11 and completely absent at 12 weeks after birth. p57KIP2 was not detected in either corneal epithelium or endothelium at P1, P11, or 12 weeks after birth. In wholemounts of corneal endothelium at 12 weeks of age, the number of endothelial nuclei in the p27KIP1-/- mice was significantly higher than that in wild-type mice in both the center and peripheral regions of the cornea. In the BrdU assay, positive cells were abundant in the corneal endothelium of p27KIP1-/- mice, whereas there were few positive cells in control mice. PCNA immunoreactivity in the endothelium of the p27 KIP1-/- mice was completely absent at 12 weeks after birth. CONCLUSIONS. These results suggest that p27KIP1 is involved in the regulation of proliferation in the endothelium of the developing cornea..
112. Makoto Urushitani, Junko Kurisu, Minako Tateno, Shigetsugu Hatakeyama, Keiichi Nakayama, Shinsuke Kato, Ryosuke Takahashi, CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70, Journal of Neurochemistry, 10.1111/j.1471-4159.2004.02486.x, 90, 1, 231-244, 2004.07, Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SODl-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and poly-ubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1G93A transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome..
113. Kenjiro Kamezaki, Kazuya Shimoda, Akihiko Numata, Tadashi Matsuda, Keiichi Nakayama, Mine Harada, The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals, International Immunology, 10.1093/intimm/dxh118, 16, 8, 1173-1179, 2004.08, Mice lacking Tyk2, Stat1 or Stat4, which are members of the Jak-Stat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-κB by LPS, and the production of TNF-α and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4 In Stat1-deficient mice, the induction of IFN-β by LPS in macrophages was severely reduced, although the serum level of IFN-γ was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-β by LPS was normal, but the serum level of IFN-γ remained low after LPS injection. Interestingly, the induction of both IFN-β and IFN-γ by LPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LPS. Tyk2 is essential for LPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4..
114. Hiroko Akiyoshi, Shigetsugu Hatakeyama, Jukka Pitkänen, Yasuhiro Mouri, Vassilis Doucas, Jun Kudoh, Kyoko Tsurugaya, Daisuke Uchida, Akemi Matsushima, Kiyotaka Oshikawa, Keiichi Nakayama, Nobuyoshi Shimizu, Pärt Peterson, Mitsuru Matsumoto, Subcellular expression of autoimmune regulator is organized in a spatiotemporal manner, Journal of Biological Chemistry, 10.1074/jbc.M400702200, 279, 32, 33984-33991, 2004.08, Autoimmune regulator (AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body (NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner..
115. Miho Terunuma, Il Sung Jang, Sang Hoon Ha, Josef T. Kittler, Takashi Kanematsu, Jasmina N. Jovanovic, Keiichi Nakayama, Norio Akaike, Sung Ho Ryu, Stephen J. Moss, Masato Hirata, GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein, Journal of Neuroscience, 10.1523/JNEUROSCI.1323-04.2004, 24, 32, 7074-7084, 2004.08, GABAA receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABAA receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAA receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABAA receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors..
116. Baojie Li, Xueying Wang, Naslin Rasheed, Yuanyu Hu, Sharon Boast, Tetsuro Ishii, Keiko Nakayama, Keiichi Nakayama, Stephen P. Golf, Distinct roles of c-Abl and Atm in oxidative stress response are mediated by protein kinase C δ, Genes and Development, 10.1101/gad.1223504, 18, 15, 1824-1837, 2004.08, c-Abl and Atm have been implicated in cell responses to DNA damage and oxidative stress. However, the molecular mechanisms by which they regulate oxidative stress response remain unclear. In this report, we show that deficiency of c-Abl and deficiency of ATM differentially altered cell responses to oxidative stress by induction of antioxidant protein peroxiredoxin I (Prx I) via Nrf2 and cell death, both of which required protein kinase C (PKC) δ activation and were mediated by reactive oxygen species. c-abl-/- osteoblasts displayed enhanced Prx I induction, elevated Nrf2 levels, and hypersusceptibility to arsenate, which were reinstated by reconstitution of c-Abl; Atm-/- osteoblasts showed the opposite. These phenotypes correlated with increased PKC δ expression in c-abl-/- osteoblasts and decreased PKC δ expression in Atm-/- cells, respectively. The enhanced responses of c-abl-/- osteoblasts could be mimicked by overexpression of PKC δ in normal cells and impeded by inhibition of PKC δ, and diminished responses of Atm-/- cells could be rescued by PKC δ overexpression, indicating that PKC δ mediated the effects of c-Abl and ATM in oxidative stress response. Hence, our results unveiled a previously unrecognized mechanism by which c-Abl and Atm participate in oxidative stress response..
117. Tatsuo Furuyama, Kazuko Kitayama, Yuri Shimoda, Minetaro Ogawa, Kiyoaki Sone, Kiyomi Yoshida-Araki, Hiroshi Hisatsune, Shin Ichi Nishikawa, Keiko Nakayama, Keiichi Nakayama, Kyoji Ikeda, Noboru Motoyama, Nozomu Mori, Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice, Journal of Biological Chemistry, 10.1074/jbc.M314214200, 279, 33, 34741-34749, 2004.08, Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhr11), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development..
118. Shigetsugu Hatakeyama, Masaki Matsumoto, Takumi Kamura, Miyuki Murayama, Du Hua Chui, Emmanuel Planel, Ryosuke Takahashi, Keiichi Nakayama, Akihiko Takashima, U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy, Journal of Neurochemistry, 10.1111/j.1471-4159.2004.02713.x, 91, 2, 299-307, 2004.10, Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons..
119. Tomoko Komatsu, Hirofumi Mizusaki, Tokuo Mukai, Hidesato Ogawa, Daichi Baba, Masahiro Shirakawa, Shigetsugu Hatakeyama, Keiichi Nakayama, Hideki Yamamoto, Akira Kikuchi, Ken-Ichirou Morohashi, Small ubiquitin-like modifier 1 (SUMO-1) modification of the synergy control motif of Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) regulates synergistic transcription between Ad4BP/SF-1 and Sox9, Molecular Endocrinology, 10.1210/me.2004-0173, 18, 10, 2451-2462, 2004.10, An orphan nuclear receptor, Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1), is essential for the development and function of steroidogenic tissues. To examine the transcriptional regulation of Ad4BP/SF-1, two-hybrid screening was performed, and the sumoylation [conjugation of a small ubiqutin-like modifier (SUMO-1)] components Ubc9, protein inhibitor of activated STAT1 (PIAS1), and protein inhibitor of activated STAT 3 (PIAS3) were isolated. Cultured cell and in vitro studies revealed that Ad4BP/SF-1 is sumoylated at K119 and K194. Because K194 lies within the synergy control (SC) motif defined to repress synergistic transcription from promoters containing multiple binding sites, correlation between the functions of the SC motif and sumoylation was investigated. The K194R mutant of Ad4BP/SF-1, which cannot be sumoylated, showed enhanced synergistic transcription from a promoter containing multiple Ad4/SF-1 sites, suggesting that sumoylation is necessary for repression of transcriptional synergy through the SC motif. It has been established that the Müllerian inhibiting substance gene is transcribed predominantly under the control of Ad4BP/SF-1 and, moreover, its transcription is regulated synergistically with Sox9, Gata4, and Wt1. Interestingly, it was found that all of these factors are sumoylated, and these sumoylation sites occur within SC motifs. Based on the observation that SC motif mutants of Ad4BP/SF-1 and Sox9 resulted in the enhancement of their synergistic transcription, it was concluded that the SC motif regulates synergistic transcription even between distinct types of transcription factors. Considering that both mutants cannot be sumoylated, it is likely that sumoylation is implicated in this regulation. Because it was revealed with an in vitro sumoylated Ad4BP/SF-1 that DNA binding activity and interaction with Sox9 were unaffected, sumoylation may regulate transcription through affecting selective and cooperative interaction among factors constituting transcriptional complexes..
120. Taku Yamaguchi, Takashi Kubota, Takashi Kanematsu, Keiichi Nakayama, Masato Hirata, Tsuneyuki Yamamoto, Hypersensitivity to pentylenetetrazol-induced convulsion in mice lacking the PLC-related inactive protein-1, Brain Research, 10.1016/j.brainres.2004.08.009, 1025, 1-2, 237-240, 2004.10, In the present study, we investigated the effects of pentylenetetrazol (PTZ), a chemical convulsant that interacts with the GABA A receptor, in mice lacking the phospholipase C (PLC)-related inactive protein-1 (PRIP-1). PRIP-1 knockout mice did not develop spontaneous behavioral seizure. PRIP-1 knockout mice had markedly shorter latencies until the first clonic convulsion (CL) and tonic extensor (TE) following PTZ administration and increased incidence of convulsion compared to those in wild-type mice. Furthermore, the mortality rate by PTZ in mice lacking the PRIP-1 was also significantly increased in comparison with that in wild-type mice. These findings suggested that mice lacking the PRIP-1 were hypersensitive to PTZ-induced convulsion, and PRIP-1 might play roles in suppressing excessive excitability via interactions with the GABA A receptor..
121. Masaaki Nishiyama, Keiko Nakayama, Ryosuke Tsunematsu, Tadasuke Tsukiyama, Akira Kikuchi, Keiichi Nakayama, Early embryonic death in mice lacking the β-catenin-binding protein duplin, Molecular and Cellular Biology, 10.1128/MCB.24.19.8386-8394.2004, 24, 19, 8386-8394, 2004.10, The Wnt signaling pathway plays a pivotal role in vertebrate early development and morphogenesis. Duplin (axis duplication inhibitor) interacts with β-catenin and prevents its binding to Tcf, thereby inhibiting downstream Wnt signaling. Here we show that Duplin is expressed predominantly from early to mid-stage mouse embryogenesis, and we describe the generation of mice deficient in Duplin. Duplin-/-embryos manifest growth retardation from embryonic day 5.5 (E5.5) and developmental arrest accompanied by massive apoptosis at E7.5. The mutant embryos develop into an egg cylinder but do not form a primitive streak or mesoderm. Expression of β-catenin target genes, including those for T (brachyury), Axin2, and cyclin Dl, was not increased in Duplin-/- embryos, suggesting that the developmental defect is not simply attributable to upregulation of Wnt signaling caused by the lack of this inhibitor. These results suggest that Duplin plays an indispensable role, likely by a mechanism independent of inhibition of Wnt signaling, in mouse embryonic growth and differentiation at an early developmental stage..
122. Sara Tokarz, Catherine Berset, Janna La Rue, Kevin Friedman, Keiichi Nakayama, Keiko Nakayama, Dong Er Zhang, Stefan Lanker, The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCF Skp2 ubiquitin ligase, Journal of Biological Chemistry, 10.1074/jbc.M403189200, 279, 45, 46424-46430, 2004.11, The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-β. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling..
123. Mimi Tamamori-Adachi, Kentaro Hayashida, Kiyoshi Nobori, Chie Omizu, Kazuhiko Yamada, Naoya Sakamoto, Takumi Kamura, Keiichi Fukuda, Satoshi Ogawa, Keiichi Nakayama, Shigetaka Kitajima, Down-regulation of p27Kip1 promotes cell proliferation of rat neonatal cardiomyocytes induced by nuclear expression of cyclin D1 and CDK4
Evidence for impaired Skp2-dependent degradation of p27 in terminal differentiation, Journal of Biological Chemistry, 10.1074/jbc.M403084200, 279, 48, 50429-50436, 2004.11, Mammalian cardiomyocytes lose their capacity to proliferate during terminal differentiation. We have previously reported that the expression of nuclear localization signal-tagged cyclin D1 (D1NLS) and its partner cyclin-dependent kinase 4 (CDK4) induces proliferation of rat neonatal cardiomyocytes. Here we show that the D1NLS/CDK4 cells, after their entry into the cell cycle, accumulated cyclin-dependent kinase inhibitor p27 in the nuclei and decreased the cyclin-dependent kinase 2 (CDK2) activity, leading to early cell cycle arrest. Biochemical analysis demonstrated that Skp2-dependent p27 ubiquitylation was remarkably suppressed in cardiomyocytes, whereas Skp2, a component of Skp1-Cullin-F-box protein ubiquitin ligase, was more actively ubiquitylated compared with proliferating rat fibroblasts. Specific degradation of p27 by co-expressing Skp2 or p27 small interfering RNA caused an increase of CDK2 activity and overrode the limited cell cycle. These data altogether indicate that the impaired Skp2-dependent p27 degradation is causally related to the loss of proliferation in cardiomyocytes. This provides a novel insight in understanding the molecular mechanism by which mammalian cardiomyocytes cease to proliferate during terminal differentiation..
124. Takumi Kamura, Katsumi Maenaka, Shuhei Kotoshiba, Masaki Matsumoto, Daisuke Kohda, Ronald C. Conaway, Joan Weliky Conaway, Keiichi Nakayama, VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases, Genes and Development, 10.1101/gad.1252404, 18, 24, 3055-3065, 2004.12, The ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS-box proteins recruit substrates to the ECS complex and are linked to Cullin-Rbx via Elongin B/C. VHL has been implicated as a SOCS-box protein, but lacks a C-terminal sequence (downstream of the BC box) of the SOCS box. We now show that VHL specifically interacts with endogenous Cul2-Rbx1 in mammalian cells, whereas SOCS-box proteins associate with Cul5-Rbx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL box, including the BC box and downstream residues) and associate with Cul2-Rbx1. ECS complexes can thus be classified into two distinct protein assemblies, that is, those that contain a subunit with a VHL box (composed of the BC box and a downstream Cul2 box) that interacts with Cul2-Rbx1, and those that contain a subunit with a SOCS box (BC box and downstream Cul5 box) that interacts with Cul5-Rbx2. Domain-swapping analyses showed that the specificity of interaction of VHL-box and SOCS-box proteins with Cullin-Rbx modules is determined by the Cul2 and Cul5 boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-Rbx1 inhibited the VHL-mediated degradation of HIF-2α, whereas knockdown of Cul5-Rbx2 did not affect it. These data suggest that the functions of the Cul2-Rbx1 and Cul5-Rbx2 modules are distinct..
125. Fumihiko Okumura, Shigetsugu Hatakeyama, Masaki Matsumoto, Takumi Kamura, Keiichi Nakayama, Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis, Journal of Biological Chemistry, 10.1074/jbc.M402916200, 279, 51, 53533-53543, 2004.12, E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Cζ (PKCζ). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCζ. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys 27 of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCζ. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation..
126. Jian Hua Mao, Jesus Perez-Iosada, Di Wu, Reyno DelRosario, Ryosuke Tsunematsu, Keiichi Nakayama, Ken Brown, Sheila Bryson, Allan Balmain, Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene, Nature, 10.1038/nature03155, 432, 7018, 775-779, 2004.12, The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identity the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7 +/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 smali interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers..
127. Takumi Kamura, Taichi Hara, Masaki Matsumoto, Noriko Ishida, Fumihiko Okumura, Shigetsugu Hatakeyama, Minoru Yoshida, Keiko Nakayama, Keiichi Nakayama, Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27Kip1 at G1 phase, Nature Cell Biology, 10.1038/ncb1194, 6, 12, 1229-1235, 2004.12, The cyclin-dependent kinase inhibitor p27Kip1 is degraded at the G0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCFSkp2 is implicated in p27Kip1 degradation, proteolysis of p27Kip1 at the G0-G1 transition proceeds normally in Skp2-/- cells. Moreover, p27Kip1 is exported from the nucleus to the cytoplasm at G0-G1 (refs 9-11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27Kip1 at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27Kip1 and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27Kip1, whereas a dominant-negative mutant of KPC1 delayed p27Kip1 degradation. The nuclear export of p27Kip1 by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27Kip1 degradation. KPC thus probably controls degradation of p27Kip1 in G1 phase after export of the latter from the nucleus..
128. Amer M. Mirza, Stephan Gysin, Nisar Malek, Keiichi Nakayama, James M. Roberts, Martin McMahon, Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT, Molecular and Cellular Biology, 10.1128/MCB.24.24.10868-10881.2004, 24, 24, 10868-10881, 2004.12, The RAS-activated RAF→MEK→extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′-kinase (PI3′-kinase) →PDK1→AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G1 cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27 Kip1 expression. Repression of p27Kip1 was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27Kip1. Consistent with these observations, pharmacological inhibition of MEK or PI3′-kinase inhibited the effects of activated RAS on the expression of p27Kip1 in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21Cip1 from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF→MEK→ERK and PI3′K→PDK→AKT signaling pathways can cooperate to promote G0→G1→S-phase cell cycle progression in both normal and cancer cells..
129. Geneviève Rodier, Constantin Makris, Philippe Coulombe, Anthony Scime, Keiko Nakayama, Keiichi Nakayama, Sylvain Meloche, p107 inhibits G1 to S phase progression by down-regulating expression of the F-box protein Skp2, Journal of Cell Biology, 10.1083/jcb.200404146, 168, 1, 55-66, 2005.01, Cell cycle progression is negatively regulated by the pocket proteins pRb, p107, and p130. However, the mechanisms responsible for this inhibition are not fully understood. Here, we show that overexpression of p107 in fibroblasts inhibits Cdk2 activation and delays S phase entry. The inhibition of Cdk2 activity is correlated with the accumulation of p27, consequent to a decreased degradation of the protein, with no change of Thr187 phosphorylation. Instead, we observed a marked decrease in the abundance of the F-box receptor Skp2 in p107-overexpressing cells. Reciprocally, Skp2 accumulates to higher levels in p107-/- embryonic fibroblasts. Ectopic expression of Skp2 restores p27 down-regulation and DNA synthesis to the levels observed in parental cells, whereas inactivation of Skp2 abrogates the inhibitory effect of p107 on S phase entry. We further show that the serum-dependent increase in Skp2 half-life observed during G1 progression is impaired in cells overexpressing p107. We propose that p107, in addition to its interaction with E2F, inhibits cell proliferation through the control of Skp2 expression and the resulting stabilization of p27..
130. Shuhei Kotoshiba, Keiichi Nakayama, The degradation of p27 and cancer, Nippon rinsho. Japanese journal of clinical medicine, 63, 11, 2047-2056, 2005.01, The cell cycle of eukaryotic cells is regulated by a series of protein complexes composed of cyclins and cyclin-dependent kinases (CDKs), the activity of which is suppressed by a group of CDK inhibitors (CKIs). Among the CKIs, p27 plays a pivotal role in the control of cell proliferation. Degradation of p27 is a critical event for reentry of cells into the cell cycle from G0 phase and occurs through ubiquitination by two ubiquitin ligase complexes (KPC and SCFSkP2) and subsequent degradation by the 26S-proteasome. A tumor suppressing function of p27 has been demonstrated in mouse models and studies of human tumors. This review will focus on the regulation of p27 proteolysis and its consequences for tumorigenesis..
131. Yojiro Kotake, Keiko Nakayama, Noriko Ishida, Keiichi Nakayama, Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation, Journal of Biological Chemistry, 10.1074/jbc.M406117200, 280, 2, 1095-1102, 2005.01, The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in iransfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 "knock-in" mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27S10A/S10A mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27S10A/S10A mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G 0-G1 transition occurred normally in p27 S10A/S10A mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process..
132. Keiichi Nakayama, Keiko Nakayama, Regulation of the cell cycle by SCF-type ubiquitin ligases, Seminars in Cell and Developmental Biology, 10.1016/j.semcdb.2005.02.010, 16, 3, 323-333, 2005.01, Regulation of the cell cycle is dependent on protein degradation by the ubiquitin-proteasome system. Two major ubiquitin ligases, the anaphase-promoting complex or cyclosome (APC/C) and SCF complex, are responsible for the periodic proteolysis of many regulators of the cell cycle. The receptor component of the SCF complex is one of many F-box proteins, three of which - Skp2, Fbw7, and β-TrCP - are well characterized and implicated in cell cycle regulation. We have generated mice deficient in Skp2, Fbw7, or β-TrCP1 and have identified the roles of these proteins in both cell cycle regulation and mouse development. Clinical evidence also suggests that dysregulation of these F-box proteins contributes to human cancers..
133. Satoru Kase, Kazuhiko Yoshida, Keiichi Nakayama, Keiko Nakayama, Hiromi Ikeda, Takayuki Harada, Chikako Harada, Kazuhiro Ohgami, Kenji Shiratori, Shigeaki Ohno, Phosphorylation of p27(KIP1) in the developing retina and retinoblastoma., International journal of molecular medicine, 16, 2, 257-262, 2005.01, Cellular distribution of the p27(KIP1) protein and its phosphorylation on threonine (T) 187 in mouse retinas from three stages of development, and retinoblastoma were examined. Retinas in C57Bl6 mice at embryonic day (E) 14, postnatal day (P) 1 and P11 were analyzed using immunohistochemistry with anti-p27(KIP1), threonine-187-phosphorylated p27(KIP1) (T187-phospho-p27), bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) antibodies, and phosphorylated histon H3 (pHiston H3), which is a marker for cells in M phase. p27(KIP1) knockout (-/-) mice and human retinoblastoma were also analyzed. T187-phospho-p27 was detected in the outermost layer of the retina, whereas several neuroblastic cells expressed p27(KIP1) at E14. Many neuroblastic cells expressed BrdU in the middle layer. At P1, p27(KIP1) was detected in the ganglion cell layer and neuroblastic layer. T187-phospho-p27 was detected in the outermost layer, and that was localized in mitotic cells that also showed pHiston H3-positive. At P11, p27(KIP1) was detected in the inner nuclear layer, whereas T187-phospho-p27-positive or mitotic cells were not. BrdU positive nuclei were not detected in wild-type but were noted in the inner nuclear layer and the outer nuclear layer of the p27(KIP1) -/- mice retina at P11. In retinoblastoma, tumor cells formed numerous rosettes with Flexner-Wintersteiner rosettes. Several pHiston H3 -positive nuclei were noted in the tumor cells forming Flexner-Wintersteiner rosettes. Several T187-phospho-p27-positive nuclei were also detected in the mitotic cells forming Flexner-Wintersteiner rosettes. PCNA was expressed in rosette-forming cells. In conclusion, T187-phospho-p27(KIP1) was correlated with M phase of the cell cycle in the developing retina and retinoblastoma..
134. Shigetsugu Hatakeyama, Masaki Matsumoto, Keiichi Nakayama, Mapping of ubiquitination sites on target proteins, Methods in enzymology, 10.1016/S0076-6879(05)99019-8, 399, 277-286, 2005.01, Although the identification of ubiquitin-conjugated lysine residues on target proteins is extremely significant, to date it is generally quite difficult to identify ubiquitinated sites by usual mutation analysis. More recently, the technology of mass spectrometry is answering these difficult questions. In this chapter, we introduce the method of purification of ubiquitinated target proteins using affinity chromatography with anti-polyubiquitin antibody and the identification of ubiquitinated lysine residues on target proteins using mass spectrometry. Using these techniques, we can obtain comprehensive information about ubiquitinated proteins in various cells and tissues..
135. Chuan Hua He, Aaron B. Waxman, Chun Geun Lee, Holger Link, Morgan E. Rabach, Bing Ma, Qingsheng Chen, Zhou Zhu, Mei Zhong, Keiko Nakayama, Keiichi Nakayama, Robert Homer, Jack A. Elias, Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury, Journal of Clinical Investigation, 10.1172/JCI23004, 115, 4, 1039-1048, 2005.01, Hyperoxic acute lung injury (HALI) is characterized by a cell death response with features of apoptosis and necrosis that is inhibited by IL-11 and other interventions. We hypothesized that Bfl-1/A1, an antiapoptotic Bcl-2 protein, is a critical regulator of HALI and a mediator of IL-11-induced cytoprotection. To test this, we characterized the expression of A1 and the oxygen susceptibility of WT and IL-11 Tg(+) mice with normal and null A1 loci. In WT mice, 100% O2 caused TUNEL+ cell death, induction and activation of intrinsic and mitochondrial-death pathways, and alveolar protein leak. Bcl-2 and Bcl-xl were also induced as an apparent protective response. A1 was induced in hyperoxia, and in A1-null mice, the toxic effects of hyperoxia were exaggerated, Bcl-2 and Bcl-xl were not induced, and premature death was seen. In contrast, IL-11 stimulated A1, diminished the toxic effects of hyperoxia, stimulated Bcl-2 and Bcl-xl, and enhanced murine survival in 100% O2. In A1-null mice, IL-11-induced protection, survival advantage, and Bcl-2 and Bcl-xl induction were significantly decreased. VEGF also conferred protection via an A1-dependent mechanism. In vitro hyperoxia also stimulated A1, and A1 overexpression inhibited oxidant-induced epithelial cell apoptosis and necrosis. A1 is an important regulator of oxidant-induced lung injury, apoptosis, necrosis, and Bcl-2 and Bcl-xl gene expression and a critical mediator of IL-11- and VEGF-induced cytoprotection..
136. Kae Harada, Hiroshi Takeuchi, Masahiro Oike, Miho Matsuda, Takashi Kanematsu, Hitoshi Yagisawa, Keiichi Nakayama, Katsumasa Maeda, Christophe Erneux, Masato Hirata, Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling, Journal of cellular physiology, 10.1002/jcp.20136, 202, 2, 422-433, 2005.02, PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-δ1 (PLC-δ1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1 -/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1 -/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,S)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1 PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P 3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3..
137. Hidehisa Takahashi, Shigetsugu Hatakeyama, Hisato Saitoh, Keiichi Nakayama, Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein, Journal of Biological Chemistry, 10.1074/jbc.M408130200, 280, 7, 5611-5621, 2005.02, SUMO-1 is a member of a family of ubiquitin-like molecules that are post-translationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation. To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait. This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb). A deletion mutant of TDGb (TDGb(Δ11)) that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an acljacent lysine residue, suggesting that such binding is required for covalent modification. In contrast, another mutant of TDGb (TDGb(KR)) in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 noncovalently. TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells. TDGb(KR) also colocalized with PML, whereas TDGb(Δ11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML. Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins. These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through post-translational modification and noncovalent protein-protein interaction..
138. Tohru Uchida, Takehiro Nakamura, Naoko Hashimoto, Tomokazu Matsuda, Ko Kotani, Hiroshi Sakaue, Yoshiaki Kido, Yoshitake Hayashi, Keiichi Nakayama, Morris F. White, Masato Kasuga, Deletion of Cdkn1b ameliorates hyperglycemia by maintaining compensatory hyperinsulinemia in diabetic mice, Nature medicine, 10.1038/nm1187, 11, 2, 175-182, 2005.02, The protein p27Kip1 regulates cell cycle progression in mammals by inhibiting the activity of cyclin-dependent kinases (CDKs). Here we show that p27Kip1 progressively accumulates in the nucleus of pancreatic beta cells in mice that lack either insulin receptor substrate 2 (Irs2-/-) or the long form of the leptin receptor (Lepr-/- or db/db). Deletion of the gene encoding p27Kip1 (Cdkn1b) ameliorated hyperglycemia in these animal models of type 2 diabetes mellitus by increasing islet mass and maintaining compensatory hyperinsulinemia, effects that were attributable predominantly to stimulation of pancreatic beta-cell proliferation. Thus, p27Kip1 contributes to beta-cell failure during the development of type 2 diabetes in Irs2-/- and Lepr-/- mice and represents a potential new target for the treatment of this condition..
139. Desmond Jackson, Yang Zheng, Donggon Lyo, Yinjie Shen, Keiko Nakayama, Keiichi Nakayama, Michael J. Humphries, Mary E. Reyland, David A. Foster, Suppression of cell migration by protein kinase Cδ, Oncogene, 10.1038/sj.onc.1208465, 24, 18, 3067-3072, 2005.04, The ability of cancer cells to migrate is strongly correlated with malignant progression and metastasis. Survival signals that suppress apoptosis have also been linked to increased cell motility. We previously reported that suppression of protein kinase Cδ (PKCδ) provided survival signals in a rat fibroblast model system. These studies have been extended to human breast cancer cells with differential cell motilities and PKCδ levels. BT-549 cells, which lack detectable expression of PKCδ, migrate very efficiently, whereas MCF-7 cells, which express high levels of PKCδ, migrate very poorly. Ectopic expression of PKCδ suppressed cell migration in the BT-549 cells, and downregulation of PKCδ enhanced cell migration in the MCF-7 cells. Downregulation of PKCδ in the MCF-7 cells also led to increased secretion of the matrix metalloprotease MMP-9. The migration of mouse embryo fibroblasts (MEFs) from wild type and PKCδ knockout mice was also examined and MEFs from PKCδ knockout mice had a five-fold increase in eel migration relative to the wild-type MEFs. These data provide evidence that PKCδ suppresses cell migration in both human breast cancer cells and in primary mouse fibroblasts, and indicate that the loss of PKCδ in human cancers could contribute to both eel survival and metastasis..
140. Shuhei Kotoshibai, Takumi Kamura, Taichi Hara, Noriko Ishida, Keiichi Nakayama, Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase, Journal of Biological Chemistry, 10.1074/jbc.M500866200, 280, 18, 17694-17700, 2005.05, The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G 0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway in a Skp2-independent manner. We recently identified a novel ubiquitin ligase, KPC (Kip1 ubiquitylation-promoting complex), consisting of KPC1 and KPC2, which regulates the ubiquitin-dependent degradation of p27 at G1 phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH2-terminal region of KPC1 was found to be responsible for binding to KPC2 and to p27. KPC1 mutants that lack this region failed to mediate polyubiquitylation of p27 in vitro and expression of one such mutant delayed p27 degradation in vivo. We also generated a series of deletion mutants of p27 and found that KPC failed to polyubiquitylate a p27 mutant that lacks the CDK inhibitory domain. Interestingly, the cyclin E-CDK2 complex prevented both the interaction of KPC with p27 as well as KPC-mediated polyubiquitylation of p27. A complex of cyclin E with a kinase-negative mutant of CDK2 also exhibited these inhibitory effects, suggesting that cyclin E-CDK2 competes with KPC1 for access to the CDK inhibitory domain of p27. These results suggest that free p27 is recognized by the NH2-terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1..
141. Hao Jiang, Fu Chung Chang, Ashley E. Ross, Jihyun Lee, Keiichi Nakayama, Keiko Nakayama, Stephen Desiderio, Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle, Molecular Cell, 10.1016/j.molcel.2005.05.011, 18, 6, 699-709, 2005.06, The periodic destruction of RAG-2 at the G1-to-S transition couples V(D)J recombination to the G0 and G1 cell cycle phases and coordinates RAG-mediated DNA cleavage with DNA repair by nonhomologous end joining. To define the mechanism by which this occurs, we reproduced cell cycle-dependent regulation of the V(D)J recombinase in a cell-free system. The ubiquitin-proteasomal pathway carries out destruction of RAG-2 in lysates of S phase cells and during S phase in vivo. Remarkably, the Skp2-SCF ubiquitin ligase, which plays a central role in cell cycle regulation through the destruction of p27, mediates ubiquitylation of RAG-2 in vitro and degradation of RAG-2 in vivo. The regulation of antigen receptor gene assembly by Skp2-SCF provides an unexpected and direct mechanistic link between DNA recombination and the cell cycle..
142. Hua Qin Wang, Yoshifumi Nakaya, Zhenyu Du, Takuya Yamane, Michiko Shirane, Takashi Kudo, Masatoshi Takeda, Koichi Takebayashi, Yoichi Noda, Keiichi Nakayama, Masaki Nishimura, Interaction of presenilins with FKBP38 promotes apoptosis by reducing mitochondrial Bcl-2, Human Molecular Genetics, 10.1093/hmg/ddi195, 14, 13, 1889-1902, 2005.07, Presenilins 1 and 2 (PS1/2), causative molecules for familial Alzheimer's disease (FAD), are multipass transmembrane proteins localized predominantly in the endoplasmic reticulum (ER) and Golgi apparatus. Heteromeric protein complexes containing PS1/2 are thought to participate in several functions, including intramembrane proteolysis mediated by their γ-secretase activities. Previous studies have shown that PS1/2 are also involved in the regulation of apoptotic cell death, although the underlying mechanism remains unknown. Here, we demonstrate that FKBP38, an immunophilin family member residing in the mitochondrial membrane, is an authentic PS1/2-interacting protein. PS1/2 and FKBP38 form macromolecular complexes together with anti-apoptotic Bcl-2. PS1/2 promote the degradation of FKBP38 and Bcl-2 and sequester these proteins in the ER/Golgi compartments, thereby inhibiting FKBP38-mediated mitochondrial targeting of Bcl-2 via a γ-secretase-independent mechanism. Thus, PS1/2 increase the susceptibility to apoptosis by antagonizing the anti-apoptotic function of FKBP38. In contrast, C-terminal fragments of caspase-processed PS1/2 redistribute Bcl-2 to the mitochondria by abrogating the activity of full-length PS1/2, resulting in a dominant-negative anti-apoptotic effect. In cultured cells and mutant PS1-knockin mice brains, FAD-linked PS1/2 mutants enhance the pro-apoptotic activity by causing a more efficient reduction in mitochondrial Bcl-2 than wild-type PS1/2. These results suggest a novel molecular mechanism for the regulation of mitochondria-mediated apoptosis by competition between PS1/2 and FKBP38 for subcellular targeting of Bcl-2. Excessive pro-apoptotic activity of PS1/2 may play a role in the pathogenesis of FAD..
143. Satoru Kase, Kazuhiko Yoshida, Hiromi Ikeda, Takayuki Harada, Chikako Harada, Junko Imaki, Kazuhiro Ohgami, Kenji Shiratori, Keiichi Nakayama, Keiko Nakayama, Shigeaki Ohno, Disappearance of p27(KIP1) and increase in proliferation of the lens cells after extraction of most of the fiber cells of the lens, Current Eye Research, 10.1080/02713680590959286, 30, 6, 437-442, 2005.07, Purpose: Proliferation of the lens epithelial cells is involved in the fibrotic changes of lens capsules after cataract extraction. However, the mechanisms of the proliferation of the lens epithelial cells are largely unknown. The purpose of this study was to examine the correlation between the expression of p27(KIP1) and cell proliferation in the lens cells after the extraction of lens fiber cells. Methods: At embryonic days (E) 14 and 18, the C57B16 mice were anesthetized and the embryos were surgically removed. The eyes were dissected from these embryos and also from mice 12 weeks after birth. The 12-week-old mice were anesthetized, and then the lens fiber cells were extracted. Normal eyes at E14 and 18 and eyes fixed at 15 min and 48 hr after the extraction of the lens fiber cells were analyzed using immunohistochemistry with anti-p27(KIP1), p57(KIP2), and phosphorylated extracellular signal-regulated kinase (phospho-ERK) 1/2 antibodies, and cells in the S phase of the cell cycle were also examined using anti-bromodeoxyuridine (BrdU) antibody. Results: p27(KIP1) and p57(KIP2)-positive cells were present in the equatorial region of E14 mice. At E18, many lens fiber cells showed nuclear immunoreactivity for p27(KIP1), whereas a small number of cells were positive for p57(KIP2) in the equatorial region. At 12 weeks of age, all nuclei of the lens epithelial cells as well as lens fiber cells showed nuclear immunoreactivity for p27(KIP1). In contrast, p57(KIP2) and phospho-ERK1/2 were not expressed in the lens cells. At 15 min after the extraction of lens fiber cells, phospho-ERK1/2 as well as p27(KIP1) were detected in the lens cells. At 48 hr after the extraction of the lens fiber cells, a few p27(KIP1)-positive nuclei were observed in the equatorial region of the lens capsule. In contrast, many lens cells showed nuclear immunoreactivity for BrdU. Conclusions: These findings suggest that degradation of p27(KIP1) mediated by phosphorylation of ERK 1/2 is correlated with proliferation of the epithelial cells after the extraction of the lens fiber cells..
144. Tatiana Pushkarsky, Vyacheslav Yurchenko, Christophe Vanpouille, Beda Brichacek, Iosif Vaisman, Shigetsugu Hatakeyama, Keiichi Nakayama, Barbara Sherry, Michael I. Bukrinsky, Cell surface expression of CD147/EMMPRIN is regulated by cyclophilin 60, Journal of Biological Chemistry, 10.1074/jbc.M503770200, 280, 30, 27866-27871, 2005.07, CD147, also known as extracellular matrix metalloproteinase inducer, is a regulator of matrix metalloproteinase production and also serves as a signaling receptor for extracellular cyclophilins. Previously, we demonstrated that cell surface expression of CD147 is sensitive to cyclophilin-binding drug cyclosporin A, suggesting involvement of a cyclophilin in the regulation of intracellular transport of CD147. In this report, we identify this cyclophilin as cyclophilin 60 (Cyp60), a distinct member of the cyclophilin family of proteins. CD147 co-immunoprecipitated with Cyp60, and confocal immunofluorescent microscopy revealed intracellular colocalization of Cyp60 and CD147. This interaction with Cyp60 involved proline 211 of CD147, which was shown previously to be critical for interaction between CD147 and another cyclophilin, cyclophilin A, in solution. Mutation of this proline residue abrogated co-immunoprecipitation of CD147 and Cyp60 and reduced surface expression of CD147 on the plasma membrane. Suppression of Cyp60 expression using RNA interference had an effect similar to that of cyclosporin A: reduction of cell surface expression of CD147. These results suggest that Cyp60 plays an important role in the translocation of CD147 to the cell surface. Therefore, Cyp60 may present a novel target for therapeutic interventions in diseases where CD147 functions as a pathogenic factor, such as cancer, human immunodeficiency virus infection, or rheumatoid arthritis..
145. Zhiqiang Chen, Matthew W. Foster, Jian Zhang, Lan Mao, Howard A. Rockman, Toshihiro Kawamoto, Kyoko Kitagawa, Keiichi Nakayama, Douglas T. Hess, Jonathan S. Stamler, An essential role for mitochondrial aldehyde dehydrogenase in nitroglycerin bioactivation, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0503723102, 102, 34, 12159-12164, 2005.08, The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALDH-/- mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NO-based cellular signals that may originate independently of NO synthase activity..
146. Shigetsugu Hatakeyama, Masashi Watanabe, Yo Fujii, Keiichi Nakayama, Targeted destruction of c-Myc by an engineered ubiquitin ligase suppresses cell transformation and tumor formation, Cancer Research, 10.1158/0008-5472.CAN-05-1581, 65, 17, 7874-7879, 2005.09, Given that expression of c-Myc is up-regulated in many human malignancies, targeted inactivation of this oncoprotein is a potentially effective strategy for cancer treatment The ubiquitin-proteasome pathway of protein degradation is highly specific and can be engineered to achieve the elimination of undesirable proteins such as oncogene products. We have now generated a fusion protein (designated Max-U) that is composed both of Max, which forms a heterodimer with c-Myc, and of CHIP, which is a U box-type ubiquitin ligase (E3). Max-U physically interacted with c-Myc in transfected cells and promoted the ubiquitylation of c-Myc in vitro. It also reduced the stability of c-Myc in vivo, resulting in suppression of transcriptional activity dependent on c-Myc. Expression of Max-U reduced both the abundance of endogenous c-Myc in and the proliferation rate of a Burkitt lymphoma cell line. Furthermore, expression of Max-U but not that of a catalytically inactive mutant thereof markedly inhibited both the anchorage-independent growth in vitro of NIH 3T3 cells that overexpress c-Myc as well as tumor formation by these cells in nude mice. These findings indicate that the targeted destruction of c-Myc by an artificial E3 may represent an effective therapeutic strategy for certain human malignancies..
147. Shin Ichiro Hino, Chie Tanji, Keiichi Nakayama, Akira Kikuchi, Phosphorylation of β-catenin by cyclic AMP-dependent protein kinase stabilizes β-catenin through inhibition of its ubiquitination, Molecular and Cellular Biology, 10.1128/MCB.25.20.9063-9072.2005, 25, 20, 9063-9072, 2005.10, The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E
1
(PGE
1
), isoproterenol, and dibutyryl cAMP (Bt
2
cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE
1
and Bt
2
cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt
2
CAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated..
148. Taichi Hara, Takumi Kamura, Shuhei Kotoshiba, Hidehisa Takahashi, Kenichiro Fujiwara, Ichiro Onoyama, Masahiro Shirakawa, Noboru Mizushima, Keiichi Nakayama, Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle, Molecular and Cellular Biology, 10.1128/MCB.25.21.9292-9303.2005, 25, 21, 9292-9303, 2005.11, KPC2 (Kip1 ubiquitylation-promoting complex 2) together with KPC1 forms the ubiquitin ligase KPC, which regulates degradation of the cyclin-dependent kinase inhibitor p27 at the G1 phase of the cell cycle. KPC2 contains a ubiquitin-Iike (UBL) domain, two ubiquitin-associated (UBA) domains, and a heat shock chaperonin-binding (STI1) domain. We now show that KPC2 interacts with KPC1 through its UBL domain, with the 26S proteasome through its UBL and NH2-terminaI UBA domains, and with polyubiquitylated proteins through its UBA domains. The association of KPC2 with KPC1 was found to stabilize KPC1 in a manner dependent on the STI1 domain of KPC2. KPC2 mutants that lacked either the NH2-terminal or the COOH-terminal UBA domain supported the polyubiquitylation of p27 in vitro, whereas a KPC2 derivative lacking the STI1 domain was greatly impaired in this regard. Depletion of KPC2 by RNA interference resulted in inhibition of p27 degradation at the G1 phase, and introduction of KPC2 derivatives into the KPC2-depleted cells revealed that the NH2-terminal UBA domain of KPC2 is essential for p27 degradation. These observations suggest that ICPC2 cooperatively regulates p27 degradation with KPC1 and that the STI1 domain as well as the UBL and UBA domains of KPC2 are indispensable for its function..
