Language：English   Publishing type：Research paper (scientific journal)  

The metabolic processes of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into PI(3,4,5)P3 and the subsequent PI(3,4,5)P3 signalling are involved in cell migration. Dysfunctions in the control of this pathway can cause human cancer cell migration and metastatic growth. Here we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a PI(4,5)P2-binding protein, regulates cancer cell migration. PRIP overexpression in MCF-7 and BT-549 human breast cancer cells inhibited cell migration in vitro and metastasis development in vivo. Overexpression of the PRIP pleckstrin homology domain, a PI(4,5)P2 binding motif, in MCF-7 cells caused significant suppression of cell migration. Consistent with these results, in comparison with wild-type cells, Prip-deficient mouse embryonic fibroblasts exhibited increased cell migration, and this was significantly attenuated upon transfection with a siRNA targeting p110α, a catalytic subunit of class I phosphoinositide 3-kinases (PI3Ks). PI(3,4,5)P3 production was decreased in Prip-overexpressing MCF-7 and BT-549 cells. PI3K binding to PI(4,5)P2 was significantly inhibited by recombinant PRIP in vitro, and thus the activity of PI3K was downregulated. Collectively, PRIP regulates the production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K, and the suppressor activity of PRIP in PI(4,5)P2 metabolism regulates the tumour migration, suggesting PRIP as a promising target for protection against metastatic progression.

DOI： 10.1038/s41598-017-05908-7

Other Link： https://www.nature.com/articles/s41598-017-05908-7

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.biocel.2015.02.013

Language：English   Publishing type：Research paper (scientific journal)  

The C2 domain is a protein module often found in molecules that regulate exocytosis. C2 domains mediate interactions between the parental molecule and Ca²⁺, phospholipids, and proteins. Although various molecules have been shown to interact with several C2 domains, no interactions between the C2 domains from different molecules have yet been reported. In the present study, we identified direct interactions between the C2 domain of PRIP (phospholipase C-related but catalytically inactive protein) and the C2 domains of other molecules. Among the C2 domains examined, those of synaptotagmin-1 (Syt1-C2A and Syt1-C2B) and phospholipase C δ-1 bound to the C2 domain of PRIP. We investigated the interactions between the C2 domain of PRIP (PRIP-C2) with Syt1-C2A and Syt1-C2B, and the mode of binding of each was Ca²⁺-dependent and -independent, respectively. We further demonstrated that the Ca²⁺ dependence of the interaction between PRIP-C2 and Syt1-C2A was attributed to Ca²⁺ binding with Syt1-C2A, but not PRIP-C2, using a series of mutants prepared from both C2 domains. We previously reported that the interaction between PRIP-C2 and the membrane fusion machinery suggested a critical role for PRIP in exocytosis; therefore, the results of the present study further support the importance of PRIP-C2 in the inhibitory function of PRIP in regulating exocytosis.

DOI： 10.1016/j.jbior.2014.09.001.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbior.2013.07.001

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M112.419317

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0057375

Language：English   Publishing type：Research paper (scientific journal)  

Exocytosis is one of the most fundamental cellular events. The basic mechanism of the final step, membrane fusion, is mediated by the formation of the SNARE complex, which is modulated by the phosphorylation of proteins controlled by the concerted actions of protein kinases and phosphatases. We have previously shown that a protein phosphatase-1 (PP1) anchoring protein, phospholipase C-related but catalytically inactive protein (PRIP), has an inhibitory role in regulated exocytosis. The current study investigated the involvement of PRIP in the phospho-dependent modulation of exocytosis. Dephosphorylation of synaptosome-associated protein of 25 kDa (SNAP-25) was mainly catalyzed by PP1, and the process was modulated by wild-type PRIP but not by the mutant (F97A) lacking PP1 binding ability in in vitro studies.We then examined the role of PRIP in phospho-dependent regulation of exocytosis in cell-based studies using pheochromocytoma cell line PC12 cells, which secrete noradrenalin. Exogenous expression of PRIP accelerated the dephosphorylation process of phosphorylated SNAP-25 after for-skolin or phorbol ester treatment of the cells. The phospho-states of SNAP-25 were correlated with noradrenalin secretion, which was enhanced by forskolin or phorbol ester treatment and modulated by PRIP expression in PC12 cells. Both SNAP-25 and PP1 were co-precipitated in anti-PRIP immunocomplex isolated from PC12 cells expressing PRIP. Collectively, together with our previous observation regarding the roles of PRIP in PP1 regulation, these results suggest that PRIP is involved in the regulation of the phospho-states ofSNAP-25by modulating the activity of PP1, thus regulating exocytosis.

