Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Nobushige Nakajo Last modified date:2024.06.26

Lecturer / Graduate school of kyusyu university, fuculty of sciences / Department of Biology / Faculty of Sciences


Papers
1. Aiba Y, Kim J, Imamura A, Okumoto K, Nakajo N., Regulation of Myt1 kinase activity via its N-terminal region in Xenopus meiosis and mitosis., Cells Dev., doi: 10.1016/j.cdev.2021.203754., 2021.10.
2. Yoshitome S, Aiba Y, Yuge M, Furuno N, Watanabe M, Nakajo N., Involvement of Myt1 kinase in the G2 phase of the first cell cycle in Xenopus laevis., Biochem Biophys Res Commun., 2019.06.
3. Kenji Nishizawa, Kei Fujiwara, Masahiro Ikenaga, Nobushige Nakajo, Miho Yanagisawa, Daisuke Mizuno, Universal glass-forming behavior of in vitro and living cytoplasm, Scientific reports, 10.1038/s41598-017-14883-y, 7, 1, 2017.12, Physiological processes in cells are performed efficiently without getting jammed although cytoplasm is highly crowded with various macromolecules. Elucidating the physical machinery is challenging because the interior of a cell is so complex and driven far from equilibrium by metabolic activities. Here, we studied the mechanics of in vitro and living cytoplasm using the particle-tracking and manipulation technique. The molecular crowding effect on cytoplasmic mechanics was selectively studied by preparing simple in vitro models of cytoplasm from which both the metabolism and cytoskeletons were removed. We obtained direct evidence of the cytoplasmic glass transition; a dramatic increase in viscosity upon crowding quantitatively conformed to the super-Arrhenius formula, which is typical for fragile colloidal suspensions close to jamming. Furthermore, the glass-forming behaviors were found to be universally conserved in all the cytoplasm samples that originated from different species and developmental stages; they showed the same tendency for diverging at the macromolecule concentrations relevant for living cells. Notably, such fragile behavior disappeared in metabolically active living cells whose viscosity showed a genuine Arrhenius increase as in typical strong glass formers. Being actively driven by metabolism, the living cytoplasm forms glass that is fundamentally different from that of its non-living counterpart..
4. Kazuhiro Suzuki, Kosuke Sako, Kazuhiro Akiyama, Michitaka Isoda, Chiharu Senoo, Nobushige Nakajo, Noriyuki Sagata, Identification of non-Ser/Thr-Pro consensus motifs for Cdk1 and their roles in mitotic regulation of C2H2 zinc finger proteins and Ect2, Scientific reports, 10.1038/srep07929, 5, 2015.01, The cyclin B-dependent protein kinase Cdk1 is a master regulator of mitosis and phosphorylates numerous proteins on the minimal consensus motif Ser/Thr-Pro (S/T-P). At least in several proteins, however, not well-defined motifs lacking a Pro in the +1 position, referred herein to as non-S/T-P motifs, have been shown to be phosphorylated by Cdk1. Here we show that non-S/T-P motifs in fact form consensus sequences for Cdk1 and probably play roles in mitotic regulation of physiologically important proteins. First, we show, by in vitro kinase assays, that previously identified non-S/T-P motifs all harbour one or more C-terminal Arg/Lys residues essential for their phosphorylation by Cdk1. Second, using Arg/Lys-scanning oriented peptide libraries, we demonstrate that Cdk1 phosphorylates a minimal sequence S/T-X-X-R/K and more favorable sequences (P)-X-S/T-X-[R/K] 2-5 as its non-S/T-P consensus motifs. Third, on the basis of these results, we find that highly conserved linkers (typically, T-G-E-K-P) of C2H2 zinc finger proteins and a nuclear localization signal-containing sequence (matching P-X-S-X-[R/K] 5) of the cytokinesis regulator Ect2 are inhibitorily phosphorylated by Cdk1, well accounting for the known mitotic regulation and function of the respective proteins. We suggest that non-S/T-P Cdk1 consensus motifs identified here may function to regulate many other proteins during mitosis..
