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1996 Fiscal Year Annual Research Report

低分子量G蛋白質による細胞機能の制御機構

Research Project

Project/Area Number 06404021
Research InstitutionOsaka University

Principal Investigator

高井 義美  大阪大学, 医学部, 教授 (60093514)

Keywords低分子量G蛋白質 / アクチン細胞骨格 / 神経伝達物質 / BNI1 / BNR1 / プロフィリン / PKC1 / Rabphilin-3A / Doc2
Research Abstract

本年度の本研究では、低分子量G蛋白質のうち、主にRhoおよびRab3Aの機能、活性化機構および作用機構について検討した。私共は、Rhoは、アクチン繊維と細胞膜との結合を制御するERM-CD44系を介してアクチン細胞骨格系の再編成を制御していることを明らかにしているが、本年度の本研究で、Rhoは、何らかの標的蛋白質を介して、ERMが細胞膜のCD44に結合して細胞質から細胞膜に移行する過程に作用することを明らかにした。さらに、このERMが、Rho-Rho GDI複合体に直接作用し、RhoをRho GDIから解離させて活性化反応を開始させる作用も有していることを明らかにした。また、酵母の遺伝学的手法を用いて、新たにRhoの標的蛋白質がBNI1とBNR1であることを明らかにすると共に、BNI1とBNR1が、アクチン結合蛋白質であるプロフィリンに直接結合することを示した。したがって、RhoはBNI1とBNR1を介してアクチン細胞骨格系に作用する可能性が出てきた。さらに、Racが、何らかの標的蛋白質を介して、E-カドヘリン系を制御することにより、細胞間接着を制御していることを明らかにした。一方、Rab3Aは神経伝達物質の放出を制御しており、私共は、Rab3Aの標的蛋白質としてRabphilin3Aを見い出している。本年度の本研究において、私共は、Rabphilin-3Aが実際に神経伝達物質の放出に働いていることを証明した。また、Rabphilin-3Aがアクチン結合蛋白質であるα-アクチニンに結合し、α-アクチニンによるF-アクチンのbundingを促進することを明らかにした。その結果、Rabphilin-3Aが細胞骨格系をも介して神経伝達物質の放出を制御している可能性が明らかになった。さらに、私共は、世界に先駆けてRab3AのGAPおよびGEPを精製すると共に、そのcDNAをクローニングして一次構造を決定した。私共は、Rabphilin-3Aと同様C2様ドメインを2個有する蛋白質としてDoc2を見い出しているが、本年度の本研究で、このDoc2も実際に神経伝達物質の放出に働いていることを証明した。さらに、新たにDoc2に結合する蛋白質としてホルボールエステル結合領域であるC1ドメインを有する新しい蛋白質を見い出した。以上の結果を得たことにより、本年度の研究計画はほぼ達成できたと考えている。

  • Research Products

    (68 results)

All Other

All Publications (68 results)

  • [Publications] Miki,H.: "Association of Ash/Grb-2 with dynamin through the Src homology 3 domain." J.Biol.Chem.269. 5489-5492 (1994)

  • [Publications] Nishimura,N.: "Molecular cloning and characterization of two rab GDI species from rat brain-Brain-specific and ubiquitous types." J.Biol.Chem.269. 14191-14198 (1994)

  • [Publications] Nakanishi,H.: "Different functions of smg GDP dissociation stimulator and mammalian counterpart of yeast Cdc25." J.Biol.Chem.269. 15085-15091 (1994)

  • [Publications] Masuda,T.: "Molecular cloning and characterization of yeast rho GDP dissociation inhibitor." J.Biol.Chem.269. 19173-19718 (1994)

  • [Publications] Shimizu,K.: "Synergistic activation by Ras and 14-3-3 protein of a mitogen-activated protein kinase kinase kinase named Ras-dependent extracellular signal-regulated kinase kinase stimulator." J.Biol.Chem.269. 22917-22920 (1994)