DOI： 10.1074/jbc.M111.294645

Other Link： http://www.jbc.org/content/287/13/10565.long

Language：English   Publishing type：Research paper (scientific journal)  

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

A well-known protein module regulating molecular interactions is the pleckstrin homology (PH) domain whose best-characterised ligand is phosphoinositide. In the present study, we analysed the PH domain from PRIP (phospholipase C-related but catalytically inactive protein, comprising types 1 and 2) regarding phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] binding employing a variety of binding assays. The PH domains prepared from PRIP-1 and -2 showed similar binding profiles to soluble ligands in vitro and showed similar plasma membrane localisation to that of PLC-δ1; however, the PH domain with the N-terminal extension of PRIP-1 but not PRIP-2 showed even distribution throughout the cytoplasm, indicating that the N-terminal extension of PRIP-1 inhibited binding to PtdIns(4,5)P₂ present in the plasma membrane. A chimeric molecule of PLC-δ1 PH domain with the N-terminal extension of PRIP-1 exhibited similar localisation to PRIP-1 PH domain with the N-terminal extension. Binding assay to liposomes containing various concentrations of PtdIns(4,5)P₂ revealed that the PH domain of PLC-δ1 bound steeply to the maximum, even at a concentration of 1.2 mol%, whereas the PH domains from PRIP-1 and -2 bound depending on the concentration up to 5 mol%. We also performed binding experiments using saponin-permeabilised PC12 cells. PH domains from PRIP increased the binding to cells preincubated with the brain cytosol extract in the presence of ATP, during which PtdIns(4,5)P₂ were probably synthesised. The binding of PH domain with the following EF hand motifs showed Ca²⁺-dependent binding. These results indicate that the PH domain of PRIP binds to PtdIns(4,5)P₂ present in the plasma membrane, depending on the concentrations of the lipid ligand and Ca²⁺, suggesting that PRIP might play physiological roles in events involved in the changes of these parameters, probably including Ins(1,4,5)P₃.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/sj.bjp.0706373

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bone.2021.116210.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1016/j.jbc.2021.100274.

Language：English   Publishing type：Research paper (scientific journal)  

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

DOI： 10.1002/jcb.29193

Language：English   Publishing type：Research paper (scientific journal)  

Osteocalcin (OC) is a bone-derived hormone that regulates energy metabolism. OC exists in two forms, carboxylated (GlaOC) and uncaboxylated (GluOC), but only the latter appears to have an endocrine function. In this study, we prepared an extract containing both Gla- and GluOC from boiled pork bone using 0.2 M carbonate buffer at pH 9.5, and tested whether the extract had beneficial effects on improving metabolic parameters in obese mice. The extract equivalent of 1.2 μg of GluOC/mouse was orally administrated to C57BL/6 female mice fed a high-fat, high-sucrose diet. Daily oral administration of the extract for four weeks decreased blood glucose levels and promoted glucose tolerance as well as insulin sensitivity. Our study shows for the first time that boiled pork bones are a source material for osteocalcin in the large-scale production of supplements designed to improve glucose metabolism.