5. Takayuki Uozumi, Masayuki Hamakawa, Yu ki Deno, Nobushige Nakajo, Takaaki Hirotsu, Voltage-dependent anion channel (VDAC-1) is required for olfactory sensing in Caenorhabditis elegans, Genes to Cells, 10.1111/gtc.12269, 20, 10, 802-816, 2015.01, The Ras-MAP kinase signaling pathway plays important roles for the olfactory reception in olfactory neurons in Caenorhabditis elegans. However, given the absence of phosphorylation targets of MAPK in the olfactory neurons, the mechanism by which this pathway regulates olfactory function is unknown. Here, we used proteomic screening to identify the mitochondrial voltage-dependent anion channel VDAC-1 as a candidate target molecule of MAPK in the olfactory system of C. elegans. We found that Amphid Wing "C" (AWC) olfactory neuron-specific knockdown of vdac-1 caused severe defects in chemotaxis toward AWC-sensed odorants. We generated a new vdac-1 mutant using the CRISPR-Cas9 system, with this mutant also showing decreased chemotaxis toward odorants. This defect was rescued by AWC-specific expression of vdac-1, indicating that functions of VDAC-1 in AWC neurons are essential for normal olfactory reception in C. elegans. We observed that AWC-specific RNAi of vdac-1 reduced AWC calcium responses to odorant stimuli and caused a decrease in the quantity of mitochondria in the sensory cilia. Behavioral abnormalities in vdac-1 knockdown animals might therefore be due to reduction of AWC response, which might be caused by loss of mitochondria in the cilia. Here, we showed that the function of VDAC-1 is regulated by phosphorylation and identified Thr175 as the potential phosphorylation site of MAP kinase..
6. Kosuke Sako, Kazuhiro Suzuki, Michitaka Isoda, Satomi Yoshikai, Chiharu Senoo, Nobushige Nakajo, Munemichi Ohe, Noriyuki Sagata, Emi2 mediates meiotic MII arrest by competitively inhibiting the binding of Ube2S to the APC/C, Nature communications, 10.1038/ncomms4667, 5, 2014.04, In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Emi2, a direct inhibitor of the APC/C ubiquitin ligase. Two different ubiquitin-conjugating enzymes, UbcH10 and Ube2S, work with the APC/C to target APC/C substrates for degradation. However, their possible roles and regulations in unfertilized/fertilized eggs are not known. Here we use Xenopus egg extracts to show that both UbcH10 and Ube2S are required for rapid cyclin B degradation at fertilization, when APC/C binding of Ube2S, but not of UbcH10, increases several fold, coincidently with (SCFβ-TrCP-dependent) Emi2 degradation. Interestingly, before fertilization, Emi2 directly inhibits APC/C-Ube2S binding via the C-terminal tail, but on fertilization, its degradation allows the binding mediated by the Ube2S C-terminal tail. Significantly, Emi2 and Ube2S bind commonly to the APC/C catalytic subunit APC10 via their similar C-terminal tails. Thus, Emi2 competitively inhibits APC/C-Ube2S binding before fertilization, while its degradation on fertilization relieves the inhibition for APC/C activation..