149. Masaki Matsumoto, Shigetsugu Hatakeyama, Koji Oyamada, Yoshiya Oda, Toshihide Nishimura, Keiichi Nakayama, Large-scale analysis of the human ubiquitin-related proteome, Proteomics, 10.1002/pmic.200401280, 5, 16, 4145-4151, 2005.11, Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions..
150. Satomi Yogosawa, Shigetsugu Hatakeyama, Keiichi Nakayama, Hiroyuki Miyoshi, Shinichi Kohsaka, Chihiro Akazawa, Ubiquitylation and degradation of serum-inducible kinase by hVPS18, a RING-H2 type ubiquitin ligase, Journal of Biological Chemistry, 10.1074/jbc.M508397200, 280, 50, 41619-41627, 2005.12, Serum-inducible kinase (SNK) is a member of polo-like kinases that serve as regulators of multiple events during cell division. Rapid changes in the activity and abundance of SNK were reported after the serum stimulation and after the activation of synaptic transmission in the brain. Yet the detailed mechanisms that control the level of SNK protein have not been fully elucidated. In this report, we show that the RING-H2 domain of hVPS18 (human vacuolar pro-tein sorting 18) has a genuine ubiquitin ligase (E3) activity. Using the yeast two-hybrid screening, we identify SNK as a candidate substrate of hVPS18. The half-life of SNK is increased in HeLa cells that down-regulated hVPS18 by lentivirus-mediated small hairpin RNA interference. Furthermore, the delayed entry into S phase is observed in HeLa cells overexpressing h VPS18. These results suggest that hVPS18 may play an important role in regulation of SNK activity through its ubiquitin ligase..
151. Chie Kaneko-Oshikawa, Tadashi Nakagawa, Mitsunori Yamada, Hiroo Yoshikawa, Masaki Matsumoto, Masayoshi Yada, Shigetsugu Hatakeyama, Keiko Nakayama, Keiichi Nakayama, Mammalian E4 is required for cardiac development and maintenance of the nervous system, Molecular and Cellular Biology, 10.1128/MCB.25.24.10953-10964.2005, 25, 24, 10953-10964, 2005.12, Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a-/- mouse embryonic fibroblasts. Furthermore, Ufd2a+/- mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress..
152. Hideo Nishitani, Nozomi Sugimoto, Vassilis Roukos, Yohsuke Nakanishi, Masafumi Saijo, Chikashi Obuse, Toshiki Tsurimoto, Keiichi Nakayama, Keiko Nakayama, Masatoshi Fujita, Zoi Lygerou, Takeharu Nishimoto, Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis, EMBO Journal, 10.1038/sj.emboj.7601002, 25, 5, 1126-1136, 2006.03, Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication..
153. Satoru Kase, Kazuhiko Yoshida, Takayuki Harada, Chikako Harada, Kazuhiko Namekata, Yukari Suzuki, Kazuhiro Ohgami, Kenji Shiratori, Keiichi Nakayama, Shigeaki Ohno, Phosphorylation of extracellular signal-regulated kinase and p27(KIP1) after retinal detachment, Graefe's Archive for Clinical and Experimental Ophthalmology, 10.1007/s00417-005-0016-5, 244, 3, 352-358, 2006.03, Purpose: The roles of the extracellular signal-regulated kinase (ERK) pathway in the expression of cyclin D1 and p27(KIP1), the phosphorylation of p27(KIP1), and proliferation activity were examined after retinal detachment. Methods: Normal eyes and eyes at 15 min, 2 and 4 days after retinal detachment in C57Bl6 mice were examined by immunohistochemistry using anti-phosphorylated (p) ERK1/2, anti-cyclin D1, anti-p27(KIP1), anti-p27(KIP1) phosphorylated at serine 10 (S10-phospho-p27), and anti-proliferating cell nuclear antigen (PCNA) antibodies with or without treatment with a specific ERK inhibitor, PD98059. Mouse Müller cells were isolated and examined for alteration of p27(KIP1) and cyclin D1 after exposure of basic fibroblast growth factor (bFGF) with and without treatment of PD98059 using Western blotting. Results: In the normal retina, nuclear immunoreactivity for p27(KIP1), but not S10-phospho-p27 or pERK1/2, was observed in the middle sublayer of the inner nuclear layer (INL), where Müller glial cells are situated. At 15 min after the retinal detachment, p27(KIP1), S10-phospho-p27 and pERK1/2-positive nuclei were noted in the INL, whereas immunoreactivity for pERK1/2 or S10-phospho-p27 was not observed after treatment with PD98095. Cyclin D1 was induced in the INL 2 days after the retinal detachment, and the induction was inhibited by PD98059. At 4 days after the detachment, p27(KIP1) immunoreactivity was not observed, and cyclin D1 and PCNA were expressed. The disappearance of p27(KIP1) was suppressed, whereas expression of cyclin D1 and PCNA was not observed in mice treated with PD98059. Exposure of bFGF relatively decreased the expression level of p27(KIP1) and increased the level of cyclin D1 in mouse Müller cells, compared with control level. Induction of cyclin D1 and decrease in p27(KIP1) were inhibited with treatment of PD98059. Conclusion: Phosphorylation of ERK and expression of p27(KIP1) and cyclin D1 are involved in the proliferation of Müller cells after retinal detachment..
154. R. Iwanaga, H. Komori, S. Ishida, N. Okamura, K. Nakayama, Keiichi Nakayama, K. Ohtani, Identification of novel E2F1 target genes regulated in cell cycle-dependent and independent manners, Oncogene, 10.1038/sj.onc.1209210, 25, 12, 1786-1798, 2006.03, The transcription factor E2F mediates cell cycle-dependent expression of genes important for cell proliferation in response to growth stimulation. To further understand the role of E2F, we utilized a sensitive subtraction method to explore new E2F1 targets, which are expressed at low levels and might have been unrecognized in previous studies. We identified 33 new E2F1-inducible genes, including checkpoint genes Claspin and Rad51ap1, and four genes with unknown function required for cell cycle progression. Moreover, we found three groups of E2F1-inducible genes that were not induced by growth stimulation. At least, two groups of genes were directly induced by E2F1, indicating that E2F1 can regulate expression of genes not induced during the cell cycle. One included Neogenin, WASF1 and SGEF genes, which may have a role in differentiation or development. The other was the cyclin-dependent kinase inhibitor p27 Kip1, which was involved in suppression of inappropriate cell cycle progression induced by deregulated E2F. E2F1-responsive regions of these genes were located more upstream than those of typical E2F targets and did not have typical E2F sites. These results indicate that there are groups of E2F1 targets, which are regulated in a distinct manner from that of typical E2F targets..
155. Satoru Kase, Kazuhiko Yoshida, Kazuhiro Ohgami, Kenji Shiratori, Yukari Suzuki, Keiichi Nakayama, Shigeaki Ohno, Expression of cdc2 and p27(KIP1) phosphorylation in mitotic cells of the human retinoblastoma, International Journal of Molecular Medicine, 17, 3, 465-468, 2006.03, It was recently demonstrated that a lack of p27(KIP1) degradation resulted in the suppression of cdc2 activity and consequent inhibition of entry into the M-phase. The aim of this study was to examine the distribution of phosphorylated p27(KIP1) on threonine 187 (T187-phospho-p27) and cdc2 in mitotic cells of human retinoblastoma, a malignant retinal neoplasm. Several T187-phospho-p27- immunopositive cells were observed in mitotic retinoblastoma cells, but not in the normal retina. Immunoreactivity for T187-phospho-p27 was located in the prophase and metaphase of mitotic tumor cells. In contrast, tumor cells in the anaphase showed no immunoreactivity for T187-phospho-p27. Nuclear expression of cdc2 was detected in many retinoblastoma cells, including mitotic cells. The immunoreactivity in mitotic cells was located in the prophase, as well as metaphase. In contrast, anaphase cells did not show immunoreactivity. Double staining demonstrated the same localization of T187-phospho-p27 and cdc2 in mitotic cells. These results suggest that p27(KIP1) interacts with cdc2 in the M-phase of human retinoblastoma cells..
156. Eiji Sugihara, Masayuki Kanai, Soichiro Saito, Takayuki Nitta, Hideo Toyoshima, Keiko Nakayama, Keiichi Nakayama, Kenji Fukasawa, Manfred Schwab, Hideyuki Saya, Masanao Miwa, Suppression of centrosome amplification after DNA damage depends on p27 accumulation, Cancer Research, 10.1158/0008-5472.CAN-05-3250, 66, 8, 4020-4029, 2006.04, The centrosome plays a fundamental role in cell division, cell polarity, and cell cycle progression. Centrosome duplication is mainly controlled by cyclin-dependent kinase 2 (CDK2)/cyclin E and cyclin A complexes, which are inhibited by the CDK inhibitors p21Cip1 and p27Kip1. It is thought that abnormal activation of CDK2 induces centrosome amplification that is frequently observed in a wide range of aggressive tumors. We previously reported that overexpression of the oncogene MYCN leads to centrosome amplification after DNA damage in neuroblastoma cells. We here show that centrosome amplification after γ-irradiation was caused by suppression of p27 expression in MYCN-overexpressing cells. We further show that p27-/- and p27+/- mouse embryonic fibroblasts and p27-silenced human cells exhibited a significant increase in centrosome amplification after DNA damage. Moreover, abnormal mitotic cells with amplified centrosomes were frequently observed in p27-silenced cells. In response to DNA damage, the level of p27 gradually increased in normal cells independently of the ataxia telangiectasia mutated/p53 pathway, whereas Skp2, an F-box protein component of an SCF ubiquitin ligase complex that targets p27, was reduced. Additionally, p27 levels in MYCN-overexpressing cells were restored by treatment with Skp2 small interfering RNA, indicating that down-regulation of p27 by MYCN was due to high expression of Skp2. These results suggest that the accumulation of p27 after DNA damage is required for suppression of centrosome amplification, thereby preventing chromosomal instability..
157. Michael J. Humphries, Kirsten H. Limesand, Jonathan C. Schneider, Keiichi Nakayama, Steven M. Anderson, Mary E. Reyland, Suppression of apoptosis in the protein kinase Cδ null mouse in vivo, Journal of Biological Chemistry, 10.1074/jbc.M507851200, 281, 14, 9728-9737, 2006.04, Protein kinase C (PKC) δ is an essential regulator of mitochondrial dependent apoptosis in epithelial cells. We have used the PKCδ -/- mouse to ask if loss of PKCδ protects salivary glands against γ-irradiation-induced apoptosis in vivo and to explore the mechanism underlying protection from apoptosis. We show that γ-irradiation in vivo results in a robust induction of apoptosis in the parotid glands of wild type mice, whereas apoptosis is suppressed by greater than 60% in the parotid glands of PKCδ-/- mice. Primary parotid cells from PKCδ-/- mice are defective in mitochondrial dependent apoptosis as indicated by suppression of etoposide-induced cytochrome c release, poly(ADP-ribose) polymerase cleavage, and caspase-3 activation. Notably, apoptotic responsiveness can be restored by re-introduction of PKCδ by adenoviral transduction. Etoposide and γ-irradiation-induced activation of p53 is similar in primary parotid cells and parotid glands from PKCδ+/+ and PKCδ-/- mice, indicating that PKCδ functions downstream of the DNA damage response. In contrast, activation of the c-Jun amino-terminal kinase is reduced in primary parotid cells from PKCδ-/- cells and in parotid C5 cells, which express a dominant inhibitory mutant of PKCδ. Similarly, c-Jun amino-terminal kinase activation is suppressed in vivo in γ-irradiated parotid glands from PKCδ-/- mice. These studies indicate an essential role for PKCδ downstream of the p53 response and upstream of the c-Jun amino-terminal kinase activation in DNA damage-induced apoptosis in vivo and in vitro..
158. Evan J. Ryer, R. Patrick Hom, Kenji Sakakibara, Keiichi Nakayama, Keiko Nakayama, Peter L. Faries, Bo Liu, K. Craig Kent, PKCδ is necessary for Smad3 expression and transforming growth factor β-induced fibronectin synthesis in vascular smooth muscle cells, Arteriosclerosis, thrombosis, and vascular biology, 10.1161/01.ATV.0000209517.00220.cd, 26, 4, 780-786, 2006.04, Objective - The purpose of these studies is to investigate the mechanism by which transforming growth factor (TGF)β1 regulates the synthesis of the extracellular matrix protein fibronectin (FN). Methods and Results - TGFβ1 elicited a time-dependent induction of FN protein and mRNA in A10 rat aortic smooth muscle cells (SMCs). Ectopic expression of Smad3 in A10 cells stimulated both basal and TGFβ1-induced FN expression, whereas expression of Smad7 eliminated the TGFβ response. Because TGFβ activated PKCδ in SMCs, we tested the role of PKCδ in regulation of FN expression. Inhibition of PKCδ activity by rottlerin or dominant-negative adenovirus (AdPKCδ DN) blocked TGFβ1's induction of FN, whereas overexpression of PKCδ enhanced TGFβ's effect. Moreover, aortic SMCs isolated from PKCδ-/- mice exhibited diminished FN induction in response to TGFβ. Furthermore, we found that Smad3 protein and mRNA were markedly reduced in AdPKCδ DN-treated A10 cells and in PKCδ null cells. Finally, restoring Smad3 in rottlerin-treated A10 and PKCδ null cells rescues the ability of TGFβ to upregulate FN protein and mRNA expression. Conclusion - Our data suggest that TGFβ-activated PKCδ is critical to maintain normal expression of Smad3, which in turn is required for the induction of fibronectin. PKCδ represents a promising target for treating the fibroproliferative response after arterial injury..
159. Abbas Fotovati, Keiko Nakayama, Keiichi Nakayama, Impaired germ cell development due to compromised cell cycle progression in Skp2-deficient mice, Cell Division, 10.1186/1747-1028-1-4, 1, 2006.04, Background: The gonads are responsible for the production of germ cells through both mitosis and meiosis. Skp2 is the receptor subunit of an SCF-type ubiquitin ligase and is a major regulator of the progression of cells into S phase of the cell cycle, which it promotes by mediating the ubiquitin-dependent degradation of p27, an inhibitor of cell proliferation. However, the role of the Skp2-p27 pathway in germ cell development remains elusive. Results: We now show that disruption of Skp2 in mice results in a marked impairment in the fertility of males, with the phenotypes resembling Sertoli cell-only syndrome in men. Testes of Skp2-/- mice manifested pronounced germ cell hypoplasia accompanied by massive apoptosis in spermatogenic cells. Flow cytometry revealed an increased prevalence of polyploidy in spermatozoa, suggesting that the aneuploidy of these cells is responsible for the induction of apoptosis. Disruption of the p27 gene of Skp2-/- mice restored germ cell development, indicating that the testicular hypoplasia of Skp2-/- animals is attributable to the antiproliferative effect of p27 accumulation. Conclusion: Our results thus suggest that compromised cell cycle progression caused by the accumulation of p27 results in aneuploidy and the induction of apoptosis in gonadal cells of Skp2-/- mice. The consequent reduction in the number of mature gametes accounts for the decreased fertility of these animals. These findings reinforce the importance of the Skp2-p27 pathway in cell cycle regulation and in germ cell development..
160. Keiichi Nakayama, Keiko Nakayama, Ubiquitin ligases
Cell-cycle control and cancer, Nature Reviews Cancer, 10.1038/nrc1881, 6, 5, 369-381, 2006.05, A driving force of the cell cycle is the activation of cyclin-dependent kinases (CDKs), the activities of which are controlled by the ubiquitin-mediated proteolysis of key regulators such as cyclins and CDK inhibitors. Two ubiquitin ligases, the SKP1-CUL1-F-box-protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C), are responsible for the specific ubiquitylation of many of these regulators. Deregulation of the proteolytic system might result in uncontrolled proliferation, genomic instability and cancer. Cumulative clinical evidence shows alterations in the ubiquitylation of cell-cycle regulators in the aetiology of many human malignancies. A better understanding of the ubiquitylation machinery will provide new insights into the regulatory biology of cell-cycle transitions and the development of anti-cancer drugs..
161. Arti Shukla, Trisha F. Barrett, Keiichi Nakayama, Kieko Nakayama, Brooke T. Mossman, Karen M. Lounsbury, Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKCδ-dependent pathway in murine lung, FASEB Journal, 10.1096/fj.05-4554fje, 20, 7, 2006.05, Asbestos is a known inflammatory, carcinogenic, and fibrotic agent, but the mechanisms leading to asbestos-induced lung diseases are unclear. Using a murine inhalation model of fibrogenesis, we show that asbestos causes significant increases in mRNA levels of lung matrix metalloproteinases (MMPs 12 and 13) and tissue inhibitor of metalloproteinases (TIMP1), as well as increased activities of MMP 2, 9, and 12 in bronchoalveolar lavage fluids (BALF). Asbestos-exposed PKCδ knockout (PKCδ-/-) mice exhibited decreased expression of lung MMP12 and MMP13 compared with asbestos-exposed wild-type mice. Studies using small molecule inhibitors in murine alveolar epithelial type II cells (C10) and primary lung fibroblasts confirmed that asbestos transcriptionally upregulates MMPs via an EGFR (or other growth factor receptors)/PI3K/PKCδ/ERK1/2 pathway. Moreover, use of a broad-spectrum MMP inhibitor showed that MMPs play an important role in further enhancing asbestos-induced signaling events by activating EGFR. These data reveal a potentially important link between asbestos signaling and integrity of the extracellular matrix (ECM) that likely contributes to asbestos-induced lung remodeling and diseases..
162. Kiyotsugu Yoshida, Tomoko Yamaguchi, Hirokuni Shinagawa, Naoe Taira, Keiichi Nakayama, Yoshio Miki, Protein kinase C δ activates topoisomerase IIα to induce apoptotic cell death in response to DNA damage, Molecular and Cellular Biology, 10.1128/MCB.26.9.3414-3431.2006, 26, 9, 3414-3431, 2006.05, DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIα is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIα is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C δ (PKCδ) interacts with topoisomerase IIα. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIα in response to DNA damage. Conversely, inhibition of PKCδ activity attenuates DNA damage-induced activation of topoisomerase IIα. Finally, aberrant activation of topoisomerase IIα by PKCδ is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCδ regulates topoisomerase IIα and thereby cell fate in the genotoxic stress response..
163. Satoru Kase, Kazuhiko Yoshida, Kazuhiro Ohgami, Kenji Shiratori, Shigeaki Ohno, Keiichi Nakayama, Phosphorylation of p27(KIP1) in the mitotic cells of the corneal epithelium, Current Eye Research, 10.1080/02713680600584687, 31, 4, 307-312, 2006.05, Purpose: The mechanism in regulation of the cell cycle and proliferation of corneal epithelium in the homeostatic ocular surface remains unclear. The aim of this study is to examine the expression of p27(KIP1) and its phosphorylation in corneal epithelium. Methods: The eyes of C57BL/6 mice (7 weeks old) were enucleated. Formalin-fixed and paraffin-embedded tissue sections were examined using immunohistochemistry with anti-p27(KIP1), threonine 187 phosphorylated p27(KIP1) (T187-phospho-p27), and phosphorylated Histon H3 (pHiston H3) antibodies. Anti-T187-phospho-p27 and anti-pHiston H3 polyclonal antibodies were used for parallel immunofluorescent staining. Results: pHiston H3-immunopositive cells were noted in basal cells of the corneal epithelium. At high magnification of DAPI nuclear staining, mitotic and non-mitotic cells were observed in corneal basal layer. p27(KIP1)-positive nuclei were detected in corneal basal cells, where non-mitotic basal cells were located. In contrast, mitotic cells showed under detectable level on p27(KIP1) immunoreactivity. Immunoreactivity for T187-phospho-p27 was detected in basal cells of the corneal epithelium. At high magnification, it was confirmed that the immunopositive cells were mitotic cells. Immunoreactivity of T187-phospho-p27 as well as pHiston H3 was localized in the same corneal basal cells using double-staining immunohistochemistry. Conclusions: These results suggested that degradation of p27(KIP1) regulates progression into mitosis in corneal basal cells..
164. Takuya Nojima, Katsuhiko Hayashi, Ryo Goitsuka, Keiko Nakayama, Keiichi Nakayama, Daisuke Kitamura, Double knockout mice show BASH and PKCδ have different epistatic relationships in B cell maturation and CD40-mediated activation, Immunology Letters, 10.1016/j.imlet.2005.12.004, 105, 1, 48-54, 2006.05, The development and survival of mature B cells requires an antigen-independent signal from the B cell receptor (BCR) through an adaptor protein containing an SH2 domain, BASH (BLNK/SLP-65). It also requires signaling through BAFF and the BAFF receptor (BAFF-R), and is negatively regulated by protein kinase Cδ (PKCδ). In PKCδ-deficient mice, B cell maturation occurs independently of the BAFF receptor (BAFF-R), indicating that BAFF-R signaling promotes maturation by inhibiting the negative function of PKCδ. To clarify which of the two signaling pathways plays the primary role in B cell maturation, we crossed BASH-deficient mice with PKCδ-deficient mice to generate BASH/PKCδ-double knockout (DKO) mice. In the DKO mice, B cell maturation was blocked at the transitional type 1 (T1) stage and B cells were prone to apoptosis, in common with BASH-deficient mice. This indicates that BASH-mediated BCR signaling primarily controls B cell survival and maturation, with BAFF-R signaling and its inhibition of PKCδ acting as a secondary regulator. By contrast, CD40-mediated proliferation and antibody production, which are low in BASH-deficient mice, were rescued in the DKO mice, indicating that the suppression of CD40-mediated B cell activation by PKCδ is epistatic to BASH-mediated promotion. The physiological relevance of these opposing hierarchical effects of BASH and PKCδ in the regulation of B cell maturation and activation is discussed..
165. Shino Niki, Kiyotaka Oshikawa, Yasuhiro Mouri, Fumiko Hirota, Akemi Matsushima, Masashi Yano, Hongwei Han, Yoshimi Bando, Keisuke Izumi, Masaki Matsumoto, Keiichi Nakayama, Noriyuki Kuroda, Mitsuru Matsumoto, Alteration of intra-pancreatic target-organ specificity by abrogation of Aire in NOD mice, Journal of Clinical Investigation, 10.1172/JCI26971, 116, 5, 1292-1301, 2006.05, Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against β cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than β cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus..
166. Arnaud Parcellier, Mathilde Brunet, Elise Schmitt, Edwige Col, Céline Didelot, Arlette Hammann, Keiko Nakayama, Keiichi Nakayama, Saadi Khochbin, Eric Solary, Carmen Garrido, HSP27 favors ubiquitination and proteasomal degradation of p27 Kip1 and helps S-phase re-entry in stressed cells, FASEB Journal, 10.1096/fj.05-4184fje, 20, 8, 2006.06, Stress-inducible HSP27 protects cells from death through various mechanisms. We have recently demonstrated that HSP27 can also enhance the degradation of some proteins through the proteasomal pathway. Here, we show that one of these proteins is the cyclin-dependent kinase (Cdk) inhibitor p27 Kip1. The ubiquitination and degradation of this protein that favors progression through the cell cycle was previously shown to involve either a Skp2-dependent mechanism, i.e., at the S-/G2-transition, or a KPC (Kip1 ubiquitination-promoting complex)-dependent mechanism, i.e., at the G 0/G1 transition. In this work, we demonstrate that, in response to serum depletion, p27Kip1 cellular content first increases then progressively decreases as cells begin to die. In this stressful condition, HSP27 favors p27Kip1 ubiquitination and degradation by the proteasome. A similar observation was made in response to stress induced by the NO donor glyceryl trinitrate (GTN). HSP27-mediated ubiquitination of p27 Kip1 does not require its phosphorylation on Thr187 or Ser-10, nor does it depend on the SCFSkp2 ubiquitin ligase E3 complex. It facilitates the G1/S transition, which suggests that, in stressful conditions, HSP27 might render quiescent cells competent to re-enter the cell cycle..
167. Takayuki Tsukuba, Shinya Yamamoto, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Keiichi Nakayama, Tomoko Kadowaki, Kenji Yamamoto, Cathepsin E-deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors, Journal of Biochemistry, 10.1093/jb/mvj132, 140, 1, 57-66, 2006.07, Cathepsin E, an intracellular aspartic proteinase, is predominantly localized in the endosomal compartments of immune system cells. In the present study, we investigated the role of cathepsin E in immune defense systems against bacterial infection. Cathepsin E-deficient (CatE-/-) mice showed dramatically increased susceptibility to infection with both the Gram-positive bacterium Staphyrococcus aureus, and the Gram-negative bacterium Porphyromonas gingivalis when compared with syngeneic wild-type mice, most likely due to impaired regulation of bacterial elimination. Peritoneal macrophages from CatE-/- mice showed significantly impaired tumor necrosis factor-α and IL-6 production in response to S. aureus and decreased bactericidal activities toward this bacterium. Moreover, the cell surface levels of Toll-like receptor-2 (TLR2) and TLR4, which recognize specific components of Gram-positive and -negative bacteria, respectively, were decreased in CatE -/- macrophages, despite no significant difference in the total cellular expression levels of these receptors between the wild-type and CatE-/- macrophages, implying trafficking defects in these surface receptors in the latter. These results indicate an essential role of cathepsin E in immune defense against invading microorganisms, most probably due to regulation of the cell surface expression of TLR family members required for innate immune responses..
168. Keiichi Nakayama, Keiko Nakayama, Ubiquitin system regulating G1 and S phases of cell cycle, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 51, 10 Suppl, 1362-1369, 2006.08.
169. Takashi Kanematsu, Atsushi Yasunaga, Yoshito Mizoguchi, Akiko Kuratani, Josef T. Kittler, Jasmina N. Jovanovic, Kei Takenaka, Keiichi Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Stephen J. Moss, Junichi Nabekura, Masato Hirata, Modulation of GABAA receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition, Journal of Biological Chemistry, 10.1074/jbc.M603118200, 281, 31, 22180-22189, 2006.08, Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases..
170. Ryosuke Tsunematsu, Masaaki Nishiyama, Shuhei Kotoshiba, Toru Saiga, Takumi Kamura, Keiichi Nakayama, Fbxw8 is essential for Cul1-Cul7 complex formation and for placental development, Molecular and Cellular Biology, 10.1128/MCB.00595-06, 26, 16, 6157-6169, 2006.08, Cullin-based ubiquitin ligases (E3s) constitute one of the largest E3 families. Fbxw8 (also known as Fbw6 or Fbx29) is an F-box protein that is assembled with Cul7 in an SCF-like E3 complex. Here we show that Cul7 forms a heterodimeric complex with Cul1 in a manner dependent on FbxwS. We generated mice deficient in Fbxw8 and found that Cul7 did not associate with Cul1 in cells of these mice. Two-thirds of Fbxw8-/- embryos die in utero, whereas the remaining one-third are born alive and grow to adulthood. Fbxw8 -/- embryos show intrauterine growth retardation and abnormal development of the placenta, characterized by both a reduced thickness of the spongiotrophoblast layer and abnormal vessel structure in the labyrinth layer. Although the placental phenotype of Fbxw8-/- mice resembles that of Cul7-/- mice, other abnormalities of Cul7-/- mice are not apparent in Fbxw8-/- mice. These results suggest that the Cul7-based SCF-like E3 complex has both Fbxw8-dependent and Fbxw8-independent functions..
171. Yo Fujii, Masayoshi Yada, Masaaki Nishiyama, Takumi Kamura, Hidehisa Takahashi, Ryosuke Tsunematsu, Etsuo Susaki, Tadashi Nakagawa, Akinobu Matsumoto, Keiichi Nakayama, Fbxw7 contributes to tumor suppression by targeting multiple proteins for ubiquitin-dependent degradation, Cancer Science, 10.1111/j.1349-7006.2006.00239.x, 97, 8, 729-736, 2006.08, Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development..
172. Yoshihiro Hiramatsu, Kyoko Kitagawa, Toru Suzuki, Chiharu Uchida, Takayuki Hattori, Hirotoshi Kikuchi, Toshiaki Oda, Shigetsugu Hatakeyama, Keiichi Nakayama, Tadashi Yamamoto, Hiroyuki Konno, Masatoshi Kitagawa, Degradation of Tob1 mediated by SCFSkp2-dependent ubiquitination, Cancer Research, 10.1158/0008-5472.CAN-06-1603, 66, 17, 8477-8483, 2006.09, Tob1, a member of the Tob/BTG family, is involved in the control of G 1-S progression by suppressing cyclin D1 expression and acts as a tumor suppressor gene. Tob1 was reported to have a quick turnover through the ubiquitin-proteasome pathway, but proteins involved in this process are still unknown. We showed that Skp2, a substrate-targeting subunit of the SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complex, was involved in ubiquitin-dependent degradation of Tob1. Skp2 interacted with Tob1 and facilitated ubiquitination of Tob1 in intact cells as well as in vitro. Skp2 mutants without the F-box or leucine rich repeat were not able to bind to Tob1 and did not enhance ubiquitination of Tob1. Tob1 was stabilized in both Skp2-/- mouse fibroblasts and Skp2 knockdown HeLa cells. Moreover, cyclin D1 expression was suppressed in Skp2 knockdown HeLa cells. These data suggest that Tob1 is a novel target for degradation by the SCF-Skp2 ubiquitin ligase..
173. T. Shimazu, Y. Komatsu, Keiichi Nakayama, H. Fukazawa, S. Horinouchi, M. Yoshida, Regulation of SV40 large T-antigen stability by reversible acetylation, Oncogene, 10.1038/sj.onc.1209731, 25, 56, 7391-7400, 2006.11, Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as α-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells..
174. Michiko Shirane, Keiichi Nakayama, Protrudin induces neurite formation by directional membrane trafficking, Science, 10.1126/science.1134027, 314, 5800, 818-821, 2006.11, Guanosine triphosphatases of the Rab family are key regulators of membrane trafficking, with Rab11 playing a specific role in membrane recycling. We identified a mammalian protein, protrudin, that promoted neurite formation through interaction with the guanosine diphosphate (GDP)-bound form of Rab11. Phosphprylation of protrudin by extracellular signal-regulated kinase (ERK) in response to nerve growth factor promoted protrudin association with Rab11-GDP. Down-regulation of protrudin by RNA interference induced membrane extension in all directions and inhibited neurite formation. Thus, protrudin regulates Rab11-dependent membrane recycling to promote the directional membrane trafficking required for neurite formation..
175. Akiko Takahashi, Naoko Ohtani, Kimi Yamakoshi, Shin Ichi Iida, Hidetoshi Tahara, Keiko Nakayama, Keiichi Nakayama, Toshinori Ide, Hideyuki Saya, Eiji Hara, Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence, Nature Cell Biology, 10.1038/ncb1491, 8, 11, 1291-1297, 2006.11, The p16INK4a cyclin-dependent kinase inhibitor has a key role in establishing stable G1 cell-cycle arrest through activating the retinoblastoma (Rb) tumour suppressor protein pRb1-5 in cellular senescence. Here, we show that the p16INK4a/ Rb-pathway also cooperates with mitogenic signals to induce elevated intracellular levels of reactive oxygen species (ROS), thereby activating protein kinase Cδ (PKCδ) in human senescent cells. Importantly, once activated by ROS, PKCδ promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCδ signalling6-8. Sustained activation of ROS-PKCδ signalling irreversibly blocks cytokinesis, at least partly through reducing the level of WARTS (also known as LATS1), a mitotic exit network (MEN) kinase required for cytokinesis9-11, in human senescent cells. This irreversible cytokinetic block is likely to act as a second barrier to cellular immortalization ensuring stable cell-cycle arrest in human senescent cells. These results uncover an unexpected role for the p16INK4a-Rb pathway and provide a new insight into how senescent cell-cycle arrest is enforced in human cells..
176. Kentaro Hara, Keiichi Nakayama, Keiko Nakayama, Geminin is essential for the development of preimplantation mouse embryos, Genes to Cells, 10.1111/j.1365-2443.2006.01019.x, 11, 11, 1281-1293, 2006.11, Replication of DNA is strictly controlled to ensure that it occurs only once per cell cycle. Geminin has been thought to serve as a central mediator of this licensing mechanism by binding to and antagonizing the function of Cdt1 and thereby preventing re-replication during S and G2 phases. We have now generated mice deficient in geminin to elucidate the physiologic role of this protein during development. Lack of geminin was shown to result in preimplantation mortality. A delay in the development of homozygous mutant embryos was first apparent at the transition from the four- to eight-cell stages, concomitant with the disappearance of maternal geminin protein, and development was arrested at the eight-cell stage. The mutant embryos manifest morphological abnormalities such as dispersed blastomeres with nuclei that are irregular both in size and shape as well as impaired cell-cell adhesion. DNA replication occurs but mitosis was not detected in the mutant embryos. The abnormal blastomeres contain damaged DNA and undergo apoptosis, likely as a consequence of the deregulation of DNA replication. Our results suggest that geminin is essential for cooperative progression of the cell cycle through S phase to M phase during the preimplantation stage of mouse development..
177. Akinobu Matsumoto, Ichiro Onoyama, Keiichi Nakayama, Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2006.09.003, 350, 1, 114-119, 2006.11, Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (α, β, and γ) of Fbxw7 are produced from mRNAs with distinct 5′ exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7α mRNA was present in all tissues examined, whereas Fbxw7β mRNA was detected only in brain and testis, and Fbxw7γ mRNA in heart and skeletal muscle. The amount of Fbxw7α mRNA was high during quiescence (G0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7α mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7β mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7β mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner..
178. Yun Gao, Kyoko Kitagawa, Yoshihiro Hiramatsu, Hirotoshi Kikuchi, Tomoyasu Isobe, Mai Shimada, Chiharu Uchida, Takayuki Hattori, Toshiaki Oda, Keiko Nakayama, Keiichi Nakayama, Tatsuo Tanaka, Hiroyuki Konno, Masatoshi Kitagawa, Up-regulation of GPR48 induced by down-regulation of p27Kip1 enhances carcinoma cell invasiveness and metastasis, Cancer Research, 10.1158/0008-5472.CAN-06-2629, 66, 24, 11623-11631, 2006.12, A reduced expression level of the cyclin-dependent kinase inhibitor p27Kip1 is associated with increased tumor malignancy and poor prognosis in individuals with various types of cancer. To investigate the basis for this relation, we applied microarray analysis to screen for genes differentially expressed between p27+/- and parental (p27 +/+) HCT116 human colon carcinoma cells. Expression of the gene for G protein-coupled receptor 48 (GPR48) was increased in the p27+/- cells. Forced expression of GPR48 increased both in vitro invasive activity and lung metastasis potency of HCT116 cells. In contrast, depletion of endogenous GPR48 by RNA interference reduced the invasive potential of HeLa and Lewis lung carcinoma cells not only in vitro but also in vivo. Moreover, GPR48 expression was significantly associated with lymph node metastasis and inversely correlated with p27 expression in human colon carcinomas. GPR48 may thus play an important role in invasiveness and metastasis of carcinoma and might therefore represent a potential prognostic marker or therapeutic target..
179. Giordano Pula, Kai Schuh, Keiko Nakayama, Keiichi Nakayama, Ulrich Walter, Alastair W. Poole, PKCδ regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation, Blood, 10.1182/blood-2006-05-023739, 108, 13, 4035-4044, 2006.12, Protein kinase Cδ (PKCδ) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCδ-/- platelets, we show that the mechanism did not involve altered inside-out signaling to integrin α IIbβ3 and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCγ2 between wild-type and PKCδ-/- platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCδ-/- platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCδ is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCδ physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP -/- platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCδ is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP..
180. Yasuhiro Itoh, Norihisa Masuyama, Keiko Nakayama, Keiichi Nakayama, Yukiko Gotoh, The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex, Journal of Biological Chemistry, 10.1074/jbc.M609944200, 282, 1, 390-396, 2007.01, Neuronal precursors remain in the proliferative zone of the developing mammalian neocortex until after they have undergone neuronal differentiation and cell cycle arrest. The newborn neurons then migrate away from the proliferative zone and enter the cortical plate. The molecules that coordinate migration with neuronal differentiation have been unclear. We have proposed in this study that the cdk inhibitors p57 and p27 play a role in this coordination. We have found that p57 and p27 mRNA increase upon neuronal differentiation of neocortical neuroepithelial cells. Knockdown of p57 by RNA interference resulted in a significant delay in the migration of neurons that entered the cortical plate but did not affect neuronal differentiation. Knockdown of p27 also inhibits neuronal migration in the intermediate zone as well as in the cortical plate, as reported by others. We have also found that knockdown of p27 increases p57 mRNA levels. These results suggest that both p57 and p27 play essential roles in neuronal migration and may, in concert, coordinate the timing of neuronal differentiation, migration, and possibly cell cycle arrest in neocortical development..
181. Tamon Sakai, Hiroshi Sakaue, Takehiro Nakamura, Mitsuru Okada, Yasushi Matsuki, Eijiro Watanabe, Ryuji Hiramatsu, Keiko Nakayama, Keiichi Nakayama, Masato Kasuga, Skp2 controls adipocyte proliferation during the development of obesity, Journal of Biological Chemistry, 10.1074/jbc.M608144200, 282, 3, 2038-2046, 2007.01, The increase in the mass of adipose tissue during the development of obesity can arise through an increase in cell size, an increase in cell number, or both. Here we show that long term maintenance of C57BL/6 mice on a high fat diet (for ∼25 weeks) induces an initial increase in adipocyte size followed by an increase in adipocyte number in white adipose tissue. The latter effect was found to be accompanied by up-regulation of expression of the gene for the F-box protein Skp2 as well as by down-regulation of the cyclin-dependent kinase inhibitor p27Kip1, a principal target of the SCFSkp2 ubiquitin ligase, in white adipose tissue. Ablation of Skp2 protected mice from the development of obesity induced either by a high fat diet or by the lethal yellow agouti (Ay) mutation, and this protective action was due to inhibition of the increase in adipocyte number without an effect on adipocyte hypertrophy. The reduction in the number of adipocyte caused by Skp2 ablation also inhibited the development of obesity-related insulin resistance in the Ay mutant mice, although the reduced number of β cells and reduced level of insulin secretion in Skp2-deficient mice resulted in glucose intolerance. Our observations thus indicate that Skp2 controls adipocyte proliferation during the development of obesity..
182. Sayuri Suzuki, Hirotaka Fukasawa, Kyoko Kitagawa, Chiharu Uchida, Takayuki Hattori, Tomoyasu Isobe, Toshiaki Oda, Taro Misaki, Naro Ohashi, Keiko Nakayama, Keiichi Nakayama, Akira Hishida, Tatsuo Yamamoto, Masatoshi Kitagawa, Renal damage in obstructive nephropathy is decreased in Skp2-deficient mice, American Journal of Pathology, 10.2353/ajpath.2007.070279, 171, 2, 473-483, 2007.01, Ubiquitin-dependent degradation of the cyclin-dependent kinase inhibitor p27 mediated by SCF-Skp2 ubiquitin ligase is involved in cell cycle regulation. Proliferation of tubular cells is a characteristic feature in obstructed kidneys of unilateral ureteral obstruction. Comparing Skp2+/+ mice with Skp2-/- mice, we investigated the involvement of Skp2, a component of SCF-Skp2 ubiquitin ligase for p27, in the progression of renal lesions in unilateral ureteral obstructed kidneys. mRNA expression of Skp2 was markedly increased in the obstructed kidneys from Skp2+/+ mice and peaked 3 days after unilateral ureteral obstruction. Renal atrophy, tubular dilatation, tubulointerstitial fibrosis, and increases in α-smooth muscle actin expression, the number of tubular cells, and proliferating tubular cells positive for Ki67 were observed in the obstructed kidneys from Skp2 +/+ mice; however, these findings were significantly attenuated in Skp2-/- mice. The p27 protein level was increased in the obstructed kidneys but was significantly greater in Skp2-/- mice. The number of Ki67-positive p27-negative cells was lower in obstructed kidneys from Skp2 -/- mice than Skp2+/+ mice, whereas that of Ki67-negative p27-positive cells was greater in Skp2-/- mice. These findings suggest that p27 accumulation, which results from SCF-Skp2 ubiquitin ligase deficiency in Skp2-/-mice, is involved in the amelioration of renal damage induced by obstructive nephropathy..