  • [Publications] Shirataki,H.: "Rabphilin-3A is associated with synaptic vesicle through a vesicle protein in a manner independent of Rab3A." J.Biol.Chem.269. 32717-32720 (1994)

  • [Publications] Nishiyama,T.: "Rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor and 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced membrane ruffling in KB cells." Mol.Cell.Biol.14. 2447-2456 (1994)

  • [Publications] Yamochi,W.: "Growth site localization of Rhol small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae." J.Cell.Biol.125. 1077-1093 (1994)

  • [Publications] Huber,L.A.: "Mapping of ras-ralated GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis." Proc.Natl.Acad.Sci.USA. 91. 7874-7878 (1994)

  • [Publications] Takaishi,K.: "Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility." Oncogene. 9. 273-279 (1994)

  • [Publications] Wada,K.: "Localization of Rabphilin-3A,a putative target for Rab3A,at the aites of Ca^<2+>-dependent cxocytosis in Pc12 cells." Biochem.Biophys.Res.Commun.198. 158-165 (1994)

  • [Publications] Yaku,H.: "The Db1 oncogene product as a GDP/GTP exchange protein for the family-Its properties in comparison with those of Smg GDS." Biochem.Biophys.Res.Commun.198. 811-818 (1994)

  • [Publications] Nakanishi,H.: "Kinetic proerties of Ash/Grb2-interacting GDP/GTP exchange protein." Biochem.Biophys.Res.Commun.198. 1255-1261 (1994)

  • [Publications] Kim.Y.S.: "Phosphoprylation and activation of mitogen-activated protein kinase by kainic acid-induced seizure in rat hippocampus." Biochem.Biophys.Res.Commun.202. 1163-1168 (1994)

  • [Publications] Mizoguchi,A.: "Localization of Rabphilin-3A on the synaptic vesicle." Biochem.Biophys.Res.Commun.202. 1235-1243 (1994)

  • [Publications] Numata,S.: "Phosphorylation of Rabphilin-3A,a putative target protein for Rab3A,by cyclic AMP-dependent protein kinase." Biochem.Biophys.Res.Commun.203. 1927-1934 (1994)

  • [Publications] Miyazaki,M.: "Identification as β-adducin of a protein interacting with Rabphilin-3A in the presence of Ca^<2+> and phosphatidylserine." Biochem.Biophys.Res.Commun.205. 460-466 (1994)

  • [Publications] Imazumi,K.: "Identification of a Rabphilin-3A-interacting protein as GTP cyclohydorase I in PC12 cells." Biochem.Biophys.Res.Commun.205. 1409-1416 (1994)

  • [Publications] Kato.M.: "Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinese II." Biochem.Biophys.Res.Commun.205. 1776-1784 (1994)

  • [Publications] Miyazaki,A.: "Comparison of kinetic properties between MSS4 and Pab3A GRF GDP/GTP exchange proteins." FEBS Lett.350. 333-336 (1994)

  • [Publications] Fujita,Y.: "GDP/GTP exchange reaction-atimulating activity of Rabphilin-3A for Rab3A small GTP-binding protein." FEBS Lett.353. 67-70 (1994)

  • [Publications] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs-Identification of REKS as a Rasdependent mitogen-activated protein kinase kinese kinese" J.Biol.Chem.270. 2460-2465 (1995)

  • [Publications] Bourgoin,S.: "Low molecular weight GTP-binding proteins in HL-60 granulocytes." J.Biol.Chem.270. 3172-3178 (1995)

  • [Publications] Yamamori,B.: "Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-raf and 14-3-3 proteins." J.Biol.Chem.270. 11723-11726 (1995)

  • [Publications] Chung,S-H.: "Evidence that the Rab3A-binding protein,Rabphilin-3A,enhances regulated secretion-Studies in adrenal chromaffin cells." J.Biol.Chem.270. 16714-16718 (1995)

  • [Publications] Kuribara,H.: "Synergistic activation of rat brain phosholipase D by ADP-ribosylation factor and rhoA p21,and its inhibition by clostridium botulinum C3 exoenzyme." J.Biol.Chem.270. 25667-25671 (1995)