7. Michitaka Isoda, Kosuke Sako, Kazuhiro Suzuki, Kazuaki Nishino, Nobushige Nakajo, Munemichi Ohe, Takanori Ezaki, Yoshinori Kanemori, Daigo Inoue, Hiroyuki Ueno, Noriyuki Sagata, Dynamic Regulation of Emi2 by Emi2-Bound Cdk1/Plk1/CK1 and PP2A-B56 in Meiotic Arrest of Xenopus Eggs, Developmental Cell, 10.1016/j.devcel.2011.06.029, 21, 3, 506-519, 2011.09, In vertebrates, unfertilized eggs are arrested at metaphase of meiosis II by Mos and Emi2, an inhibitor of the APC/C ubiquitin ligase. In Xenopus, Cdk1 phosphorylates Emi2 and both destabilizes and inactivates it, whereas Mos recruits PP2A phosphatase to antagonize the Cdk1 phosphorylation. However, how Cdk1 phosphorylation inhibits Emi2 is largely unknown. Here we show that multiple N-terminal Cdk1 phosphorylation motifs bind cyclin B1-Cdk1 itself, Plk1, and CK1δ/ε to inhibit Emi2. Plk1, after rebinding to other sites by self-priming phosphorylation, partially destabilizes Emi2. Cdk1 and CK1δ/ε sequentially phosphorylate the C-terminal APC/C-docking site, thereby cooperatively inhibiting Emi2 from binding the APC/C. In the presence of Mos, however, PP2A-B56β/ε bind to Emi2 and keep dephosphorylating it, particularly at the APC/C-docking site. Thus, Emi2 stability and activity are dynamically regulated by Emi2-bound multiple kinases and PP2A phosphatase. Our data also suggest a general role for Cdk1 substrate phosphorylation motifs in M phase regulation..
8. Nobushige Nakajo, Yu Ki Deno, Hiroyuki Ueno, Chihiro Kenmochi, Ken Shimuta, Noriyuki Sagata, Temporal and spatial expression patterns of Cdc25 phosphatase isoforms during early xenopus development, International Journal of Developmental Biology, 10.1387/ijdb.113287nn, 55, 6, 627-632, 2011.09, In early animal development, cell proliferation and differentiation are tightly linked and coordinated. It is important, therefore, to know how the cell cycle is controlled during early development. Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell-cycle progression. In Xenopus laevis, three isoforms of cdc25 have been identified, viz. cdc25A, cdc25B and cdc25C. In this study, we isolated a cDNA encoding a novel Xenopus Cdc25 phosphatase (named cdc25D). We investigated the temporal and spatial expression patterns of the four cdc25 isoforms during early Xenopus development, using RT-PCR and whole-mount in situ hybridization. cdc25A and cdc25C were expressed both maternally and zygotically, whereas cdc25B and cdc25D were expressed zygotically. Both cdc25A and cdc25C were expressed mainly in prospective neural regions, whereas cdc25B was expressed preferentially in the central nervous system (CNS), such as the spinal cord and the brain. Interestingly, cdc25D was expressed in the epidermal ectoderm of the late-neurula embryo, and in the liver diverticulum endoderm of the midtailbud embryo. In agreement with the spatial expression patterns in whole embryos, inhibition of bone morphogenetic protein (BMP), a crucial step for neural induction, induced an upregulation of cdc25B, but a downregulation of cdc25D in animal cap assays. These results indicate that different cdc25 isoforms are differently expressed and play different roles during early Xenopus development..
9. Munemichi Ohe, Yoshiko Kawamura, Hiroyuki Ueno, Daigo Inoue, Yoshinori Kanemori, Chiharu Senoo, Michitaka Isoda, Nobushige Nakajo, Noriyuki Sagata, Emi2 inhibition of the anaphase-promoting complex/cyclosome absolutely requires Emi2 binding via the C-terminal RL tail, Molecular biology of the cell, 10.1091/mbc.E09-11-0974, 21, 6, 905-913, 2010.03, Emi2 (also called Erp1) inhibits the anaphase-promoting complex/cyclosome (APC/C) and thereby causes metaphase II arrest in unfertilized vertebrate eggs. Both the D-box and the zinc-binding region (ZBR) of Emi2 have been implicated in APC/C inhibition. However, it is not well known how Emi2 interacts with and hence inhibits the APC/C. Here we show that Emi2 binds the APC/C via the C-terminal tail, termed here the RL tail. When expressed in Xenopus oocytes and egg extracts, Emi2 lacking the RL tail fails to interact with and inhibit the APC/C. The RL tail itself can directly bind to the APC/C, and, when added to egg extracts, either an excess of RL tail peptides or anti-RL tail peptide antibody can dissociate endogenous Emi2 from the APC/C, thus allowing APC/C activation. Furthermore, and importantly, the RL tail-mediated binding apparently promotes the inhibitory interactions of the D-box and the ZBR (of Emi2) with the APC/C. Finally, Emi1, a somatic paralog of Emi2, also has a functionally similar RL tail. We propose that the RL tail of Emi1/Emi2 serves as a docking site for the APC/C, thereby promoting the interaction and inhibition of the APC/C by the D-box and the ZBR..