183. Toyoyoshi Uchida, Noseki Iwashita, Mica Ohara-Imaizumi, Takeshi Ogihara, Shintaro Nagai, Jong Bock Choi, Yoshifumi Tamura, Norihiro Tada, Ryuzo Kawamori, Keiichi Nakayama, Shinya Nagamatsu, Hirotaka Watada, Protein kinase Cδ plays a non-redundant role in insulin secretion in pancreatic β cells, Journal of Biological Chemistry, 10.1074/jbc.M610482200, 282, 4, 2707-2716, 2007.01, Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic β cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCδ, a major isoform expressed in pancreatic β cells on β cell function. Here, we showed that PKCδ null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCδ null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into β cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in β cells of PKCδ null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCδ null mice. We conclude that PKCδ plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCδ null mice is associated with reduced phosphorylation of Munc18-1..
184. Xiaolin Tu, Kyu Sang Joeng, Keiichi Nakayama, Keiko Nakayama, Jayaraj Rajagopal, Thomas J Carroll, Andrew P. McMahon, Fanxin Long, Noncanonical Wnt Signaling through G Protein-Linked PKCδ Activation Promotes Bone Formation, Developmental Cell, 10.1016/j.devcel.2006.11.003, 12, 1, 113-127, 2007.01, Wnt signaling regulates a variety of developmental processes in animals. Although the β-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Gαq/11 subunits of G proteins to activate phosphatidylinositol signaling and PKCδ in the murine ST2 cells. Gαq/11-PKCδ signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCδ homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCδ-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation..
185. Michiyo Yanagawa, Takayuki Tsukuba, Tsuyoshi Nishioku, Yoshiko Okamoto, Kuniaki Okamoto, Ryosuke Takii, Yoshihiro Terada, Keiichi Nakayama, Tomoko Kadowaki, Kenji Yamamoto, Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages, Journal of Biological Chemistry, 10.1074/jbc.M604143200, 282, 3, 1851-1862, 2007.01, Cathepsin E, an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, has an important role in immune responses. However, little is known about the precise roles of cathepsin E in this system. Here we report that cathepsin E deficiency (CatE-/-) leads to a novel form of lysosome storage disorder in macrophages, exhibiting the accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 and the elevation of lysosomal pH. These striking features were also found in wild-type macrophages treated with pepstatin A and Ascaris inhibitor. Whereas there were no obvious differences in their expression, biosynthesis, and trafficking between wild-type and CatE-/- macrophages, the degradation rates of these two membrane proteins were apparently decreased as a result of cathepsin E deficiency. Because there was no difference in the vacuolar-type H +-ATPase activity in both cell types, the elevated lysosomal pH in CatE-/- macrophages is most likely due to the accumulation of these lysosomal membrane glycoproteins highly modified with acidic monosaccharides, thereby leading to the disruption of non-proton factors controlling lysosomal pH. Furthermore, the selective degradation of LAMP-1 and LAMP-2, as well as LIMP-2, was also observed by treatment of the lysosomal membrane fraction isolated from wild-type macrophages with purified cathepsin E at pH 5. Our results thus suggest that cathepsin E is important for preventing the accumulation of these lysosomal membrane sialoglycoproteins that can induce a new form of lysosomal storage disorder..
186. Arti Shukla, Karen M. Lounsbury, Trisha F. Barrett, Joanna Gell, Mercedes Rincon, Kelly J. Butnor, Douglas J. Taatjes, Gerald S. Davis, Pamela Vacek, Keiichi Nakayama, Keiko Nakayama, Chad Steele, Brooke T. Mossman, Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-δ knockout mice, American Journal of Pathology, 10.2353/ajpath.2007.060381, 170, 1, 140-151, 2007.01, The signaling pathways leading to the development of asbestos-associated diseases are poorly understood. Here we used normal and protein kinase C (PKC)-δ knockout (PKCδ-/-) mice to demonstrate multiple roles of PKC-δ in the development of cell proliferation and inflammation after inhalation of chrysotile asbestos. At 3 days, asbestos-induced peribronchiolar cell proliferation in wild-type mice was attenuated in PKCδ-/- mice. Cytokine profiles in bronchoalveolar lavage fluids showed increases in interleukin (IL)-1β, IL-4, IL-6, and IL-13 that were decreased in PKCδ-/- mice. At 9 days, microarray and quantitative reverse transcriptase-polymerase chain reaction analysis of lung tissues revealed increased mRNA levels of the profibrotic cytokine, IL-4, in asbestos-exposed wildtype mice but not PKCδ-/- mice. PKCδ-/- mice also exhibited decreased lung infiltration of polymorphonuclear cells, natural killer cells, and macrophages in bronchoalveolar lavage fluid and lung, as well as increased numbers of B lymphocytes and plasma cells. These changes were accompanied by elevated mRNA levels of immunoglobulin chains. These data show that modulation of PKC-δ has multiple effects on peribronchiolar cell proliferation, proinflammatory and profibrotic cytokine expression, and immune cell profiles in lung. These results also implicate targeted interruption of PKC-δ as a potential therapeutic option in asbestos-induced lung diseases..
187. Akiko Mizokami, Takashi Kanematsu, Hitoshi Ishibashi, Taku Yamaguchi, Isei Tanida, Kei Takenaka, Keiichi Nakayama, Kiyoko Fukami, Tadaomi Takenawa, Eiki Kominami, Stephen J. Moss, Tsuneyuki Yamamoto, Junichi Nabekura, Masato Hirata, Phosholipase C-related inactive protein is involved in trafficking of γ2 subunit-containing GABAA receptors to the cell surface, Journal of Neuroscience, 10.1523/JNEUROSCI.3155-06.2007, 27, 7, 1692-1701, 2007.02, The subunit composition of GABAA receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABAA receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABAA receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABAA receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABAA receptors and GABAA receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABAA receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABAA receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABAA receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors..
188. Zhixue Liu, Xia Liu, Keiichi Nakayama, Keiko Nakayama, Keqiang Ye, Protein kinase C-δ phosphorylates Ebp1 and prevents its proteolytic degradation, enhancing cell survival, Journal of Neurochemistry, 10.1111/j.1471-4159.2006.04313.x, 100, 5, 1278-1288, 2007.03, ErbB3-binding protein (Ebp1) promotes cell survival by preventing apoptotic DNA fragmentation through a complex with active nuclear Akt. Ebp1 phosphorylation by protein kinase C (PKC)-δ mediates its binding to nuclear Akt. In this study, we show that Ebp1 itself acts as a substrate of active caspase 3 during the programmed cell death. PKC-δ phosphorylation on Ebp1 protects it from apoptotic degradation initiated in cell-free apoptotic solution. Moreover, Ebp1 is evidently cleaved in PKC-δ-deficient cells but not in wild-type cells. Ebp1 translated from first ATG is resistant to apoptotic cleavage; by contrast, Ebp1 from second and third ATG demonstrates robust degradation, and PKC phosphorylation on S360 suppresses its cleavage by active caspase 3. Ebp1 can be digested at both D53 and D196 sites, but cleavage at D196 appears to be a prerequisite for its further degradation at D53 site. Compared with wild-type Ebp1, D196A mutant markedly protects cells from apoptosis. Thus, PKC-δ antagonizes apoptosis through phosphorylating Ebp1 and protects it from apoptotic degradation..
189. Christine Möller, Mats Karlberg, Åbrink Magnus, Keiichi Nakayama, Noboru Motoyama, Gunnar Nilsson, Bcl-2 and Bcl-XL are indispensable for the late phase of mast cell development from mouse embryonic stem cells, Experimental Hematology, 10.1016/j.exphem.2006.11.008, 35, 3, 385-393, 2007.03, Objective: The aim of this study was to determine the importance of the prosurvival factors Bcl-2 and Bcl-XL for mast cell development and survival. Methods: bcl-x-/- and bcl-2-/- mouse embryonic stem cells were maintained in medium supplemented with either interleukin (IL)-3 or IL-3 in combination with stem cell factor (SCF) to favor mast cell development. The development of Bcl-2 family deficient embryonic stem cell-derived mast cells (ESMCs) was monitored and Bcl-2 family gene expression and cell numbers were analyzed. Results: Deficiency in either bcl-x or bcl-2 totally inhibited the development of ESMCs when IL-3 alone was used as a mast cell growth factor. Intriguingly, when IL-3 was used in combination with SCF, the ESMCs developed normally the first 2 weeks but thereafter the cell numbers dropped drastically. The remaining ESMCs express mouse mast cell protease 1, suggesting a mucosal-like phenotype. ESMCs lacking bcl-x or bcl-2 exhibited strong expression of A1, another prosurvival Bcl-2 family member. Conclusion: For the first time we provide direct evidence that both bcl-x and bcl-2 are indispensable for mast cell survival during the late phase of their development..
190. Wei Li, Hisakata Yamada, Toshiki Yajima, Ryusuke Nakagawa, Kazuya Shimoda, Keiichi Nakayama, Yasunobu Yoshikai, Tyk2 signaling in host environment plays an important role in contraction of antigen-specific CD8+ T cells following a microbial infection, Journal of Immunology, 10.4049/jimmunol.178.7.4482, 178, 7, 4482-4488, 2007.04, Tyrosine kinase 2 (Tyk2), a member of JAK signal transducer family contributes to the signals triggered by IL-12 for IFN-γ production. To elucidate potential roles of Tyk2 in generation and maintenance of Ag-specific CD8+ T cells, we followed the fate of OVA-specific CD8+ T cells in Tyk2-deficient (-/-) mice after infection with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). Results showed that the numbers of OVA257-264/Kb tetramer-positive CD8+ T cells in Tyk2-/- mice were almost the same as those in Tyk2+/+ mice at the expansion phase on day 7 but were significantly larger in Tyk2 -/- mice than those in Tyk2+/+ mice at the contraction phase on day 10 and at the memory phase on day 60 after infection. The intracellular expression level of active caspase-3 was significantly decreased in the OVA-specific CD8+ T cells of Tyk2-/- mice on day 7 compared with those of Tyk2+/+ mice. Adaptive transfer experiments revealed that Tyk2 signaling in other factors rather than CD8+ T cells played a regulatory role in CD8+ T cell contraction following infection. Administration of exogenous IFN-γ from day 6 to day 9 restored the CD8+ T cell contraction in Tyk2-/- mice after infection with rLM-OVA. These results suggest that Tyk2 signaling for IFN-γ production in host environment plays an important role in contraction of effector CD8+ T cells following a microbial infection..
191. Paul S. Cooke, Denise R. Holsberger, Melissa A. Cimafranca, Daryl D. Meling, Charity M. Beals, Keiko Nakayama, Keiichi Nakayama, Hiroaki Kiyokawa, The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo, Obesity, 10.1038/oby.2007.168, 15, 6, 1400-1408, 2007.06, Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin-dependent kinase inhibitor p27 regulates activity of cyclin/cyclin- dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1-Cullin-F-box protein) complex targets proteins such as p27 for ubiquitin-proteosome degradation; the F box protein S phase kinase-associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2
-/-
) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild-type (WT), Skp2
-/-
, and p27
-/-
Skp2
-/-
mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2
-/-
fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2
-/-
mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27
-/-
Skp2
-/-
double knockout mice. The Skp2
-/-
decrements in adipocyte number and fat pad mass were totally reversed in p27
-/-
Skp2
-/-
mice. Adipogenesis was inhibited in MEFs from Skp2
-/-
vs. WT mice, and this inhibition was absent in MEFs from p27
-/-
Skp2
-/-
mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia..
192. Kana Miyamoto, Kiyomi Y. Araki, Kazuhito Naka, Fumio Arai, Keiyo Takubo, Satoshi Yamazaki, Sahoko Matsuoka, Takeshi Miyamoto, Keisuke Ito, Masako Ohmura, Chen Chen, Kentaro Hosokawa, Hiromitsu Nakauchi, Keiko Nakayama, Keiichi Nakayama, Mine Harada, Noboru Motoyama, Toshio Suda, Atsushi Hirao, Foxo3a Is Essential for Maintenance of the Hematopoietic Stem Cell Pool, Cell stem cell, 10.1016/j.stem.2007.02.001, 1, 1, 101-112, 2007.06, Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a-/- mice and showed that, although the proliferation and differentiation of Foxo3a-/- hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a-/- bone marrow cells and stromal cells was reduced. The ability of Foxo3a-/- HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a-/- HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a-/- mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool..
193. Tadashi Nakagawa, Michiko Shirane, Shun Ichiro Lemura, Tohru Natsume, Keiichi Nakayama, Anchoring of the 26S proteasome to the organellar membrane by FKBP38, Genes to Cells, 10.1111/j.1365-2443.2007.01086.x, 12, 6, 709-719, 2007.06, FK506-binding protein 38 (FKBP38) is a member of the immunophilin family that resides in the mitochondrial outer membrane and the endoplasmic reticulum (ER) membrane. To investigate the physiological function of FKBP38, we performed a comprehensive search for proteins with which it interacts in human cells by liquid chromatographic and mass spectrometric analysis of FKBP38 immunoprecipitates. Almost all subunits of the 26S proteasome were thus found to interact with FKBP38. In vivo co-immunoprecipitation analyses confirmed that FKBP38 indeed associates with the 26S proteasome via its three tandem tetratricopeptide repeats (TPRs). Binding assays in vitro also revealed that FKBP38 directly interacts with the S4 subunit of the 19S proteasome. Immunofluorescence analysis demonstrated that the subcellular distributions of FKBP38 and the 26S proteasome partially overlapped at mitochondria. Both the abundance and activity of the proteasome in a membrane fraction were markedly reduced for mouse embryonic fibroblasts prepared from Fkbp38-/- mice compared with those prepared from wild-type mice. These results suggest that FKBP38 functions to anchor the 26S proteasome at the organellar membrane..
194. Etsuo Susaki, Keiko Nakayama, Keiichi Nakayama, Cyclin D2 translocates p27 out of the nucleus and promotes its degradation at the G0-G1 transition, Molecular and Cellular Biology, 10.1128/MCB.00862-06, 27, 13, 4626-4640, 2007.07, The nuclear export and cytoplasmic degradation of the cyclin-dependent kinase inhibitor p27 are required for effective progression of the cell cycle through the G0-G1 transition. The mechanism responsible for this translocation of p27 has remained unclear, however. We now show that cyclin D2 directly links growth signaling with the nuclear export of p27 at the G0-G1 transition in some cell types. The up-regulation of cyclin D2 in response to mitogenic stimulation was found to occur earlier than that of other D-type cyclins and in parallel with down-regulation of p27 at the G0-G1 transition. RNA interference-mediated depletion of cyclin D2 inhibited the nuclear export of p27 and delayed its degradation at the G0-G1 transition. In contrast, overexpression of cyclin D2 in G0 phase shifted the localization of p27 from the nucleus to the cytoplasm and reduced the stability of p27. Overexpression of the cyclin D2(T280A) mutant, whose export from the nucleus is impaired, prevented the translocation and degradation of p27. These results indicate that cyclin D2 translocates p27 from the nucleus into the cytoplasm for its KPC-dependent degradation at the G0-G1 transition..
195. Rui Sheng Wang, Katsumi Ohtani, Megumi Suda, Kyoko Kitagawa, Keiichi Nakayama, Toshihiro Kawamoto, Tamie Nakajima, Reproductive toxicity of ethylene glycol monoethyl ether in Aldh2 knockout mice, Industrial health, 10.2486/indhealth.45.574, 45, 4, 574-578, 2007.08, Ethylene glycol monoethyl ether (EGEE) can cause damage to testes and sperm, and its metabolites are believed to play an important role in its toxicity. Aldehyde dehydrogenase 2 (ALDH2) is involved in the metabolism of this chemical. To investigate whether and how the enzyme affects the toxicity of EGEE, we conducted experiments comparing Aldh2 knockout mice with wild-type mice. Administration of EGEE at 100 and 600 mg/kg/day for one week did not induce any significant change in the weight and body weight ratios of testes, prostate and epididymides in either Aldh2 knockout or wild-type mice. However, motion of sperm from the spermaduct, as analyzed with a Hamilton-Thorne Sperm analyzer, was slightly decreased in the low dose group, and significantly lower in the high dose group; and the percentage of progressive sperm was also reduced in the two EGEE groups. This effect of EGEE treatment was observed in the wild-type, but not in the Aldh2 knockout mice. Sperm motion from the cauda epididymides was not affected. On the other hand, the concentration of ethoxyacetic acid, a metabolite of EGEE, in 24 h pooled urine of EGEE-treated Aldh2 knockout mice was not significantly lower than that of the wild-type mice on most days of urine sampling. These results suggest that inactivation of the ALDH2 enzyme due to gene mutation may be linked to differences in the susceptibility to EGEE-induced sperm toxicity..
196. Hidetoshi Wakeyama, Toru Akiyama, Katsuhiko Takahashi, Hitoshi Amano, Yuho Kadono, Masaki Nakamura, Yasushi Oshima, Hiroyuki Itabe, Keiichi Nakayama, Keiko Nakayama, Kozo Nakamura, Sakae Tanaka, Negative feedback loop in the Bim-caspase-3 axis regulating apoptosis and activity of osteoclasts, Journal of Bone and Mineral Research, 10.1359/jbmr.070619, 22, 10, 1631-1639, 2007.10, Proapoptotic Bcl-2 family member Bim plays an essential role in the osteoclast apoptosis and is degraded through ubiquitin/proteasome pathways in a caspase-3-dependent manner. This negative feedback loop in the Bim-caspase-3 axis is important for regulating the survival and activity of osteoclasts. Introduction: Bim is a member of the proapoptotic Bcl-2 family and regulates the mitochondrial apoptosis pathway. Bim expression is post-translationally regulated in osteoclasts (OCs) through ubiquitin/proteasome pathways, and Bim is critical for their survival and activity. Materials and Methods: Time-course of change in the expression of Bim in the course of OC apoptosis was examined, and the effect of various proteinase inhibitors on the degradation of Bim was analyzed. The role of caspase-3 and caspase-7 on Bim degradation was studied using RNA interference technique and caspase-3-/- mice. Results: Bim was degraded after caspase-3 activation, which was suppressed by a caspase inhibitor and a proteasome inhibitor. Bim degradation was suppressed by gene knockdown of caspase-3 or in caspase-3-/- OCs but not by caspase-7 knockdown. OCs generated from caspase-3-/- bone marrow cells exhibited a shorter life span and higher bone-resorbing activity than normal OCs. Association of Bim with E3 ubiquitin ligase c-Cbl was suppressed by gene knockdown of caspase-3 or in caspase-3-/- OCs. Actin ring formation and cathepsin K expression were promoted in caspase-3-/- OCs. Conclusions: Caspase-3 negatively regulates Bim expression by stimulating its degradation through ubiquitin/ proteasome pathways, thus creating a negative feedback loop in the Bim-caspase axis..
197. Lingwen Zhong, Senta Georgia, Shuen Ing Tschen, Keiko Nakayama, Keiichi Nakayama, Anil Bhushan, Essential role of Skp2-mediated p27 degradation in growth and adaptive expansion of pancreatic β cells, Journal of Clinical Investigation, 10.1172/JCI32198, 117, 10, 2869-2876, 2007.10, Diabetes results from an inadequate mass of functional β cells, due to either β cell loss caused by immune assault or the lack of compensation to overcome insulin resistance. Elucidating the mechanisms that regulate β cell mass has important ramifications for fostering β cell regeneration and the treatment of diabetes. We report here that Skp2, a substrate recognition component of Skp1-Cul1-F-box (SCF) ubiquitin ligase, played an essential and specific role in regulating the cellular abundance of p27 and was a critical determinant of β cell proliferation. In Skp2-/- mice, accumulation of p27 resulted in enlarged polyploid β cells as a result of endoreduplication replacing proliferation. Despite β cell hypertrophy, Skp2-/- mice exhibited diminished β cell mass, hypoinsulinemia, and glucose intolerance. Increased insulin resistance resulting from diet-induced obesity caused Skp2-/- mice to become overtly diabetic, because β cell growth in the absence of cell division was insufficient to compensate for increased metabolic demand. These results indicate that the Skp2-mediated degradation pathway regulating the cellular degradation of p27 is essential for establishing β cell mass and to respond to increased metabolic demand associated with insulin resistance..
198. Camille Ehre, Yunxiang Zhu, Lubna H. Abdullah, John Olsen, Keiichi Nakayama, Keiko Nakayama, Robert O. Messing, C. William Davis, nPKCε, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells, American Journal of Physiology - Cell Physiology, 10.1152/ajpcell.00051.2007, 293, 5, 2007.11, Airway goblet cell mucin secretion is controlled by agonist activation of P2Y2 purinoceptors, acting through Gq/PLC, inositol-1,4,5- trisphosphate (IP3), diacylglycerol, Ca2+ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCα, nPKCβ, nPKCε, and nPKCη; of these, only nPKCδ translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCα, nPKCδ, and nPKCη had the same levels of ATPγS- and phorbol-1,2-myristate-13- acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPγS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCε exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPγS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y2-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCδ knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCε KO mice relative to its WT littermates. We conclude that nPKCε is the effector isoform downstream of P2Y2-R activation in the goblet cell secretory response. The translocation of nPKCδ observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology..
199. Tomonari Matsuda, Akiko Matsumoto, Mitsuhiro Uchida, Robert A. Kanaly, Kentaro Misaki, Shinya Shibutani, Toshihiro Kawamoto, Kyoko Kitagawa, Keiichi Nakayama, Katsumaro Tomokuni, Masayoshi Ichiba, Increased formation of hepatic N2 -ethylidene-2′-deoxyguanosine DNA adducts in aldehyde dehydrogenase 2-knockout mice treated with ethanol, Carcinogenesis, 10.1093/carcin/bgm057, 28, 11, 2363-2366, 2007.11, N2-ethylidene-2′-deoxyguanosine (N2 -ethylidene-dG) is a major DNA adduct induced by acetaldehyde. Although it is unstable in the nucleoside form, it is relatively stable when present in DNA. In this study, we analyzed three acetaldehyde-derived DNA adducts, N2-ethylidene-dG, N2-ethyl-2′-deoxyguanosine (N2-Et-dG) and α-methyl-γ-hydroxy-1, N2 -propano-2′-deoxyguanosine (α-Me-γ-OH-PdG) in the liver DNA of aldehyde dehydrogenase (Aldh)-2-knockout mice to determine the influence of alcohol consumption and the Aldh2 genotype on the levels of DNA damage. In control Aldh2+/+ mice, the level of N2-ethylidene-dG adduct in liver DNA was 1.9 ± 0.7 adducts per 107 bases and was not significantly different than that of Aldh2+/- and -/- mice. In alcohol-fed mice (20% ethanol for 5 weeks), the adduct levels of Aldh2+/+, +/- and -/- mice were 7.9 ± 1.8, 23.3 ± 4.0 and 79.9 ± 14.2 adducts per 107 bases, respectively, and indicated that adduct level was alcohol and Aldh2 genotype dependent. In contrast, an alcohol- or Aldh2 genotype-dependent increase was not observed for α-Me-γ-OH-PdG, and N2-Et-dG was not detected in any of the analyzed samples. In conclusion, the risk of formation of N2-ethylidene-dG in model animal liver in vivo is significantly higher in the Aldh2-deficient population and these results may contribute to our understanding of in vivo adduct formation in humans..
200. Ichiro Onoyama, Ryosuke Tsunematsu, Akinobu Matsumoto, Taichi Kimura, Ignacio Moreno De Alborán, Keiko Nakayama, Keiichi Nakayama, Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis, Journal of Experimental Medicine, 10.1084/jem.20062299, 204, 12, 2875-2888, 2007.11, Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4+ CD8+ stage, but the mechanism underlying such differentiation- specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4+ CD8+ stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells. JEM.
201. Tomoyo Kawakubo, Kuniaki Okamoto, Jun Ichi Iwata, Masashi Shin, Yoshiko Okamoto, Atsushi Yasukochi, Keiichi Nakayama, Tomoko Kadowaki, Takayuki Tsukuba, Kenji Yamamoto, Cathepsin E prevents tumor growth and metastasis by catalyzing the proteolytic release of soluble TRAIL from tumor cell surface, Cancer Research, 10.1158/0008-5472.CAN-07-2048, 67, 22, 10869-10878, 2007.11, The aspartic proteinase cathepsin E is expressed predominantly in cells of the immune system and highly secreted by activated phagocytes, and deficiency of cathepsin E in mice results in a phenotype affecting immune responses. However, because physiologic substrates for cathepsin E have not yet been identified, the relevance of these observations to the physiologic functions of this protein remains speculative. Here, we show that cathepsin E specifically induces growth arrest and apoptosis in human prostate carcinoma tumor cell lines without affecting normal cells by catalyzing the proteolytic release of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from the cell surface. The antitumor activity of cathepsin E was corroborated by in vivo studies with mice bearing human and mouse tumor transplants. Administration of purified cathepsin E into human tumor xenografts in nude mice dose-dependently induced apoptosis in the tumor cells to inhibit tumor growth. The growth, viability, and metastasis of mouse B16 melanoma cells were also more profound in cathepsin E-deficient mice compared with those in the syngeneic wild-type and transgenic mice overexpressing cathepsin E. Taken together, the number of apoptotic tumor cells, as well as tumor-infiltrating activated macrophages, was apparently reduced in cathepsin E-deficient mice compared with those in the other two groups, implying the positive correlation of endogenous cathepsin E levels with the extent of tumor suppression in vivo. These results thus indicate that cathepsin E plays a substantial role in host defense against tumor cells through TRAIL-dependent apoptosis and/or tumor-associated macrophage-mediated cytotoxicity..
202. Chihiro Ueyama, Hiroki Akashiba, Keiko Nakayama, Keiichi Nakayama, Nobuyoshi Nishiyama, Norio Matsuki, Ablation of p27 enhance kainate-induced seizure and hippocampal degeneration, NeuroReport, 10.1097/WNR.0b013e3282f16df6, 18, 17, 1781-1785, 2007.11, p27 is a cyclin-dependent kinase inhibitor which arrests cell cycle at G1-S phase. Using RNA interference method, we previously showed that reduction of endogenous p27 expression induces cell death through cell cycle progression in cultured cortical neurons. In this study, we investigated responses to kainate treatment using p27 knockout mice. Injection of kainic acid induced p27 downregulation and retinoblastoma protein phosphorylation in wild-type mouse hippocampus. No change was observed in hippocampal cell viability in untreated adult p27 heterozygous and homozygous mice compared with wild type (+/+). p27 homozygous mice, however, displayed enhanced seizure and hippocampal degeneration after kainic acid treatment. This study first suggests that ablation of p27 enhance kainate-induced seizure and hippocampal cell death in vivo..
203. Etsuo Susaki, Keiichi Nakayama, Multiple mechanisms for p27Kip1 translocation and degradation, Cell Cycle, 10.4161/cc.6.24.5087, 6, 24, 3015-3020, 2007.12, The nuclear export and rapid degradation of p27Kip1 at the G0-G1 transition are critical events for effective progression of the cell cycle. Several pathways have been proposed at the molecular level for the export of this cyclin-dependent kinase inhibitor from the nucleus. However, the addition of each new pathway renders the situation more complicated. We recently showed that cyclin D2 links growth signals to the cytoplasmic translocation and degradation of p27 at the G0-G 1 transition. Here we describe our findings and discuss how the multiple potential mechanisms for p27 translocation that precedes its degradation might be integrated in the context of growth stimulation and G 1 progression..
204. Jayme R. Gallegos, Joel Litersky, Hunjoo Lee, Yi Sun, Keiichi Nakayama, Keiko Nakayama, Hua Lu, SCFβTrCP1 activates and ubiquitylates TAp63γ, Journal of Biological Chemistry, 10.1074/jbc.M704686200, 283, 1, 66-75, 2008.01, p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and also has an important role in cancer progression. Currently, the molecular mechanisms governing regulation of p63 function remain largely unclear. This study identifies a unique E3 ubiquitin ligase for p63, SCFβTrCP1. SCFβTrCP1 is able to bind p63γ isoforms, with a higher affinity for the TAp63γ isoform. Strikingly, co-expression of TAp63γ and βTrCP1 leads to the stabilization of TAp63γ. This stabilization of TAp63γ leads to up-regulation of p21 at the mRNA and protein level by increased binding of TAp63γ at the p21 promoter. The up-regulation of p21 causes a subsequent increase in G1 phase cell cycle arrest. Last, SCFβTrCP1 is able to ubiquitylate TAp63γ, and this ubiquitylation, as well as the increased activity of TAp63γ, is ablated with the expression of a ubiquitin-deficient mutant of βTrCP1 (ΔFβTrCP1). Therefore, our study reveals that SCF βTrCP1 is an E3 ligase that activates p63 through ubiquitylation..
205. Sahoko Matsuoka, Yuichi Oike, Ichiro Onoyama, Atsushi Iwama, Fumio Arai, Keiyo Takubo, Yoichi Mashimo, Hideyuki Oguro, Eriko Nitta, Keisuke Ito, Kana Miyamoto, Hiroki Yoshiwara, Kentaro Hosokawa, Yuka Nakamura, Yumiko Gomei, Hiroko Iwasaki, Yasuhide Hayashi, Yumi Matsuzaki, Keiko Nakayama, Yasuo Ikeda, Akira Hata, Shigeru Chiba, Keiichi Nakayama, Toshio Suda, Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL, Genes and Development, 10.1101/gad.1621808, 22, 8, 986-991, 2008.04, Common molecular machineries between hematopoietic stem cell (HSC) maintenance and leukemia prevention have been highlighted. The tumor suppressor Fbxw7 (F-box and WD-40 domain protein 7), a subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle. We demonstrate that inactivation of Fbxw7 in hematopoietic cells causes premature depletion of HSCs due to active cell cycling and p53-dependent apoptosis. Interestingly, Fbxw7 deletion also confers a selective advantage to cells with suppressed p53 function, eventually leading to development of T-cell acute lymphoblastic leukemia (TALL). Thus, Fbxw7 functions as a fail-safe mechanism against both premature HSC loss and leukemogenesis..
206. Akiko Mukai, Emi Mizuno, Kaoru Kobayashi, Masaki Matsumoto, Keiichi Nakayama, Naomi Kitamura, Masayuki Komada, Dynamic regulation of ubiquitylation and deubiquitylation at the central spindle during cytokinesis, Journal of Cell Science, 10.1242/jcs.027417, 121, 8, 1325-1333, 2008.04, During cytokinesis, the central spindle, a bundle of interdigitated anti-parallel microtubules between separating chromosomes, recruits various cytokinetic regulator proteins to the cleavage region. Here, we show that the level of protein ubiquitylation is strikingly and transiently elevated in Aurora B kinase-positive double-band regions of the central spindle during cytokinesis. Two deubiquitylating enzymes UBPY and AMSH, which act on endosomes in interphase, were also recruited to the cleavage region. Whereas UBPY was detected only in the final stage of cytokinesis at the midbody, AMSH localized to a ring structure surrounding the mitotic kinesin MKLP1-positive region of the central spindle and midbody throughout cytokinesis. Depletion of cellular UBPY or AMSH led to defects in cytokinesis. VAMP8, a v-SNARE required for vesicle fusion in cytokinesis, localized to the central spindle region positive for ubiquitylated proteins, and underwent ubiquitylation and deubiquitylation by both UBPY and AMSH. Our results thus implicate the ubiquitylation/deubiquitylation of proteins including VAMP8 in cytokinesis..
207. Shin Ichiro Inoue, Toshinori Kinoshita, Masaki Matsumoto, Keiichi Nakayama, Michio Doi, Ken Ichiro Shimazaki, Blue light-induced autophosphorylation of phototropin is a primary step for signaling, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0709189105, 105, 14, 5626-5631, 2008.04, Phototropins are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatography-tandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events..
208. Qing Chen, Weilin Xie, Deborah J. Kuhn, Peter M. Voorhees, Antonia Lopez-Girona, Derek Mendy, Laura G. Corral, Veronique Plantevin Krenitsky, Weiming Xu, Laure Moutouh De Parseval, David R. Webb, Frank Mercurio, Keiichi Nakayama, Keiko Nakayama, Robert Z. Orlowski, Targeting the p27 E3 ligase SCF Skp2 results in p27and Skp2-mediated cell-cycle arrest and activation of autophagy, Blood, 10.1182/blood-2007-09-112904, 111, 9, 4690-4699, 2008.05, Decreased p27 KlP1 levels are a poor prognostic factor in many malignancies, and can occur through up-regulation of SCF skP2 E3 ligase function, resulting in enhanced p27 ubiquitination and proteasome-mediated degradation. While proteasome inhibitors stabilize p27 KiP1 agents inhibiting SCF skp2 may represent more directly targeted drugs with the promise of enhanced efficacy and reduced toxicity. Using high-throughput screening, we identified Compound A (CpdA), which interfered with SCF skP2 ligase function in vitro, and induced specific accumulation of p21 and other SCF skp2 substrates in cells without activating a heatshock protein response. CpdA prevented incorporation of Skp2 into the SCF skp2 ligase, and induced G- 1/S cell-cycle arrest as well as SCF skp2and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF skp2 inhibitors as a novel class of antitumor agents..
209. M. S. Song, S. J. Song, S. J. Kim, K. Nakayama, Keiichi Nakayama, D. S. Lim, Skp2 regulates the antiproliferative function of the tumor suppressor RASSF1A via ubiquitin-mediated degradation at the G1-S transition, Oncogene, 10.1038/sj.onc.1210971, 27, 22, 3176-3185, 2008.05, The tumor suppressor RASSF1A is inactivated in many human cancers and is implicated in regulation of microtubule stability, cell cycle progression and apoptosis. However, the precise mechanisms of RASSF1A action and their regulation remain unclear. Here we show that Skp2, an oncogenic subunit of the Skp1-Cul1-F-box ubiquitin ligase complex, interacts with, ubiquitinates, and promotes the degradation of RASSF1A at the G1-S transition of the cell cycle. This Skp2-dependent destruction of RASSF1A requires phosphorylation of the latter on serine-203 by cyclin D-cyclin-dependent kinase 4. Interestingly, mutation of RASSF1A-phosphorylation site Ser203 to alanine results in a delay in cell cycle progression from G1 to S phase. Moreover, enforced expression of Skp2 abolishes the inhibitory effect of RASSF1A on cell proliferation. Finally, the delay in G1-S progression after Skp2 removal is normalized by depletion of RASSF1A. These findings suggest that the Skp2-mediated degradation of RASSF1A plays an important role in cell proliferation and survival..
210. Naoki Shigematsu, Takaichi Fukuda, Tsuneyuki Yamamoto, Tsuyoshi Nishioku, Taku Yamaguchi, Masaru Himeno, Keiichi Nakayama, Takayuki Tsukuba, Tomoko Kadowaki, Kuniaki Okamoto, Shun Higuchi, Kenji Yamamoto, Association of cathepsin E deficiency with the increased territorial aggressive response of mice, Journal of Neurochemistry, 10.1111/j.1471-4159.2008.05242.x, 105, 4, 1394-1404, 2008.05, Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE-/-) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE-/- mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE-/- mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE-/- mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge..
211. Michiko Shirane, Masaharu Ogawa, Jun Motoyama, Keiichi Nakayama, Regulation of apoptosis and neurite extension by FKBP38 is required for neural tube formation in the mouse, Genes to Cells, 10.1111/j.1365-2443.2008.01194.x, 13, 6, 635-651, 2008.06, FKBP38 (also known as FKBP8) is a transmembrane chaperone protein that inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-x L to mitochondria. We have now generated mice harboring a loss-of-function mutation in Fkbp38. The Fkbp38-/- mice die soon after birth manifesting defects in neural tube closure in the thoraco-lumbar-sacral region (spina bifida) as well as skeletal defects including scoliosis, rib deformities, club foot and curled tail. The neuroepithelium is disorganized and that formation of dorsal root ganglia is defective in Fkbp38-/- embryos, likely as a result of an increased frequency of apoptosis and aberrant migration of neuronal cells. Furthermore, the extension of nerve fibers in the spinal cord is abnormal in the mutant embryos. To explore the mechanisms underlying these characteristics, we screened for proteins that interact with FKBP38 in the yeast two-hybrid system and thereby identified protrudin, a protein that promotes process formation by regulating membrane trafficking. Protrudin was found to be hyperphosphorylated in the brain of Fkbp38-/- mice, suggesting that FKBP38 regulates protrudin-dependent membrane recycling and neurite outgrowth. Together, our findings suggest that FKBP38 is required for neuroectodermal organization during neural tube formation as a result of its anti-apoptotic activity and regulation of neurite extension..
212. Risa Nakamura, Kensuke Shibata, Hisakata Yamada, Kazuya Shimoda, Keiichi Nakayama, Yasunobu Yoshikai, Tyk2-signaling plays an important role in host defense against Escherichia coli through IL-23-induced IL-17 production by γδ T cells, Journal of Immunology, 181, 3, 2071-2075, 2008.08, Tyrosine kinase 2 (Tyk2), a member of the JAK-signal transducer family, is involved in intracellular signaling triggered by various cytokines, including IL-23. We have recently reported that resident γδ T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23. In this study, we examined importance of Tyk2-mediated signaling in the IL-17 production by γδ T cells using Tyk2 deficient (-/-) mice. γ T cells in the peritoneal cavity of Tyk2-/- mice displayed effecter/memory phenotypes and TCR V repertoire similar to those in Tyk2+/+ mice and produced comparable level of IL-17 to those in Tyk2+/+ mice in response to PMA and ionomycin, indicating normal differentiation to IL-17-producing effectors in the absence of Tyk2-signaling. However, γδ T cells in Tyk2-/- mice produced less amount of IL-17 in response to IL-23 in vitro than those in Tyk2+/+ mice. Similarly, γδ T cells in the peritoneal cavity of Tyk2-/- mice showed severely impaired IL-17 production after an i.p. infection with E. coli despite comparable level of IL-23 production to Tyk2+/+ mice. As a consequence, Tyk2-/- mice showed a reduced infiltration of neutrophils and severely impaired bacterial clearance after Escherichia coli infection. These results indicate that Tyk2-signaling is critical for IL-23-induced IL-17 production by γδ T cells, which is involved in the first line of host defense by controlling neutrophil-mediated immune responses..
213. Ichiro Onoyama, Keiichi Nakayama, Regulation of cell-cycle exit and G0-phase maintenance during differentiation by Fbxw7, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 53, 10, 1217-1224, 2008.08.
214. Anupriya Agarwal, Thomas G P Bumm, Amie S. Corbin, Thomas O'Hare, Marc Loriaux, Jonathan Van Dyke, Stephanie G. Willis, Jutta Deininger, Keiichi Nakayama, Brian J. Druker, Michael W. Deininger, Absence of SKP2 expression attenuates BCR-ABL-induced myeloproliferative disease, Blood, 10.1182/blood-2007-09-113860, 112, 5, 1960-1970, 2008.09, BCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCF SKP2 (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G 1 arrest, downregulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2 V617F, and TEL-PDGFRβ, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL-infected SKP2 -/- marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2 +/+ marrow. SKP2 -/- leukemic cells demonstrated higher levels of nuclear p27 than SKP2 +/+ counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL-induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCF SKP2 may be therapeutically useful..
215. Y. Ishikawa, Ichiro Onoyama, Keiichi Nakayama, K. Nakayama, Notch-dependent cell cycle arrest and apoptosis in mouse embryonic fibroblasts lacking Fbxw7, Oncogene, 10.1038/onc.2008.216, 27, 47, 6164-6174, 2008.10, The F-box protein Fbxw7 mediates the ubiquitylation and consequent degradation of proteins that regulate cell cycle progression, including cyclin E, c-Myc, c-Jun and Notch. Moreover, certain human cancer cell lines harbor loss-of-function mutations in FBXW7 that result in excessive accumulation of Fbxw7 substrates, implicating Fbxw7 in tumor suppression. To elucidate the physiological function of Fbxw7, we conditionally ablated Fbxw7 in mouse embryonic fibroblasts (MEFs). Unexpectedly, loss of Fbxw7 induced cell cycle arrest and apoptosis that were accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1). Forced expression of NICD1 in wild-type MEFs recapitulated the phenotype of the Fbxw7-deficient (Fbxw7
Δ/Δ
) MEFs. Conversely, deletion of Rbpj normalized the phenotype of Fbxw7
Δ/Δ
MEFs, indicating that this phenotype is dependent on the Notch1-RBP-J signaling pathway. Deletion of the p53 gene prevented cell cycle arrest but not the induction of apoptosis in Fbxw7
Δ/Δ
cells. These observations suggest that Fbxw7 does not function as an oncosuppressor in MEFs. Instead, it promotes cell cycle progression and cell survival through degradation of Notch1, with loss of Fbxw7 resulting in NICD1 accumulation, cell cycle arrest and apoptosis..