DOI： 10.1080/09168451.2016.1214530

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cellsig.2015.12.014

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cellsig.2014.12.018

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bi2018128

Language：English   Publishing type：Research paper (scientific journal)  

Other Link： http://www.sciencedirect.com/science/article/pii/S0065257111000549

Event date： 2018.9 - 2019.9

Language：Japanese  

Venue：九州大学病院キャンパス　百年講堂、　福岡   Country：Japan  

肥満や糖尿病などのメタボリック症候群は世界的な健康問題であるが、特に２型糖尿病の発症基盤の一つであるインスリンシグナル伝達経路異常の分子メカニズムについては、未だ不明な点も多い。イノシトール1,4,5- 三リン酸結合性タンパク質であるPRIP（PLC-Related but catalytically Inactive Protein）はインスリンシグナル伝達経路の中心的分子であるAktと結合する。今回我々はPRIP欠損(KO)マウスを用いて、インスリンシグナリングの調節におけるPRIPの役割について検討を行なった。マウスにインスリン投与後に摘出した脂肪組織のインスリン受容体（IR）とAktのリン酸化レベルを比較すると、KOマウス由来の脂肪組織でIR、Aktともにリン酸化レベルが低下していた。骨格筋と肝臓では差が認められなかった。器官培養した脂肪組織をインスリン刺激すると、KOマウス由来のものでIRおよびAktのリン酸化が抑えられていただけでなく、糖取り込みやGLUT４の細胞膜へのトランスロケーションも抑制されていた。また、KOマウス由来のMEFから分化した脂肪細胞では、IRの膜発現量が野生型と比べて減少していた。インスリンシグナルはIRのクラスリン依存性のエンドサイトーシスにより負に制御される。そこで、脂肪組織を用いて免疫沈降を行ったところPRIPがClathrin Heavy Chain（CLTC）およびIRと結合することがわかった。siRNAを用いてCLTCの発現を抑制すると、KO由来脂肪細胞でIRの膜発現量がWTと同等まで回復した。以上の結果から、PRIPはIRのエンドサイトーシスの調節を介してIRの膜発現量を調節し、脂肪細胞におけるインスリンシグナルを制御していることが示唆された。

Event date： 2017.12

Language：Japanese  

Venue：日本・神戸神戸ポートアイランド   Country：Japan  

Numerous studies have demonstrated a reduced activity of insulin signaling, but the molecular mechanism of triggering events remains incompletely understood. We previously reported that PRIP (phospholipase C-related but catalytically inactive protein) interacts with Akt, the center molecule of insulin signaling pathway. In the present study, we investigated whether PRIP is involved in the regulation of insulin signaling. We found that the phosphorylation of of Akt and even upstream molecules, IR (insulin receptor) and IRS-1 (insulin receptor substrate) were inhibited in PRIP-KO (Knockout) MEFs (mouse embryonic fibroblasts), and that glucose uptake was inhibited in adipocytes differentiated from PRIP-KO MEF, indicating insulin insensitivity in PRIP-KO cells. We further examined the serine phosphorylation of IRS-1 which inhibits insulin signaling. We found that insulin triggered more phosphorylation of Ser1101 of IRS-1 in PRIP-KO MEF than WT, and that dephosphorylation process of Ser1101 of IRS-1 was blocked by calyculin A, an inhibitor for the protein phosphatase1 (PP1) and 2A (PP2A). Furthermore, we detected the interaction of IRS-1 and PP2A catalytic subunit (PP2Ac) using in situ Proximity Ligation Assay (PLA) in WT MEF cells, which was diminished in PRIP-KO cells. Combined with our previous finding that PRIP binds to PP2Ac, we speculate that serine phosphorylation of IRS was enhanced due to the decreased dephosphorylation process in PRIP-KO MEF, which in turn inhibited insulin signaling. These results suggest that PRIP is involved in the regulation of insulin signaling, likely through modulating serine phosphorylation of IRS-1.