10. Ueno, H. Nakajo, N. Watanabe, M. Isoda, M. Sagata, N., FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos, Development, 135,11,2023-30, 2009.10.
11. Michitaka Isoda, Yoshinori Kanemori, Nobushige Nakajo, Sanae Uchida, Katsumi Yamashita, Hiroyuki Ueno, Noriyuki Sagata, The extracellular signal-regulated kinase-mitogen-activated protein kinase pathway phosphorylates and targets Cdc25A for SCFβ-TrCP- dependent degradation for cell cycle arrest, Molecular biology of the cell, 10.1091/mbc.E09-01-0008, 20, 8, 2186-2195, 2009.04, The extracellular signal-regulated kinase (ERK) pathway is generally mitogenic, but, upon strong activation, it causes cell cycle arrest by a not-yet fully understood mechanism. In response to genotoxic stress, Chkl hyperphosphorylates Cdc25A, a positive cell cycle regulator, and targets it for Skpl/Cullinl/F-box protein (SCF)β-TrCP ubiquitin ligase-dependent degradation, thereby leading to cell cycle arrest. Here, we show that strong ERK activation can also phosphorylate and target Cdc25A for SCFβ-TrCP-dependent degradation. When strongly activated in Xenopus eggs, the ERK pathway induces prominent phosphorylation and SCF β-TrCP-dependent degradation of Cdc25A. p90rsk, the kinase downstream of ERK, directly phosphorylates Cdc25A on multiple sites, which, interestingly, overlap with Chkl phosphorylation sites. Furthermore, ERK itself phosphorylates Cdc25A on multiple sites, a major site of which apparently is phosphorylated by cyclin-dependent kinase (Cdk) in Chkl-induced degradation. p90rsk phosphorylation and ERK phosphorylation contribute, roughly equally and additively, to the degradation of Cdc25A, and such Cdc25A degradation occurs during oocyte maturation in which the endogenous ERK pathway is fully activated. Finally, and importantly, ERK-induced Cdc25A degradation can elicit cell cycle arrest in early embryos. These results suggest that strong ERK activation can target Cdc25A for degradation in a manner similar to, but independent of, Chkl for cell cycle arrest..
12. Masateru Hiyoshi, Nobushige Nakajo, Sin Ichi Abe, Kazufumi Takamune, Involvement of Xtr (Xenopus tudor repeat) in microtubule assembly around nucleus and karyokinesis during cleavage in Xenopus laevis, Development Growth and Differentiation, 10.1111/j.1440-169x.2005.00787.x, 47, 2, 109-117, 2005.02, We have previously shown that the transcriptional product of the novel gene, Xenopus tudor repeat (Xtr), occurred exclusively in germline cells and early embryonic cells and that the putative Xtr contained plural tudor domains which are thought to play a role in the protein-protein interactions. To understand the role of Xtr, we produced an antibody against a polypeptide containing Xtr tudor domains as an antigen and investigated the distribution and the function of the Xtr. Immunoprecipitation/Western blot and immunohistochemical analyses indicated a similar occurrence of the Xtr to the mRNA except for a slightly different profile of its amount during spermatogenesis. In spite of a large amount of Xtr mRNA at late-secondary spermatogonial stage, the amount of Xtr was kept at a low level until this stage and increased after entering into the meiotic phase. Depletion of the Xtr function in the activated eggs by injection of the anti-Xtr antibody caused the inhibition both of microtubule assembly around nucleus and of karyokinesis progression after prophase, but not of the oscillation of H1 kinase activity. These results suggest that the karyokinesis of at least early embryonic cells are regulated by unique mechanisms in which the Xtr is involved..