216. Nobutaka Sakae, Nobuyuki Yamasaki, Kiyoyuki Kitaichi, Takaichi Fukuda, Mitsunori Yamada, Hiroo Yoshikawa, Takato Hiranita, Yoshiki Tatsumi, Jun-Ichi Kira, Tsuneyuki Yamamoto, Tsuyoshi Miyakawa, Keiichi Nakayama, Mice lacking the schizophrenia-associated protein FEZ1 manifest hyperactivity and enhanced responsiveness to psychostimulants, Human Molecular Genetics, 10.1093/hmg/ddn215, 17, 20, 3191-3203, 2008.10, FEZ1 (fasciculation and elongation protein zeta 1), a mammalian ortholog of Caenorhabditis elegans UNC-76, interacts with DISC1 (disrupted in schizophrenia 1), a schizophrenia susceptibility gene product, and polymorphisms of human FEZ1 have been associated with schizophrenia. We have now investigated the role of FEZ1 in brain development and the pathogenesis of schizophrenia by generating mice that lack Fez1. Immunofluorescence staining revealed FEZ1 to be located predominantly in γ-aminobutyric acid-containing interneurons. The Fez1-/- mice showed marked hyperactivity in a variety of behavioral tests as well as enhanced behavioral responses to the psychostimulants MK-801 and methamphetamine. In vivo microdialysis revealed that the methamphetamine-induced release of dopamine in the nucleus accumbens was exaggerated in the mutant mice, suggesting that enhanced mesolimbic dopaminergic transmission contributes to their hyperactivity phenotype. These observations implicate impairment of FEZ1 function in the pathogenesis of schizophrenia..
217. Akiko Matsumoto, Toshihiro Kawamoto, Fumihiro Mutoh, Toyohi Isse, Tsunehiro Oyama, Kyoko Kitagawa, Keiichi Nakayama, Masayoshi Ichiba, Effects of 5-week ethanol feeding on the liver of aldehyde dehydrogenase 2 knockout mice, Pharmacogenetics and Genomics, 10.1097/FPC.0b013e328307a0a9, 18, 10, 847-852, 2008.10, Objective The polymorphism of aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2, is very common in East Asian countries, and the mutated ALDH2 protein derived from ALDH2*2 lacks the ability of acetaldehyde metabolization. Our aim was to determine the consequences of the ALDH2 polymorphism on ethanol-administered liver tissue. Methods Aldh2+/+, +/-, and -/- mice were fed with ethanol solution and standard hard feed for 5 weeks. Results The serum alanine aminotransferase (ALT) level in the Aldh2-/- mice clearly decreased upon ethanol feeding, in contrast to Aldh2+/+ mice, in which the ALT level was unchanged. The levels of malondialdehyde, phospho extracellular signal-regulated kinase 2, and tumor necrosis factor-alpha in the liver tissue all correlated with the ALT level. Conclusion These findings suggest that ethanol intake in the presence of inactive ALDH2 decreases the serum ALT level following a decrease in oxidative stress and tumor necrosis factor-alpha secretion..
218. Kanae Ohsaki, Katsutaka Oishi, Yuko Kozono, Keiko Nakayama, Keiichi Nakayama, Norio Ishida, The role of β-TrCP1 and β-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2, Journal of biochemistry, 10.1093/jb/mvn112, 144, 5, 609-618, 2008.11, The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein β-TrCP1, a component of ubiquitin E3 ligase. Here, we show that β-TrCP2 as well as β-TrCP1 target PER2 protein in vitro. We also identified β-TrCP binding site (m2) of PER2 being recognized by both β-TrCP1 and β-TrCP2. Luciferase-PER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of β-TrCP1 and β-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of β-TrCP1 and/or β-TrCP2 attenuate circadian oscillations in NIH3T3 cell. β-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that β-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that β-TrCP1 and β-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of β-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated..
219. Akinobu Matsumoto, Keiichi Nakayama, Regulation of cell proliferation and tumorigenesis by ubiquitin-proteasome pathway, Biotherapy, 22, 6, 363-370, 2008.11, The strict regulation of protein degradation by the ubiquitin-proteasome pathway is pivotal for cell cycle progression. Skp2 and Fbw7 are F-box proteins that are substrate-recognition subunits of the SCF-type ubiquitin ligase complex and involved in cell cycle entry and exit. Skp2 promotes the degradation of negative regulators of the cell cycle, such as p27, and facilitates the cell cycle entry. On the other hand, Fbw7 promotes the degradation of positive regulators of the cell cycle, such as c-Myc, and induces the cell cycle exit. We have demonstrated the in vivo roles of Skp2 and Fbw7 in cell cycle regulation using gene-targeting technology in mice. Consistent with our animal models, clinical studies have suggested Skp2 as an oncogene and Fbw7 as an oncosuppressor gene. These accumulating lines of evidence support the notion that Skp2 and Fbw7 are involved in cell cycle regulation and tumorigenesis..
220. Ichiro Onoyama, Keiichi Nakayama, Fbxw7 in cell cycle exit and stem cell maintenance
Insight from gene-targeted mice, Cell Cycle, 10.4161/cc.7.21.6931, 7, 21, 3307-3313, 2008.11, Regulation of the exit of cells from the cell cycle is important in the development of multicellular organisms and is also implicated in the maintenance of stem cells. Furthermore, defects in cell cycle exit are thought to be a major cause of cancer. However, the mechanisms responsible for regulation of cell cycle exit have remained largely unknown. Fbxw7 is the F-box protein subunit of an SCF-type ubiquitin ligase complex that targets positive regulators of the cell cycle - including cyclin E, c-Myc, Notch and c-Jun - for ubiquitylation and subsequent degradation by the 26S proteasome in order to promote cell cycle exit. Consistent with such a function, mutations of the Fbxw7 gene have been detected in various human malignancies. We have recently generated conventional and conditional Fbxw7 knockout mice and examined stem cells, progenitor cells and differentiated cells in the mutant animals for cell cycle defects. Here we summarize the pleiotropic phenotypes of Fbxw7 deficiency in various cell types including T cells, hematopoietic stem cells and embryonic fibroblasts. Such phenotypes have provided insight into the biological roles of Fbxw7 in cell cycle exit, stem cell maintenance and oncosuppression..
221. Chie Kanei-Ishii, Teruaki Nomura, Tsuyoshi Takagi, Nobumoto Watanabe, Keiichi Nakayama, Shunsuke Ishii, Fbxw7 acts as an E3 ubiquitin ligase that targets c-Myb for Nemo-like kinase (NLK)-induced degradation, Journal of Biological Chemistry, 10.1074/jbc.M804340200, 283, 45, 30540-30548, 2008.11, The c-myb proto-oncogene product (c-Myb) is degraded in response to Wnt-1 signaling via a pathway involving TAK1 (transforming growth factor-β- activated kinase 1), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). NLK directly binds to c-Myb, which results in the phosphorylation of c-Myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. Here, we report that Fbxw7, the F-box protein of an SCF complex, targets c-Myb for degradation in a Wnt-1- and NLK-dependent manner. Fbxw7α directly binds to c-Myb via its C-terminal WD40 domain and induces the ubiquitination of c-Myb in the presence of NLK in vivo and in vitro. The c-Myb phosphorylation site mutant failed to interact with Fbxw7α, suggesting that the c-Myb/Fbxw7α interaction is enhanced by NLK phosphorylation of c-Myb. Treatment of M1 cells with Fbxw7 small interfering RNA (siRNA) rescued the Wnt-induced c-Myb degradation and also the Wnt-induced inhibition of cell proliferation. NLK bound to Cul1, a component of the SCF complex, while HIPK2 interacted with both Fbxw7α and Cul1, suggesting that both kinases enhance the c-Myb/SCF interaction. In contrast to c-Myb, the v-myb gene product (v-Myb) encoded by the avian myeloblastosis virus was resistant to NLK/Fbxw7α-induced degradation. Thus, Fbxw7 is an E3 ubiquitin ligase of c-Myb, and the increased c-Myb levels may contribute, at least partly, to transformation induced by mutation of Fbxw7..
222. Mimi Tamamori-Adachi, Hiromitsu Takagi, Kimio Hashimoto, Kazumichi Goto, Toshinori Hidaka, Uichi Koshimizu, Kazuhiko Yamada, Ikuko Goto, Yasuhiro Maejima, Mitsuaki Isobe, Keiichi Nakayama, Norio Inomata, Shigetaka Kitajima, Cardiomyocyte proliferation and protection against post-myocardial infarction heart failure by cyclin D1 and Skp2 ubiquitin ligase, Cardiovascular research, 10.1093/cvr/cvn183, 80, 2, 181-190, 2008.11, Aims: Cyclins and other cell-cycle regulators have been used in several studies to regenerate cardiomyocytes in ischaemic heart failure. However, proliferation of cardiomyocytes induced by nuclear-targeted cyclin D1 (D1NLS) stops after one or two rounds of cell cycles due in part to accumulation of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK). Thus, expression of S-phase kinase-associated protein 2 (Skp2), a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on cardiomyocyte proliferation in vitro. Here, we examined whether Skp2 can also improve cardiomyocyte regeneration and post-ischaemic cardiac performance in vivo. Methods and results: Wistar rats underwent ischaemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs. 4.00%), and mitotic phosphorylated histone H3 (0.24% vs. 0.58%). Compared with rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio. Conclusion: Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischaemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischaemic regeneration of cardiomyocytes in vivo..
223. Gustavo A. Miranda-Carboni, Susan A. Krum, Kathleen Yee, Miguel Nava, Qiming E. Deng, Shehla Pervin, Alicia Collado-Hidalgo, Zoran Galić, Jerome A. Zack, Keiko Nakayama, Keiichi Nakayama, Timothy F. Lane, A functional link between Wnt signaling and SKP2-independent p27 turnover in mammary tumors, Genes and Development, 10.1101/gad.1692808, 22, 22, 3121-3134, 2008.11, Loss of the CDK inhibitor p27KIP1 is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27KIP1 were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27KIP1. Interestingly we found that Wnt-induced turnover of p27 KIP1 was independent from classical SCFSKP2-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27KIP1 in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27KIP1 degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27 KIP1 levels and cell cycle progression in mammalian cells..
224. Yu Lu, Olasunkanmi A.J. Adegoke, Alain Nepveu, Keiichi Nakayama, Nathalie Bedard, Dongmei Cheng, Junmin Peng, Simon S. Wing, USP19 deubiquitinating enzyme supports cell proliferation by stabilizing KPC1, a ubiquitin ligase for p27Kip1, Molecular and Cellular Biology, 10.1128/MCB.00329-08, 29, 2, 547-558, 2009.01, p27Kip1 is a cyclin-dependent kinase inhibitor that regulates the G1 transition. Increased degradation of p27Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCFSkp2 ubiquitinate p27 Kip1 in G1 and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G0/G1 to S phase, and accumulated p27Kip1. This increase in p27Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27-/- cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G1 to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion..
225. Masaaki Nishiyama, Kiyotaka Oshikawa, Yuichi Tsukada, Tadashi Nakagawa, Shun Ichiro Iemura, Tohru Natsume, Yuhong Fan, Akira Kikuchi, Arthur I. Skoultchi, Keiichi Nakayama, CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis, Nature Cell Biology, 10.1038/ncb1831, 11, 2, 172-182, 2009.01, The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8-/- mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1..
226. Mohd Minhajuddin, Kaiser M. Bijli, Fabeha Fazal, Antonella Sassano, Keiichi Nakayama, Nissim Hay, Leonidas C. Platanias, Arshad Rahman, Protein kinase C-δ and phosphatidylinositol 3-Kinase/Akt activate mammalian target of rapamycin to modulate NF-κB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells, Journal of Biological Chemistry, 10.1074/jbc.M805032200, 284, 7, 4052-4061, 2009.02, We have shown that the mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by suppressing the activation of NF-κB. However, the mechanisms by which mTOR is activated to modulate these responses remain to be addressed. Here, we show that thrombin engages protein kinase C (PKC)-δ and phosphattidylinositol 3-kinase (PI3K)/Akt pathways to activate mTOR and thereby dampens NF-κB activation and intercellular adhesion molecule 1 (ICAM-1) expression. Stimulation of human vascular endothelial cells with thrombin induced the phosphorylation of mTOR and its downstream target p70 S6 kinase in a PKC-δ- and PI3K/Akt-dependent manner. Consistent with this, thrombin-induced phosphorylation of p70 S6 kinase was defective in embryonic fibroblasts from mice with targeted disruption of PKC-δ (Pkc-δ-/-), p85α and p85β subunits of the PI3K (p85α-/-β-/-), or Akt1 and Akt2 (Akt1-/-2-/-). Furthermore, we observed that expression of the constitutively active form of PKC-δ or Akt was sufficient to induce NF-κB activation and ICAM-1 expression, and that co-expression of mTOR suppressed these responses. In reciprocal experiments, inhibition/depletion of mTOR augmented NF-κB activation and ICAM-1 expression induced by PKC-δ or Akt. In control experiments, increasing or impairing mTOR signaling by the above approaches produced similar effects on NF-κB activation and ICAM-1 expression induced by thrombin. Thus, these data reveal an important role of PKC-δ and PI3K/Akt pathways in activating mTOR as an endogenous modulator to ensure a tight regulation of NF-κB signaling of ICAM-1 expression in endothelial cells..
227. Hui Kuan Lin, Guocan Wang, Zhenbang Chen, Julie Teruya-Feldstein, Yan Liu, Chia Hsin Chan, Wei Lei Yang, Hediye Erdjument-Bromage, Keiichi Nakayama, Stephen Nimer, Paul Tempst, Pier Paolo Pandolfi, Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB, Nature Cell Biology, 10.1038/ncb1849, 11, 4, 420-432, 2009.03, Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 are critical for Skp2-mediated entry to the cell cycle, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis, although the functional significance of the latter is still unknown. Here we show that Akt/ protein kinase B (PKB) interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. A phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3β-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of activation of Akt correlate with the cytosolic accumulation of Skp2 in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signalling pathway..
228. Akinori Endo, Masaki Matsumoto, Toshifumi Inada, Akitsugu Yamamoto, Keiichi Nakayama, Naomi Kitamura, Masayuki Komada, Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36, Journal of Cell Science, 10.1242/jcs.044461, 122, 5, 678-686, 2009.03, The nucleolus is a subnuclear compartment and the site of ribosome biogenesis. Previous studies have implicated protein ubiquitylation in nucleolar activity. Here we show that USP36, a deubiquitylating enzyme of unknown function, regulates nucleolar activity in mammalian cells. USP36 localized to nucleoli via the C-terminal region, which contains basic amino acid stretches. Dominant-negative inhibition of USP36 caused the accumulation of ubiquitin-protein conjugates in nucleoli, suggesting that nucleoli are the site of USP36 action. USP36 deubiquitylated the nucleolar proteins nucleophosmin/B23 and fibrillarin, and stabilized them by counteracting ubiquitylation-mediated proteasomal degradation. RNAi-mediated depletion of cellular USP36 resulted in reduced levels of rRNA transcription and processing, a less-developed nucleolar morphology and a slight reduction in the cytoplasmic ribosome level, which eventually led to a reduced rate of cell proliferation. We conclude that by deubiquitylating various nucleolar substrate proteins including nucleophosmin/B23 and fibrillarin, USP36 plays a crucial role in regulating the structure and function of nucleoli..
229. Etsuo Susaki, Keiko Nakayama, Lili Yamasaki, Keiichi Nakayama, Common and specific roles of the related CDK inhibitors p27 and p57 revealed by a knock-in mouse model, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0811712106, 106, 13, 5192-5197, 2009.03, Although p27 and p57 are structurally related cyclin-dependent kinase inhibitors (CKIs), and are thought to perform similar functions, p27 knockout (p27 KO) and p57 KO mice show distinct phenotypes. Toelucidate theinvivo functionsofthese CKIs, wehave now generated a knock-in mouse model (p57 p27KI), in which the p57 gene has been replaced with the p27 gene. The p57 27KI mice are viable and appear healthy, with mostof the developmental defects characteristic ofp57 KOmice having been correctedbyp27 knock-in. Such developmental defects of p57 KO mice were also ameliorated in mice deficient in both p57 and the transcription factor E2F1, suggesting that loss of p57 promotes E2F1-dependent apoptosis. The developmental defects apparent in a few tissues of p57 KO mice were unaffected or only partially corrected by knock-in expression of p27. Thus, these observations indicate that p57 and p27 share many characteristics in vivo, but that p57 also performs specific functions not amenable to substitution with p27..
230. Ameno Kiyoshi, Wang Weihuan, Jamal Mostofa, Kumihashi Mitsuru, Isse Toyoshi, Kawamoto Toshihiro, Kitagawa Kyoko, Keiichi Nakayama, Ijiri Iwao, Kinoshita Hiroshi, Ethanol metabolism in ALDH2 knockout mice - Blood acetate levels, Legal Medicine, 10.1016/j.legalmed.2009.02.043, 11, SUPPL. 1, 2009.04, We described here blood acetate levels in aldehyde dehydrogenase 2 knockout (ALDH2 KO) male mice based on C57BL/6 J strain after ethanol (EtOH) dosing (2 g/kg). Blood samples were collected at 30, 60, 90, 120 180, and 240 min after decapitation, and then EtOH, acetaldehyde (AcH) and acetate were determined by head-space gas chromatography. We found that blood acetate levels in ALDH2 KO mice were slightly lower than those in wild type (WT), whereas EtOH and AcH levels in ALDH2 KO were significantly higher than those in WT. These observations indicate that high EtOH, AcH and low acetate in the blood of ALDH2 KO are due to the deficient effect of ALDH2 enzyme activity..
231. Shotaro Saita, Michiko Shirane, Tohru Natume, Shun Ichiro Iemura, Keiichi Nakayama, Promotion of neurite extension by protrudin requires its interaction with vesicle-associated membrane protein-associated protein, Journal of Biological Chemistry, 10.1074/jbc.M807938200, 284, 20, 13766-13777, 2009.05, Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth..
232. Takehiko Yokobori, Koshi Mimori, Masaaki Iwatsuki, Hideshi Ishii, Ichiro Onoyama, Takeo Fukagawa, Hiroyuki Kuwano, Keiichi Nakayama, Masaki Mori, P53-altered FBXW7 expression determines poor prognosis in gastric cancer cases, Cancer Research, 10.1158/0008-5472.CAN-08-2846, 69, 9, 3788-3794, 2009.05, A molecular target associated with the progression of gastric cancer has not yet been uncovered. FBXW7 is a tumor suppressor gene transcriptionally controlled by p53 that plays a role in the regulation of cell cycle exit and reentry via c-Myc degradation. Few studies have addressed the clinical significance of FBXW7 expression in gastric cancer. Therefore, we examined FBXW7 mRNA expression to determine its clinicopathologic significance in 100 cases of gastric cancer. Low expression levels of FBXW7 in primary gastric cancer contributed to malignant potential, such as lymph node metastasis (P = 0.0012), tumor size (P = 0.0003), and poor prognosis (P = 0.018). In comparison with 52 cases of gastric cancer without the p53 mutation, 29 cases with the mutation exhibited lower expression levels of FBXW7 (P = 0.0034), revealing a significant relationship between p53 mutation and FBXW7 expression. Furthermore, we found that gastric cancer patients who had low FBXW7 expression levels and p53 mutation had a distinctively poor prognosis in comparison with other subgroups (P = 0.0033). In conclusion, we showed a role for p53 in the transcriptional regulation of FBXW7 expression in clinical gastric cancer cases and showed that disruption of both p53 and FBXW7 contributes to poor prognosis..
233. Takayuki Tsukuba, Michiyo Yanagawa, Kuniaki Okamoto, Yoshiko Okamoto, Yoshiyuki Yasuda, Keiichi Nakayama, Tomoko Kadowaki, Kenji Yamamoto, Impaired chemotaxis and cell adhesion due to decrease in several cell-surface receptors in cathepsin E-deficient macrophages, Journal of biochemistry, 10.1093/jb/mvp016, 145, 5, 565-573, 2009.05, Cathepsin E is an endo-lysosomal aspartic proteinase exclusively present in immune system cells. Previous studies have shown that cathepsin E-deficient (CatE /) mice display aberrant immune responses such as atopic dermatitis and higher susceptibility to bacterial infection. However, the mechanisms underlying abnormal immune responses induced by cathepsin E deficiency are still unclear. In this study, we found that the cell-surface levels of chemotactic receptors, including chemokine receptor (CCR)-2 and N-formyl peptide receptors (FPRs), were clearly diminished in CatE /macrophages compared with those in wild-type cells. Consistently, chemotaxis of CatE /macrophages to MCP-1 and N-formyl-methionyl- leucyl-phenylalanine was also decreased. Similar to the chemotactic receptors, the surface expressions of the adhesion receptors CD18 (integrin β2) and CD 29 (integrin β1) in CatE / macrophages were significantly decreased, thereby reducing cell attachment of CatE / macrophages. These results indicate that the defects in chemotaxis and cell adhesion are likely to be involved in the imperfect function of CatE /macrophages..
234. Partha Mitra, Prachi N. Ghule, Margaretha Van Der Deen, Ricardo Medina, Rong Lin Xie, William F. Holmes, Xin Ye, Keiichi Nakayama, J. Wade Harper, Janet L. Stein, Gary S. Stein, Andre J. Van Wijnen, CDK inhibitors selectively diminish cell cycle controlled activation of the histone H4 gene promoter by p220NPAT and HiNF-P, Journal of cellular physiology, 10.1002/jcp.21687, 219, 2, 438-448, 2009.05, Cell cycle progression into S phase requires the induction of histone gene expression to package newly synthesized DNA as chromatin. Cyclin E stimulation of CDK2 at the Restriction point late in G1 controls both histone gene expression by the p220NPAT/HiNF-P pathway and initiation of DNA replication through the pRB/E2F pathway. The three CDK inhibitors (CKIs) p21CIP1/WAF1, p27KIP1, and p57KIP2 attenuate CDK2 activity. Here we find that g-irradiation induces p21CIP1/WAF1 but not the other two CKIs, while reducing histone H4 mRNA levels but not histone H4 gene promoter activation by the p220NPAT/HiNF-P complex. We also show that p21CIP1/WAF1 is less effective than p27 KIP1 and p57KIP2 in inhibiting the CDK2 dependent phosphorylation of p220NPAT at subnuclear foci and transcriptional activation of histone H4 genes. The greater effectiveness of p57KIP2 in blocking the p220NPAT/HiNF-P pathway is attributable in part to its ability to form a specific complex with p220NPAT that may suppress CDK2/cyclin E phosphorylation through direct substrate inhibition. We conclude that CKIs selectively control stimulation of the histone H4 gene promoter by the p220NPAT/HiNF-P complex..
235. Yu Lu, Olasunkanmi A.J. Adegoke, Alain Nepveu, Keiichi Nakayama, Nathalie Bedard, Dongmei Cheng, Junmin Peng, Simon S. Wing, USP19 deubiquitinating enzyme supports cell proliferation by stabilizing KPC1, a ubiquitin ligase for p27Kip1 (Molecular and Cellular Biology (2009) 29, 2, (547-558)), Molecular and Cellular Biology, 10.1128/MCB.00437-09, 29, 11, 2009.06.
236. Taichi Kimura, Mieko Sakai, Kouichi Tabu, Lei Wang, Ryosuke Tsunematsu, Masumi Tsuda, Hirofumi Sawa, Kazuo Nagashima, Hiroshi Nishihara, Shigetsugu Hatakeyama, Keiko Nakayama, Marc Ladanyi, Shinya Tanaka, Keiichi Nakayama, Human synovial sarcoma proto-oncogene Syt is essential for early embryonic development through the regulation of cell migration, Laboratory Investigation, 10.1038/labinvest.2009.25, 89, 6, 645-656, 2009.06, SYT-SSX protein, resulted from chromosomal translocation, causes synovial sarcoma, which is a malignant tumor accounting for 10% of soft tissue sarcoma. However, biological functions of SYT (synovial sarcoma translocation), also known as SS18, are largely unclear, whereas it has been proven that Syt-null mice die at early stages of embryonic development. Here, we generated Syt-deficient mice and confirmed the reported phenotypes, including growth retardation, open neural tube and haplo-insufficient lethality, and therefore, there is no doubt that Syt is essential for embryonic development. However, placental defects, described in the earlier report, were rarely seen in our mice and we frequently observed cardiac defect in Syt-deficient mice. As the mechanisms responsible for embryonic lethality seem to be complicate, we performed additional experiments. By using primary cultured embryonic fibroblasts, we showed that Syt / MEFs deregulate actin organization and suppressed cell migration. These observations suggest that Syt may contribute to the signaling pathway important for various cellular functions in vivo and in vitro, and we propose that Syt-deficient MEFs would be a powerful means to understand the biological roles of SYT in vitro..
237. Masaki Matsumoto, Koji Oyamada, Hidehisa Takahashi, Takamichi Sato, Shigetsugu Hatakeyama, Keiichi Nakayama, Large-scale proteomic analysis of tyrosine-phosphorylation induced by T-cell receptor or B-cell receptor activation reveals new signaling pathways, Proteomics, 10.1002/pmic.200900011, 9, 13, 3549-3563, 2009.07, Activation of the T-cell receptor (TCR) and that of the B-cell receptor (BCR) elicits tyrosinephosphorylation of proteins that belongs to similar functional categories, but result in distinct cellular responses. Large-scale analyses providing an overview of the signaling pathways downstream of TCR or BCR have not been described, so it has been unclear what components of these pathways are shared and which are specific. We have now performed a systematic analysis and provide a comprehensive list of tyrosine-phosphorylated proteins (PY proteome) with quantitative data on their abundance in T cell, B cell, and nonlymphoid cell lines. Our results led to the identification of novel tyrosine-phosphorylated proteins and signaling pathways not previously implicated in immunoreceptor signal transduction, such as clathrin, zonula occludens 2, eukaryotic translation initiation factor 3, and RhoH, suggesting that TCR or BCR signaling may be linked to downstream processes such as endocytosis, cell adhesion, and translation. Thus comparative and quantitative studies of tyrosine-phosphorylation have the potential to expand knowledge of signaling networks and to promote understanding of signal transduction at the system level..
238. Toru Saiga, Takaichi Fukuda, Masaki Matsumoto, Hirobumi Tada, Hirotaka James Okano, Hideyuki Okano, Keiichi Nakayama, Fbxo45 forms a novel ubiquitin ligase complex and is required for neuronal development, Molecular and Cellular Biology, 10.1128/MCB.00364-09, 29, 13, 3529-3543, 2009.07, Fbxo45 is an F-box protein that is restricted to the nervous system. Unlike other F-box proteins, Fbxo45 was found not to form an SCF complex as a result of an amino acid substitution in the consensus sequence for Cul1 binding. Proteomics analysis revealed that Fbxo45 specifically associates with PAM (protein associated with Myc), a RING finger-type ubiquitin ligase. Mice deficient in Fbxo45 were generated and found to die soon after birth as a result of respiratory distress. Fbxo45-/- embryos show abnormal innervation of the diaphragm, impaired synapse formation at neuromuscular junctions, and aberrant development of axon fiber tracts in the brain. Similar defects are also observed in mice lacking Phr1 (mouse ortholog of PAM), suggesting that Fbxo45 and Phr1 function in the same pathway. In addition, neuronal migration was impaired in Fbxo45-/- mice. These results suggest that Fbxo45 forms a novel Fbxo45-PAM ubiquitin ligase complex that plays an important role in neural development..
239. Etsuo Susaki, Keiichi Nakayama, Functional similarities and uniqueness of p27 and p57
Insight from a knock-in mouse model, Cell Cycle, 10.4161/cc.8.16.9330, 8, 16, 2497-2501, 2009.08, The cyclin-dependent kinase inhibitors (CKIs) p27 and p57 are structurally similar, and their biochemical and cellular functions have been thought to be equivalent. However, mice deficient in either p27 or p57 exhibit markedly different phenotypes, suggesting that the in vivo roles of these two proteins might differ. To address this apparent discrepancy, we have generated a knock-in mouse model in which the endogenous p57 gene is replaced by the p27 gene, with p27 thus being expressed instead of p57. This mouse model has provided evidence that p57 functions as a bona fide CKI in vivo and that most of its roles can be performed by p27. Our findings also highlight and provide insight into the question of what determines the distinct cellular responses to abnormal cell cycling induced by the loss of CKIs..
240. Jindan Zhang, Amos C. Hung, Poh Yong Ng, Keiichi Nakayama, Yuanyu Hu, Baojie Li, Alan G. Porter, Saravanakumar Dhakshinamoorthy, PKCδ mediates Nrf2-dependent protection of neuronal cells from NO-induced apoptosis, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2009.06.129, 386, 4, 750-756, 2009.09, A chemical inhibitor library of 84 compounds was screened to investigate the signaling pathway(s) leading to activation of Nrf2 in response to nitric oxide (NO). We identified the protein kinase C delta (PKCδ) inhibitor rottlerin as the only compound that reduced NO-induced ARE-luciferase reporter activity and diminished NO-induced up-regulation of two Nrf2/ARE-regulated proteins - NAD(P)H:quinone oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO-1) in SH-Sy5y cells. Rottlerin also sensitized neuroblastoma cells and mouse primary cortical neurons to NO-induced apoptosis. Stable over-expression of PKCδ augmented NO-induced, ARE-dependent gene expression of HO-1 in SH-Sy5y cells, which were more protected from NO killing. Conversely, NO-induced ARE-dependent gene expression was reduced in PKCδ-knockdown SH-EP cells, which displayed greater sensitivity to apoptosis. PKCδ-/- cortical neurons exhibited increased NO-induced apoptosis and less HO-1 mRNA and protein induction compared with wild type neurons. Hence, PKCδ is an important positive modulator of NO-induced Nrf2/ARE-dependent signaling that counteracts NO-mediated apoptosis in neuronal cells..
241. Xiaogang Jiang, Paul F. Austin, Robert A. Niederhoff, Scott R. Manson, Jacob J. Riehm, Brian L. Cook, Gina Pengue, Kanchan Chitaley, Keiko Nakayama, Keiichi Nakayama, Steven J. Weintraub, Mechanoregulation of proliferation, Molecular and Cellular Biology, 10.1128/MCB.00465-09, 29, 18, 5104-5114, 2009.09, The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation..
242. Mostofa Jamal, Kiyoshi Ameno, Takanori Miki, Weihuan Wang, Mitsuru Kumihashi, Toyohi Isse, Toshihiro Kawamoto, Kyoko Kitagawa, Keiichi Nakayama, Iwao Ijiri, Hiroshi Kinoshita, Cholinergic alterations following alcohol exposure in the frontal cortex of Aldh2-deficient mice models, Brain Research, 10.1016/j.brainres.2009.07.099, 1295, 37-43, 2009.10, We investigated the effects of alcohol (EtOH) and acetaldehyde (ACe) on choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) in the frontal cortex of Aldh2-/- (KO) mice. KO mice were used as models of Aldh2-deficient humans to examine ACe effects. Brain samples were analyzed at 40 and 120 min after 2- and 4-g/kg intraperitoneal EtOH administration by RT-PCR and Western blot. Wild-type (WT) mice exhibited a remarkable decrease in ChAT and AChE mRNA expression at both time points only after 4-g/kg EtOH treatment compared with the naive control, whereas KO mice showed a considerable reduction in cholinergic markers after 2- and 4-g/kg EtOH treatment. The 4-g/kg EtOH-induced decrease in ChAT and AChE RNA expression at both time points was significantly greater than that in obtained with the administration of 2-g/kg at 40 min in WT mice. KO mice showed a significant difference in ChAT mRNA at 40 min between the EtOH groups. The findings regarding the ChAT mRNA levels are consistent with the results of Western blot in both types of mice, with some exceptions. EtOH-induced ChAT and AChE expression in KO mice was significantly lower than that in WT mice. This genotype effect occurred mostly at 40 min after EtOH dosing. Only ACe was quantified in the brains of KO mice, whereas EtOH was detected in both types of mice in vivo. These results suggest that EtOH and ACe combined or high EtOH alone alters cholinergic markers expression via changes in presynaptic and postsynaptic processes in the mice frontal cortex, thus indicating that central cholinergic neurons may be sensitive to EtOH and ACe..
243. Isidora Ranchal, Raúl González, Rosario I. Bello, Gustavo Ferrín, Ana B. Hidalgo, Clara I. Linares, Patricia Aguilar-Melero, Sandra González-Rubio, Pilar Barrera, Trinidad Marchal, Keiichi Nakayama, Manuel De La Mata, Jordi Muntané, The reduction of cell death and proliferation by p27 Kip1 minimizes DNA damage in an experimental model of genotoxicity, International Journal of Cancer, 10.1002/ijc.24621, 125, 10, 2270-2280, 2009.11, Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer worldwide. The expression of p27 has been related to reduced severity of tumor grade and recurrence of HCC. The study assessed the role of p27 on the cell proliferation and death, and DNA mutagenesis in experimental genotoxicity induced by aflatoxin B1 (AFB 1) in cultured hepatocytes obtained from control and p27 Kip1 deficient mice. The overexpression of p27 was assessed with wild type p27 Kip1 expression vector in HepG2 cells. The expression of p27, p21 and p53 was assessed in well and poorly-differentiated liver tumors. DNA damage and cell death induced by AFB 1 were related to a reduction of p27 and p21 expression in cultured hepatocytes. AFB 1-induced nuclear phosphorylated (Ser 10) p27 degradation was related to a rise of nuclear KIST, Rsk-1 and Rsk-2 expression and cytoplasm phosphorylated (Thr 198) p27 expression. The overexpression of p27 reduced cell proliferation, cell death and DNA damage in AFB 1-treated hepatocytes. The enhanced survival of patients with well differentiated compared to poorly-differentiated tumors was related to high expression of p27, p21 and p53 in liver sections. The study showed that the p27 reduced cell proliferation and death, as well as the accumulation of DNA damage in hepatocarcinogenesis..
244. Yih Jer Wu, Graciela B. Sala-Newby, Kuo Tung Shu, Hung I. Yeh, Keiichi Nakayama, Keiko Nakayama, Andrew C. Newby, Mark Bond, S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo, Journal of Vascular Surgery, 10.1016/j.jvs.2009.07.066, 50, 5, 1135-1142, 2009.11, Objective: Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo. Methods and Results: Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening. Conclusion: This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases..
245. Akiko Oyamada, Hiori Ikebe, Momoe Itsumi, Hirokazu Saiwai, Seiji Okada, Kazuya Shimoda, Yoichiro Iwakura, Keiichi Nakayama, Yukihide Iwamoto, Yasunobu Yoshikai, Hisakata Yamada, Tyrosine kinase 2 plays critical roles in the pathogenic CD4 T cell responses for the development of experimental autoimmune encephalomyelitis, Journal of Immunology, 10.4049/jimmunol.0902740, 183, 11, 7539-7546, 2009.12, Tyrosine kinase 2 (Tyk2), a member of the JAK family, is involved in IL-12- and IL-23-mediated signaling. In the present study, we examined the roles of Tyk2 in the development of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by using Tyk2 knockout (KO) mice. In vitro differentiation of Th1 but not Th17 cells was severely impaired in Tyk2 KO CD4 T cells, although Tyk2 KO Th17 cells did not respond to IL-23. Tyk2 KO mice showed complete resistance against EAE with no infiltration of CD4 T cells in the spinal cord. Surprisingly, the number of MOG-specific Th17 cells in the periphery was comparable between KO and wild-type (WT) mice, whereas Th1 cells were greatly reduced in Tyk2 KO mice. Adoptive transfer of MOG-primed WT T cells induced EAE in Tyk2 KO recipients, indicating that Tyk2 in the environment was dispensable for the infiltration of effector T cells into the spinal cord. A reduced but significant number of Tyk2 KO T cells were detected in the spinal cord of mice with EAE, which had been reconstituted with bone marrow cells of WT and KO mice. Furthermore, MOG-immunized Tyk2 KO mice developed EAE after adoptive transfer of MOG-primed WT Th1 cells, which might trigger local inflammation that recruits Th17 cells. Taken together, these results indicate that Tyk2 is critically involved in the pathogenic CD4 T cell responses and thus could be a target molecule for the treatment of autoimmune diseases..
246. Miho Matsuda, Koushirou Tsutsumi, Takashi Kanematsu, Kiyoko Fukami, Yoshihiro Terada, Tadaomi Takenawa, Keiichi Nakayama, Masato Hirata, Involvement of phospholipase C-related inactive protein in the mouse reproductive system through the regulation of gonadotropin levels, Biology of Reproduction, 10.1095/biolreprod.109.076760, 81, 4, 681-689, 2009.12, Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and PRIP-2 [officially designated PLCL1 and PLCL2]) was first identified in our laboratory, but the biological functions have remained elusive. Therefore, we generated Plcll and Plcl2 double-knockout mice (Plcl1 tm1Mh; Plcl2tm1Tta) to gain insight into the biological function. Double-knockout mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments to discrim-inate whether males or females were defective indicated that the cause appeared to be on the female side. Mutant female mice had an apparently smaller uterus by gross anatomical observation and had more estrous days during the cycles. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone were measured for 5-6 consecutive days and were significantly higher in the mutant, which was also confirmed by examining the secretion of LH from the explant culture of anterior pituitary glands of wild-type and double-knockout mice. These results suggest that through gonadotropin secretion, PRIP plays an important role in female reproduction..
247. Hongbo Wang, Frederick Bauzon, Peng Ji, Xiaoliang Xu, Daqian Sun, Joseph Locker, Rani S. Sellers, Keiko Nakayama, Keiichi Nakayama, David Cobrinik, Liang Zhu, Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1
+/
mice, Nature genetics, 10.1038/ng.498, 42, 1, 83-88, 2010.01, Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1
+/
mice. Here we report that inactivation of pRb target Skp2 (refs. 7,8) completely prevents spontaneous tumorigenesis in Rb1
+/
mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knock-in mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCF Skp2 ubiquitin ligase as the underlying mechanism for Skp2's essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency..
248. B. L. Allen-Petersen, M. R. Miller, M. C. Neville, S. M. Anderson, Keiichi Nakayama, M. E. Reyland, Loss of protein kinase C delta alters mammary gland development and apoptosis, Cell Death and Disease, 10.1038/cddis.2009.20, 1, 1, 2010.01, As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKC) / mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKC / mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKC / mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKC / mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKC / mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKC in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo..
249. Etsuo Susaki, Keiichi Nakayama, An animal model manifesting neurodegeneration and obesity, Aging, 10.18632/aging.100172, 2, 7, 453-456, 2010.01, Although the existence of a link between neurodegenerative diseases and obesity has been suggested, a causal relation between neural degeneration and obesity has remained to be demonstrated experimentally. We recently showed that neurodegeneration in the hypothalamic satiety center results in obesity in mice transgenic for E4B (also known as UFD2a), a mammalian ubiquitin elongation factor (E4). Increased expression of E4B in neurons of the transgenic mice results in the formation of ubiquitin-positive aggregates similar to those apparent in many human neurodegenerative diseases as well as in degeneration of hypothalamic neurons responsible for the regulation of food intake and energy expenditure. We thus propose that neurodegeneration is a possible cause of human obesity and related metabolic diseases, which have become a serious public health problem worldwide. Our animal model is thus a powerful tool for studies of the relation between neurodegeneration and obesity..
250. Makoto Fujii, Takashi Kanematsu, Hitoshi Ishibashi, Kiyoko Fukami, Tadaomi Takenawa, Keiichi Nakayama, Stephen J. Moss, Junichi Nabekura, Masato Hirata, Phospholipase C-related but catalytically inactive protein is required for insulin-induced cell surface expression of γ-aminobutyric acid type A receptors, Journal of Biological Chemistry, 10.1074/jbc.M109.070045, 285, 7, 4837-4846, 2010.02, The γ-aminobutyric acid type A (GABAA) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABAA receptor insertion. Insulin potentiated the GABA-induced Cl- current (IGABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABAA receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in IGABA. We also revealed that PRIP recruited active Akt to the GABAA receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABAA receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of IGABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABAA receptor insertion by recruiting active Akt to the receptor complex..
251. Hirobumi Tada, Hirotaka James Okano, Hiroshi Takagi, Shinsuke Shibata, Ikuko Yao, Masaki Matsumoto, Toru Saiga, Keiichi Nakayama, Haruo Kashima, Takuya Takahashi, Mitsutoshi Setou, Hideyuki Okano, Fbxo45, a novel ubiquitin ligase, regulates synaptic activity, Journal of Biological Chemistry, 10.1074/jbc.M109.046284, 285, 6, 3840-3849, 2010.02, Neurons communicate with each other through synapses. To establish the precise yet flexible connections that make up neural networks in the brain, continuous synaptic modulation is required. The ubiquitin-proteasome system of protein degradation is one of the critical mechanisms that underlie this process, playing crucial roles in the regulation of synaptic structure and function. We identified a novel ubiquitin ligase, Fbxo45, that functions at synapses. Fbxo45 is evolutionarily conserved and selectively expressed in the nervous system. We demonstrated that the knockdown of Fbxo45 in primary cultured hippocampal neurons resulted in a greater frequency of miniature excitatory postsynaptic currents. We also found that Fbxo45 induces the degradation of a synaptic vesicle-priming factor, Munc13-1.We propose that Fbxo45 plays an important role in the regulation of neurotransmission by modulating Munc13-1 at the synapse..