Event date： 2016.12

Language：English  

Venue：Moscone Center, San Francisco   Country：United States  

Insulin resistance is strongly associated with obesity and other symptoms related to the metabolic syndrome (MS). Numerous studies using insulin-resistant animal models and humans have consistently demonstrated a reduced activity of insulin signaling via the insulin receptor substrate (IRS-1)/phosphatidylinositol (PI) 3-kinase/Akt pathway, but the molecular mechanism of triggering events remains incompletely understood. We previously reported that PRIP (phospholipase C-related but catalytically inactive protein) interacts with Akt, the center molecule of insulin signaling pathway. In the present study, we investigated whether PRIP is involved in the regulation of insulin signaling using mouse embryonic fibroblasts (MEF) from wild type (WT) and PRIP-Knockout (KO) mice. We found that the phosphorylation of Thr308 and Ser473 of Akt and tyrosine phosphorylation of even upstream molecules, IR (insulin receptor) and IRS-1 (insulin receptor substrate-1) were inhibited in MEF cells from PRIP-KO mice, and that glucose uptake was also inhibited in adipocytes differentiated from PRIP-KO MEF, indicating insulin insensitivity in PRIP-KO cells. We then investigated how the insulin signaling via the IR/IRS-1/Akt pathway was inhibited in PRIP-KO MEF cells. Since serine phosphorylation of IRS proteins inhibits insulin signaling, we further examined the serine phosphorylation of IRS-1 using MEF cells. We found that stimulation of PRIP-KO MEF with insulin for 30 min triggered more phosphorylation of Ser1101 of IRS-1 than WT, and that the dephosphorylation process of Ser1101 of IRS-1 after insulin stimulation in MEF cells from WT mice was blocked by calyculin A, an inhibitor for the protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A). Furthermore, we detected the interaction of IRS-1 and protein phosphatase 2A catalytic subunit (PP2Ac), but not PP1 and PP2A subunit A using in situ Proximity Ligation Assay (PLA) in WT MEF cells, whereas PLA signals was dramatically diminished in PRIP-KO cells. Based on these results and our previous finding that PRIP binds with PP2Ac, we speculated that serine phosphorylation of IRS-1 was enhanced due to the decreased dephosphorylation process in PRIP-KO MEF cells compared to WT cells, which in turn inhibited the IRS-1/PI3K/Akt insulin signaling. Collectively, these results suggest that PRIP is involved in the regulation of insulin signaling, likely through modulating the serine phosphorylation of IRS-1.

Event date： 2016.10

Language：English  

Venue：Fukuoka   Country：Japan  

We studied the functional consequences of SNAP-25 phosphorylation by protein kinase A (PKA) and protein kinase C (PKC). Phosphorylation of SNAP-25 at Thr138 by PKA inhibited SNARE complex formation and functional exocytosis in PC12 cells. Phosphorylation of SNAP-25 at Ser187 by PKC enhanced SNARE complex formation and functional exocytosis in PC12 cells.