13. Katsuhiro Uto, Daigo Inoue, Ken Shimuta, Nobushige Nakajo, Noriyuki Sagata, Chk1, but not Chk2, inhibits Cdc25 phosphatases by a novel common mechanism, EMBO Journal, 10.1038/sj.emboj.7600328, 23, 16, 3386-3396, 2004.08, Cdc25 phosphatases activate cyclin-dependent kinases (Cdks) and thereby promote cell cycle progression. In vertebrates, Chk1 and Chk2 phosphorylate Cdc25A at multiple N-terminal sites and target it for rapid degradation in response to genotoxic stress. Here we show that Chk1, but not Chk2, phosphorylates Xenopus Cdc25A at a novel C-terminal site (Thr504) and inhibits it from C-terminally interacting with various Cdk-cyclin complexes, including Cdk1-cyclin A, Cdk1-cyclin B, and Cdk2-cyclin E. Strikingly, this inhibition, rather than degradation itself, of CdcZSA is essential for the Chk1-induced cell cycle arrest and the DNA replication checkpoint in early embryos. 14-3-3 proteins bind to Chk1-phosphorylated Thr504, but this binding is not required for the inhibitory effect of Thr504 phosphorylation. A C-terminal site presumably equivalent to Thr504 exists in all known Cdc25 family members from yeast to humans, and its phosphorylation by Chk1 (but not Chk2) can also inhibit all examined Cdc25 family members from C-terminally interacting with their Cdk-cyclin substrates. Thus, Chk1 but not Chk2 seems to inhibit virtually all Cdc25 phosphatases by a novel common mechanism..
14. Satoshi Yoshitome, Hiroyasu Nakamura, Nobushige Nakajo, Kengo Okamoto, Isamu Sugimoto, Hiromi Kohara, Kaori Kitayama, Kazuaki Igarashi, Susumu Ito, Noriyuki Sagata, Eikichi Hashimoto, Mr 25 000 protein, a substrate for protein serine/threonine kinases, is identified as a part of Xenopus laevis vitellogenin B1, Development Growth and Differentiation, 10.1046/j.1524-4725.2003.696.x, 45, 3, 283-294, 2003.06, A phosphorylated protein with a molecular mass of 25 000 (pp25) previously purified from the cytosolic fraction of Xenopus laevis oocytes is an effective phosphate acceptor for casein kinases and protein kinase C. In this study, based on the partial amino acid sequence of pp25, a cDNA was isolated that encodes a new yolk precursor protein, Xenopus vitellogenin B1, which contained the sequence encoding pp25. Both mRNA and protein of vitellogenin B1 were expressed in all of the female organs examined. In agreement with a previous report, the amount of vitellogenin B1 protein in the liver increased after stimulation with estrogen. These results suggest that pp25 is a cytosolic non-crystallized yolk protein nutnent source, but it might also play a role in rapid development..
15. Tadahiro Goda, Takashi Ishii, Nobushige Nakajo, Noriyuki Sagata, Hideki Kobayashi, The RRASK motif in Xenopus cyclin B2 is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex, Journal of Biological Chemistry, 10.1074/jbc.M300210200, 278, 21, 19032-19037, 2003.05, The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex..