252. Hui Kuan Lin, Zhenbang Chen, Guocan Wang, Caterina Nardella, Szu Wei Lee, Chan Hsin Chan, Wei Lei Yang, Jing Wang, Ainara Egia, Keiichi Nakayama, Carlos Cordon-Cardo, Julie Teruya-Feldstein, Pier Paolo Pandolfi, Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence, Nature, 10.1038/nature08815, 464, 7287, 374-379, 2010.03, Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19 Arf ĝ€"p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19Arf-p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19Arf-p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy..
253. Jennifer B. Old, Susanne Kratzat, Alexander Hoellein, Steffi Graf, Jonas A. Nilsson, Lisa Nilsson, Keiichi Nakayama, Christian Peschel, John L. Cleveland, Ulrich B. Keller, Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis, Molecular Cancer Research, 10.1158/1541-7786.MCR-09-0232, 8, 3, 353-362, 2010.03, The universal cyclin-dependent kinase inhibitor p27Kip1 functions as a tumor suppressor, and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E-cyclin-dependent kinase 2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27 Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27Kip1, are critical for Myc-driven proliferation and tumorigenesis..
254. Yuichi Tsukada, Tohru Ishitani, Keiichi Nakayama, KDM7 is a dual demethylase for histone H3 Lys 9 and Lys 27 and functions in brain development, Genes and Development, 10.1101/gad.1864410, 24, 5, 432-437, 2010.03, Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono- or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development..
255. K. Masuda, Y. Ishikawa, Ichiro Onoyama, M. Unno, I. M. De Alborán, Keiichi Nakayama, K. Nakayama, Complex regulation of cell-cycle inhibitors by Fbxw7 in mouse embryonic fibroblasts, Oncogene, 10.1038/onc.2009.469, 29, 12, 1798-1809, 2010.03, The F-box protein Fbxw7 (also known as Fbw7, SEL-10, hCdc4 or hAgo) mediates the ubiquitylation and thereby contributes to the degradation of proteins that positively regulate cell cycle. Conditional ablation of Fbxw7 in mouse embryonic fibroblasts (MEFs) induces cell-cycle arrest accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1) and c-Myc. However, the molecular mechanisms by which the accumulation of NICD1 and c-Myc induces cell-cycle arrest have remained unclear. We have now examined the expression of cell-cycle inhibitors in Fbxw7-deficient MEFs and found that the abundance of p27 Kip1 and p57 Kip2 is paradoxically decreased. This phenomenon appears to be attributable to the accumulation of NICD1, given that it was recapitulated by overexpression of NICD1 and blocked by ablation of RBP-J. Conversely, the expression of p16 Ink4a and p19 ARF was increased in an NICD1-independent manner in Fbxw7-null MEFs. The increased expression of p19 ARF was recapitulated by overexpression of c-Myc and abolished by ablation of c-Myc, suggesting that the accumulation of c-Myc is primarily responsible for that of p19 ARF. In contrast, the upregulation of p16 Ink4a appeared to be independent of c-Myc. These results indicate that cell-cycle inhibitors undergo complex regulation by the Fbxw7-mediated proteolytic system..
256. Masaaki Iwatsuki, Koshi Mimori, Hideshi Lshii, Takehiko Yokobori, Yasushi Takatsuno, Tetsuya Sato, Hiroyuki Toh, Ichiro Onoyama, Keiichi Nakayama, Hideo Baba, Masaki Mori, Loss of FBXW7, a cell cycle regulating gene, in colorectal cancer
Clinical significance, International Journal of Cancer, 10.1002/ijc.24879, 126, 8, 1828-1837, 2010.04, This study focused on a cell cycle regulatory gene, FBXW7, which ubiquitinates c-Myc and cyclin E and promotes exit from the cell cycle. We determined the expression level of FBXW7 in colorectal cancer (CRC) cases, correlated those values with clinicopathologic features, and characterized the molecular mechanism of reduced expression of FBXW7 in CRC cells in vitro. FBXW7 mRNA and protein expression were evaluated in 93 CRC cases. Using CGH array, the copy number aberrations of the flanking region of FBXW7 were evaluated in another 130 CRC specimens. In vitro analysis of FBXW7 gene silencing in CRC cells was conducted. FBXW7 mRNA expression was significantly tower in tumor tissues than the corresponding normal tissues. The low FBXW7 expression group showed a significantly poorer prognosis than patients in the high expression group. A concordant relationship was observed between the incidence of KXIRH repression and the genetic alteration. The incidence of genetic alteration was associated with the stage of disease progression. In vitro, FBXW7-specific siRNA enhanced expression of c-MYC and cyclin E proteins and up-regulated cell proliferation. Genetic alterations in tumors led to the loss of FBXW7 expression and increased cell proliferation. FBXW7 expression provides a prognostic factor for patients with CRC..
257. Etsuo Susaki, Chie Kaneko-Oshikawa, Keishi Miyata, Mitsuhisa Tabata, Tetsuya Yamada, Yuichi Oike, Hideki Katagiri, Keiichi Nakayama, Increased E4 activity in mice leads to ubiquitin-containing aggregates and degeneration of hypothalamic neurons resulting in obesity, Journal of Biological Chemistry, 10.1074/jbc.M110.105841, 285, 20, 15538-15547, 2010.05, Obesity has become a serious worldwide public health problem. Although neural degeneration in specific brain regions has been suggested to contribute to obesity phenotype in humans, a causal relationship between these two conditions has not been demonstrated experimentally. We now show that E4B (also known as UFD2a), a mammalian ubiquitin chain elongation factor (E4), induces the formation of intracellular aggregates positive for ubiquitin and the adaptor protein p62 when overexpressed in cultured cells or the brain. Mice transgenic for E4B manifested neural degeneration in association with aggregate formation, and they exhibited functional impairment specifically in a subset of hypothalamic neurons that regulate food intake and energy expenditure, resulting in development of hyperphagic obesity and related metabolic abnormalities. The neural pathology of E4B transgenic mice was similar to that of human neurodegenerative diseases associated with the formation of intracellular ubiquitin-positive deposits, indicating the existence of a link between such diseases and obesity and related metabolic disorders. Our findings thus provide experimental evidence for a role of hypothalamic neurodegeneration in obesity, and the E4B transgenic mouse should prove to be a useful animal model for studies of the relationship between neurodegenerative diseases and obesity..
258. Chia Hsin Chan, Szu Wei Lee, Chien Feng Li, Jing Wang, Wei Lei Yang, Ching Yuan Wu, Juan Wu, Keiichi Nakayama, Hong Yo Kang, Hsuan Ying Huang, Mien Chie Hung, Pier Paolo Pandolfi, Hui Kuan Lin, Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis, Nature Cell Biology, 10.1038/ncb2047, 12, 5, 457-467, 2010.05, The RhoA GTPase is crucial in numerous biological functions and is linked to cancer metastasis. However, the understanding of the molecular mechanism responsible for RhoA transcription is still very limited. Here we show that RhoA transcription is orchestrated by the Myc-Skp2-Miz1-p300 transcriptional complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of Skp1-Cullin-F-box protein containing complex (SCF)-Skp2 E3 ligase activity. Deficiency of this complex results in impairment in RhoA expression, cell migration, invasion, and breast cancer metastasis, recapitulating the phenotypes observed in RhoA knockdown, and RhoA restoration rescues the defect in cell invasion. Overexpression of the Myc-Skp2-Miz1 complex is found in metastatic human cancers and is correlated with RhoA expression. Our study provides insight into how oncogenic Skp2 and Myc coordinate to induce RhoA transcription and establishes a novel SCF-Skp2 E3-ligase-independent function for oncogenic Skp2 in transcription and cancer metastasis..
259. Atsushi Hatano, Masaki Matsumoto, Toru Higashinakagawa, Keiichi Nakayama, Phosphorylation of the chromodomain changes the binding specificity of Cbx2 for methylated histone H3, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2010.05.074, 397, 1, 93-99, 2010.06, The chromatin organizer modifier domain (chromodomain) is present in proteins that contribute to chromatin organization and mediates their binding to methylated histone H3. Despite a high level of sequence conservation, individual chromodomains manifest substantial differences in binding preference for methylated forms of histone H3, suggesting that posttranslational modification of the chromodomain might be an important determinant of binding specificity. We now show that mouse Cbx2 (also known as M33), a homolog of Drosophila Polycomb protein, is highly phosphorylated in some cell lines. A low-mobility band of Cbx2 observed on SDS-polyacrylamide gel electrophoresis was thus converted to a higher-mobility band by treatment with alkaline phosphatase. Mass spectrometric analysis revealed serine-42, a conserved amino acid in the chromodomain, as a phosphorylation site of Cbx2. Phosphorylation of the chromodomain of Cbx2 on this residue in vitro resulted in a reduced level of binding to an H3 peptide containing trimethylated lysine-9 as well as an increase in the extent of binding to an H3 peptide containing trimethylated lysine-27, suggesting that such phosphorylation changes the binding specificity of Cbx2 for modified histone H3. Phosphorylation of the chromodomain of Cbx2 may therefore serve as a molecular switch that affects the reading of the histone modification code and thereby controls epigenetic cellular memory..
260. Chiho Ohzono, Sachise Etoh, Masaki Matsumoto, Keiichi Nakayama, Yuko Hirota, Yoshitaka Tanaka, Hideaki Fujita, Nedd4-interacting protein 2, a short half-life membrane protein degraded in lysosomes, negatively controls down-regulation of connexin43, Biological and Pharmaceutical Bulletin, 10.1248/bpb.33.951, 33, 6, 951-957, 2010.06, Nedd4-interacting protein 2 (NDFIP2) has three transmembrane domains and interacts with multiple Nedd4 family ubiquitin ligases through polyprolinetyrosine (PY) motifs located in its N-terminal cytoplasmic domain. It has been postulated that NDFIP2 acts as an adaptor for the ubiquitylation of substrates with Nedd4 ubiquitin ligase. However, whether NDFIP2 promotes or inhibits the ubiquitylation of Nedd4 substrates is still under debate. We show here that although NDFIP2 is detected in the Golgi/frans-Golgi network (TGN) area, it is rapidly delivered to and degraded in lysosomes with its half-life ca. 1.5 h. Intriguingly, knockdown (KD) of NDFIP2 with small interfering RNA (siRNA) impaired both the formation and function of gap junctions. Indeed, KD of NDFIP2 destabilized the gap junction protein connexin43 that contains PY motif. In support of this, over-expression of NDFIP2 stabilized connexin43 and enhanced the formation of gap junctions. Furthermore, the PY motifs of NDFIP2, which are required for its interaction with Nedd4, Atrophin-1 interacting protein (AIP) 4 (AIP4)/Itch, and AIP2/WWP2, were necessary for the targeting of NDFIP2 to lysosomes and/or the stability of connexin43 and gap junctions. Collectively these findings suggest that NDFIP2 may inhibit the Nedd4-dependent ubiquitylation of membrane proteins containing PY motifs, such as connexin43, in a competitive manner..
261. Yun Gao, Chunyu Gu, Shaoyi Li, Tsutomu Tokuyama, Naoki Yokota, Keiichi Nakayama, Masatoshi Kitagawa, Hiroki Namba, p27 modulates tropism of mesenchymal stem cells toward brain tumors, Experimental and Therapeutic Medicine, 10.3892/etm_00000107, 1, 4, 695-699, 2010.07, Mesenchymal stem cells (MSCs) have inherent tumor-tropic properties in the brain and seem to be a useful tool for cellular therapy for brain tumors. However, the mechanisms involved in MSC migration are not fully understood. The tumor suppressor p27, an inhibitor of cyclin-dependent kinase complexes, not only plays a crucial role in cell cycle regulation but also has cell cycle-independent functions, such as differentiation and migration of cells. In fact, p27 has been alternatively reported to inhibit or stimulate cell migration in cells of different types. Therefore, in the present study, we investigated whether p27 is involved in the tumor-tropic activity of MSCs using MSCs from p27-null mice. It was found that p27-/- MSCs showed a decreased motility in the wound healing assay and displayed increased numbers of stress fibers. To compare the in vivo migratory activity of p27-/- and p27+/+ MSCs toward glioma, we injected C6 glioma cells into one side of the mouse brain and BrdU-labeled p27-/- or p27+/+ MSCs into the other side. Significantly fewer labeled p27-/- MSCs were observed in the tumor area compared with p27+/+ MSCs. The present study suggests that p27 works as a stimulator of the in vitro and in vivo migration process of MSCs toward tumors. These findings are important when the efficacy of stem cell-based strategies for glioma therapy is considered..
262. Fumihiko Okumura, Yui Matsunaga, Yuta Katayama, Keiichi Nakayama, Shigetsugu Hatakeyama, TRIM8 modulates STAT3 activity through negative regulation of PIAS3, Journal of Cell Science, 10.1242/jcs.068981, 123, 13, 2238-2245, 2010.07, TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3..
263. Akiko Mizokami, Hiroto Tanaka, Hitoshi Ishibashi, Hisanori Umebayashi, Kiyoko Fukami, Tadaomi Takenawa, Keiichi Nakayama, Takeshi Yokoyama, Junichi Nabekura, Takashi Kanematsu, Masato Hirata, GABAA receptor subunit alteration-dependent diazepam insensitivity in the cerebellum of phospholipase C-related inactive protein knockout mice, Journal of Neurochemistry, 10.1111/j.1471-4159.2010.06754.x, 114, 1, 302-310, 2010.07, The GABAA receptor, a pentamer composed predominantly of α, β, and γ subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABAA receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The α subunit affects diazepam sensitivity; α1, 2, 3, and 5 subunits assemble with any form of β and the γ2 subunits to produce diazepam-sensitive receptors, whereas α4 or α6/β/γ2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the α6 subunit. The expression of α1/β/γ2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the α6 subunit were increased, the α1/β/γ2 receptors might be replaced with α6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of α6/δ receptors was decreased in cerebellar granule neurons, while that of α6/γ2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (α1/γ2) and increase in diazepam-insensitive (α6/γ2) GABAA receptors in the cerebellar granule cells..
264. Hui Kuan Lin, Zhenbang Chen, Guocan Wang, Caterina Nardella, Szu Wei Lee, Chia Hsin Chan, Wei Lei Yang, Jing Wang, Ainara Egia, Keiichi Nakayama, Carlos Cordon-Cardo, Julie Teruya-Feldstein, Pier Paolo Pandolfi, Erratum
Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence (Nature (2010) 464 (374-379)), Nature, 10.1038/nature09280, 466, 7304, 2010.07.
265. Robert C. Benirschke, James R. Thompson, Yves Nominé, Emeric Wasielewski, Nenad Juranić, Slobodan Macura, Shigetsugu Hatakeyama, Keiichi Nakayama, Maria Victoria Botuyan, Georges Mer, Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4, Structure, 10.1016/j.str.2010.04.017, 18, 8, 955-965, 2010.08, Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles..
266. Heng Ma, Lu Yu, Emily A. Byra, Nan Hu, Kyoko Kitagawa, Keiichi Nakayama, Toshihiro Kawamoto, Jun Ren, Aldehyde dehydrogenase 2 knockout accentuates ethanol-induced cardiac depression
Role of protein phosphatases, Journal of Molecular and Cellular Cardiology, 10.1016/j.yjmcc.2010.03.017, 49, 2, 322-329, 2010.08, Alcohol consumption leads to myocardial contractile dysfunction possibly due to the toxicity of ethanol and its major metabolite acetaldehyde. This study was designed to examine the influence of mitochondrial aldehyde dehydrogenase-2 (ALDH2) knockout (KO) on acute ethanol exposure-induced cardiomyocyte dysfunction. Wild-type (WT) and ALDH2 KO mice were subjected to acute ethanol (3. g/kg, i.p.) challenge and cardiomyocyte contractile function was assessed 24. h later using an IonOptix edge detection system. Western blot analysis was performed to evaluate ALDH2, protein phosphatase 2A (PP2A), phosphorylation of Akt, and glycogen synthase kinase-3β (GSK-3β). ALDH2 KO accentuated ethanol-induced elevation in cardiac acetaldehyde levels. Ethanol exposure depressed cardiomyocyte contractile function including decreased cell shortening amplitude and maximal velocity of shortening/relengthening as well as prolonged relengthening duration and a greater decline in peak shortening in response to increasing stimulus frequency, the effect of which was significantly exaggerated by ALDH2 KO. ALDH2 KO also unmasked an ethanol-induced prolongation of shortening duration. In addition, short-term in vitro incubation of ethanol-induced cardiomyocyte mechanical defects was exacerbated by the ALDH inhibitor cyanamide. Ethanol treatment dampened phosphorylation of Akt and GSK-3β associated with upregulated PP2A, which was accentuated by ALDH2 KO. ALDH2 KO aggravated ethanol-induced decrease in mitochondrial membrane potential. These results suggested that ALDH2 deficiency led to worsened ethanol-induced cardiomyocyte function, possibly due to upregulated expression of protein phosphatase, depressed Akt activation, and subsequently impaired mitochondrial function. These findings depict a critical role of ALDH2 in the pathogenesis of alcoholic cardiomyopathy..
267. Yuichi Tsukada, Keiichi Nakayama, In vitro histone demethylase assay, Cold Spring Harbor Protocols, 10.1101/pdb.prot5512, 5, 10, 2010.10.
268. Hiroyuki Takeda, Yoshifumi Kawamura, Aya Miura, Masatoshi Mori, Ai Wakamatsu, Jun Ichi Yamamoto, Takao Isogai, Masaki Matsumoto, Keiichi Nakayama, Tohru Natsume, Nobuo Nomura, Naoki Goshima, Comparative analysis of human Src-family kinase substrate specificity in vitro, Journal of Proteome Research, 10.1021/pr100773t, 9, 11, 5982-5993, 2010.11, Src family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase-substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction..
269. Sakae Tanaka, Hidetoshi Wakeyama, Toru Akiyama, Katsuhiko Takahashi, Hitoshi Amano, Keiichi Nakayama, Kozo Nakamura, Regulation of osteoclast apoptosis by bcl-2 family protein bim and caspase-3, Osteoimmunology Interactions of the Immune and Skeletal Systems II, 10.1007/978-1-4419-1050-9_12, 111-116, 2010.12, Apopotosis of osteoclasts is regulated by the Bcl-2 family protein Bim. Bim is degraded in the course of osteoclast apoptosis, which is regulated by Caspase-3. Osteoclasts generated from caspase-3 -/- mice exhibited a shorter life span and a higher bone-resorbing activity than those generated from normal littermates. These results suggest the important role of Caspase-3-Bim axis in regulating both apoptosis and activation of osteoclasts..
270. Hiroshi Hayakawa, Aya Fujikane, Riyoko Ito, Masaki Matsumoto, Keiichi Nakayama, Mutsuo Sekiguchi, Human proteins that specifically bind to 8-oxoguanine-containing RNA and their responses to oxidative stress, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2010.11.011, 403, 2, 220-224, 2010.12, Exposure of cells to oxygen radicals damage various biologically important molecules. Among the oxidized bases produced in nucleic acids, 8-oxo-7,8-dihydroguanine (8-oxoguanine) is particularly important since it causes base mispairing. To ensure accurate gene expression, organisms must have a mechanism to discriminate 8-oxoguanine-containing RNA from normal transcripts. We searched for proteins that specifically bind to 8-oxoguanine-containing RNA from human HeLa cell extracts, and the candidate proteins were identified using mass spectrometry. Among the identified candidates, splicing isoform 1 of heterogeneous nuclear ribonucleoprotein D0 (HNRNPD) and splicing isoform C1 of heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) exhibited strong abilities to bind to oxidized RNA. The amount of HNRNPD protein rapidly decreased when cells were exposed to hydrogen peroxide, an agent that enhances oxidative stress. Moreover, the suppression of HNRNPD expression by siRNA caused cells to exhibit an increased sensitivity to hydrogen peroxide. The application of siRNA against HNRNPC also caused an increase in sensitivity to hydrogen peroxide. Since no additive effect was observed with a combined addition of siRNAs for HNRNPD and HNRNPC, we concluded that the two proteins may function in the same mechanism for the accurate gene expression..
271. Christopher Sistrunk, Everardo MacIas, Keiichi Nakayama, Yongbaek Kim, Marcelo L. Rodriguez-Puebla, Skp2 is necessary for Myc-induced keratinocyte proliferation but dispensable for Myc oncogenic activity in the oral epithelium, American Journal of Pathology, 10.1016/j.ajpath.2011.02.034, 178, 6, 2470-2477, 2011.01, The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27 Kip1. Moreover, p27Kip1 protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCFSkp2. Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27Kip1 levels and further inhibition of Myc-mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5-Myc) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5-Myc- transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc-mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc-driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5-Myc/Skp2+/+ and K5-Myc/Skp2-/- mice. Altogether, these findings suggest that Skp2 and p27Kip1 are critical for Myc-driven keratinocyte proliferation; however, Myc-mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27Kip1 axis..
272. Miyuki Bohgaki, Masaki Matsumoto, Tatsuya Atsumi, Takeshi Kondo, Shinsuke Yasuda, Tetsuya Horita, Keiichi Nakayama, Fumihiko Okumura, Shigetsugu Hatakeyama, Takao Koike, Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin, Journal of Cellular and Molecular Medicine, 10.1111/j.1582-4934.2009.00940.x, 15, 1, 141-151, 2011.01, Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β2-glycoprotein I (β2GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen-activated protein kinase (MAPK) pathway plays an important role in aPL-induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β2GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β2GPI interacts with plasma gelsolin, which binds to integrin a5β1 through fibronectin. The tethering of β2GPI to monoclonal anti-β2GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti-β2GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti-integrin a5β1 antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin-integrin signalling pathway, was phosphorylated by anti-β2GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti-β2GPI antibody-induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS..
273. Ichiro Onoyama, Atsushi Suzuki, Akinobu Matsumoto, Kengo Tomita, Hideki Katagiri, Yuichi Oike, Keiko Nakayama, Keiichi Nakayama, Fbxw7 regulates lipid metabolism and cell fate decisions in the mouse liver, Journal of Clinical Investigation, 10.1172/JCI40725, 121, 1, 342-354, 2011.01, E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box- and WD repeat domain-containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7-/- embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver..
274. Koko Katagiri, Yoshihiro Ueda, Takashi Tomiyama, Kaneki Yasuda, Yoshinobu Toda, Susumu Ikehara, Keiichi Nakayama, Tatsuo Kinashi, Deficiency of Rap1-Binding Protein RAPL Causes Lymphoproliferative Disorders through Mislocalization of p27kip1, Immunity, 10.1016/j.immuni.2010.12.010, 34, 1, 24-38, 2011.01, RAPL (an alternative spliced form of Rassf5) is a critical Ras-related protein1 (Rap1) effector that regulates lymphocyte adhesion. Here, we have shown that in addition to this previously described function, RAPL also negatively controls lymphocyte proliferation and prevents autoimmunity and lymphoma. RAPL-deficient mice experienced age-related lupus-like glomerulonephritis and developed B cell lymphomas. RAPL-deficient lymphocytes showed hyperproliferation by enhanced S phase entry after antigen receptor ligation. Compared to wild-type cells, RAPL-deficient naive lymphocytes had a 2- to 3-fold increase in Cdk2 kinase activity with a cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27kip1. RAPL was found to suppress the phosphorylation of p27kip1 on serine 10 (S10) and promoted p27kip1 nuclear translocation. An S10A mutation in p27kip1 corrected its cytoplasmic accumulation, reduced hyperproliferation in RAPL-deficient lymphocytes, and suppressed glomerulonephritis and development of B cell lymphoma. Thus, RAPL serves as a checkpoint for S phase entry to prevent lymphoproliferative disorders through the spatial regulation of p27kip1..
275. Hirono Iriuchishima, Keiyo Takubo, Sahoko Matsuoka, Ichiro Onoyama, Keiichi Nakayama, Yoshihisa Nojima, Toshio Suda, Ex vivo maintenance of hematopoietic stem cells by quiescence induction through Fbxw7α overexpression, Blood, 10.1182/blood-2010-07-294801, 117, 8, 2373-2377, 2011.02, Cell-cycle quiescence in hematopoietic stem cells (HSCs) is essential for maintaining stemness by protecting cells from differentiation or senescence. F-box and WD-40 domain protein 7 (Fbxw7) maintains HSCs and suppresses leukemogenesis by mediating ubiquitin-dependent degradation of cell-cycle activators and oncoproteins. Fbxw7α was shown to be the preferentially expressed Fbxw7 isoform in primitive HSCs. Forced Fbxw7α expression in lineage marker Sca-1+ c-Kit+ cells led to cell-cycle dormancy by reducing the protein levels of the Fbxw7 substrates c-Myc, Notch1, and phosphorylated S6 (a key downstream element of mTOR). Hypoxia, an essential factor for HSC quiescence, suppressed c-Myc in an Fbxw7α-dependent manner. Fbxw7α-overexpressing lineage marker Sca-1+c-Kit+ cells sustained high reconstitution capacities during in vitro culture. These data suggest that Fbxw7α sustains HSC dormancy through c-Myc, Notch1, and the mTOR pathways. The modulation of Fbxw7α expression or activity represents a promising new tool for ex vivo HSC maintenance..
276. Hong Wu, Scott L. Pomeroy, Manuel Ferreira, Natalia Teider, Juliana Mariani, Keiichi Nakayama, Shigetsugu Hatakeyama, Victor A. Tron, Linda F. Saltibus, Leo Spyracopoulos, Roger P. Leng, UBE4B promotes Hdm2-mediated degradation of the tumor suppressor p53, Nature medicine, 10.1038/nm.2283, 17, 3, 347-355, 2011.03, The TP53 gene (encoding the p53 tumor suppressor) is rarely mutated, although frequently inactivated, in medulloblastoma and ependymoma. Recent work in mouse models showed that the loss of p53 accelerated the development of medulloblastoma. The mechanism underlying p53 inactivation in human brain tumors is not completely understood. We show that ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, physically interacts with p53 and Hdm2 (also known as Mdm2 in mice). UBE4B promotes p53 polyubiquitination and degradation and inhibits p53-dependent transactivation and apoptosis. Notably, silencing UBE4B expression impairs xenotransplanted tumor growth in a p53-dependent manner and overexpression of UBE4B correlates with decreased expression of p53 in these tumors. We also show that UBE4B overexpression is often associated with amplification of its gene in human brain tumors. Our data indicate that amplification and overexpression of UBE4B represent previously undescribed molecular mechanisms of inactivation of p53 in brain tumors..
277. Hiroyuki Inuzuka, Shavali Shaik, Ichiro Onoyama, Daming Gao, Alan Tseng, Richard S. Maser, Bo Zhai, Lixin Wan, Alejandro Gutierrez, Alan W. Lau, Yonghong Xiao, Amanda L. Christie, Jon Aster, Jeffrey Settleman, Steven P. Gygi, Andrew L. Kung, Thomas Look, Keiichi Nakayama, Ronald A. Depinho, Wenyi Wei, SCFFBW7 regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction, Nature, 10.1038/nature09732, 471, 7336, 104-109, 2011.03, The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCFFBW7 (a SKP1-cullin-1-F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL..
278. Changming Lu, Xin Huang, Xiaoxiao Zhang, Kristin Roensch, Qing Cao, Keiichi Nakayama, Bruce R. Blazar, Yan Zeng, Xianzheng Zhou, miR-221 and miR-155 regulate human dendritic cell development, apoptosis, and IL-12 production through targeting of p27 kip1, KPC1, and SOCS-1, Blood, 10.1182/blood-2010-12-322503, 117, 16, 4293-4303, 2011.04, Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitor cells and circulating monocytes. To investigate the role of microRNAs (miRNAs) during DC differentiation, maturation, and function, we profiled miRNA expression in human monocytes, immature DCs (imDCs), and mature DCs (mDCs). Stage-specific, differential expression of 27 miRNAs was found during monocyte differentiation into imDCs and mDCs. Among them, decreased miR-221 and increased miR-155 expression correlated with p27 kip1 accumulation in DCs. Silencing of miR-221 or overexpressing of miR-155 in DCs resulted in p27 kip1 protein increase and DC apoptosis. Moreover, mDCs from miR-155 -/- mice were less apoptotic than those from wild-type mice. Silencing of miR-155 expression had little effect on DC maturation but reduced IL-12p70 production, whereas miR-155 overexpression in mDCs enhanced IL-12p70 production. Kip1 ubiquitination-promoting complex 1, suppressor of cytokine signaling 1, and CD115 (M-CSFR) were functional targets of miR-155. Furthermore, we provide evidence that miR-155 indirectly regulated p27 kip1 protein level by targeting Kip1 ubiquitination-promoting complex 1. Thus, our study uncovered miRNA signatures during monocyte differentiation into DCs and the new regulatory role of miR-221 and miR-155 in DC apoptosis and IL-12p70 production..
279. Ryo Tachiyama, Daisuke Ishikawa, Masaki Matsumoto, Keiichi Nakayama, Tamotsu Yoshimori, Sadaki Yokota, Masaru Himeno, Yoshitaka Tanaka, Hideaki Fujita, Proteome of ubiquitin/MVB pathway
Possible involvement of iron-induced ubiquitylation of transferrin receptor in lysosomal degradation, Genes to Cells, 10.1111/j.1365-2443.2011.01499.x, 16, 4, 448-466, 2011.04, Ubiquitylation of membrane proteins triggers their endocytosis at the plasma membrane and subsequent lysosomal degradation through multivesicular bodies (MVBs). A dominant-negative mutant SKD1/Vps4B caused an accumulation of ubiquitylated membrane proteins in MVBs. We have identified 22 membrane proteins whose trafficking is potentially regulated by ubiquitylation. Nine of them, including transferrin receptor (TfR), are indeed ubiquitylated and/or accumulated in MVBs in the cells expressing mutant Vps4. While the recycling route and iron-regulated expression of TfR are well characterized, the mechanism by which the degradation of TfR is regulated is largely unknown. We show that an excess of iron enhances both TfR's ubiquitylation and degradation in lysosomes. Probably, the up-regulated expression of ferritin, an endogenous iron-chelating molecule, attenuated the iron-induced degradation of TfR. Exogenously introduced lysine-less TfR, compared to the wild-type one, showed resistance to the iron-induced ubiquitylation and degradation, when endogenous TfR, which most certainly heterodimerizes with exogenous ones, was depleted with siRNA. These data suggest that the iron-induced ubiquitylation and degradation of TfR along with MVB pathway physiologically plays an important role in iron homeostasis..
280. Akinobu Matsumoto, Yuki Tateishi, Ichiro Onoyama, Yasutaka Okita, Keiko Nakayama, Keiichi Nakayama, Fbxw7β resides in the endoplasmic reticulum membrane and protects cells from oxidative stress, Cancer Science, 10.1111/j.1349-7006.2011.01851.x, 102, 4, 749-755, 2011.04, Oxidative stress has been implicated in cancer initiation and progression. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of oncoproteins such as c-Myc, c-Jun, Notch, and cyclin E. Fbxw7 is therefore thought to function as a tumor suppressor, and indeed the Fbxw7 gene is frequently mutated in many human malignancies. The Fbxw7 gene locus encodes three protein isoforms: Fbxw7α, Fbxw7β, and Fbxw7γ. Whereas Fbxw7α and Fbxw7γ are resident in the nucleus, Fbxw7β shows a cytoplasmic distribution suggestive of localization to the endoplasmic reticulum (ER). The specific function of Fbxw7β has remained unknown, however. We now show that Fbxw7β contains a putative transmembrane domain near its NH2-terminus, and topological analysis revealed that Fbxw7β is inserted in the ER membrane. Fbxw7β assembled with Skp1, Cul1, and Rbx1 to form an SCF complex, although the efficiency of this process appeared lower than that for Fbxw7α or Fbxw7γ. To explore the physiological role of Fbxw7β, we generated mice specifically lacking this isoform of Fbxw7. Although these animals did not exhibit any apparent abnormalities in development, primary cultures of neurons prepared from the mutant mice were more vulnerable to oxidative stress than were those prepared from wild-type mice. Conversely, overexpression of Fbxw7β rendered cells resistant to oxidative stress, without affecting sensitivity to ER stress or other apoptosis-inducing agents. Our results thus suggest that Fbxw7β contributes to the protection of cells from oxidative stress..
281. Akinobu Matsumoto, Ichiro Onoyama, Takehiko Sunabori, Ryoichiro Kageyama, Hideyuki Okano, Keiichi Nakayama, Fbxw7-dependent degradation of notch is required for control of "Stemness" and neuronal-glial differentiation in neural stem cells, Journal of Biological Chemistry, 10.1074/jbc.M110.194936, 286, 15, 13754-13764, 2011.04, Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL- 10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1- Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7F/F mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain..
282. Shin ichiro Inoue, Tomonao Matsushita, Yuta Tomokiyo, Masaki Matsumoto, Keiichi Nakayama, Toshinori Kinoshita, Ken ichiro Shimazaki, Functional analyses of the activation loop of phototropin2 in Arabidopsis, Plant physiology, 10.1104/pp.111.175943, 156, 1, 117-128, 2011.05, Phototropins (phot1 and phot2) are autophosphorylating blue-light receptor kinases that mediate blue-light responses such as phototropism, chloroplast accumulation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Only phot2 induces the chloroplast avoidance response under strong blue light. The serine (Ser) residues of the kinase activation loop in phot1 are autophosphorylated by blue light, and autophosphorylation is essential for the phot1-mediated responses. However, the role of autophosphorylation in phot2 remains to be determined. In this study, we substituted the conserved residues of Ser-761 and Ser-763 with alanine (S761A S763A) in the phot2 activation loop and analyzed their function by investigating the phot2- mediated responses after the transformation of phot1 phot2 double mutant with this mutant phot2 gene. Transgenic plants expressing the mutant phot2 protein exhibited impaired responses in chloroplast movement, stomatal opening, phototropic bending, leaf flattening, and plant growth; and those expressing phot2 with S761D S763D mutations showed the normal responses. Substitution of both Ser-761 and Ser-763 with alanine in phot2 did not significantly affect the kinase activity in planta. From these results, we conclude that phosphorylation of Ser-761 and Ser-763 in the activation loop may be a common primary step for phot2-mediated responses..
283. Sonia Rodriguez, Lin Wang, Christen Mumaw, Edward F. Srour, Cristina Lo Celso, Keiichi Nakayama, Nadia Carlesso, The SKP2 E3 ligase regulates basal homeostasis and stress-induced regeneration of HSCs, Blood, 10.1182/blood-2010-11-321521, 117, 24, 6509-6519, 2011.06, Exit from quiescence and reentry into cell cycle is essential for HSC self-renewal and regeneration. Skp2 is the F-box unit of the SCF E3-ligase that targets the CDK inhibitors (CKIs) p21Cip1, p27Kip1, p57Kip2, and p130 for degradation. These CKIs inhibit the G 1 to S-phase transition of the cell cycle, and their deletion results in increased cell proliferation and decreased stem cell self-renewal. Skp2 deletion leads to CKIs stabilization inducing cell-cycle delay or arrest, and conversely, increased Skp2 expression is often found in cancers. Here, we show that SKP2 expression is increased in HSC and progenitors in response to hematopoietic stress from myelosuppression or after transplantation. At steady state, SKP2 deletion decreased the mitotic activity of HSC and progenitors resulting in enhanced HSC quiescence, increased HSC pool size, and maintenance. However, the inability to rapidly enter cell cycle greatly impaired the short-term repopulating potential of SKP2 null HSC and their ability to regenerate after myeloablative stress. Mechanistically, deletion of SKP2 in HSC and progenitors stabilized CKIs in vivo, particularly p27Kip1, p57Kip2, and p130. Our results demonstrate a previously unrecognized role for SKP2 in regulating HSC and progenitor expansion and hematopoietic regeneration after stress..
284. Abbas Fotovati, Samah Abu-Ali, Keiko Nakayama, Keiichi Nakayama, Impaired ovarian development and reduced fertility in female mice deficient in Skp2, Journal of Anatomy, 10.1111/j.1469-7580.2011.01370.x, 218, 6, 668-677, 2011.06, p27 is a major negative regulator of somatic cellular proliferation, and its down-regulation has been shown to be associated with cancer development. Targeted disruption ofp27 results in complete loss of fertility in female mice, suggesting that it plays a significant role in the development of female gametes and the surrounding environment. We have now investigated the effect of loss of Skp2, an F-box protein that mediates ubiquitin-dependent degradation of p27, on female gamete production. The female Skp2-deficient mice showed accumulation of p27 in the ovary and severely compromised gamete development from the embryonic stage to follicular growth in the adult ovary, eventually leading to a decreased functional gamete reserve. Additional deletion of p27 resulted in relatively normal ovarian folliculogenesis, suggesting that accumulating p27 is primarily responsible for the compromised ovarian development. Embryonic ovaries of Skp2-/- mice manifested massive apoptosis as evidenced by cleavage of pro-caspase 3 and poly(ADP-ribose) polymerase-1. This in turn resulted in a significant decrease in the remaining pool of functional gametes in Skp2-/- mice shortly after sexual maturity and premature ovarian failure. The increased apoptosis seemed to be attributable to the polyploidy of granulosa cells. These results suggest that proper progression of the cell cycle, regulated by the p27-Skp2 axis, is pivotal for the maintenance of fertility, and that defects in this system may underlie the pathogenesis of abnormal gamete production and premature ovarian failure during the reproductive life of women..
285. Eun Joo Shin, Chu Xuan Duong, Xuan Khanh Thi Nguyen, Guoying Bing, Jae Hyung Bach, Dae Hun Park, Keiichi Nakayama, Syed F. Ali, Anumantha G. Kanthasamy, Jean L. Cadet, Toshitaka Nabeshima, Hyoung Chun Kim, PKCδ inhibition enhances tyrosine hydroxylase phosphorylation in mice after methamphetamine treatment, Neurochemistry International, 10.1016/j.neuint.2011.03.022, 59, 1, 39-50, 2011.08, The present study was designed to evaluate the specific role of protein kinase C (PKC) δ in methamphetamine (MA)-induced dopaminergic toxicity. A multiple-dose administration regimen of MA significantly increases PKCδ expression, while rottlerin, a PKCδ inhibitor, significantly attenuates MA-induced hyperthermia and behavioral deficits. These behavioral effects were not significantly observed in PKCδ antisense oligonucleotide (ASO)-treated- or PKCδ knockout (-/-)-mice. There were no MA-induced significant decreases of dopamine (DA) content or tyrosine hydroxylase (TH) expression in the striatum in rottlerin-treated-, ASO-treated- or PKCδ (-/-)-mice. The administration of MA also results in a significant decrease of TH phosphorylation at ser 40, but not ser 31, while the inhibition of PKCδ consistently and significantly attenuates MA-induced reduction in the phosphorylation of TH at ser 40. Therefore, these results suggest that the MA-induced enhancement of PKCδ expression is a critical factor in the impairment of TH phosphorylation at ser 40 and that pharmacological or genetic inhibition of PKCδ may be protective against MA-induced dopaminergic neurotoxicity in vivo..
286. Zhiqian Yu, Chiaki Ono, Helen B. Kim, Hiroshi Komatsu, Yoichiro Tanabe, Nobutaka Sakae, Keiichi Nakayama, Hiroo Matsuoka, Ichiro Sora, William E. Bunney, Hiroaki Tomita, Four mood stabilizers commonly induce FEZ1 expression in human astrocytes, Bipolar Disorders, 10.1111/j.1399-5618.2011.00946.x, 13, 5-6, 486-499, 2011.08, Objectives: Mood stabilizers influence the morphology, chemotaxis, and survival of neurons, which are considered to be related to the mood-stabilizing effects of these drugs. Although previous studies suggest glial abnormalities in patients with bipolar disorder and an effect of mood stabilizers on certain genes in astrocytes, less is known about the effects of mood stabilizers in astrocytes than in neurons. The present study identifies a common underlying response to mood stabilizers in astrocytes. Methods: Human astrocyte-derived cells (U-87 MG) were treated with the four most commonly used mood stabilizers (lithium, valproic acid, carbamazepine, and lamotrigine) and subjected to microarray gene expression analyses. The most prominently regulated genes were validated by qRT-PCR and western blot analysis. The intercellular localization of one of these regulated genes, fasciculation and elongation protein zeta 1 (FEZ1), was evaluated by immunofluorescence staining. Results: The microarray data indicated that FEZ1 was the only gene commonly induced by the four mood stabilizers in human astrocyte-derived cells. An independent experiment confirmed astrocytic FEZ1 induction at both the transcript and protein levels following mood stabilizer treatments. FEZ1 localized to the cytoplasm of transformed and primary astrocytes from the human adult brain. Conclusions: Our data suggest that FEZ1 may play important roles in human astrocytes, and that mood stabilizers might exert their cytoprotective and mood-stabilizing effects by inducing FEZ1 expression in astrocytes..