Event date： 2016.8

Language：Japanese  

Venue：札幌コンベンションセンター   Country：Japan  

SNARE 複合体は syntaxin、SNAP-25 、VAMPよりなるが、それらのタンパク質のリン酸化によって開口分泌は調節される。PKA および PKC による SNAP-25 のリン酸化による調節はよく知られているが、リン酸化された SNAP-25 の SNARE 複合体の形成における役割は明らかではない。そこで、本研究では、SNAP-25 の PKA および PKC によるリン酸化の SNARE 複合体の形成に及ぼす影響を検討した。精製した SNAP-25、syntaxin-1、VAMP-2 を用いた実験の結果、in vitro において SNARE 複合体の形成は PKA による SNAP-25 の Thr138 のリン酸化によって抑えられ、PKC による Ser187 のリン酸化によって促進された。また、PC12 細胞における SNARE 複合体形成は PKA 活性化剤であるホルスコリン刺激によって阻害され、PKC の活性化剤であるPMA によって促進された。高カリウムイオン刺激による PC12 からのノルアドレナリン分泌は、ホルスコリン、PMA いずれの刺激によっても増加した。SNAP-25 を欠損させた PC12 細胞では高濃度カリウムイオン刺激によるノルアドレナリン分泌は抑制されたが、そこに野生型 SNAP-25 の遺伝子を導入するとノルアドレナリン分泌能が回復した。SNAP-25 を欠損させた PC12 細胞に SNAP-25 のリン酸化されない変異体である T138A または S187A 変異体を発現させても高濃度カリウムイオン刺激による分泌応答は回復したが、ホルスコリン刺激による分泌増強は野生型 SNAP-25 を発現させたものと比べて T138A 変異体を発現させた細胞においてより明白に認められた。一方、S187A を発現させた細胞における PMA の効果は野生型を発現させたものと比べて抑えられていた。以上のことから、PKA および PKC による SNAP-25 のリン酸化は SNARE 複合体の形成を、それぞれ抑制的、あるいは促進的に調節し、PC12 細胞における開口分泌を効果的に調節していることが示唆された。

Event date： 2015.12

Language：English  

Venue：神戸ポートアイランド   Country：Japan  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学箱崎キャンパス   Country：Japan  

Event date： 2014.10

Language：English  

Venue：国立京都国際会館   Country：Japan  

Event date： 2013.9

Language：English   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2013.9

Language：English  

Venue：Yokohama   Country：Japan  

The 87th Annual Meeting of the Japanese Biochemical Society

Event date： 2012.12

Language：English  

Venue：福岡国際会議場・マリンメッセ福岡   Country：Japan  

Event date： 2012.9

Language：English  

Venue：Seville Conference and Exhibition Centre (FIBES)   Country：Spain  

Event date： 2012.2

Presentation type：Oral presentation (general)  

Venue：Ulsan   Country：Korea, Republic of  

Roles of PRIP in phospho-regulation of exocytosis through the interaction with protein phosphatases

Event date： 2011.11

Venue：Fukuoka   Country：Japan  

Phospho-dependent modulation of exocytosis by PRIP

Other Link： http://www.mcb.dent.kyushu-u.ac.jp/News/News_2011/FukuokaSymposiumProgram.pdf

Event date： 2011.9

Venue：京都   Country：Japan  

The 84th Annual Meeting of the Japanese Biochemical Society

Other Link： http://www.aeplan.co.jp/jbs2011/outline.html

Event date： 2010.12

Venue：神戸ポートアイランド   Country：Japan  

The 33rd Annual Meeting of the molecular Biology Society of Japan
The 83rd Annual Meeting of the Japanese Biochemical Society

Event date： 2010.9 - 2010.10

Venue：Melbourne Convention and Exhibition Centre   Country：Australia  

Event date： 2022.9

Language：Japanese  

Venue：徳島県、徳島市、徳島大学キャンパス   Country：Japan  

Event date： 2022.9

Language：English  

Venue：徳島県、徳島市、徳島大学キャンパス   Country：Japan  

Event date： 2021.10

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン開催   Country：Japan  

Event date： 2021.10

Language：English  

Venue：オンライン開催   Country：Japan  

Event date： 2019.10

Language：Japanese  

Venue：東京歯科大学   Country：Japan  

Event date： 2018.9

Language：Japanese  

Venue：九州大学病院キャンパス　百年講堂、　福岡   Country：Japan  

Event date： 2016.7

Language：English  

Venue：Ewa Womans University, Seoul   Country：Korea, Republic of  

Event date： 2013.11

Language：English  

Venue：Fukuoka   Country：Japan  

Event date： 2013.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Fukuoka   Country：Japan  

Event date： 2013.7

Language：English   Presentation type：Oral presentation (general)  

Venue：UNIST,Ulsan   Country：Korea, Republic of  

Language：English   Presentation type：Oral presentation (general)  

Venue：Mont Saint-Odile, Alsace   Country：France