16. Ken Shimuta, Nobushige Nakajo, Katsuhiro Uto, Yoshimasa Hayano, Kenji Okazaki, Noriyuki Sagata, Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition, EMBO Journal, 10.1093/emboj/cdf357, 21, 14, 3694-3703, 2002.07, In Xenopus embryos, cell cycle elongation and degradation of Cdc25A (a Cdk2 Tyr15 phosphatase) occur naturally at the midblastula transition (MBT), at which time a physiological DNA replication checkpoint is thought to be activated by the exponentially increased nucleo-cytoplasmic ratio. Here we show that the checkpoint kinase Chk1, but not Cds1 (Chk2), is activated transiently at the MBT in a maternal/zygotic gene product-regulated manner and is essential for cell cycle elongation and Cdc25A degradation at this transition. A constitutively active form of Chk1 can phosphorylate Cdc25A in vitro and can target it rapidly for degradation in pre-MBT embryos. Intriguingly, for this degradation, however, Cdc25A also requires a prior Chk1-independent phosphorylation at Ser73. Ectopically expressed human Cdc25A can be degraded in the same way as Xenopus Cdc25A. Finally, Cdc25A degradation at the MBT is a prerequisite for cell viability at later stages. Thus, the physiological replication checkpoint is activated transiently at the MBT by developmental cues, and activated Chkl, only together with an unknown kinase, targets Cdc25A for degradation to ensure later development..
17. Kengo Okamoto, Nobushige Nakajo, Noriyuki Sagata, The existence of two distinct Wee1 isoforms in Xenopus
Implications for the developmental regulation of the cell cycle, EMBO Journal, 10.1093/emboj/21.10.2472, 21, 10, 2472-2484, 2002.05, In eukaryotic cells, the Wee1 protein kinase phosphorylates and inhibits Cdc2, thereby creating an interphase of the cell cycle. In Xenopus, the conventional Wee1 homolog (termed Xe-Wee1A, or Wee1A for short) is maternally expressed and functions in pregastrula embryos with rapid cell cycles. Here, we have isolated a second, zygotic isoform of Xenopus Wee1, termed Xe-Wee1B (or Wee1B for short), that is expressed in postgastrula embryos and various adult tissues. When ectopically expressed in immature oocytes, Wee1B inhibits Cdc2 activity and oocyte maturation (or entry into M phase) much more strongly than Wee1A, due to its short C-terminal regulatory domain. Moreover, ectopic Wee1B, unlike Wee1A, is very labile during meiosis II and cannot accumulate in mature oocytes due to the presence of PEST-like sequences in its N-terminal regulatory domain. Finally, when expressed in fertilized eggs, ectopic Wee1B but not Wee1A does affect cell division and impair cell viability in early embryos, due primarily to its very strong kinase activity. These results suggest strongly that the differential expression of Wee1A and Wee1B is crucial for the developmental regulation of the cell cycle in Xenopus..
18. Tomoya Oe, Nobushige Nakajo, Yoshinori Katsuragi, Kenji Okazaki, Noriyuki Sagata, Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes, Developmental Biology, 10.1006/dbio.2000.9968, 229, 1, 250-261, 2001.01, Chk1, a nuclear DNA damage/replication G2 checkpoint kinase, phosphorylates Cdc25 and causes its nuclear exclusion in yeast and mammalian cells, thereby arresting the cell at the G2 phase until DNA repair/replication is completed. Chk1 is also involved, at least in part, in the natural G2 arrest of immature Xenopus oocytes, but it is unknown how Chk1 inhibits Cdc25 function and undergoes regulation during oocyte maturation. By using enucleated oocytes, we show here that Chk1 inhibits Cdc25 function in the cytoplasm of G2-arrested oocytes and that Cdc25 is activated exclusively in the cytoplasm of maturing oocytes. Moreover, we show that Chk1 activity is not appreciably altered during maturation, being maintained at basal levels, and that C-terminal truncation mutants of Chk1 have very high kinase activities, strong abilities to inhibit maturation, and altered subcellular localization in oocytes. These results, together with other results, suggest that the Chk1/Cdc25 pathway is involved cytoplasmically in G2 arrest of Xenopus oocytes, but moderately and independent of the G2 checkpoint, and that the C-terminal region of Chk1 negatively regulates its kinase activity and also determines its subcellular localization. Based on these results, we discuss the possibility that Chk1 (with the basal activity) may function as an ordinary regulator of Cdc25 in oocytes (and in other cell types) and that Chk1 might be hyperactivated in response to the G2 checkpoint via its dramatic conformational change..