287. Toshiro Moroishi, Masaaki Nishiyama, Yukiko Takeda, Kazuhiro Iwai, Keiichi Nakayama, The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo, Cell metabolism, 10.1016/j.cmet.2011.07.011, 14, 3, 339-351, 2011.09, Iron-dependent degradation of iron-regulatory protein 2 (IRP2) is a key event for maintenance of an appropriate intracellular concentration of iron. Although FBXL5 (F box and leucine-rich repeat protein 5) is thought to mediate this degradation, the role of FBXL5 in the control of iron homeostasis in vivo has been poorly understood. We have now found that mice deficient in FBXL5 died in utero, associated with excessive iron accumulation. This embryonic mortality was prevented by additional ablation of IRP2, suggesting that impaired IRP2 degradation is primarily responsible for the death of Fbxl5 - /- mice. We also found that liver-specific deletion of Fbxl5 resulted in deregulation of both hepatic and systemic iron homeostasis, leading to the development of steatohepatitis. The liver-specific mutant mice died with acute liver failure when fed a high-iron diet. Thus, our results uncover a major role for FBXL5 in ensuring an appropriate supply of iron to cells..
288. Akinobu Matsumoto, Shoichiro Takeishi, Tomoharu Kanie, Etsuo Susaki, Ichiro Onoyama, Yuki Tateishi, Keiko Nakayama, Keiichi Nakayama, P57 Is required for quiescence and maintenance of adult hematopoietic stem cells, Cell stem cell, 10.1016/j.stem.2011.06.014, 9, 3, 262-271, 2011.09, Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G 0 phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs..
289. Peng Zou, Hiroki Yoshihara, Kentaro Hosokawa, Ikue Tai, Kaori Shinmyozu, Fujiko Tsukahara, Yoshiro Maru, Keiko Nakayama, Keiichi Nakayama, Toshio Suda, P57 Kip2 and p27 Kip1 cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70, Cell stem cell, 10.1016/j.stem.2011.07.003, 9, 3, 247-261, 2011.09, Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Combined deficiency of p57 and p27 in HSCs resulted in nuclear import of an Hsc70/cyclin D1 complex, concomitant with Rb phosphorylation, and elicited severe defects in maintaining HSC quiescence. Taken together, these data suggest that regulation of cytoplasmic localization of Hsc70/cyclin D1 complex by p57 and p27 is a key intracellular mechanism in controlling HSC dormancy..
290. Fumihiko Okumura, Akiko J. Okumura, Masaki Matsumoto, Keiichi Nakayama, Shigetsugu Hatakeyama, TRIM8 regulates Nanog via Hsp90β-mediated nuclear translocation of STAT3 in embryonic stem cells, Biochimica et Biophysica Acta - Molecular Cell Research, 10.1016/j.bbamcr.2011.05.013, 1813, 10, 1784-1792, 2011.10, TRIM8 is a member of a protein family defined by the presence of a common domain structure composed of a tripartite motif including a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with Hsp90β, which interacts with STAT3 and selectively downregulates transcription of Nanog in embryonic stem cells. Knock-down of TRIM8 increased phosphorylated STAT3 in the nucleus and also enhanced transcription of Nanog. These findings suggest that TRIM8 modulates translocation of phosphorylated STAT3 into the nucleus through interaction with Hsp90β and consequently regulates transcription of Nanog in embryonic stem cells..
291. Akinobu Matsumoto, Etsuo Susaki, Ichiro Onoyama, Keiko Nakayama, Mikio Hoshino, Keiichi Nakayama, Deregulation of the p57-E2F1-p53 axis results in nonobstructive hydrocephalus and cerebellar malformation in mice, Molecular and Cellular Biology, 10.1128/MCB.05370-11, 31, 20, 4176-4192, 2011.10, The cyclin-dependent kinase inhibitor (CKI) p57 Kip2 plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27 Kip1 at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1..
292. José J. Fuster, Herminia González-Navarro, Angela Vinué, Pedro Molina-Sànchez, Maria J. Andrés-Manzano, Keiichi Nakayama, Keiko Nakayama, Antonio Díez-Juan, Antonio Bernad, Cristina Rodríguez, José Martínez-González, Vicente Andrés, Deficient p27 phosphorylation at serine 10 increases macrophage foam cell formation and aggravates atherosclerosis through a proliferation-independent mechanism, Arteriosclerosis, Thrombosis, and Vascular Biology, 10.1161/ATVBAHA.111.235580, 31, 11, 2455-2463, 2011.11, Objective-: Genetic ablation of the growth suppressor p27 Kip1 (p27) in the mouse aggravates atherosclerosis coinciding with enhanced arterial cell proliferation. However, it is unknown whether molecular mechanisms that limit p27's protective function contribute to atherosclerosis development and whether p27 exerts proliferation-independent activities in the arterial wall. This study aims to provide insight into both questions by investigating the role in atherosclerosis of p27 phosphorylation at serine 10 (p27-phospho-Ser10), a major posttranslational modification of this protein. Methods and Results-: Immunoblotting studies revealed a marked reduction in p27-phospho-Ser10 in atherosclerotic arteries from apolipoprotein E-null mice, and expression of the nonphosphorylatable mutant p27Ser10Ala, either global or restricted to bone marrow, accelerated atherosclerosis. p27Ser10Ala expression did not affect cell proliferation in early and advanced atheroma but activated RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) signaling and promoted macrophage foam cell formation in a ROCK-dependent manner. Supporting the clinical relevance of these findings, human atherosclerotic coronary arteries exhibited a prominent reduction in p27-phospho-Ser10 and increased ezrin/radixin/moesin protein phosphorylation, a marker of RhoA/ROCK activation. Conclusion-: Scarce phosphorylation of p27 at Ser10 is a hallmark of human and mouse atherosclerosis and promotes disease progression in mice. This proatherogenic effect is mediated by a proliferation-independent mechanism that involves augmented foam cell formation owing to increased RhoA/ROCK activity. These findings unveil a new atheroprotective action of p27 and identify p27-phospho-Ser10 as an attractive target for the treatment of atherosclerosis..
293. Fumiko Matsuzaki, Michiko Shirane, Masaki Matsumoto, Keiichi Nakayama, Protrudin serves as an adaptor molecule that connects KIF5 and its cargoes in vesicular transport during process formation, Molecular Biology of the Cell, 10.1091/mbc.E11-01-0068, 22, 23, 4602-4620, 2011.12, Neurons are highly polarized cells with long neurites. Vesicular transport is required for neurite extension. We recently identified protrudin as a key regulator of vesicular transport during neurite extension. Expression of protrudin in nonneuronal cells thus induces formation of neurite-like membrane protrusions. We adopted a proteomics approach to identify proteins that associate with protrudin. Among the protrudin-associated proteins, including many with a function related to intracellular trafficking, we focused on KIF5, a motor protein that mediates anterograde vesicular transport in neurons. A coimmunoprecipitation assay confirmed that endogenous protrudin and KIF5 interact in mouse brain. Overexpression of KIF5 induced the formation of membrane protrusions in HeLa cells, reminiscent of the effect of protrudin overexpression. Forced expression of both protrudin and KIF5 promoted protrusion extension in a synergistic manner, whereas depletion of either protein attenuated protrusion formation. Protrudin facilitated the interaction of KIF5 with Rab11, VAP-A and -B, Surf4, and RTN3, suggesting that protrudin serves as an adaptor protein and that the protrudin-KIF5 complex contributes to the transport of these proteins in neurons. Given that mutation of protrudin or KIF5 is a cause of human hereditary spastic paraplegia, the protrudin-KIF5 axis appears to be integral to neuronal function..
294. Hirokazu Nakatsumi, Masaki Matsumoto, Keiichi Nakayama, New horizons in biological research developed by nextgeneration proteomics
Say good-bye to western blotting, Seikagaku. The Journal of Japanese Biochemical Society, 84, 1, 53-57, 2012.
295. Masaaki Nishiyama, Arthur I. Skoultchi, Keiichi Nakayama, Histone H1 recruitment by CHD8 is essential for suppression of the Wnt-β-catenin signaling pathway, Molecular and Cellular Biology, 10.1128/MCB.06409-11, 32, 2, 501-512, 2012.01, Members of the chromodomain helicase DNA-binding (CHD) family of proteins are thought to regulate gene expression. Among mammalian CHD proteins, CHD8 was originally isolated as a negative regulator of the Wnt-β-catenin signaling pathway that binds directly toβ-catenin and suppresses its transactivation activity. The mechanism by which CHD8 inhibits β-catenin-dependent transcription has been unclear, however. Here we show that CHD8 promotes the association ofβ-catenin and histone H1, with formation of the trimeric complex on chromatin being required for inhibition ofβ-catenin-dependent transactivation. A CHD8 mutant that lacks the histone H1 binding domain did not show such inhibitory activity, indicating that histone H1 recruitment is essential for the inhibitory effect of CHD8. Furthermore, either depletion of histone H1 or expression of a dominant negative mutant of this protein resulted in enhancement of the response to Wnt signaling. These observations reveal a new mode of regulation of the Wnt signaling pathway by CHD8, which counteractsβ-catenin function through recruitment of histone H1 to Wnt target genes. Given that CHD8 is expressed predominantly during embryogenesis, it may thus contribute to setting a threshold for responsiveness to Wnt signaling that operates in a development-dependent manner..
296. Paola Bargagna-Mohan, Riya R. Paranthan, Adel Hamza, Chang Guo Zhan, Do Min Lee, Kyung Bo Kim, Daniel L. Lau, Cidambi Srinivasan, Keiko Nakayama, Keiichi Nakayama, Harald Herrmann, Royce Mohan, Corneal antifibrotic switch identified in genetic and pharmacological deficiency of vimentin, Journal of Biological Chemistry, 10.1074/jbc.M111.297150, 287, 2, 989-1006, 2012.01, The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim -/-) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim -/- mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim -/- corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27 Kip1 and p21 Cip1. In cell culture models, WFA exerts G 2/M cell cycle arrest in a p27 Kip1- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim -/- mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development..
297. Kiyotaka Oshikawa, Masaki Matsumoto, Koji Oyamada, Keiichi Nakayama, Proteome-wide identification of ubiquitylation sites by conjugation of engineered lysine-less ubiquitin, Journal of Proteome Research, 10.1021/pr200668y, 11, 2, 796-807, 2012.02, Ubiquitin conjugation (ubiquitylation) plays important roles not only in protein degradation but also in many other cellular functions. However, the sites of proteins that are targeted for such modification have remained poorly characterized at the proteomic level. We have now developed a method for the efficient identification of ubiquitylation sites in target proteins with the use of an engineered form of ubiquitin (K0-Ub), in which all seven lysine residues are replaced with arginine. K0-Ub is covalently attached to lysine residues of target proteins via an isopeptide bond, but further formation of a polyubiquitin chain does not occur on K0-Ub. We identified a total of 1392 ubiquitylation sites of 794 proteins from HEK293T cells. Profiling of ubiquitylation sites indicated that the sequences surrounding lysine residues targeted for ubiquitin conjugation do not share a common motif or structural feature. Furthermore, we identified a critical ubiquitylation site of the cyclin-dependent kinase inhibitor p27 Kip1. Mutation of this site thus inhibited ubiquitylation of and stabilized p27 Kip1, suggesting that this lysine residue is the target site of p27 Kip1 for ubiquitin conjugation in vivo. In conclusion, our method based on K0-Ub is a powerful tool for proteome-wide identification of ubiquitylation sites of target proteins..
298. Tomoharu Kanie, Ichiro Onoyama, Akinobu Matsumoto, Masanori Yamada, Hirokazu Nakatsumi, Yuki Tateishi, So Yamamura, Ryosuke Tsunematsu, Masaki Matsumoto, Keiichi Nakayama, Genetic reevaluation of the Role of F-Box proteins in cyclin D1 degradation, Molecular and Cellular Biology, 10.1128/MCB.06570-11, 32, 3, 590-605, 2012.02, D-type cyclins play a pivotal role in G1-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4-/-mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4-/-, Fbxw8-/-, and Fbxo4-/-; Fbxw8-/- mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4-/-; Fbxw8-/- MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis..
299. Sayuri Suzuki, Hirotaka Fukasawa, Taro Misaki, Akashi Togawa, Naro Ohashi, Kyoko Kitagawa, Yojiro Kotake, Ning Liu, Hiroyuki Niida, Keiko Nakayama, Keiichi Nakayama, Tatsuo Yamamoto, Masatoshi Kitagawa, The amelioration of renal damage in Skp2-deficient mice canceled by p27Kip1 deficiency in Skp2-/- p27-/- mice, PloS one, 10.1371/journal.pone.0036249, 7, 4, 2012.04, SCF-Skp2 E3 ubiquitin ligase (Skp2 hereafter) targets several cell cycle regulatory proteins for degradation via the ubiquitin-dependent pathway. However, the target-specific physiological functions of Skp2 have not been fully elucidated in kidney diseases. We previously reported an increase in Skp2 in progressive nephropathy and amelioration of unilateral ureteral obstruction (UUO) renal injury associated with renal accumulation of p27 in Skp2-/- mice. However, it remains unclear whether the amelioration of renal injury in Skp2-/- mice is solely caused by p27 accumulation, since Skp2 targets several other proteins. Using Skp2-/-p27-/- mice, we investigated whether Skp2 specifically targets p27 in the progressive nephropathy mediated by UUO. In contrast to the marked suppression of UUO renal injury in Skp2-/- mice, progression of tubular dilatation associated with tubular epithelial cell proliferation and tubulointerstitial fibrosis with increased expression of collagen and α-smooth muscle actin were observed in the obstructed kidneys in Skp2-/-p27-/- mice. No significant increases in other Skp2 target proteins including p57, p130, TOB1, cyclin A and cyclin D1 were noted in the UUO kidney in Skp2-/- mice, while p21, c-Myc, b-Myb and cyclin E were slightly increased. Contrary to the ameliorated UUO renal injure by Skp2-deficiency, the amelioration was canceled by the additional p27-deficiency in Skp2-/-p27-/- mice. These findings suggest a pathogenic role of the reduction in p27 targeted by Skp2 in the progression of nephropathy in UUO mice..
300. C. Chow, N. Wong, M. Pagano, S. W.M. Lun, Keiichi Nakayama, K. Nakayama, K. W. Lo, Regulation of APC/C Cdc20 activity by RASSF1A-APC/C Cdc20 circuitry, Oncogene, 10.1038/onc.2011.372, 31, 15, 1975-1987, 2012.04, RASSF1A is a key tumor-suppressor gene that is often inactivated in a wide variety of solid tumors. Studies have illustrated that RASSF1A plays vital roles in the regulation of cell-cycle progression and functions as a guardian of mitosis. Nevertheless, the precise mechanism of RASSF1A-dependent regulation of mitosis remains largely unclear. APC/C Cdc20 is the master switch and regulator of mitosis. The activity of APC/C Cdc20 is tightly controlled by phosphorylation and specific inhibitors to ensure the sequential ubiquitination of downstream targets. Here, we report on the novel finding of a regulated circuitry that controls the timely expression and hence activity of APC/C Cdc20 during mitosis. Our study showed that RASSF1A and APC/C Cdc20 form a molecular relay that regulates the APC/C Cdc20 activity at early mitosis. We found that RASSF1A inhibits APC/C Cdc20 function through its D-box motifs. Paradoxically, RASSF1A was also demonstrated to be ubiquitinated by APC/C Cdc20 in vitro and degraded at prometaphase despite of active spindle checkpoint presence. The first two unique D-boxes at the N-terminal of RASSF1A served as specific degron recognized by APC/C Cdc20. Importantly, we found that Aurora A and Aurora B directly phosphorylate RASSF1A, a critical step by which RASSF1A switches from being an inhibitor to a substrate of APC/C Cdc20 during the course of mitotic progression. As a result of RASSF1A degradation, APC/C Cdc20 can then partially activate the ubiquitination of Cyclin A in the presence of spindle checkpoint. This circuitry is essential for the timely degradation of Cyclin A. To conclude, our results propose a new model for RASSF1A-APC/C Cdc20 interaction in ensuring the sequential progression of mitosis..
301. Ning Liu, Masaki Matsumoto, Kyoko Kitagawa, Yojiro Kotake, Sayuri Suzuki, Senji Shirasawa, Keiichi Nakayama, Makoto Nakanishi, Hiroyuki Niida, Masatoshi Kitagawa, Chk1 phosphorylates the tumour suppressor Mig-6, regulating the activation of EGF signalling, EMBO Journal, 10.1038/emboj.2012.88, 31, 10, 2365-2377, 2012.05, The tumour suppressor gene product Mig-6 acts as an inhibitor of epidermal growth factor (EGF) signalling. However, its posttranslational modifications and regulatory mechanisms have not been elucidated. Here, we investigated the phosphorylation of human Mig-6 and found that Chk1 phosphorylated Mig-6 in vivo as well as in vitro. Moreover, EGF stimulation promoted phosphorylation of Mig-6 without DNA damage and the phosphorylation was inhibited by depletion of Chk1. EGF also increased Ser280-phosphorylated Chk1, a cytoplasmic-tethering form, via PI3K pathway. Mass spectrometric analyses suggested that Ser 251 of Mig-6 was a major phosphorylation site by Chk1 in vitro and in vivo. Substitution of Ser 251 to alanine increased inhibitory activity of Mig-6 against EGF receptor (EGFR) activation. Moreover, EGF-dependent activation of EGFR and cell growth were inhibited by Chk1 depletion, and were rescued by co-depletion of Mig-6. Our results suggest that Chk1 phosphorylates Mig-6 on Ser 251, resulting in the inhibition of Mig-6, and that Chk1 acts as a positive regulator of EGF signalling. This is a novel function of Chk1..
302. Michael B. Ellman, Jae Sung Kim, Howard S. An, Jeffrey S. Kroin, Xin Li, Di Chen, Dongyao Yan, Doug D. Buechter, Keiichi Nakayama, Bo Liu, Stephanie Morgan, Hee Jeong Im, The pathophysiologic role of the protein kinase Cδ pathway in the intervertebral discs of rabbits and mice
In vitro, ex vivo, and in vivo studies, Arthritis and rheumatism, 10.1002/art.34337, 64, 6, 1950-1959, 2012.06, Objective Protein kinase Cδ (PKCδ) activation has been shown to be a principal rate-limiting step in matrix-degrading enzyme production in human articular chondrocytes. The aim of this study was to assess the role of the PKC pathways, specifically PKCδ, in intervertebral disc tissue homeostasis. Methods Using in vitro, ex vivo, and in vivo techniques, we evaluated the pathophysiologic role of the PKCδ pathway by examining 1) proteoglycan deposition, 2) matrix-degrading enzyme production and activity, 3) downstream signaling pathways regulated by PKCδ, and 4) the effect on in vivo models of disc degeneration in genetically engineered PKCδ-knockout mice. Results Studies of pathway-specific inhibitors revealed a vital role of the PKCδ/MAPK (ERK, p38, JNK) axis and NF-κB in disc homeostasis. Accordingly, in an in vivo model of disc injury, PKCδ-knockout mice were markedly resistant to disc degeneration. Conclusion Suppression of the PKCδ pathway may be beneficial in the prevention and/or treatment of disc degeneration. The results of this study provide evidence for a potential therapeutic role of pathway-specific inhibitors of the PKCδ cascade in the future..
303. Eun Joo Shin, Chu Xuan Duong, Xuan Khanh Thi Nguyen, Zhengyi Li, Guoying Bing, Jae Hyung Bach, Dae Hun Park, Keiichi Nakayama, Syed F. Ali, Anumantha G. Kanthasamy, Jean Lud Cadet, Toshitaka Nabeshima, Hyoung Chun Kim, Role of oxidative stress in methamphetamine-induced dopaminergic toxicity mediated by protein kinase Cδ, Behavioural Brain Research, 10.1016/j.bbr.2012.04.001, 232, 1, 98-113, 2012.06, This study examined the role of protein kinase C (PKC) isozymes in methamphetamine (MA)-induced dopaminergic toxicity. Multiple-dose administration of MA did not significantly alter PKCα, PKCβI, PKCβII, or PKCζ expression in the striatum, but did significantly increase PKCδ expression. Gö6976 (a co-inhibitor of PKCα and -β), hispidin (PKCβ inhibitor), and PKCζ pseudosubstrate inhibitor (PKCζ inhibitor) did not significantly alter MA-induced behavioral impairments. However, rottlerin (PKCδ inhibitor) significantly attenuated behavioral impairments in a dose-dependent manner. In addition, MA-induced behavioral impairments were not apparent in PKCδ knockout (-/-) mice. MA-induced oxidative stress (. i.e., lipid peroxidation and protein oxidation) was significantly attenuated in rottlerin-treated mice and was not apparent in PKCδ (-/-) mice. Consistent with this, MA-induced apoptosis (. i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells) was significantly attenuated in rottlerin-treated mice. Furthermore, MA-induced increases in the dopamine (DA) turnover rate and decreases in tyrosine hydroxylase (TH) activity and the expression of TH, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) were not significantly observed in rottlerin-treated or PKCδ (-/-) mice. Our results suggest that . PKCδ gene expression is a key mediator of oxidative stress and dopaminergic damage induced by MA. Thus, inhibition of PKCδ may be a useful target for protection against MA-induced neurotoxicity..
304. Jonathan E. Grim, Sue E. Knoblaugh, Katherine A. Guthrie, Amanda Hagar, Jherek Swanger, Jessica Hespelt, Jeffrey J. Delrow, Tom Small, William M. Grady, Keiichi Nakayama, Bruce E. Clurman, Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer, Molecular and Cellular Biology, 10.1128/MCB.00305-12, 32, 11, 2160-2167, 2012.06, Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7-/-; p53-/- carcinomas exhibit a CIN+phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations..
305. Kanae Yumimoto, Masaki Matsumoto, Koji Oyamada, Toshiro Moroishi, Keiichi Nakayama, Comprehensive identification of substrates for f-box proteins by differential proteomics analysis, Journal of Proteome Research, 10.1021/pr201216u, 11, 6, 3175-3185, 2012.06, Although elucidation of enzyme-substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases..
306. Yasuyuki Kita, Masaaki Nishiyama, Keiichi Nakayama, Identification of CHD7 S as a novel splicing variant of CHD7 with functions similar and antagonistic to those of the full-length CHD7 L, Genes to Cells, 10.1111/j.1365-2443.2012.01606.x, 17, 7, 536-547, 2012.07, CHD7 is one of the nine members of the chromodomain helicase DNA-binding family of ATP-dependent chromatin remodeling enzymes. Mutations in CHD7 give rise to CHARGE syndrome, a human condition characterized by malformation of various organs. We have now identified a novel transcript of CHD7 that is generated by alternative splicing of exon 6. The protein encoded by this variant transcript (termed CHD7 S) lacks one of the two chromodomains as well as the helicase/ATPase domain, DNA-binding domain and BRK domains of the full-length protein (CHD7 L). CHD7 S was found to localize specifically to the nucleolus in a manner dependent on a nucleolar localization signal. Over-expression of CHD7 S, as well as that of CHD7 L, resulted in an increase in 45S precursor rRNA production. Conversely, depletion of both CHD7 S and CHD7 L by RNA interference inhibited both 45S precursor rRNA production and cell proliferation to a greater extent than did depletion of CHD7 L alone. Furthermore, we found that, like CHD7 L, CHD7 S binds to Sox2 in the nucleoplasm. Unexpectedly, however, whereas over-expression of CHD7 L promoted Sox2-mediated transcriptional regulation, over-expression of CHD7 S suppressed it. These results indicate that CHD7 S functions cooperatively or antagonistically with CHD7 L in the nucleolus and nucleoplasm, respectively..
307. Takehiko Yokobori, Koshi Mimori, Masaaki Iwatsuki, Hideshi Ishii, Fumiaki Tanaka, Tetsuya Sato, Hiroyuki Toh, Tomoya Sudo, Takeshi Iwaya, Yoichi Tanaka, Ichiro Onoyama, Hiroyuki Kuwano, Keiichi Nakayama, Masaki Mori, Copy number loss of FBXW7 is related to gene expression and poor prognosis in esophageal squamous cell carcinoma, International journal of oncology, 10.3892/ijo.2012.1436, 41, 1, 253-259, 2012.07, FBXW7 is a tumor suppressor gene that plays a role in cell cycle regulation via Myc degradation. However, the clinical significance of FBXW7 in esophageal squamous cell carcinoma (ESCC) has not been evaluated. The purpose of this study was to assess the clinical significance of FBXW7 for prognosis in human ESCC. Real-time RT-PCR was used to examine the expression of FBXW7 to determine its clinicopathological significance in 75 cases of ESCC. Overall survival rate was calculated using the Kaplan-Meier method, while multivariate survival was analyzed with the Cox hazard model. FBXW7 suppression analysis was performed to examine proliferation potency and Myc expression in the FBXW7 siRNA groups. The relationship between FBXW7 expression and the copy number loss of FBXW7 was examined in clinical samples of ESCC. Finally, FBXW7 copy number loss was linked to prognosis in 42 ESCC patients. FBXW7 expression in cancer was lower compared to non-cancer tissues (P=0.003) and is an independent prognostic factor. The proliferation rates and Myc protein expression were significantly enhanced in FBXW7 siRNA cells compared to the controls. Cases with a loss of FBXW7 copy number had low FBXW7 expression and a poorer prognosis than cases with no loss of copy number. Genetic alterations in esophageal cancer lead to the loss of FBXW7 expression and increased cell proliferation. These genetic alterations of FBXW7 status may provide a prognostic factor for ESCC patients..
308. Yasutaka Okita, Akinobu Matsumoto, Kanae Yumimoto, Rieko Isoshita, Keiichi Nakayama, Increased efficiency in the generation of induced pluripotent stem cells by Fbxw7 ablation, Genes to Cells, 10.1111/j.1365-2443.2012.01626.x, 17, 9, 768-777, 2012.09, Induced pluripotent stem cells (iPSCs) share many biological properties with embryonic stem cells (ESCs), and are generated from somatic cells by expression of some transcription factors such as Oct3/4, Sox2, Klf4 and c-Myc. Among these factors, the abundance of c-Myc is strictly regulated by Fbxw7, a subunit of Skp1-Cul1-F-box protein-type ubiquitin ligase. We have now shown that the expression of Fbxw7 was increased as ESCs differentiated. To investigate the role of Fbxw7 in the ESCs/iPSCs, we examined the impact of Fbxw7 ablation in the efficiency in iPSC generation. The frequency of iPSC generation from mouse embryonic fibroblasts (MEFs) lacking Fbxw7 was markedly greater than that from control MEFs. Depletion of Fbxw7 also resulted in promotion of iPSC generation. Morphology of iPSC clonies from Fbxw7-depleted MEFs appeared more undifferentiated than that from MEFs overexpressing c-Myc. Additional depletion of c-Myc did not abrogate the effect of Fbxw7 depletion, suggesting that c-Myc accumulation is not necessarily required for the increased efficiency in iPSC generation by Fbxw7 ablation. Substrates of Fbxw7 other than c-Myc might therefore play a key role in iPSC generation. These results suggest that transient inhibition of Fbxw7 would be a more promising approach to efficient generation of iPSCs than c-Myc over-expression..
309. Hiroko Hoshi, Wu Hao, Yoshinari Fujita, Atsushi Funayama, Yoshiteru Miyauchi, Kazuaki Hashimoto, Kana Miyamoto, Ryotaro Iwasaki, Yuiko Sato, Tami Kobayashi, Hiroya Miyamoto, Shigeyuki Yoshida, Tomoaki Mori, Hiroya Kanagawa, Eri Katsuyama, Atsuhiro Fujie, Kyoko Kitagawa, Keiichi Nakayama, Toshihiro Kawamoto, Motoaki Sano, Keiichi Fukuda, Ikuroh Ohsawa, Shigeo Ohta, Hideo Morioka, Morio Matsumoto, Kazuhiro Chiba, Yoshiaki Toyama, Takeshi Miyamoto, Aldehyde-stress resulting from Aldh2 mutation promotes osteoporosis due to impaired osteoblastogenesis, Journal of Bone and Mineral Research, 10.1002/jbmr.1634, 27, 9, 2015-2023, 2012.09, Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics, and aging. Here we show that a dominant-negative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2*2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol-detoxification by acetaldehyde-detoxification; however, transgenic mice expressing Aldh2*2 (Aldh2*2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild-type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2, and osterix expression, or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2*2 transgene or acetaldehyde treatment induced accumulation of the lipid-oxidant 4-hydroxy-2-nonenal (4HNE) and expression of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Antioxidant treatment inhibited acetaldehyde-induced proliferation-loss, apoptosis, and PPARγ expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARγ inhibitor also restored acetaldehyde-mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2*2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases..
310. Yuki Tateishi, Akinobu Matsumoto, Tomoharu Kanie, Eiji Hara, Keiko Nakayama, Keiichi Nakayama, Development of mice without Cip/Kip CDK inhibitors, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2012.09.041, 427, 2, 285-292, 2012.10, Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G0 to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage)..
311. Chia Hsin Chan, Chien Feng Li, Wei Lei Yang, Yuan Gao, Szu Wei Lee, Zizhen Feng, Hsuan Ying Huang, Kelvin K.C. Tsai, Leo G. Flores, Yiping Shao, John D. Hazle, Dihua Yu, Wenyi Wei, Dos Sarbassov, Mien Chie Hung, Keiichi Nakayama, Hui Kuan Lin, Erratum
The Skp2-SCF E3 ligase regulates akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis (Cell (2012) 149 (1098-1111)), Cell, 10.1016/j.cell.2012.10.025, 151, 4, 913-914, 2012.11.
312. Ryohei Narumi, Tatsuo Murakami, Takahisa Kuga, Jun Adachi, Takashi Shiromizu, Satoshi Muraoka, Hideaki Kume, Yoshio Kodera, Masaki Matsumoto, Keiichi Nakayama, Yasuhide Miyamoto, Makoto Ishitobi, Hideo Inaji, Kikuya Kato, Takeshi Tomonaga, A strategy for large-scale phosphoproteomics and SRM-based validation of human breast cancer tissue samples, Journal of Proteome Research, 10.1021/pr3005474, 11, 11, 5311-5322, 2012.11, Protein phosphorylation is a key mechanism of cellular signaling pathways and aberrant phosphorylation has been implicated in a number of human diseases. Thus, approaches in phosphoproteomics can contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets. Moreover, careful validation of large-scale phosphoproteome analysis, which is lacking in the current protein-based biomarker discovery, significantly increases the value of identified biomarkers. Here, we performed large-scale differential phosphoproteome analysis using IMAC coupled with the isobaric tag for relative quantification (iTRAQ) technique and subsequent validation by selected/multiple reaction monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk recurrence groups. We identified 8309 phosphorylation sites on 3401 proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were able to be quantified and 133 phosphopeptides (117 phosphoproteins) were differentially expressed between the two groups. Among them, 19 phosphopeptides were selected for further verification and 15 were successfully quantified by SRM using stable isotope peptides as a reference. The ratio of phosphopeptides between high- and low-risk groups quantified by SRM was well correlated with iTRAQ-based quantification with a few exceptions. These results suggest that large-scale phosphoproteome quantification coupled with SRM-based validation is a powerful tool for biomarker discovery using clinical samples..
313. Viviana Cremasco, Corinne E. Decker, Deborah Stumpo, Perry J. Blackshear, Keiichi Nakayama, Keiko Nakayama, Traian S. Lupu, Daniel B. Graham, Deborah V. Novack, Roberta Faccio, Protein kinase C-delta deficiency perturbs bone homeostasis by selective uncoupling of cathepsin K secretion and ruffled border formation in osteoclasts, Journal of Bone and Mineral Research, 10.1002/jbmr.1701, 27, 12, 2452-2463, 2012.12, Bone homeostasis requires stringent regulation of osteoclasts, which secrete proteolytic enzymes to degrade the bone matrix. Despite recent progress in understanding how bone resorption occurs, the mechanisms regulating osteoclast secretion, and in particular the trafficking route of cathepsin K vesicles, remain elusive. Using a genetic approach, we describe the requirement for protein kinase C-delta (PKCδ) in regulating bone resorption by affecting cathepsin K exocytosis. Importantly, PKCδ deficiency does not perturb formation of the ruffled border or trafficking of lysosomal vesicles containing the vacuolar-ATPase (v-ATPase). Mechanistically, we find that cathepsin K exocytosis is controlled by PKCδ through modulation of the actin bundling protein myristoylated alanine-rich C-kinase substrate (MARCKS). The relevance of our finding is emphasized in vivo because PKCδ-/- mice exhibit increased bone mass and are protected from pathological bone loss in a model of experimental postmenopausal osteoporosis. Collectively, our data provide novel mechanistic insights into the pathways that selectively promote secretion of cathepsin K lysosomes independently of ruffled border formation, providing evidence of the presence of multiple mechanisms that regulate lysosomal exocytosis in osteoclasts. © 2012 American Society for Bone and Mineral Research..
314. Kiyotaka Oshikawa, Masaki Matsumoto, Keiichi Nakayama, Comprehensive study of protein ubiquitylation sites by conjugation of engineered lysine-less ubiquitin, Seikagaku. The Journal of Japanese Biochemical Society, 84, 6, 479-487, 2012.12.
315. Tadashi Yamamoto, Keiichi Nakayama, Hisashi Hirano, Takeshi Tomonaga, Yasushi Ishihama, Tetsushi Yamada, Tadashi Kondo, Yoshio Kodera, Yuichi Sato, Norie Araki, Hiroshi Mamitsuka, Naoki Goshima, Integrated view of the human chromosome x-centric proteome project, Journal of Proteome Research, 10.1021/pr300844p, 12, 1, 58-61, 2013.01, This article introduces how the human chromosome Xcentric proteome project is carried out by the Japan Chromosome X Project Consortium. The inactivation of one of two chromosomes in female mammals and accumulation of genes related to neural/immune systems/tumor/testis are characteristic of chromosome X. In this Chromosome X Project, information on proteins translated from genes on chromosome X is collected by both mass spectrometry-and antibody-based proteomics. Information on the following resources is also provided: antibodies to proteins translated and full-length cDNAs transcripted from the chromosome X genes for recombinant proteins. The consortium aims to provide the following tools to search useful antibodies in the literature (Antibody Ranker), to find gene expression sites in microarray databases (Transcript Localizer) and to do advanced MRM analysis (information-based MRM)..
316. Akinobu Matsumoto, Keiichi Nakayama, Role of key regulators of the cell cycle in maintenance of hematopoietic stem cells, Biochimica et Biophysica Acta - General Subjects, 10.1016/j.bbagen.2012.07.004, 1830, 2, 2335-2344, 2013.02, Background: Hematopoietic stem cells (HSCs) are characterized by pluripotentiality and self-renewal ability. To maintain a supply of mature blood cells and to avoid HSC exhaustion during the life span of an organism, most HSCs remain quiescent, with only a limited number entering the cell cycle. Scope of review: The molecular mechanisms by which quiescence is maintained in HSCs are addressed, with recent genetic studies having provided important insight into the relation between the cell cycle activity and stemness of HSCs. Major conclusions: The cell cycle is tightly regulated in HSCs by complex factors. Key regulators of the cell cycle in other cell types - including cyclins, cyclin-dependent kinases (CDKs), the retinoblastoma protein family, the transcription factor E2F, and CDK inhibitors - also contribute to such regulation in HSCs. Most, but not all, of these regulators are necessary for maintenance of HSCs, with abnormal activation or suppression of the cell cycle resulting in HSC exhaustion. The cell cycle in HSCs is also regulated by external factors such as cytokines produced by niche cells as well as by the ubiquitin-proteasome pathway. General significance: Studies of the cell cycle in HSCs may shed light on the pathogenesis of hematopoietic disorders, serve as a basis for the development of new therapeutic strategies for such disorders, prove useful for the expansion of HSCs in vitro as a possible replacement for blood transfusion, and provide insight into stem cell biology in general. This article is part of a Special Issue entitled Biochemistry of Stem Cells..
317. Shungo Adachi, Masae Homoto, Rikou Tanaka, Yusaku Hioki, Hiroshi Murakami, Hiroaki Suga, Masaki Matsumoto, Keiichi Nakayama, Tomohisa Hatta, Shun Ichiro Iemura, Tohru Natsume, ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway, Nucleic acids research, 10.1093/nar/gku652, 42, 15, 10037-10049, 2014.01, Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signalregulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide based therapeutics..
318. Mito Kanatsu-Shinohara, Ichiro Onoyama, Keiichi Nakayama, Takashi Shinohara, Skp1-Cullin-F-box (SCF)-type ubiquitin ligase FBXW7 negatively regulates spermatogonial stem cell self-renewal, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1401837111, 111, 24, 8826-8831, 2014.01, Spermatogonial stem cells (SSCs) undergo self-renewal divisions to support spermatogenesis throughout life. Although several positive regulators of SSC self-renewal have been discovered, little is known about the negative regulators. Here, we report that F-box and WD-40 domain protein 7 (FBXW7), a component of the Skp1-Cullin-F-box-type ubiquitin ligase, is a negative regulator of SSC self-renewal. FBXW7 is expressed in undifferentiated spermatogonia in a cell cycle-dependent manner. Although peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1), essential for spermatogenesis, is thought to destroy FBXW7, Pin1 depletion decreased FBXW7 expression. Spermatogonial transplantation showed that Fbxw7 overexpression compromised SSC activity whereas Fbxw7 deficiency enhanced SSC colonization and caused accumulation of undifferentiated spermatogonia, suggesting that the level of FBXW7 is critical for self-renewal and differentiation. Screening of putative FBXW7 targets revealed that Fbxw7 deficiency up-regulated myelocytomatosis oncogene (MYC) and cyclin E1 (CCNE1). Although depletion of Myc/Mycn or Ccne1/Ccne2 compromised SSC activity, overexpression of Myc, but not Ccne1, increased colonization of SSCs. These results suggest that FBXW7 regulates SSC self-renewal in a negative manner by degradation of MYC..
319. S. Takeishi, Keiichi Nakayama, Role of Fbxw7 in the maintenance of normal stem cells and cancer-initiating cells., British journal of cancer, 10.1038/bjc.2014.259, 111, 6, 1054-1059, 2014.01, In addition to the properties of self-renewal and multipotency, stem cells are characterised by their distinct cell cycle status. Somatic stem cells are maintained in a quiescent state but switch reversibly from quiescence to proliferation as needed. On the other hand, embryonic stem cells and induced pluripotent stem cells proliferate rapidly until the induction of differentiation results in inhibition of cell cycle progression. Uncovering the mechanisms underlying cell cycle control in stem cells should thus provide insight into regulation of the balance between self-renewal and differentiation, a key goal of stem cell biology. Recent research has shown that cancer-initiating cells (CICs), a cell population with stem cell-like properties in cancer, are also quiescent, with this characteristic conferring resistance to anticancer therapies that target dividing cells. Elucidation of the mechanisms of CIC quiescence might therefore be expected to provide a basis for the eradication of cancer. This review summarises our current understanding of the role of F-box and WD40 repeat domain-containing 7 (Fbxw7), a key regulator of the cell cycle, in the maintenance of normal stem cells and CICs, as well as attempts to define future challenges in this field..
320. Michiko Shirane, Keiichi Nakayama, Mitochondria
FKBP38 and mitochondrial degradation, International Journal of Biochemistry and Cell Biology, 10.1016/j.biocel.2014.03.007, 51, 1, 19-22, 2014.01, FK506-binding protein 38 (FKBP38) is a membrane chaperone that is localized predominantly to mitochondria and contains a COOH-terminal tail anchor. FKBP38 also harbors an FKBP domain that confers peptidyl-prolyl cis-trans isomerase activity, but it differs from other FKBP family members in that this activity is dependent on the binding of Ca2+-calmodulin. FKBP38 inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-xL to mitochondria. Mice deficient in FKBP38 die soon after birth manifesting a defect in neural tube closure that results in part from unrestrained apoptosis. We recently found that FKBP38 and Bcl-2 translocate from mitochondria to the endoplasmic reticulum during mitophagy, a form of autophagy responsible for the elimination of damaged mitochondria. FKBP38 and Bcl-2 thus escape the degradative fate of most mitochondrial proteins during mitophagy. This escape of FKBP38 is dependent on the low basicity of its COOH-terminal sequence and is essential for the suppression of apoptosis during mitophagy. FKBP38 thus plays a key role in the regulation of apoptosis under normal and pathological conditions..