  • [Publications] McGeady,P.: "The farnesyl group of H-ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase." J.Biol.Chem.270. 26347-26351 (1995)

  • [Publications] Gotoh,T.: "Identification of Rapl as a target for the Crk SH3 Domain-binding guanine nucleotide-releasing factor C3G." Mol.Cell.Biol.15. 6746-6753 (1995)

  • [Publications] Nonaka,H.: "A downstream target of RHO1 small GTP-binding protein is PKC1,a homolog of protein kinase C,which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae." EMBO.J.14. 5931-5938 (1995)

  • [Publications] Takaishi,K.: "Translocation of activated Rho from the cytoplasm to membrane ruffling area,cell-cell adhesion sites and cleavage furrows." Oncogene. 11. 39-48 (1995)

  • [Publications] Nishimura,N.: "Cloning of a brain-type isoform of human rab GDI and its expression in human neuroblastoma cell lines and tumor specimens." Cancer Res.55. 5445-5450 (1995)

  • [Publications] Orita,S.: "Doc2: a novel brain protein having two repeated C_2-like domains." Biochem.Biophys.Res.Commun.206. 439-448 (1995)

  • [Publications] Araki,K.: "Purification and characterization of Rab GDIβ from rat brain." Biochem.Biophys.Res.Commun.211. 296-305 (1995)

  • [Publications] Takahashi,K.: "Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules." Biochem.Biophys.Res.Commun.217. 979-986 (1995)

  • [Publications] Sakaguchi,G.: "Molecular cloning of an isoform of Doc2 having two C_2-like domains." Biochem.Biophys.Res.Commun.217. 1053-1061 (1995)

  • [Publications] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine-and Ca^<2+>-dependent manner." Mol.Brain.Res.28. 29-36 (1995)

  • [Publications] Regazzi,R.: "Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells-comparison with the effect of Rab3AL peptide." Biochim.Biophys.Acta. 1268. 269-278 (1995)

  • [Publications] Takai,Y.: "Rho as a regulator of the cytoskeleton." Trends in Biochemical Sciences. 20. 227-231 (1995)

  • [Publications] Ohtsuka,T.: "Activation of brain B-Raf protein kinase by RaplB small GTP-binding protein." J.Biol.Chem.271. 1258-1261 (1996)

  • [Publications] Orita,S.: "Doc2 enhances Ca^<2+>-dependent exocytosis from PC12 cells." J.Biol.Chem.271. 7257-7260 (1996)

  • [Publications] Fujita,Y.: "Phosphorylation of Munc-18/n-Secl/rbSecl by protein kinase C-Its implication in regulating the interaction of Munc-18/n-Secl/rbSecl with Syntaxin." J.Biol.Chem.271. 7265-7268 (1996)

  • [Publications] Kuroda,S.: "Different effects of various phospholipids on Ki-Ras-,Ha-Ras-,and Rap1B-induced B-Raf activation." J.Biol.Chem.271. 14680-14683 (1996)

  • [Publications] Shimizu,K.: "SMAP,an Smg GDS-associating protein having Arm repeats and phosphorylated by Src tyrosine kinese." J.Biol.Chem.271. 27013-27017 (1996)

  • [Publications] Kato,M.: "Physical and functional interaction of Rabphilin-3A with α-Actinin." J.Biol.Chem.271. 31775-31778 (1996)

  • [Publications] Hirano,H.: "ROM7/BEM4 encodes a novel protein that interacts with the Rholp small GTP-binding protein in Saccharomyces ceravisiae." Mol.Cell.Biol.16. 4396-4403 (1996)

  • [Publications] Hirao,M.: "Regulation mechanism of ERM(Ezrin/Radixin/Moesin)protein/plasma membrane association-Possible involvement of phosphatldyiinositol turnover and Rho-dependent signaling pathway." J.Cell.Biol.135. 37-51 (1996)

  • [Publications] Masumoto,N.: "Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs." J.Cell.Biol.135. 1741-1747 (1996)