19. Nakajo, N., Yoshitome, S., Iwashita, J., Iida, M., Uto, K., Ueno, S., Okamoto, K., and Sagata N. , Absence of Wee1 ensures the meiotic cell cycle in Xenopus oocytes., Genes & Development, 14, 3, 328-338, 14巻3号328-338, 2000.02.
20. Katsuhiro Uto, Nobushige Nakajo, Noriyuki Sagata, Two structural variants of Nek2 kinase, termed Nek2A and Nek2B, are differentially expressed in Xenopus tissues and development, Developmental Biology, 10.1006/dbio.1999.9231, 208, 2, 456-464, 1999.04, Nek2 kinase, a NIMA-related kinase, has been suggested to play both meiotic and mitotic roles in mammals, but its function(s) during development is poorly understood. We have isolated here cDNAs encoding a Xenopus homolog of mammalian Nek2 and have shown that Xenopus Nek2 has two structural variants, termed Nek2A and Nek2B. Nek2A, most likely a C-terminally spliced form, corresponds to the previously described human and mouse Nek2, while Nek2B is most probably a novel, C-terminally unspliced form of Nek2. As a consequence of this (probable) alternative splicing, Nek2B lacks the C- terminal 70-amino-acid sequence of Nek2A, which contains a PEST sequence (or a motif for rapid degradation). Western blot analysis reveals that Nek2A is expressed predominantly in the testis (presumably in spermatocytes) and very weakly in the stomach and, during development, only after the neurula stage. By contrast, Nek2B is expressed mainly in the ovary and in both primary and secondary oocytes and early embryos up to the neurula stage. These results suggest that Nek2A and Nek2B may play both meiotic and mitotic roles, but in a spatially and temporally complementary manner during Xenopus development, and thai: Nek2B, rather than Nek2A (or the conventional form of Nek2), may play an important role in early development. We discuss the possibility that a counterpart of Xenopus Nek2B might also exist and function in early mammalian development..
21. Nobushige Nakajo, Tomoya Oe, Katsuhiro Uto, Noriyuki Sagata, Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes, Developmental Biology, 10.1006/dbio.1998.9178, 207, 2, 432-444, 1999.03, Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes..
22. Nobuaki Furuno, Yasuki Ogawa, Jun Iwashita, Nobushige Nakajo, Noriyuki Sagata, Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase, EMBO Journal, 10.1093/emboj/16.13.3860, 16, 13, 3860-3865, 1997.07, In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21(Cip) in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21(Cip) can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21(Cip) are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles..