321. Kyoko Kitagawa, Kiyoshi Shibat, Akinobu Matsumoto, Masaki Matsumoto, Tatsuya Ohhata, Keiichi Nakayama, Hiroyuki Niida, Masatoshi Kitagawa, Fbw7 targets GATA3 through cyclin-dependent kinase 2-dependent proteolysis and contributes to regulation of T-cell development, Molecular and Cellular Biology, 10.1128/MCB.01549-13, 34, 14, 2732-2744, 2014.01, Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4+ and CD8+ cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages..
322. T. Shimazu, Y. Komatsu, Keiichi Nakayama, H. Fukazawa, S. Horinouchi, M. Yoshida, Erratum
Regulation of SV40 large T-Antigen stability by reversible acetylation (Oncogene (2006) 25 (7391-7400) DOI: 10.1038/sj.onc.1209731), Oncogene, 10.1038/onc.2014.176, 33, 29, 2014.07.
323. Katsuyuki Yugi, Hiroyuki Kubota, Yu Toyoshima, Rei Noguchi, Kentaro Kawata, Yasunori Komori, Shinsuke Uda, Katsuyuki Kunida, Yoko Tomizawa, Yosuke Funato, Hiroaki Miki, Masaki Matsumoto, Keiichi Nakayama, Kasumi Kashikura, Keiko Endo, Kazutaka Ikeda, Tomoyoshi Soga, Shinya Kuroda, Reconstruction of insulin signal flow from phosphoproteome and metabolome data, Cell Reports, 10.1016/j.celrep.2014.07.021, 8, 4, 1171-1183, 2014.08, Cellular homeostasis is regulated by signals through multiple molecular networks that include protein phosphorylation and metabolites. However, where and when the signal flows through a network and regulates homeostasis has not been explored. We have developed a reconstruction method for the signal flow based on time-course phosphoproteome and metabolome data, using multiple databases, and have applied it to acute action of insulin, an important hormone for metabolic homeostasis. An insulin signal flows through a network, through signaling pathways that involve 13 protein kinases, 26 phosphorylated metabolic enzymes, and 35 allosteric effectors, resulting in quantitative changes in 44 metabolites. Analysis of the network reveals that insulin induces phosphorylation and activation of liver-type phosphofructokinase 1, thereby controlling a key reaction in glycolysis. We thus provide a versatile method of reconstruction of signal flow through the network using phosphoproteome and metabolome data..
324. Wenfu Lu, Shenji Liu, Bo Li, Yingqiu Xie, Christine Adhiambo, Qing Yang, Billy R. Ballard, Keiichi Nakayama, Robert J. Matusik, Zhenbang Chen, SKP2 inactivation suppresses prostate tumorigenesis by mediating JARID1B ubiquitination, Oncotarget, 10.18632/oncotarget.2718, 6, 2, 771-788, 2015.01, Aberrant elevation of JARID1B and histone H3 lysine 4 trimethylation (H3K4me3) is frequently observed in many diseases including prostate cancer (PCa), yet the mechanisms on the regulation of JARID1B and H3K4me3 through epigenetic alterations still remain poorly understood. Here we report that Skp2 modulates JARID1B and H3K4me3 levels in vitro in cultured cells and in vivo in mouse models. We demonstrated that Skp2 inactivation decreased H3K4me3 levels, along with a reduction of cell growth, cell migration and malignant transformation of Pten/Trp53 double null MEFs, and further restrained prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, Skp2 decreased the K63-linked ubiquitination of JARID1B by E3 ubiquitin ligase TRAF6, thus decreasing JARID1B demethylase activity and in turn increasing H3K4me3. In agreement, Skp2 deficiency resulted in an increase of JARID1B ubiquitination and in turn a reduction of H3K4me3, and induced senescence through JARID1B accumulation in nucleoli of PCa cells and prostate tumors of mice. Furthermore, we showed that the elevations of Skp2 and H3K4me3 contributed to castration-resistant prostate cancer (CRPC) in mice, and were positively correlated in human PCa specimens. Taken together, our findings reveal a novel network of SKP2- JARID1B, and targeting SKP2 and JARID1B may be a potential strategy for PCa control..
325. Noriko Ishida, Taichi Hara, Takumi Kamura, Minoru Yoshida, Keiko Nakayama, Keiichi Nakayama, Phosphorylation of p27Kip1on serine 10 is required for its binding to CRM1 and nuclear export, Journal of Biological Chemistry, 10.1074/jbc.A114.100762, 290, 11, 2015.03.
326. Shohei Furutachi, Hiroaki Miya, Tomoyuki Watanabe, Hiroki Kawai, Norihiko Yamasaki, Yujin Harada, Itaru Imayoshi, Mark Nelson, Keiichi Nakayama, Yusuke Hirabayashi, Yukiko Gotoh, Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells, Nature Neuroscience, 10.1038/nn.3989, 18, 5, 657-665, 2015.04, The mechanism by which adult neural stem cells (NSCs) are established during development is unclear. In this study, analysis of cell cycle progression by examining retention of a histone 2B (H2B)-GFP fusion protein revealed that, in a subset of mouse embryonic neural progenitor cells (NPCs), the cell cycle slows between embryonic day (E) 13.5 and E15.5 while other embryonic NPCs continue to divide rapidly. By allowing H2B-GFP expressed at E9.5 to become diluted in dividing cells until the young adult stage, we determined that a majority of NSCs in the young adult subependymal zone (SEZ) originated from these slowly dividing embryonic NPCs. The cyclin-dependent kinase inhibitor p57 is highly expressed in this embryonic subpopulation, and the deletion of p57 impairs the emergence of adult NSCs. Our results suggest that a substantial fraction of adult SEZ NSCs is derived from a slowly dividing subpopulation of embryonic NPCs and identify p57 as a key factor in generating this embryonic origin of adult SEZ NSCs..
327. Tomomi Nakajima, Kyoko Kitagawa, Tatsuya Ohhata, Satoshi Sakai, Chiharu Uchida, Kiyoshi Shibata, Naoko Minegishi, Kanae Yumimoto, Keiichi Nakayama, Kazuma Masumoto, Fuminori Katou, Hiroyuki Niida, Masatoshi Kitagawa, Regulation of GATA-binding protein 2 levels via ubiquitin-dependent degradation by Fbw7
Involvement of cyclin B-cyclin-dependent kinase 1-mediated phosphorylation of Thr176 in GATA-binding protein 2, Journal of Biological Chemistry, 10.1074/jbc.M114.613018, 290, 16, 10368-10381, 2015.04, AGATA family transcription factor, GATA-binding protein 2 (GATA2), participates in cell growth and differentiation of various cells, such as hematopoietic stem cells. Although its expression level is controlled by transcriptional induction and proteolytic degradation, the responsible E3 ligase has not been identified. Here, we demonstrate that F-box/WD repeat-containing protein 7 (Fbw7/Fbxw7), a component of Skp1, Cullin 1, F-box-containing complex (SCF)-type E3 ligase, is an E3 ligase for GATA2. GATA2 contains a cell division control protein 4 (Cdc4) phosphodegron (CPD), a consensus motif for ubiquitylation by Fbw7, which includes Thr176. Ectopic expression of Fbw7 destabilized GATA2 and promoted its proteasomal degradation. Substitution of threonine 176 to alanine in GATA2 inhibited binding with Fbw7, and the ubiquitylation and degradation of GATA2 by Fbw7 was suppressed. The CPD kinase, which mediates the phosphorylation of Thr176, was cyclin B-cyclin-dependent kinase 1 (CDK1). Moreover, depletion of endogenous Fbw7 stabilized endogenous GATA2 in K562 cells. Conditional Fbw7 depletion in mice increased GATA2 levels in hematopoietic stem cells and myeloid progenitors at the early stage. Increased GATA2 levels in Fbw7-conditional knock-out mice were correlated with a decrease in a c-Kit high expressing population of myeloid progenitor cells. Our results suggest that Fbw7 is a bona fide E3 ubiquitin ligase for GATA2 in vivo..
328. Hiroyuki Hosokawa, Miki Kato, Hiroyuki Tohyama, Yuuki Tamaki, Yusuke Endo, Motoko Y. Kimura, Damon John Tumes, Shinichiro Motohashi, Masaki Matsumoto, Keiichi Nakayama, Tomoaki Tanaka, Toshinori Nakayama, Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells, Journal of Biological Chemistry, 10.1074/jbc.M114.621524, 290, 21, 13095-13103, 2015.05, acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the inter-leukin- 5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methyla-tion- mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3..
329. Kanae Yumimoto, Keiichi Nakayama, Fbxw7 suppresses cancer metastasis by inhibiting niche formation, OncoImmunology, 10.1080/2162402X.2015.1022308, 4, 8, 2015.08, Fbxw7 has been identified as an oncosuppressor protein in many types of cancer. We have recently shown that loss of Fbxw7 in bone marrow-derived stromal cells (BMSCs) promotes cancer metastasis by increasing production of the chemokine CCL2, which attracts monocytic myeloid-derived suppressor cells (Mo-MDSCs) and macrophages to the metastatic niche..
330. Koki Watanabe, Kanae Yumimoto, Keiichi Nakayama, FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1, Genes to Cells, 10.1111/gtc.12260, 20, 8, 667-674, 2015.08, Although identification of substrates for ubiquitin ligase (E3) is important for understanding its biological functions, detection of the interaction between an E3 and its substrates has remained challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of a given E3 ligase. We have now applied this approach to an uncharacterized human F-box protein, FBXO21, which serves as the substrate-recognition subunit of a SKP1-CUL1-F-box protein (SCF)-type E3, thereby identifying EID1 (EP300-interacting inhibitor of differentiation 1) as a candidate substrate. The central and COOH-terminal portion of FBXO21 was found to interact with the COOH-terminal region of EID1 in transfected cells. Over-expression of FBXO21 resulted in the down-regulation of EID1, whereas disruption of the FBXO21 gene with the CRISPR/Cas9 system stabilized EID1 and led to its accumulation in both the cytoplasm and nucleus. An in vitro ubiquitylation assay showed that EID1 is a direct substrate of SCFFBXO 21. Collectively, our results suggest that EID1 is a bona fide substrate of FBXO21 and that the control of EID1 abundance by SCFFBXO 21 might affect the transcriptional repression activity of EID1..
331. Shoji Tane, Hitomi Okayama, Aiko Ikenishi, Yuki Amemiya, Keiichi Nakayama, Takashi Takeuchi, Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2015.08.102, 466, 2, 147-154, 2015.10, Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21Cip1 and p27Kip1, regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21Cip1 and p27Kip1 also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21Cip1 knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21Cip1 knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21Cip1 and p27Kip1) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21Cip1 inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle..
332. Ayaka Yanagida, Hiromi Chikada, Keiichi Ito, Ayumi Umino, Megumi Kato-Itoh, Yuji Yamazaki, Hideyuki Sato, Toshihiro Kobayashi, Tomoyuki Yamaguchi, Keiichi Nakayama, Hiromitsu Nakauchi, Akihide Kamiya, Liver maturation deficiency in p57Kip2-/- mice occurs in a hepatocytic p57Kip2 expression-independent manner, Developmental Biology, 10.1016/j.ydbio.2015.07.004, 407, 2, 331-343, 2015.11, Fetal hepatic stem/progenitor cells, hepatoblasts, are highly proliferative cells and the source of both hepatocytes and cholangiocytes. In contrast, mature hepatocytes have a low proliferative potency and high metabolic functions. Cell proliferation is regulated by cell cycle-related molecules. However, the correlation between cell cycle regulation and hepatic maturation are still unknown. To address this issue, we revealed that the cell cycle inhibitor p57Kip2 was expressed in the hepatoblasts and mesenchymal cells of fetal liver in a spatiotemporal manner. In addition, we found that hepatoblasts in p57Kip2-/- mice were highly proliferative and had deficient maturation compared with those in wild-type (WT) mice. However, there were no remarkable differences in the expression levels of cell cycle- and bipotency-related genes except for Ccnd2. Furthermore, p57Kip2-/- hepatoblasts could differentiate into mature hepatocytes in p57Kip2-/- and WT chimeric mice, suggesting that the intrinsic activity of p57Kip2 does not simply regulate hepatoblast maturation..
333. Daichi Inoue, Masaki Matsumoto, Reina Nagase, Makoto Saika, Takeshi Fujino, Keiichi Nakayama, Toshio Kitamura, Truncation mutants of ASXL1 observed in myeloid malignancies are expressed at detectable protein levels, Experimental Hematology, 10.1016/j.exphem.2015.11.011, 44, 3, 172-176.e1, 2016.03, Recent progress in deep sequencing technologies has revealed many novel mutations in a variety of genes in patients with myelodysplastic syndromes (MDS). Most of these mutations are thought to be loss-of-function mutations, with some exceptions, such as the gain-of-function IDH1/2 and SRSF2 mutations. Among the mutations, ASXL1 mutations attract much attention; the ASXL1 mutations are identified in a variety of hematologic malignancies and always predicts poor prognosis. It was found that the C-terminal truncating mutants of the ASXL1 or ASXL1 deletion induced MDS-like diseases in mouse. In addition, it has recently been reported that ASXL1 mutations are frequently found in clonal hematopoiesis in healthy elderly people, who frequently progress to hematologic malignancies. However, the underlying molecular mechanisms by which ASXL1 mutations induce hematologic malignancies are not fully understood. Moreover, whether ASXL1 mutations are loss-of-function mutations or dominant-negative or gain-of-function mutations remains a matter of controversy. We here present solid evidence indicating that the C-terminal truncating ASXL1 protein is indeed expressed in cells harboring homozygous mutations of ASXL1, indicating the ASXL1 mutations are dominant-negative or gain-of-function mutations; for the first time, we detected the truncated ASXL1 proteins in two cell lines lacking the intact ASXL1 gene by mass spectrometry and Western blot analyses. Thus, together with our previous results, the present results indicate that the truncating ASXL1 mutant is indeed expressed in MDS cells and may play a role in MDS pathogenesis not previously considered..
334. Masako Tanaka, Masayuki Shiota, Takafumi Nakao, Ryo Uemura, Satoshi Nishi, Yasuyuki Ohkawa, Masaki Matsumoto, Maki Yamaguchi, Mayuko Osada-Oka, Azusa Inagaki, Katsuyuki Takahashi, Keiichi Nakayama, Min Gi, Yasukatsu Izumi, Katsuyuki Miura, Hiroshi Iwao, Identification of low-abundance proteins in serum via the isolation of HSP72 complexes, Journal of Proteomics, 10.1016/j.jprot.2016.01.008, 136, 214-221, 2016.03, Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC-MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers..
335. Yuichiro Fujieda, Olga Amengual, Masaki Matsumoto, Kimiko Kuroki, Hidehisa Takahashi, Michihito Kono, Takashi Kurita, Kotaro Otomo, Masaru Kato, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Katsumi Maenaka, Shigetsugu Hatakeyama, Keiichi Nakayama, Tatsuya Atsumi, Ribophorin ii is involved in the tissue factor expression mediated by phosphatidylserinedependent antiprothrombin antibody on monocytes, Rheumatology (United Kingdom), 10.1093/rheumatology/kew005, 55, 6, 1117-1126, 2016.06, Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine prothrombin complex, which is associated with APS. We have previously reported that aPSPT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS..
336. Shoichiro Takeishi, Keiichi Nakayama, To wake up cancer stem cells, or to let them sleep, that is the question, Cancer Science, 10.1111/cas.12958, 107, 7, 875-881, 2016.07, Cancer stem cells (CSCs) generate transient-amplifying cells and thereby contribute to cancer propagation. A fuller understanding of the biological features of CSCs is expected to lead to the development of new anticancer therapies capable of eradicating this life-threatening disease. Cancer stem cells are known to maintain a non-proliferative state and to enter the cell cycle only infrequently. Given that conventional anticancer therapies preferentially target dividing cells, CSCs are resistant to such treatments, with those remaining after elimination of bulk cancer cells potentially giving rise to disease relapse and metastasis as they re-enter the cell cycle after a period of latency. Targeting of the switch between quiescence and proliferation in CSCs is therefore a potential strategy for preventing the reinitiation of malignancy, underscoring the importance of elucidation of the mechanisms by which these cells are maintained in the quiescent state. The fundamental properties of CSCs are thought to be governed cooperatively by internal molecules and cues from the external microenvironment (stem cell niche). Several such intrinsic and extrinsic regulators are responsible for the control of cell cycle progression in CSCs. In this review, we address two opposite approaches to the therapeutic targeting of CSCs – wake-up and hibernation therapies – that either promote or prevent the entry of CSCs into the cell cycle, respectively, and we discuss the potential advantages and risks of each strategy..
337. Katsuyuki Takahashi, Masako Tanaka, Masakazu Yashiro, Masaki Matsumoto, Asuka Ohtsuka, Keiichi Nakayama, Yasukatsu Izumi, Katsuya Nagayama, Katsuyuki Miura, Hiroshi Iwao, Masayuki Shiota, Protection of stromal cell-derived factor 2 by heat shock protein 72 prevents oxaliplatin-induced cell death in oxaliplatin-resistant human gastric cancer cells, Cancer Letters, 10.1016/j.canlet.2016.05.002, 378, 1, 8-15, 2016.08, Heat shock protein 72 (Hsp72) is a molecular chaperone that assists in the folding of nascent polypeptides and in the refolding of denatured proteins. In many cancers, Hsp72 is constitutively expressed at elevated levels, which can result in enhanced stress tolerance. Similarly, following treatment with anticancer drugs, Hsp72 binds to denatured proteins that may be essential for survival. We therefore hypothesized that Hsp72 client proteins may play a crucial role in drug resistance. Here, we aimed to identify proteins that are critical for oxaliplatin (OXA) resistance by analyzing human gastric cancer cell lines, as well as OXA-resistant cells via a mass spectrometry-based proteomic approach combined with affinity purification using anti-Hsp72 antibodies. Stromal cell-derived factor 2 (SDF-2) was identified as an Hsp72 client protein unique to OCUM-2M/OXA cells. SDF-2 was overexpressed in OXA-resistant cells and SDF-2 silencing promoted the apoptotic effects of OXA. Furthermore, Hsp72 prevented SDF-2 degradation in a chaperone activity-dependent manner. Together, our data demonstrate that Hsp72 protected SDF-2 to avoid OXA-induced cell death. We propose that inhibition of SDF-2 may comprise a novel therapeutic strategy to counteract OXA-resistant cancers..
338. Akinobu Matsumoto, Alessandra Pasut, Masaki Matsumoto, Ryu Yamashita, jacqueline Fung, Emanuele Monteleone, Alan Saghatelian, Keiichi Nakayama, John Clohessy, Pier P. Pandolfi, mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide., Nature, 541, 7636, 228, 2017, Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide 'small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile..
339. Masaki Matsumoto, Fumiko Matsuzaki, Kiyotaka Oshikawa, Naoki Goshima, Masatoshi Mori, Yoshifumi Kawamura, Koji Ogawa, Eriko Fukuda, Hirokazu Nakatsumi, Tohru Natsume, Kazuhiko Fukui, Katsuhisa Horimoto, Takeshi Nagashima, Ryo Funayama, Keiko Nakayama, Keiichi Nakayama, A large-scale targeted proteomics assay resource based on an in vitro human proteome, Nature Methods, 10.1038/nmeth.4116, 14, 3, 251-258, 2017.02, Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements..
340. Kentaro Kawata, Atsushi Hatano, Katsuyuki Yugi, Takanori Sano, Masashi Fujii, Yoko Tomizawa, Toshiya Kokaji, Kaori Y. Tanaka, Shinsuke Uda, Y. Suzuki, Masaki Matsumoto, Keiichi Nakayama, Kaori Saitoh, Keiko Kato, Ayano Ueno, Maki Ohishi, Akiyoshi Hirayama, Tomoyoshi Soga, Shinya Kuroda, Trans-omic Analysis Reveals Selective Responses to Induced and Basal Insulin across Signaling, Transcriptional, and Metabolic Networks, iScience, 7, 212-229, 2018, The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions..
341. Yuichi Abe, Masanori Honsho, Ryota Itoh, Ryoko Kawaguchi, Masashi Fujitani, Kazushirou Fujiwara, Masaaki Hirokane, Takashi Matsuzaki, Keiko Nakayama, Ryohei Ohgi, Toshihiro Marutani, Keiichi Nakayama, Toshihide Yamashita, Yukio Fujiki, Peroxisome biogenesis deficiency attenuates the BDNF-TrkB pathway-mediated development of the cerebellum, Life Science Alliance, 10.26508/lsa.201800062, 1, 6, 2018.01, Peroxisome biogenesis disorders (PBDs) manifest as neurological deficits in the central nervous system, including neuronal migration defects and abnormal cerebellum development. However, the mechanisms underlying pathogenesis remain enigmatic. Here, to investigate how peroxisome deficiency causes neurological defects of PBDs, we established a new PBD model mouse defective in peroxisome assembly factor Pex14p, termed Pex14
Δ
C/ΔC mouse. Pex14
Δ
C/Δ
C
mouse manifests a severe symptom such as disorganization of cortical laminar structure and dies shortly after birth, although peroxisomal biogenesis and metabolism are partially defective. The Pex14
Δ
C/Δ
C
mouse also shows malformation of the cerebellum including the impaired dendritic development of Purkinje cells. Moreover, extracellular signal-regulated kinase and AKT signaling are attenuated in this mutant mouse by an elevated level of brain-derived neurotrophic factor (BDNF) together with the enhanced expression of TrkB-T1, a dominant-negative isoform of the BDNF receptor. Our results suggest that dysregulation of the BDNF-TrkB pathway, an essential signaling for cerebellar morphogenesis, gives rise to the pathogenesis of the cerebellum in PBDs..
342. Kelvin Hui, Yuta Katayama, Keiichi Nakayama, Jun Nomura, Takeshi Sakurai, Characterizing vulnerable brain areas and circuits in mouse models of autism
Towards understanding pathogenesis and new therapeutic approaches, Neuroscience and Biobehavioral Reviews, 10.1016/j.neubiorev.2018.08.001, 2018.01, Recent human genetics studies have identified many genetic variants that may be responsible for autism spectrum disorder (ASD). ASD mouse models with genetic modifications mimicking these rare genetic variants have provided invaluable mechanistic insights into the disruption of various biological processes and brain areas/circuitry affected in ASD patients. In this review, we begin by reviewing several mouse models for ASD-associated copy number variations (CNVs) to illustrate how they have been employed to establish causal links between their behavioral phenotypes and the affected genes. We then focus on studies using one of the principal behavioral abnormalities associated with ASD, social behavior, to identify the molecular and circuit-level deficits involved. Finally, we end by discussing other mouse models designed to probe how the disruption of specific biological processes such as autophagy and neurogenesis may contribute to ASD pathogenesis. By achieving a greater understanding of the pathophysiology and pathogenic mechanisms involved in ASD and related disorders, novel therapeutic strategies may be devised for ASD patients in the near future..
343. Junichi Matsumoto, Shingo Takada, Shintaro Kinugawa, Takaaki Furihata, Hideo Nambu, Naoya Kakutani, Masaya Tsuda, Arata Fukushima, Takashi Yokota, Shinya Tanaka, Hidehisa Takahashi, Masashi Watanabe, Shigetsugu Hatakeyama, Masaki Matsumoto, Keiichi Nakayama, Yutaro Otsuka, Hisataka Sabe, Hiroyuki Tsutsui, Toshihisa Anzai, Brain-derived neurotrophic factor improves limited exercise capacity in mice with heart failure, Circulation, 10.1161/CIRCULATIONAHA.118.035212, 138, 18, 2064-2066, 2018.01.
344. Hiroyuki Hosokawa, Jonas Ungerbäck, Xun Wang, Masaki Matsumoto, Keiichi Nakayama, Sarah M. Cohen, Tomoaki Tanaka, Ellen V. Rothenberg, Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding, Immunity, 10.1016/j.immuni.2018.04.024, 48, 6, 1119-1134.e7, 2018.06, Transcription factors normally regulate gene expression through their action at sites where they bind to DNA. However, the balance of activating and repressive functions that a transcription factor can mediate is not completely understood. Here, we showed that the transcription factor PU.1 regulated gene expression in early T cell development both by recruiting partner transcription factors to its own binding sites and by depleting them from the binding sites that they preferred when PU.1 was absent. The removal of partner factors Satb1 and Runx1 occurred primarily from sites where PU.1 itself did not bind. Genes linked to sites of partner factor “theft” were enriched for genes that PU.1 represses despite lack of binding, both in a model cell line system and in normal T cell development. Thus, system-level competitive recruitment dynamics permit PU.1 to affect gene expression both through its own target sites and through action at a distance. Transcription factors regulate target genes via sequence-specific DNA binding. They may collaborate when bound together, but are assumed to be independent at sites where they bind alone. Hosokawa, Ungerbäck et al. show that PU.1 broadly shifts the genome-wide site choice of Runx1 DNA binding, enabling PU.1 to repress some target genes at a distance..
345. Hirokazu Nakatsumi, Takeru Oka, Tsunaki Higa, Michiko Shirane, Keiichi Nakayama, Nuclear–cytoplasmic shuttling protein PP2AB56 contributes to mTORC1-dependent dephosphorylation of FOXK1, Genes to Cells, 10.1111/gtc.12597, 23, 7, 599-605, 2018.07, Mammalian target of rapamycin complex 1 (mTORC1) kinase is a master regulator of the cellular response to nutrition-related signals such as insulin and amino acids. mTORC1 is activated on the lysosomal membrane and induces phosphorylation of a variety of downstream molecules. We previously showed that activated mTORC1 induces protein phosphatase 2A (PP2A)-mediated dephosphorylation of the transcription factor forkhead box K1 (FOXK1). The mechanism underlying the signal transduction from the cytoplasmic mTORC1 to the nuclear FOXK1 has remained unclear, however, we now show that a nuclear–cytoplasmic transport system is necessary for the mTORC1-FOXK1 signal transduction. This reaction is mediated by a shuttling protein B56, which is a regulatory subunit of PP2A and plays an essential role in the mTORC1-dependent dephosphorylation of FOXK1. These results suggest that PP2AB56 phosphatase contributes to the signaling for mTORC1-dependent transcriptional regulation..
346. Shinya Watanabe, Hiroki Fujiyama, Takuya Takafuji, Kota Kayama, Masaki Matsumoto, Keiichi I. Nakayama, Kazumasa Yoshida, Nozomi Sugimoto, Masatoshi Fujita, GRWD1 regulates ribosomal protein L23 levels via the ubiquitin-proteasome system, Journal of Cell Science, 10.1242/jcs.213009, 131, 15, 2018.08, Glutamate-rich WD40 repeat-containing 1 (GRWD1) is a Cdt1- binding protein that promotes mini-chromosome maintenance (MCM) loading through its histone chaperone activity. GRWD1 acts as a tumor-promoting factor by downregulating p53 (also known as TP53) via the RPL11-MDM2-p53 axis. Here, we identified GRWD1- interacting proteins using a proteomics approach and showed that GRWD1 interacts with various proteins involved in transcription, translation, DNA replication and repair, chromatin organization, and ubiquitin-mediated proteolysis. We focused on the ribosomal protein ribosomal protein L23 (RPL23), which positively regulates nucleolar stress responses through MDM2 binding and inhibition, thereby functioning as a tumor suppressor. Overexpression of GRWD1 decreased RPL23 protein levels and stability; this effect was restored upon treatment with the proteasome inhibitor MG132. EDD (also known as UBR5), an E3 ubiquitin ligase that interacts with GRWD1, also downregulated RPL23, and the decrease was further enhanced by co-expression of GRWD1. Conversely, siRNA-mediated GRWD1 knockdown upregulated RPL23. Co-expression of GRWD1 and EDD promoted RPL23 ubiquitylation. These data suggest that GRWD1 acts together with EDD to negatively regulate RPL23 via the ubiquitinproteasome system. GRWD1 expression reversed the RPL23- mediated inhibition of anchorage-independent growth in cancer cells. Our data suggest that GRWD1-induced RPL23 proteolysis plays a role in downregulation of p53 and tumorigenesis..
347. Hiroyuki Hosokawa, Jonas Ungerbäck, Xun Wang, Masaki Matsumoto, Keiichi Nakayama, Sarah M. Cohen, Tomoaki Tanaka, Ellen V. Rothenberg, Erratum
Transcription Factor PU.1 Represses and Activates Gene Expression in Early T Cells by Redirecting Partner Transcription Factor Binding (Immunity (2018) 48(6) (1119–1134.e7), (S1074761318301936) (10.1016/j.immuni.2018.04.024)), Immunity, 10.1016/j.immuni.2018.09.019, 49, 4, 2018.10, (Immunity 48, 1119–1134.e1–e7; June 19, 2018) Main Text The Cas9-GFP retroviral vector used for these studies was in the pQCXIN backbone, not in pMIG as originally stated. This error was inadvertent, and the correct information is now provided in the updated STAR Methods. The authors apologize for the error..
348. Asuka Ito, Mostofa Jamal, Kiyoshi Ameno, Naoko Tanaka, Ayaka Takakura, Toshihiro Kawamoto, Kyoko Kitagawa, Keiichi Nakayama, Akiko Matsumoto, Takanori Miki, Hiroshi Kinoshita, Acetaldehyde administrationinduces salsolinol formation in vivo in the dorsal striatum of Aldh2-knockout and C57BL/6N mice, Neuroscience Letters, 10.1016/j.neulet.2018.07.032, 685, 50-54, 2018.10, Acetaldehyde (AcH) and salsolinol play important roles in the central effects of ethanol. This study aimed to investigate the effect of administration of AcH on dopamine (DA), DA-derived salsolinol and serotonin (5-HT) levels in the dorsal striatum of Aldh2-knockout (Aldh2-KO) and C57BL/6 N (WT) mice. Animals were treated with AcH (50, 100 and 200 mg/kg) intraperitoneally and dialysate levels of DA, 5-HT and salsolinol were determined using in vivo microdialysis coupled with HPLC-ECD. Salsolinol was first detected at 20 min after AcH administration, and reached its peak concentration (WT mice: 0.29 ± 0.22 pg/μl; Aldh2-KO mice: 0.63 ± 0.17 pg/μl) at 25 min in the 200 mg/kg AcH group, before decreasing rapidly and reaching zero at approximately 55–80 min. Treatment with 100 and 200 mg/kg AcH increased levels of salsolinol in both WT and Aldh2-KO mice, with 200 mg/kg AcH inducing a higher level of salsolinol in Aldh2-KO mice than in WT mice. Treatment with 50 mg/kg AcH produced a small increase in salsolinol levels in Aldh2-KO mice, whereas no elevation of salsolinol was detected in WT mice. The increase in salsolinol formation was found to occur a dose-dependent manner in both genotypes. Administration of AcH and the subsequent changes in salsolinol concentrations did not change DA or 5-HT levels in either genotype. Our study suggests that AcH dose-dependently increases the formation of salsolinol in the dorsal striatum of mice, which provides further support for the role of AcH in salsolinol formation in the animal brain..
349. Hiroyuki Hosokawa, Maile Romero-Wolf, Mary A. Yui, Jonas Ungerbäck, Maria L.G. Quiloan, Masaki Matsumoto, Keiichi Nakayama, Tomoaki Tanaka, Ellen V. Rothenberg, Bcl11b sets pro-T cell fate by site-specific cofactor recruitment and by repressing Id2 and Zbtb16, Nature Immunology, 10.1038/s41590-018-0238-4, 19, 12, 1427-1440, 2018.12, Multipotent progenitor cells confirm their T cell–lineage identity in the CD4

CD8

double-negative (DN) pro-T cell DN2 stages, when expression of the essential transcription factor Bcl11b begins. In vivo and in vitro stage-specific deletions globally identified Bcl11b-controlled target genes in pro-T cells. Proteomics analysis revealed that Bcl11b associated with multiple cofactors and that its direct action was needed to recruit those cofactors to selective target sites. Regions near functionally regulated target genes showed enrichment for those sites of Bcl11b-dependent recruitment of cofactors, and deletion of individual cofactors relieved the repression of many genes normally repressed by Bcl11b. Runx1 collaborated with Bcl11b most frequently for both activation and repression. In parallel, Bcl11b indirectly regulated a subset of target genes by a gene network circuit via the transcription inhibitor Id2 (encoded by Id2) and transcription factor PLZF (encoded by Zbtb16); Id2 and Zbtb16 were directly repressed by Bcl11b, and Id2 and PLZF controlled distinct alternative programs. Thus, our study defines the molecular basis of direct and indirect Bcl11b actions that promote T cell identity and block alternative potentials..
350. Kentaro Kawata, Katsuyuki Yugi, Atsushi Hatano, Toshiya Kokaji, Yoko Tomizawa, Masashi Fujii, Shinsuke Uda, Hiroyuki Kubota, Masaki Matsumoto, Keiichi Nakayama, Shinya Kuroda, Reconstruction of global regulatory network from signaling to cellular functions using phosphoproteomic data, Genes to Cells, 10.1111/gtc.12655, 24, 1, 82-93, 2019.01, Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase–substrate relationships (KSRs). We used phosphoproteomic data from insulin-stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over-represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k-Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data..
351. Hideyuki Shimizu, Shoichiro Takeishi, Hirokazu Nakatsumi, Keiichi Nakayama, Prevention of cancer dormancy by Fbxw7 ablation eradicates disseminated tumor cells, JCI insight, 10.1172/jci.insight.125138, 4, 4, 2019.02, Dormant cancer cells known as disseminated tumor cells (DTCs) are often present in bone marrow of breast cancer patients. These DTCs are thought to be responsible for the incurable recurrence of breast cancer. The mechanism underlying the long-term maintenance of DTCs remains unclear, however. Here, we show that Fbxw7 is essential for the maintenance of breast cancer dormancy. Genetic ablation of Fbxw7 in breast cancer cells disrupted the quiescence of DTCs, rendering them proliferative, in mouse xenograft and allograft models. Fbxw7-deficient DTCs were significantly depleted by treatment with paclitaxel, suggesting that cell proliferation induced by Fbxw7 ablation sensitized DTCs to chemotherapy. The combination of Fbxw7 ablation and chemotherapy reduced the number of DTCs even when applied after tumor cell dissemination. Mice injected with Fbxw7-deficient cancer cells survived longer after tumor resection and subsequent chemotherapy than did those injected with wild-type cells. Furthermore, database analysis revealed that breast cancer patients whose tumors expressed FBXW7 at a high level had a poorer prognosis than did those with a low FBXW7 expression level. Our results suggest that a wake-up strategy for DTCs based on Fbxw7 inhibition might be of value in combination with conventional chemotherapy for the treatment of breast cancer..
352. Yoshiharu Muto, Toshiro Moroishi, Kazuya Ichihara, Masaaki Nishiyama, Hideyuki Shimizu, Hidetoshi Eguchi, Kyoji Moriya, Kazuhiko Koike, Koshi Mimori, Masaki Mori, Yuta Katayama, Keiichi Nakayama, Disruption of FBXL5-mediated cellular iron homeostasis promotes liver carcinogenesis, Journal of Experimental Medicine, 10.1084/jem.20180900, 216, 4, 950-965, 2019.04, Hepatic iron overload is a risk factor for progression of hepatocellular carcinoma (HCC), although the molecular mechanisms underlying this association have remained unclear. We now show that the iron-sensing ubiquitin ligase FBXL5 is a previously unrecognized oncosuppressor in liver carcinogenesis in mice. Hepatocellular iron overload elicited by FBXL5 ablation gave rise to oxidative stress, tissue damage, inflammation, and compensatory proliferation of hepatocytes and to consequent promotion of liver carcinogenesis induced by exposure to a chemical carcinogen. The tumor-promoting outcome of FBXL5 deficiency in the liver was also found to be effective in a model of virus-induced HCC. FBXL5-deficient mice thus constitute the first genetically engineered mouse model of liver carcinogenesis promoted by iron overload. In addition, dysregulation of FBXL5-mediated cellular iron homeostasis was found to be associated with poor prognosis in human HCC, suggesting that FBXL5 plays a key role in defense against hepatocarcinogenesis..
353. Yutaka Hashimoto, Michiko Shirane, Keiichi Nakayama, TMEM55B contributes to lysosomal homeostasis and amino acid-induced mTORC1 activation, Genes to Cells, 10.1111/gtc.12583, 2018.01, Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) responds to growth factors and nutrient availability. Amino acids induce the recruitment of mTORC1 to the lysosomal membrane and its consequent activation, but the molecular mechanism of such activation has remained unclear. We have now examined the role of TMEM55B, a lysosomal protein of unknown molecular function, in this process on the basis of the results of proteomics and immunofluorescence analyses showing that TMEM55B interacts with many proteins that participate in mTORC1 activation including components of the vacuolar-type proton ATPase (V-ATPase) and Ragulator complexes at the lysosomal membrane. The amino acid-induced phosphorylation of the mTORC1 substrates S6K and 4E-BP was attenuated in TMEM55B-depleted cells compared with control cells. Depletion of TMEM55B was also found to evoke lysosomal stress as showed by translocation of the transcription factor TFEB to the nucleus. Furthermore, recruitment of the V1 domain subcomplex of V-ATPase to lipid rafts was abrogated in TMEM55B-depleted cells. Collectively, our results suggest that TMEM55B contributes to assembly of the V-ATPase complex in lipid rafts of the lysosomal membrane and to subsequent activation of mTORC1..
354. Akinobu Matsumoto, Keiichi Nakayama, Hidden peptides encoded by putative noncoding RNAs, Cell Structure and Function, 10.1247/csf.18005, 43, 1, 75-83, 2018.01, Although the definition of a noncoding RNA (ncRNA) is an RNA molecule that does not encode a protein, recent evidence has revealed that some ncRNAs are indeed translated to give rise to small polypeptides (usually containing fewer than 100 amino acids). Despite their small size, however, these peptides are often biologically relevant in that they are required for a variety of cellular processes. In this review, we summarize the production and functions of peptides that have been recently identified as translation products of putative ncRNAs..
355. Shogo Nakayama, Kanae Yumimoto, Atsuki Kawamura, Keiichi Nakayama, Degradation of the endoplasmic reticulum–anchored transcription factor MyRF by the ubiquitin ligase SCFFbxw7 in a manner dependent on the kinase GSK-3, Journal of Biological Chemistry, 10.1074/jbc.RA117.000741, 293, 15, 5705-5714, 2018.01, The ubiquitin-proteasome system regulates the abundance of many cellular proteins by mediating their targeted degradation. We previously developed a method— differential proteomics–based identification of ubiquitylation substrates (DiPIUS)—for the comprehensive identification of substrates for a given F-box protein subunit of SCF-type ubiquitin ligases. We have now applied DiPIUS to the F-box protein Fbxw7 in three cell lines (mHepa, Neuro2A, and C2C12) and thereby identified myelin regulatory factor (MyRF), an endoplasmic reticulum–anchored transcription factor that is essential for myelination of nerves in the central nervous system, as a candidate substrate of Fbxw7 specifically in mHepa cells. Co-immunoprecipitation analysis confirmed that the NH2-terminal cytoplasmic domain of MyRF interacted with Fbxw7 in these cells. Furthermore, an in vitro ubiquitylation assay revealed that MyRF undergoes polyubiqui-tylation in the presence of purified recombinant SCFFbxw7. In addition, the stability of MyRF in mHepa cells was increased by mutation of a putative phosphodegron sequence or by exposure of the cells to an inhibitor of glycogen synthase kinase-3 (GSK-3). We found that MyRF mRNA is not restricted to the central nervous system but is instead distributed widely among mouse tissues. Furthermore, with the use of RNA sequencing in mHepa cells overexpressing or depleted of MyRF, we identified many novel potential target genes of MyRF. Our results thus suggest that Fbxw7 controls the transcription of MyRF target genes in various tissues through regulation of MyRF protein stability in a manner dependent on MyRF phosphorylation by GSK-3..
356. Masashi Mikamo, Kyoko Kitagawa, Satoshi Sakai, Chiharu Uchida, Tatsuya Ohhata, Koji Nishimoto, Hiroyuki Niida, Sayuri Suzuki, Keiichi Nakayama, Naoki Inui, Takafumi Suda, Masatoshi Kitagawa, Inhibiting Skp2 E3 ligase suppresses bleomycin-induced pulmonary fibrosis, International Journal of Molecular Sciences, 10.3390/ijms19020474, 19, 2, 2018.02, Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and no curative therapies. SCF-Skp2 E3 ligase is a target for cancer therapy, but there have been no reports about Skp2 as a target for IPF. Here we demonstrate that Skp2 is a promising therapeutic target for IPF. We examined whether disrupting Skp2 suppressed pulmonary fibrosis in a bleomycin (BLM)-induced mouse model and found that pulmonary fibrosis was significantly suppressed in Skp2-deficient mice compared with controls. The pulmonary accumulation of fibrotic markers such as collagen type 1 and fibronectin in BLM-infused mice was decreased in Skp2-deficient mice. Moreover, the number of bronchoalveolar lavage fluid cells accompanied with pulmonary fibrosis was significantly diminished. Levels of the Skp2 target p27 were significantly decreased by BLM-administration in wild-type mice, but recovered in Skp2−/− mice. In vimentin-positive mesenchymal fibroblasts, the decrease of p27-positive cells and increase of Ki67-positive cells by BLM-administration was suppressed by Skp2-deficency. As these results suggested that inhibiting Skp2 might be effective for BLM-induced pulmonary fibrosis, we next performed a treatment experiment using the Skp2 inhibitor SZL-P1-41. As expected, BLM-induced pulmonary fibrosis was significantly inhibited by SZL-P1-41. Moreover, p27 levels were increased by the SZL-P1-41 treatment, suggesting p27 may be an important Skp2 target for BLM-induced pulmonary fibrosis. Our study suggests that Skp2 is a potential molecular target for human pulmonary fibrosis including IPF..