  • [Publications] Drgonova,J.: "Rholp,a yeast protein at the interface between cell polarization and morphogenesis." Science. 272. 277-279 (1996)

  • [Publications] Ozaki,K.: "Rom1p and Rom2p are GDP/GTP exchange proteins(GEPs)for the Rholp small GTP binding protein in Saccharomyces cerevisiae." EMBO J.15. 2196-2207 (1996)

  • [Publications] Kohno,H.: "Bnilp implicated in cytoskeletal control is a putative target of Rholp small GTP binding protein in Saccharomyces cerevisiae." EMBO J.15. 6060-6068 (1996)

  • [Publications] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein-Partial purification and characterization from rat brain." Oncogene. 12. 915-920 (1996)

  • [Publications] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate repid formation of actin stress fibers." Oncogene. 12. 1357-1360 (1996)

  • [Publications] Komura,R.: "Involvement of rabphilin-3A in Ca^<2+>-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.219. 435-440 (1996)

  • [Publications] Hotta,K.: "Interaction of the Rho family small G proteins with kinectin,an anchoring protein of kinesin motor." Biochem.Biophys.Res.Commun.225. 69-74 (1996)

  • [Publications] Senbonmatsu,T.: "Interaction of Rabphilin3 with aynaptic vesicles through multiple regions." Biochem.Biophys.Res.Commun.228. 567-572 (1996)

  • [Publications] Oishi,H.: "Interaction of both the C2A and C2B domains of rabphilin3 with Ca^<2+> and pospholipid." Biochem.Biophys.Res.Commun.229. 498-503 (1996)

  • [Publications] Hori,Y.: "Possible involvement of Rabll p24,a ras-like small GTP-binding protein,in intracellular vesicular transport of isolated pancreatic acini." Digestive Diseases and Sciences. 41. 133-138 (1996)

  • [Publications] Regazzi,R.: "Expression,localization and functional role of small GTPases of the Rab3 family in insulinsecreting cells." J.Cell.Sci.109. 2265-2273 (1996)

  • [Publications] Komuro,R.: "Involvement of Rho and Rac small G proetins and Rho GDI in Ca^<2+>-dependent exocytosis from PC12 cells." Genes to Cells. 1. 943-951 (1996)

  • [Publications] Takai,Y.: "Rab3A amall GTP-binding protein in Ca^<2+> -dependent exocytosis." Genes to Cells. 1. 615-632 (1996)

  • [Publications] Wada,M.: "Isolation and characterization of a GDP/GTP exchange protein specific for the Rab3 subfamily small G proteins." J.Biol.Chem.272. 3875-3878 (1997)

  • [Publications] Fukui,K.: "Isolation and characterization of a GDPase activating protein specific for the Rab3 subfamily of small G proteins." J.Biol.Chem.272. 4655-4658 (1997)

  • [Publications] Takeuchi,M.: "SAPAPs-A family of PSD-95/SAP90-associated proteins localized at postsynaptic density." J.Biol.Chem.(in press). (1997)

  • [Publications] Imamura,H.: "Bnilp and Bnrlp-Downstream targets of the Rho family small G proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae." EMBO J.(in press). (1997)

  • [Publications] Kotani,H.: "Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells." Oncogene. (in press). (1997)

  • [Publications] Naito,A.: "Molecular cloning of mouse Doc2α and distribution of its mRNA in adult mouse brain." Mol.Brain.Res.(in press). (1997)

  • [Publications] Jin-no,Y.: "A novel function of the C-terminal liped moieties of Rab3A small G protein implicated in Ca^<2+>-dependent exocytosis-Inhibition of interaction with GTP and reduction of this inhibition by phospholipid." Genes to Cells. (in press). (1997)

  • [Publications] Koch,G.A.: "Association of the Rho family small GTP-binding proteins with Rho GDP dissociation inhibitor(Rho GDI)in Saccharomyces cerevisiae." Oncogene. (in press). (1997)

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Published: 1999-03-08   Modified: 2016-04-21  

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