23. Kazuya Nomura, Nobushige Nakajo, K. L.P.J. Hidari, H. Nomura, M. Murata, M. Suzuki, K. Yamana, Y. Hirabayashi, Occurrence of a novel fucose-containing pentaglycosylceramide with blood-group-B active determinant in Xenopus blastula cells
Its possible involvement in cell-cell adhesion, Biochemical Journal, 10.1042/bj3060821, 306, 3, 821-827, 1995.01, For understanding of the biological function of glycoconjugates during embryogenesis and morphogenesis, Xenopus laevis is considered a very useful animal model. We have found that blood-group-active molecules characteristically were distributed in the cell-cell contact region of Xenopus blastula cells. The chemical nature of blood-group-active glycoconjugates, including glycosphingolipids, is little known. T.l.c.-immunostaining using anti-blood-group-antigen antibodies showed that many species of blood group-B-active glycosphingolipids existed in the neutral glycosphingolipid fraction extracted from Xenopus laevis eggs. Among the B-active glycosphingolipids detected, two major components with the fastest mobility on a t.l.c. plate, tentatively termed XN-1 and XN-2, were isolated, and their chemical structures were characterized by gas chromatography-mass spectrometry, immunological analysis, fast-atom-bombardment mass spectrometry and 1H-n.m.r, spectroscopy. Both XN-1 and XN-2 had an identical pentaoligosaccharide structure, but differed in their ceramide moiety. The chemical structure is: Galα1-3 Galβ1-3Galβ1-4Glcβ1-1'Cer Fucα1-2 This is a novel type of pentaglycosylceramide with blood-group B activity, in that it lacks N-acetylhexosamine in its core carbohydrate structure, In this paper, a possible involvement of the blood-group antigen in the cell-adhesion process of Xenopus embryonic cells is discussed..
24. Nobuaki Furuno, Mayumi Nishizawa, Kenji Okazaki, Hirotaka Tanaka, Jun Iwashita, Nobushige Nakajo, Yasuki Ogawa, Noriyuki Sagata, Suppression of DNA replication via mos function during meiotic divisions in xenopus oocytes, EMBO Journal, 13, 10, 2399-2410, 1994.06, Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc-2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc-2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc-2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc-2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization..
25. Shinichi Ichikawa, Nobushige Nakajo, Hisako Sakiyama, Yoshio Hirabayashi, A mouse B16 melanoma mutant deficient in glycolipids, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.91.7.2703, 91, 7, 2703-2707, 1994.03, Mouse B16 melanoma cell line, GM-95 (formerly designated as MEC-4), deficient in sialyllactosylceramide was examined for its primary defect. Glycolipids from the mutant cells were analyzed by high-performance TLC. No glycolipid was detected in GM-95 cells, even when total lipid from 107 cells was analyzed. In contrast, the content of ceramide, a precursor lipid molecule of glycolipids, was normal. Thus, the deficiency of glycolipids was attributed to the first glucosylation step of ceramide. The ceramide glucosyltransferase (EC 2.4.1.80) activity was not detected in GM-95 cells. There was no significant difference of sialyllactosylceramide synthase activity, however, between GM-95 and the parental cells. The deficiency of glycolipids in GM-95 cells was associated with changes of the cellular morphology and growth rate. The parental cells showed irregular shapes and tended to overlap each other. On the other hand, GM-95 cells exhibited an elongated fibroblastic morphology and parallel arrangement. The population- doubling times of GM-95 and the parental cells in serum-free medium were 28 hr and 19 hr, respectively..
26. Kazuya Nomura, Tatsuya Tajima, Hajime Nomura, Kiyomi Tsuno, Yasuo Fujimura, Nobushige Nakajo, Kiyotaka Yamana, A Novel Monoclonal Antibody disrupting Cell Type Specific Substratum Adhesion of Frog (Xenopus laevis) Epithelial Cells and Endothelial Cells
(frog, Xenopus laevis/cell substratum adhesion/cell adhesion/endothelial cells), Development, Growth & Differentiation, 10.1111/j.1440-169X.1991.00639.x, 33, 6, 639-649, 1991.01, We isolated a mouse monoclonal antibody (FAD‐II) that disrupts cell‐substratum adhesion of amphibian (Xenopus laevis) epithelial cells and endothelial cells. The effect of the antibody was cell‐type specific, and the antibody had no effect on fibroblastic cells while fibronectin peptide blocked cell‐substratum adhesion of all the cell types examined. In developing frog embryos, the epitopes recognized by the antibody were detected in pronephrotic ducts and in other tissue cells of embryos (from stage 33/34 afterwards). In adult tissues, the antibody mainly recognized antigens in extracelluar matrices. The antigens recognized by the antibody seems to be novel glycoepitopes in frog cells..