357. Hideo Hagihara, Vibeke S. Catts, Yuta Katayama, Hirotaka Shoji, Tsuyoshi Takagi, Freesia L. Huang, Akito Nakao, Yasuo Mori, Kuo Ping Huang, Shunsuke Ishii, Isabella A. Graef, Keiichi Nakayama, Cynthia Shannon Weickert, Tsuyoshi Miyakawa, Decreased Brain pH as a Shared Endophenotype of Psychiatric Disorders, Neuropsychopharmacology, 10.1038/npp.2017.167, 43, 3, 459-468, 2018.02, Although the brains of patients with schizophrenia and bipolar disorder exhibit decreased brain pH relative to those of healthy controls upon postmortem examination, it remains controversial whether this finding reflects a primary feature of the diseases or is a result of confounding factors such as medication and agonal state. To date, systematic investigation of brain pH has not been undertaken using animal models that can be studied without confounds inherent in human studies. In the present study, we first reevaluated the pH of the postmortem brains of patients with schizophrenia and bipolar disorder by conducting a meta-analysis of existing data sets from 10 studies. We then measured pH, lactate levels, and related metabolite levels in brain homogenates from five neurodevelopmental mouse models of psychiatric disorders, including schizophrenia, bipolar disorder, and autism spectrum disorder. All mice were drug naive with the same agonal state, postmortem interval, and age within each strain. Our meta-analysis revealed that brain pH was significantly lower in patients with schizophrenia and bipolar disorder than in control participants, even when a few potential confounding factors (postmortem interval, age, and history of antipsychotic use) were considered. In animal experiments, we observed significantly lower pH and higher lactate levels in the brains of model mice relative to controls, as well as a significant negative correlation between pH and lactate levels. Our findings suggest that lower pH associated with increased lactate levels is not a mere artifact, but rather implicated in the underlying pathophysiology of schizophrenia and bipolar disorder..
358. Mami Morita, Taku Sato, Miyuki Nomura, Yoshimi Sakamoto, Yui Inoue, Ryota Tanaka, Shigemi Ito, Koreyuki Kurosawa, Kazunori Yamaguchi, Yuki Sugiura, Hiroshi Takizaki, Yoji Yamashita, Ryuichi Katakura, Ikuro Sato, Masaaki Kawai, Yoshinori Okada, Hitomi Watanabe, Gen Kondoh, Shoko Matsumoto, Ayako Kishimoto, Miki Obata, Masaki Matsumoto, Tatsuro Fukuhara, Hozumi Motohashi, Makoto Suematsu, Masaaki Komatsu, Keiichi Nakayama, Toshio Watanabe, Tomoyoshi Soga, Hiroshi Shima, Makoto Maemondo, Nobuhiro Tanuma, PKM1 Confers Metabolic Advantages and Promotes Cell-Autonomous Tumor Cell Growth, Cancer Cell, 10.1016/j.ccell.2018.02.004, 33, 3, 355-367.e7, 2018.03, Expression of PKM2, which diverts glucose-derived carbon from catabolic to biosynthetic pathways, is a hallmark of cancer. However, PKM2 function in tumorigenesis remains controversial. Here, we show that, when expressed rather than PKM2, the PKM isoform PKM1 exhibits a tumor-promoting function in KRASG12D-induced or carcinogen-initiated mouse models or in some human cancers. Analysis of Pkm mutant mouse lines expressing specific PKM isoforms established that PKM1 boosts tumor growth cell intrinsically. PKM1 activated glucose catabolism and stimulated autophagy/mitophagy, favoring malignancy. Importantly, we observed that pulmonary neuroendocrine tumors (NETs), including small-cell lung cancer (SCLC), express PKM1, and that PKM1 expression is required for SCLC cell proliferation. Our findings provide a rationale for targeting PKM1 therapeutically in certain cancer subtypes, including pulmonary NETs. The relative importance of PKM isoforms in tumor growth has been controversial. Morita et al. show that PKM1 promotes the growth of multiple tumor models using mouse lines expressing PKM1 or PKM2 from the endogenous Pkm locus. PKM1 is expressed in human SCLC, and it is important for SCLC cell proliferation..
359. Eri Sakai, Mizuho Nakayama, Hiroko Oshima, Yuta Kouyama, Atsushi Niida, Satoshi Fujii, Atsushi Ochiai, Keiichi Nakayama, Koshi Mimori, Yutaka Suzuki, Chang Pyo Hong, Chan Young Ock, Seong Jin Kim, Masanobu Oshima, Combined mutation of Apc, Kras, and Tgfbr2 effectively drives metastasis of intestinal cancer, Cancer Research, 10.1158/0008-5472.CAN-17-3303, 78, 5, 1334-1346, 2018.03, Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations (Apc, Kras, Tgfbr2, Trp53, Fbxw7) in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. Apcδ716 mutation caused intestinal adenomas and combination with Trp53R270 mutation or Tgfbr2 deletion induced submucosal invasion. The addition of KrasG12D mutation yielded epithelial-mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of Apcδ716 with KrasG12D and Fbxw7 mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that KrasG12D was critical for liver metastasis following splenic transplantation, when this mutation was combined with either Apcδ716 plus Trp53R270H or Tgfbr2 deletion, with the highest incidence of metastasis displayed by tumors with a Apcδ716 KrasG12D Tgfbr2-/- genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upre-gulated genes in Apcδ716 KrasG12D Tgfbr2-/- tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies..
360. Ryoichi Matsunuma, Hiroyuki Niida, Tatsuya Ohhata, Kyoko Kitagawa, Satoshi Sakai, Chiharu Uchida, Bunsyo Shiotani, Masaki Matsumoto, Keiichi Nakayama, Hiroyuki Ogura, Norihiko Shiiya, Masatoshi Kitagawa, Correction
"UV damage-induced phosphorylation of HBO1 triggers CRL4DDB2-mediated degradation to regulate cell proliferation" [Molecular and Cellular Biology 36, 3, (2016) (394-406)] doi 10.1128/MCB.00809-15, Molecular and Cellular Biology, 10.1128/MCB.00572-17, 38, 7, 2018.04.
361. Yasuyuki Kita, Yuta Katayama, Taichi Shiraishi, Takeru Oka, Tetsuya Sato, Mikita Suyama, Yasuyuki Ohkawa, Keishi Miyata, Yuichi Oike, Michiko Shirane, Masaaki Nishiyama, Keiichi Nakayama, The Autism-Related Protein CHD8 Cooperates with C/EBPβ to Regulate Adipogenesis, Cell Reports, 10.1016/j.celrep.2018.04.050, 23, 7, 1988-2000, 2018.05, The gene encoding the chromatin remodeler CHD8 is the most frequently mutated gene in individuals with autism spectrum disorder (ASD). Heterozygous mutations in CHD8 give rise to ASD that is often accompanied by macrocephaly, gastrointestinal complaints, and slender habitus. Whereas most phenotypes of CHD8 haploinsufficiency likely result from delayed neurodevelopment, the mechanism underlying slender habitus has remained unknown. Here, we show that CHD8 interacts with CCAAT/enhancer-binding protein β (C/EBPβ) and promotes its transactivation activity during adipocyte differentiation. Adipogenesis was impaired in Chd8-deleted preadipocytes, with the upregulation of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), two master regulators of this process, being attenuated in mutant cells. Furthermore, mice with CHD8 ablation in white preadipocytes had a markedly reduced white adipose tissue mass. Our findings reveal a mode of C/EBPβ regulation by CHD8 during adipogenesis, with CHD8 deficiency resulting in a defect in the development of white adipose tissue. Kita et al. show that autism-related protein CHD8 is essential for adipogenesis and the development of white adipose tissue. Moreover, they demonstrate that CHD8 cooperates with C/EBPβ to regulate transactivation of the genes for C/EBPα and PPARγ during adipogenesis..
362. Masaki Matsumoto, Keiichi Nakayama, The promise of targeted proteomics for quantitative network biology, Current Opinion in Biotechnology, 10.1016/j.copbio.2018.02.014, 54, 88-97, 2018.12, Proteomics is a powerful tool for obtaining information on a large number of proteins with regard to their expression levels, interactions with other molecules, and posttranslational modifications. Whereas nontargeted, discovery proteomics uncovers differences in the proteomic landscape under different conditions, targeted proteomics has been developed to overcome the limitations of this approach with regard to quantitation. In addition to technical advances in instruments and informatics tools, the advent of the synthetic proteome composed of synthetic peptides or recombinant proteins has advanced the adoption of targeted proteomics across a wide range of research fields. Targeted proteomics can now be applied to measurement of the dynamics of any proteins of interest under a variety of conditions as well as to estimation of the absolute abundance or stoichiometry of proteins in a given network. Multiplexed targeted proteomics assays of high reproducibility and accuracy can provide insight at the quantitative level into entire networks that govern biological phenomena or diseases. Such assays will establish a new paradigm for data-driven science..
363. Yoshiharu Muto, Masaaki Nishiyama, Akihiro Nita, Toshiro Moroishi, Keiichi Nakayama, Essential role of FBXL5-mediated cellular iron homeostasis in maintenance of hematopoietic stem cells, Nature Communications, 10.1038/ncomms16114, 8, 2017.07, Hematopoietic stem cells (HSCs) are maintained in a hypoxic niche to limit oxidative stress. Although iron elicits oxidative stress, the importance of iron homeostasis in HSCs has been unknown. Here we show that iron regulation by the F-box protein FBXL5 is required for HSC self-renewal. Conditional deletion of Fbxl5 in mouse HSCs results in cellular iron overload and a reduced cell number. Bone marrow transplantation reveals that FBXL5-deficient HSCs are unable to reconstitute the hematopoietic system of irradiated recipients as a result of stem cell exhaustion. Transcriptomic analysis shows abnormal activation of oxidative stress responses and the cell cycle in FBXL5-deficient mouse HSCs as well as downregulation of FBXL5 expression in HSCs of patients with myelodysplastic syndrome. Suppression of iron regulatory protein 2 (IRP2) accumulation in FBXL5-deficient mouse HSCs restores stem cell function, implicating IRP2 as a potential therapeutic target for human hematopoietic diseases associated with FBXL5 downregulation..
364. Hirokazu Nakatsumi, Masaki Matsumoto, Keiichi Nakayama, Noncanonical Pathway for Regulation of CCL2 Expression by an mTORC1-FOXK1 Axis Promotes Recruitment of Tumor-Associated Macrophages, Cell Reports, 10.1016/j.celrep.2017.11.014, 21, 9, 2471-2486, 2017.11, C-C chemokine ligand 2 (CCL2) plays pivotal roles in tumor formation, progression, and metastasis. Although CCL2 expression has been found to be dependent on the nuclear factor (NF)-κB signaling pathway, the regulation of CCL2 production in tumor cells has remained unclear. We have identified a noncanonical pathway for regulation of CCL2 production that is mediated by mammalian target of rapamycin complex 1 (mTORC1) but independent of NF-κB. Multiple phosphoproteomics approaches identified the transcription factor forkhead box K1 (FOXK1) as a downstream target of mTORC1. Activation of mTORC1 induces dephosphorylation of FOXK1, resulting in transactivation of the CCL2 gene. Inhibition of the mTORC1-FOXK1 axis attenuated insulin-induced CCL2 production as well as the accumulation of tumor-associated monocytes-macrophages and tumor progression in mice. Our results suggest that FOXK1 directly links mTORC1 signaling and CCL2 expression in a manner independent of NF-κB and that CCL2 produced by this pathway contributes to tumor progression. Nakatsumi et al. show that mTORC1 regulates CCL2 expression in a manner independent of NF-κB signaling by dephosphorylating the transcription factor FOXK1. Moreover, they demonstrate that hyperactivation of mTORC1 results in attraction of M2-type tumor-associated macrophages and promotes tumor growth in vivo via the mTORC1-FOXK1-CCL2 pathway..
365. Hidefumi Fukushima, Kouhei Shimizu, Asami Watahiki, Seira Hoshikawa, Tomoki Kosho, Daiju Oba, Seiji Sakano, Makiko Arakaki, Aya Yamada, Katsuyuki Nagashima, Koji Okabe, Satoshi Fukumoto, Eijiro Jimi, Anna Bigas, Keiichi Nakayama, Keiko Nakayama, Yoko Aoki, Wenyi Wei, Hiroyuki Inuzuka, NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis, Molecular Cell, 10.1016/j.molcel.2017.10.018, 68, 4, 645-658, 2017.11, Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS. Fukushima et al. demonstrated that the sustained osteoclast activity in Hajdu-Cheney syndrome (HCS) is largely due to elevated protein abundance of the C terminus truncating NOTCH2 mutant that escapes FBW7-mediated ubiquitination and proteolysis, suggesting that the FBW7/NOTCH2 signaling pathway is a potential therapeutic target for osteolytic bone disorders, including HCS..
366. Ryo Yonehara, Shigeyuki Nada, Tomokazu Nakai, Masahiro Nakai, Ayaka Kitamura, Akira Ogawa, Hirokazu Nakatsumi, Keiichi Nakayama, Songling Li, Daron M. Standley, Eiki Yamashita, Atsushi Nakagawa, Masato Okada, Structural basis for the assembly of the Ragulator-Rag GTPase complex, Nature Communications, 10.1038/s41467-017-01762-3, 8, 1, 2017.12, The mechanistic target of rapamycin complex 1 (mTORC1) plays a central role in regulating cell growth and metabolism by responding to cellular nutrient conditions. The activity of mTORC1 is controlled by Rag GTPases, which are anchored to lysosomes via Ragulator, a pentameric protein complex consisting of membrane-anchored p18/LAMTOR1 and two roadblock heterodimers. Here we report the crystal structure of Ragulator in complex with the roadblock domains of RagA-C, which helps to elucidate the molecular basis for the regulation of Rag GTPases. In the structure, p18 wraps around the three pairs of roadblock heterodimers to tandemly assemble them onto lysosomes. Cellular and in vitro analyses further demonstrate that p18 is required for Ragulator-Rag GTPase assembly and amino acid-dependent activation of mTORC1. These results establish p18 as a critical organizing scaffold for the Ragulator-Rag GTPase complex, which may provide a platform for nutrient sensing on lysosomes..
367. Hiroaki Yaguchi, Ichiro Yabe, Hidehisa Takahashi, Masashi Watanabe, Taichi Nomura, Takahiro Kano, Masaki Matsumoto, Keiichi Nakayama, Masahiko Watanabe, Shigetsugu Hatakeyama, Sez6l2 regulates phosphorylation of ADD and neuritogenesis, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.10.047, 494, 1-2, 234-241, 2017.12, Increasing evidence shows that immune-mediated mechanisms may contribute to the pathogenesis of central nervous system disorders including cerebellar ataxias, as indicated by the aberrant production of neuronal surface antibodies. We previously reported a patient with cerebellar ataxia associated with production of a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2). Sez6l2 is a type 1 membrane protein that is highly expressed in the hippocampus and cerebellar cortex and mice lacking Sez6l2 protein family members develop ataxia. Here we used a proteomics-based approach to show that serum derived from this patient recognizes the extracellular domain of Sez6l2 and that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1). Our results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD. Furthermore, Sez6l2 overexpression upregulates ADD phosphorylation, whereas siRNA-mediated downregulation of Sez612 prevents ADD phosphorylation, suggesting that Sez6l2 modulates AMPA-ADD signal transduction..
368. Keiichi Nakayama, Robotic crowd biology with Maholo LabDroids, 2017.07.
369. Keiichi Nakayama, Hippo signaling suppresses cell ploidy and tumorigenesis through Skp2, CANCER CELL, 10.1016/j.ccell.2017.04.004, 31, 5, 669-+, 2017.05.
370. Keiichi Nakayama, FBXL5 inactivation in mouse brain induces aberrant proliferation of neural stem progenitor cells, MOLECULAR AND CELLULAR BIOLOGY, 10.1128/MCB.00470-16, 37, 8, 2017.04.
371. Keiichi Nakayama, A large-scale targeted proteomics assay resource based on an in vitro human proteome, NATURE METHODS, 10.1038/NMETH.4116, 14, 3, 251-+, 2017.03.
372. Keiichi Nakayama, GRWD1 negatively regulates p53 via the RPL11-MDM2 pathway and promotes tumorigenesis, EMBO REPORTS, 10.15252/embr.201642444, 18, 1, 123-137, 2017.01.
373. Keiichi Nakayama, mTORC1 and muscle regeneration are regulated by the LINC00961-encoded SPAR polypeptide, NATURE, 10.1038/nature21034, 541, 7636, 228-+, 2017.01.
374. Keiichi Nakayama, SRRM4-dependent neuron-specific alternative splicing of protrudin transcripts regulates neurite outgrowth, SCIENTIFIC REPORTS, 10.1038/srep41130, 7, 2017.01.
375. Keiichi Nakayama, The SCF beta-TRCP E3 ubiquitin ligase complex targets Lipin1 for ubiquitination and degradation to promote hepatic lipogenesis, SCIENCE SIGNALING, 10.1126/scisignal.aah4117, 10, 460, 2017.01.
376. Keiichi Nakayama, The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, 10.1016/j.yjmcc.2016.09.013, 100, 43-53, 2016.11.
377. Keiichi Nakayama, Phosphoproteomics analyses show subnetwork systems in T-cell receptor signaling, GENES TO CELLS, 10.1111/gtc.12406, 21, 10, 1095-1112, 2016.10.
378. Keiichi Nakayama, FBXW7 is involved in the acquisition of the malignant phenotype in epithelial ovarian tumors, CANCER SCIENCE, 10.1111/cas.13026, 107, 10, 1399-1405, 2016.10.
379. Keiichi Nakayama, Role of apolipoprotein B100 and oxidized low-density lipoprotein in the monocyte tissue factor induction mediated by anti-2 glycoprotein I antibodies, LUPUS, 10.1177/0961203316638165, 25, 12, 1288-1298, 2016.10.
380. Keiichi Nakayama, CHD8 haploinsufficiency results in autistic-like phenotypes in mice, NATURE, 10.1038/nature19357, 537, 7622, 675-+, 2016.09.
381. Keiichi Nakayama, p57(Kip2) knock-in mouse reveals CDK-independent contribution in the development of Beckwith-Wiedemann syndrome, JOURNAL OF PATHOLOGY, 10.1002/path.4721, 239, 3, 250-261, 2016.07.
382. Keiichi Nakayama, FBXL12 regulates T-cell differentiation in a cell-autonomous manner, STEM CELLS, 10.1111/gtc.12360, 21, 5, 517-524, 2016.05.
383. Keiichi Nakayama, Akt1-mediated Gata3 phosphorylation controls the repression of IFN gamma in memory-type Th2 cells, NATURE COMMUNICATIONS, 10.1038/ncomms11289, 7, 2016.04.
384. Keiichi Nakayama, UV damage-induced phosphorylation of HBO1 triggers CRL4(DDB2)-mediated degradation to regulate cell proliferation, MOLECULAR AND CELLULAR BIOLOGY, 10.1128/MCB.00809-15, 36, 3, 394-406, 2016.02.
385. Keiichi Nakayama, FBXL12-mediated degradation of ALDH3 is essential for trophoblast differentiation during placental development, STEM CELLS, 10.1002/stem.2088, 33, 11, 3327-3340, 2015.11.
386. Keiichi Nakayama, Maternal TET3 is dispensable for embryonic development but is required for neonatal growth, SCIENTIFIC REPORTS, 10.1038/srep15876, 5, 15876, 2015.09.
387. Keiichi Nakayama, Role of the Atg9a gene in intrauterine growth and survival of fetal mice, REPRODUCTIVE BIOLOGY, 10.1016/j.repbio.2015.05.001, 15, 3, 131-138, 2015.09.
388. Keiichi Nakayama, Tissue-specific expression of histone H3 variants diversified after species separation, EPIGENETICS & CHROMATIN, 10.1186/s13072-015-0027-3, 8, 2015.09.
389. Keiichi Nakayama, K63-linked JARID1B ubiquitination by TRAF6 contributes to aberrant elevation of JARID1B in prostate cancer, CANCER RESEARCH, 10.1158/1538-7445.AM2015-90, 75, 2015.08.
390. Keiichi Nakayama, F-box protein FBXW7 inhibits-cancer metastasis in a non-cell-autonomous manner, JOURNAL OF CLINICAL INVESTIGATION, 10.1172/JCI78782, 125, 2, 621-635, 2015.02, 有名ながん抑制遺伝子産物であるFbxw7が、がん細胞内部だけでなく、がんニッチにおいてもがん転移を抑制することを明らかにした。Fbxw7はNotchを分解することによって、ケモカインCCL2の発現を負に制御しているが、骨髄細胞特異的Fbxw7欠損マウスでは間葉系幹細胞におけるCCL2が上昇しており、がんニッチにおいて単球/マクロファージの浸潤を促す結果、がん転移が促進する。この現象は、マウスだけではなくヒト乳がん患者における末梢血中のFbxw7 mRNA量と予後における相関があることからも、種間を超えて保存されていることが推測される。またマウスにおいてCCL2を抑制する既存薬プロパゲルマニウムを投与すると、がん転移が強力に阻害された。このことはヒトにおけるがん治療にプロパゲルマニウムががん転移抑制剤として利用できることを示唆するものである。.
391. Keiichi Nakayama, MDM2 Mediates Nonproteolytic Polyubiquitylation of the DEAD-Box RNA Helicase DDX24, MOLECULAR AND CELLULAR BIOLOGY, 10.1128/MCB.00320-14, 34, 17, 3321-3340, 2014.09.
392. Keiichi Nakayama, Role of the ANKMY2-FKBP38 Axis in Regulation of the Sonic Hedgehog (Shh) Signaling Pathway, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M114.558635, 289, 37, 25639-25654, 2014.09.
393. Keiichi Nakayama, HERC2 Targets the Iron Regulator FBXL5 for Degradation and Modulates Iron Metabolism, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M113.541490, 289, 23, 16430-16441, 2014.06.
394. Yutaka Hashimoto, Michiko Shirane, Fumiko Matsuzaki, Takafumi Ohnishi, Keiichi Nakayama, Protrudin Regulates Endoplasmic Reticulum Morphology and Function Associated with the Pathogenesis of Hereditary Spastic Paraplegia, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M113.528687, 289, 19, 12946-12961, 2014.05.
395. Keiichi Nakayama, Tyk2-Dependent Bystander Activation of Conventional and Nonconventional Th1 Cell Subsets Contributes to Innate Host Defense against Listeria monocytogenes Infection, JOURNAL OF IMMUNOLOGY, 10.4049/jimmunol.1303067, 192, 10, 4739-4747, 2014.05.
396. Matsumoto Akinobu, Shoichiro Takeishi, Keiichi Nakayama, p57 regulates T-cell development and prevents lymphomagenesis by balancing p53 activity and pre-TCR signaling, BLOOD, 10.1182/blood-2013-10-532390, 123, 22, 3429-3439, 2014.05, T cells are key components of the immune system, playing a central role in cell-mediated immunity. The sequential differentiation of T cells is associated with strict regulation of the cell cycle at each developmental stage. A balance between p53 activity and pre-T cell receptor (TCR) signaling regulates proliferation and differentiation decisions made by these cells. The relation between maintenance of this balance and the function of cell cycle regulators has remained largely unknown, however. We now show that mice with T cell-specific deficiency of the cyclin-dependent kinase inhibitor p57 manifest a differentiation block at the early stage of T cell maturation. Further genetic analysis showed that this defect is attributable to an imbalance between p53 activity and pre-TCR signaling caused by hyperactivation of the E2F-p53 pathway. Moreover, ablation of both p57 and p53 in T cells led to the development of aggressive thymic lymphomas with a reduced latency compared with that apparent for p53-deficient mice, whereas ablation of p57 alone did not confer susceptibility to this hematologic malignancy. Our results thus show that the p57-E2F-p53 axis plays a pivotal role in the proper development of T cells as well as in the prevention of lymphomagenesis..
397. Keiichi Nakayama, Skp2 suppresses apoptosis in Rb1-deficient tumours by limiting E2F1 activity, NATURE COMMUNICATIONS, 10.1038/ncomms4463, 5, 2014.03.
398. Takafumi Ohnishi, Michiko Shirane, Yutaka Hashimoto, Keiichi Nakayama, Identification and characterization of a neuron-specific isoform of protrudin, GENES TO CELLS, 10.1111/gtc.12109, 19, 2, 97-111, 2014.02.
399. Keiichi Nakayama, p27 is regulated independently of Skp2 in the absence of Cdk2, BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH, 10.1016/j.bbamcr.2013.11.005, 1843, 2, 436-445, 2014.02.
400. Keiichi Nakayama, Identification of anti-Sez612 antibody in a patient with cerebellar ataxia and retinopathy, JOURNAL OF NEUROLOGY, 10.1007/s00415-013-7134-5, 261, 1, 224-226, 2014.01.
401. Keiichi Nakayama, CDK inhibitors, p21(Cip1) and p27(Kip1), participate in cell cycle exit of mammalian cardiomyocytes, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2013.12.109, 443, 3, 1105-1109, 2014.01.
402. Keiichi Nakayama, Skp2 Deletion Unmasks a p27 Safeguard that Blocks Tumorigenesis in the Absence of pRb and p53 Tumor Suppressors, CANCER CELL, 10.1016/j.ccr.2013.09.021, 24, 5, 645-659, 2013.11.
403. Kanae Yumimoto, Tetsuya Muneoka, Tomohiro Tsuboi, Keiichi Nakayama, Substrate Binding Promotes Formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) Protein Complex, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M113.511303, 288, 45, 32766-32776, 2013.11.
404. Kanae Yumimoto, Masaki Matsumoto, Keiichi Nakayama, F-box and WD Repeat Domain-containing-7 (Fbxw7) Protein Targets Endoplasmic Reticulum-anchored Osteogenic and Chondrogenic Transcriptional Factors for Degradation, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M113.465179, 288, 40, 28488-28502, 2013.10.
405. Keiichi Nakayama, Phosphorylation of Noxo1 at threonine 341 regulates its interaction with Noxa1 and the superoxide-producing activity of Nox1, FEBS JOURNAL, 10.1111/febs.12489, 280, 20, 5145-5159, 2013.10.
406. Keiichi Nakayama, CELL CYCLE AND CANCER STEM CELLS: ABROGATION OF QUIESCENCE BY FBW7 ABLATION ELIMINATES LEUKEMIA STEM CELLS, EXPERIMENTAL HEMATOLOGY, 41, 8, S3-S3, 2013.08.
407. Keiichi Nakayama, Loss of Protein Kinase C-delta Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1371/journal.pone.0070815, 8, 8, 2013.08.
408. Keiichi Nakayama, Opposing functions of Fbxw7 in keratinocyte growth, differentiation and skin tumorigenesis mediated through negative regulation of c-Myc and Notch, ONCOGENE, 10.1038/onc.2012.213, 32, 15, 1921-1932, 2013.04.
409. Keiichi Nakayama, p57 controls adult neural stem cell quiescence and modulates the pace of lifelong neurogenesis, EMBO JOURNAL, 10.1038/emboj.2013.50, 32, 7, 970-981, 2013.04.
410. Linsey Reavie, Shannon M. Buckley, Evangelia Loizou, Shoichiro Takeishi, Beatriz Aranda-Orgilles, Delphine Ndiaye-Lobry, Omar Abdel-Wahab, Sherif Ibrahim, Keiichi Nakayama, Iannis Aifantis, Regulation of c-Myc Ubiquitination Controls Chronic Myelogenous Leukemia Initiation and Progression, CANCER CELL, 10.1016/j.ccr.2013.01.025, 23, 3, 362-375, 2013.03.
411. Shoichiro Takeishi, Matsumoto Akinobu, Ichiro Onoyama, Ichiro Naka, Atsushi Hirao, Keiichi Nakayama, Ablation of Fbxw7 Eliminates Leukemia-Initiating Cells by Preventing Quiescence, CANCER CELL, 10.1016/j.ccr.2013.01.026, 23, 3, 347-361, 2013.03, Imatinib eradicates dividing progenitor cells of chronic myeloid leukemia (CML) but does not effectively target nondividing leukemia-initiating cells (LICs); thus, the disease often relapse after its discontinuation. We now show that Fbxw7 plays a pivotal role in maintenance of quiescence in LICs of CML by reducing the level of c-Myc. Abrogation of quiescence in LICs by Fbxw7 ablation increased their sensitivity to imatinib, and the combination of Fbxw7 ablation with imatinib treatment resulted in a greater depletion of LICs than of normal hematopoietic stem cells in mice. Purging of LICs by targeting Fbxw7 to interrupt their quiescence and subsequent treatment with imatinib may thus provide the basis for a promising therapeutic approach to CML..
412. Arisa Hirano, Kanae Yumimoto, Ryosuke Tsunematsu, Masaki Matsumoto, Masaaki Oyama, Hiroko Kozuka-Hata, Tomoki Nakagawa, Darin Lanjakornsiripan, Keiichi Nakayama, Yoshitaka Fukada, FBXL21 Regulates Oscillation of the Circadian Clock through Ubiquitination and Stabilization of Cryptochromes, CELL, 10.1016/j.cell.2013.01.054, 152, 5, 1106-1118, 2013.02, In the mammalian circadian clockwork, CRY1 and CRY2 repressor proteins are regulated by posttranslational modifications for temporally coordinated transcription of clock genes. Previous studies revealed that FBXL3, an F-box-type E3 ligase, ubiquitinates CRYs and mediates their degradation. Here, we found that FBXL21 also ubiquitinates CRYs but counteracts FBXL3. Fbxl21(-/-) mice exhibited normal periodicity of wheel-running rhythms with compromised organization of daily activities, while an extremely long-period phenotype of Fbxl3(-/-) mice was attenuated in Fbxl3/Fbxl21 double-knockout mice. The double knockout destabilized the behavioral rhythms progressively and sometimes elicited arrhythmicity. Surprisingly, FBXL21 stabilized CRYs and antagonized the destabilizing action by FBXL3. Predominantly cytosolic distribution of FBXL21 contrasts with nuclear localization of FBXL3. These results emphasize the physiological importance of antagonizing actions between FBXL21 and FBXL3 on CRYs, and their combined actions at different subcellular locations stabilize oscillation of the circadian clock..
413. Shotaro Saita, Michiko Shirane, Keiichi Nakayama, Selective escape of proteins from the mitochondria during mitophagy, NATURE COMMUNICATIONS, 10.1038/ncomms2400, 4, 2013.01.
414. Yasutaka Okita, Keiichi Nakayama, UPS Delivers Pluripotency, CELL STEM CELL, 10.1016/j.stem.2012.11.009, 11, 6, 728-730, 2012.12.
415. Hidefumi Fukushima, Matsumoto Akinobu, Hiroyuki Inuzuka, Bo Zhai, Alan W. Lau, Lixin Wan, Daming Gao, Shavali Shaik, Min Yuan, Steven P. Gygi, Eijiro Jimi, John M. Asara, Keiko Nakayama, Wenyi Wei, Keiichi Nakayama, SCFFbw7 Modulates the NF kappa B Signaling Pathway by Targeting NF kappa B2 for Ubiquitination and Destruction, CELL REPORTS, 10.1016/j.celrep.2012.04.002, 1, 5, 434-443, 2012.05.
416. Chia-Hsin Chan, Chien-Feng Li, Wei-Lei Yang, Yuan Gao, Szu-Wei Lee, Zizhen Feng, Hsuan-Ying Huang, Leo G. Flores, Kelvin K.C. Tsai, Yiping Shao, John D. Hazle, Dihua Yu, Wenyi Wei, Dos Sarbassov, Mien-Chie Hung, Keiichi Nakayama, Hui-Kuan Lin, The Skp2-SCF E3 Ligase Regulates Akt Ubiquitination, Glycolysis, Herceptin Sensitivity, and Tumorigenesis, CELL, 10.1016/j.cell.2012.02.065, 149, 5, 1098-1111, 2012.05.
417. Moroishi, T., Nishiyama, M., Takeda, Y., Iwai, K., Nakayama, K.I., The FBXL5-IRP2 axis is integral to control of iron metabolism in vivo, Cell Metabolism, 14: 339-51, 2011.09, Iron-dependent degradation of iron-regulatory protein 2 (IRP2) is a key event for maintenance of an appropriate intracellular concentration of iron. Although FBXL5 (F-box and leucine-rich repeat protein 5) is thought to mediate this degradation, the role of FBXL5 in the control of iron homeostasis in vivo has been poorly understood. We have now found that mice deficient in FBXL5 died in utero associated with excessive iron accumulation. This embryonic mortality was prevented by additional ablation of IRP2, suggesting that impaired IRP2 degradation is primarily responsible for the death of Fbxl5–/– mice. We also found that liver-specific deletion of Fbxl5 resulted in deregulation of both hepatic and systemic iron homeostasis, leading to the development of steatohepatitis. The liver-specific mutant mice died with acute liver failure when fed a high iron diet. Our results thus uncover a major role for FBXL5 in ensuring an appropriate supply of iron to cells..
418. Matsumoto, A., Takeishi, S., Kanie, T., Susaki, E., Onoyama, I., Tateishi, Y., Nakayama, K., Nakayama, K.I., p57 is required for quiescence and maintenance of adult hematopoietic stem cells, Cell Stem Cell, 9: 262-71, 2011.09, Quiescence is required for maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in quiescence of HSCs, but loss of p21 or p27 in mice affects neither quiescence nor stemness of HSCs except under certain conditions. Although p57 is most abundant in the quiescent HSCs, its function in HSCs has remained uncharacterized. Here we show that the self-renewal capacity of p57-deficient HSCs was severely impaired, and the proportion of the cells in G0 phase was also reduced. Additional ablation of p21 on the p57-null background resulted in a further decrease in colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knock-in of the gene for p27 at the p57 locus. Our results thus suggest that, among Cip/Kip CDK inhibitors, p57 plays the predominant role in the quiescence and maintenance of adult HSCs..
419. Onoyama, I., Suzuki, A., Matsumoto, A., Tomita, K., Katagiri, H., Oike, Y., Nakayama, K., Nakayama, K.I., Fbxw7 regulates lipid metabolism and cell fate decisions in the mouse liver, J. Clin. Invest., 121, 342-354, 2011.01, E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box- and WD repeat domain-containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7-/- embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver..
420. Liu Z, Liu X, Nakayama KI, Nakayama K, Ye K., Protein kinase C-delta phosphorylates Ebp1 and prevents its proteolytic degradation, enhancing cell survival., J Neurochem., 100(5):1278-88., 2007.03.
421. Moller C, Karlberg M, Abrink M, Nakayama KI, Motoyama N, Nilsson G., Bcl-2 and Bcl-XL are indispensable for the late phase of mast cell development from mouse embryonic stem cells., Exp Hematol., 35(3):385-93., 2007.03.
422. Tu X, Joeng KS, Nakayama KI, Nakayama K, Rajagopal J, Carroll TJ, McMahon A., Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation., Dev Cell. , 12(1):113-27., 2007.01.
423. Uchida T, Iwashita N, Ohara-Imaizumi M, Ogihara T, Nagai S, Choi JB, Tamura Y, Tada N, Kawamori R, Nakayama KI, Nagamatsu S, Watada H., Protein kinase Cdelta plays a non-redundant role in insulin secretion in pancreatic beta cells., J Biol Chem., 282(4):2707-16., 2007.01.
424. Shukla A, Lounsbury KM, Barrett TF, Gell J, Rincon M, Butnor KJ, Taatjes DJ, Davis GS, Vacek P, Nakayama KI, Nakayama K, Steele C, Mossman BT., Asbestos-induced peribronchiolar cell proliferation and cytokine production are attenuated in lungs of protein kinase C-knockout mice., J Biol Chem., 170(1):140-51., 2007.01.
425. Sakai T, Sakaue H, Nakamura T, Okada M, Matsuki Y, Watanabe E, Hiramatsu R, Nakayama K, Nakayama KI, Kasuga M., Skp2 controls adipocyte proliferation during the development of obesity., J Biol Chem., 282(3):2038-46., 2007.01.
426. Yanagawa M, Tsukuba T, Nishioku T, Okamoto Y, Okamoto K, Takii R, Terada Y, Nakayama KI, Kadowaki T, Yamamoto K., Cathepsin E deficiency induces a novel form of lysosomal storage disorder showing the accumulation of lysosomal membrane sialoglycoproteins and the elevation of lysosomal pH in macrophages., J Biol Chem., 82(3):1851-62., 2007.01.
427. Itoh Y, Masuyama N, Nakayama K, Nakayama KI, Gotoh Y., The cyclin-dependent kinase inhibitors p57 and p27 regulate neuronal migration in the developing mouse neocortex., J Biol Chem., 282(1):390-6., 2007.01.
428. Shirane M, Nakayama KI., Protrudin induces neurite formation by directional membrane trafficking., Science., 314(5800):818-21., 2006.11.
429. Matsumoto A, Onoyama I, Nakayama KI., Expression of mouse Fbxw7 isoforms is regulated in a cell cycle- or p53-dependent manner., Biochem Biophys Res Commun., 3350(1):114-9., 2006.11.
430. Hara K, Nakayama KI, Nakayama K., Geminin is essential for the development of preimplantation mouse embryos., Genes Cells., 11(11):1281-93., 2006.11.
431. Tsunematsu R, Nishiyama M, Kotoshiba S, Saiga T, Kamura T, Nakayama KI., Fbxw8 is essential for Cul1-Cul7 complex formation and for placental development., Mol Cell Biol., 26(16):6157-69., 2006.10.
432. Fujii Y, Yada M, Nishiyama M, Kamura T, Takahashi H, Tsunematsu R, Susaki E, Nakagawa T, Matsumoto A, Nakayama KI., Fbxw7 contributes to tumor suppression by targeting multiple proteins for ubiquitin-dependent degradation., Cancer Sci., 97(8):729-36., 2006.10.
433. Parcellier A, Brunet M, Schmitt E, Col E, Didelot C, Hammann A, Nakayama K, Nakayama KI, Khochbin S, Solary E, Garrido C., HSP27 favors ubiquitination and proteasomal degradation of p27Kip1 and helps S-phase re-entry in stressed cells., FASEB J., 20(8):1179-81., 2006.06.
434. Yoshida K, Yamaguchi T, Shinagawa H, Taira N, Nakayama KI, Miki Y., Protein kinase C delta activates topoisomerase IIalpha to induce apoptotic cell death in response to DNA damage., Mol Cell Biol., 26(9):3414-31., 2006.06.
435. Fotovati A, Nakayama K, Nakayama KI., Impaired germ cell development due to compromised cell cycle progression in Skp2-deficient mice., Cell Div., 1:4., 2006.04.
436. Humphries MJ, Limesand KH, Schneider JC, Nakayama KI, Anderson SM, Reyland ME., Suppression of apoptosis in the protein kinase Cdelta null mouse in vivo., J Biol Chem., 281(14):9728-37., 2006.04.
437. Kase S, Yoshida K, Ohgami K, Shiratori K, Ohno S, Nakayama KI., phorylation of p27(KIP1) in the mitotic cells of the corneal epithelium., Curr Eye Res., 31(4):307-12., 2006.04.
438. Nishitani H, Sugimoto N, Roukos V, Nakanishi Y, Saijo M, Obuse C, Tsurimoto T, Nakayama KI, Nakayama K, Fujita M, Lygerou Z, Nishimoto T., Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis., EMBO J., 25(5):1126-36., 2006.03.
439. Kase S, Yoshida K, Ohgami K, Shiratori K, Suzuki Y, Nakayama KI, Ohno S., Expression of cdc2 and p27(KIP1) phosphorylation in mitotic cells of the human retinoblastoma., Int J Mol Med., 17(3):465-8., 2006.03.
440. Kase S, Yoshida K, Harada T, Harada C, Namekata K, Suzuki Y, Ohgami K, Shiratori K, Nakayama KI, Ohno S., Phosphorylation of extracellular signal-regulated kinase and p27(KIP1) after retinal detachment., Graefes Arch Clin Exp Ophthalmol., 244(3):352-8, 2006.03.
441. Shirane M, Nakayama KI., Inherent calcineurin inhibitor FKBP38 targets Bcl-2 to mitochondria and inhibits apoptosis., Nature Cell Biol., 10.1038/ncb894, 5, 1, 28-37, 5(1): 28-37, 2003.01.