1994 年度実績報告書

低分子量G蛋白質による細胞機能の制御機構

研究課題

研究課題/領域番号	06404021
研究種目	一般研究(A)
研究機関	大阪大学
研究代表者	高井義美大阪大学, 医学部, 教授 (60093514)
研究分担者	白瀧博通岡崎国立共同研究機構, 生理学研究所, 助手 (90249962) 佐々木卓也大阪大学, 医学部, 助手 (40241278) 田中一馬大阪大学, 医学部, 講師 (60188290)
キーワード	低分子量G蛋白質 / Ras / Rho / Rab3A / MAPキナーゼ / ERM-CD44系 / Rabphi7in-3A
研究概要	本研究では、低分子量G蛋白質のうち、Ras、Rho、およびRab3Aに焦点をあてて、これらの低分子G蛋白質の機能を作用機構について検討した。RasはMAPキナーゼ系を介して種々の遺伝子発現を制御していることが明らかとなっている。そこで、私共は既に世界に先駆けて開発した無細胞系でRasによりMAPキナーゼを活性化するアッセイ系を用いて、Rasの標的蛋白質をウシ大脳可溶性画分よりほぼ均一な蛋白質まで精製し、それがB-Rafと14-3-3蛋白質の複合体であることを明らかにした。さらに、14-3-3蛋白質がRasと共にB-Bafの活性を制御していることを明らかにした。一方、私共を含めたいくつかのグループによって、Rhoがアクチン系を介して種々の細胞機能を制御していることが明らかにされている。本年度、私共は、RhoがHGFやTPAの下流で細胞膜ラッフリングと細胞運動を制御していることを示した。さらに、種々の細胞刺激によりRhoが細胞質から細胞膜ラッフリング領域、細胞間接着部位、細胞質分裂の収縮環等の細胞膜部位へ移行することを明らかにした。これらの部位には、アクチン系と細胞膜との結合を制御するERM-CD44系が存在しており、RhoがERM-CD44系を介してアクチン系の再編成に関与している可能性を示した。Rab3Aは神経伝達物質の放出を制御していると考えられているが、私共は、Rab3Aの標的蛋白質Rabphilin3AがRab3Aと共にシナプス小胞に局在していること、その際、Rabphilin-3AはRab3A以外のシナプス小胞上のアンカーリング蛋白質を介してシナプス小胞に結合していることを示した。さらに、Rabphilin-3AがAキナーゼやカルモジュリンキナーゼIIによってリン酸化されることを明らかにし、これらのキナーゼがRab3A-Rabphilin-3A系を介して神経伝達物質の放出を制御している可能性を示した。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。

研究成果

(25件)

すべてその他

すべて文献書誌 (25件)

[文献書誌] Miki,H.: "Association of Ash/Grb-2 with dynamin through the src homology 3 domain." J.Biol.Chem.269. 5489-5492 (1994)
[文献書誌] 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)
[文献書誌] Nakanishi,H.: "Different functions of smg GDP dissociation stimulator and mammalian countelart of yeast Cdc25." J.Biol.Chem.269. 15085-15091 (1994)
[文献書誌] Masuda,T.: "Molecular cloning and characterization of yeast rho GDP dissociation inhibitor." J.Biol.Chem.269. 19713-19718 (1994)
[文献書誌] 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. 2917-22920 (1994)
[文献書誌] Shirataki,H.: "Rabphilin-3A is associated with synaptic vesicle through a vesicle protein in a manner independent of Red3A." J.Biol.Chem.269. 32717-32720 (1994)
[文献書誌] Nishiyama,T.: "Rac p21 is involved in insulin -induced membrane ruffling and rho p21 is involved in hepatccyte growth factor-and 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced membrane ruffling in KB cells." Mol.Cell.Biol.14. 2447-2456 (1994)
[文献書誌] Yamochi,W.: "Growth site-localization of Rhol small GTP-binding protein and its involvemment in bud formation in Saccharomyces cerevisiae." J.Cell Biol.125. 1077-1093 (1994)
[文献書誌] Huber,L.A.: "Mapping of ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis." Proc.Natl.Acad.Sci.USA. 91. 7874-7878 (1994)
[文献書誌] Takaishi,K.: "Involvement of Rho p21 small GTP-binding protein and its requlator in the HGF-induced cell motility." Oncogene. 9. 273-279 (1994)
[文献書誌] Wada,K.: "Localization of Rabphilin-3A,a putative target for Rab3A,at the sotes of Ca^<2+>-dependent exocytosis in Pc12 cells." Biochem.:Biophys.Res.Commun.198. 158-165 (1994)
[文献書誌] Yaku,H.: "The Dbl oncogene product as a GDP/GTP exchange protein for the Rho family:Its properties in comparison with those of Smg GDS." Biochem.Biophys.Res.Commun.198. 811-818 (1994)
[文献書誌] Nakanishi,H.: "Kinetic properties of Ash/Grb2-interacting GDP/GTP exchange protein." Biochem.:Biophys.Res.Commun.198. 1255-1261 (1994)
[文献書誌] Kim,Y.S.: "Phosphorylation and activation of mitogen-activated protein kinase by kainic acid-induced seizure in rat hippocampus." Biochem.:Biophys.Res.Commun.202. 1163-1168 (1994)
[文献書誌] Mizoguchi,A.: "Localization of Rabphilin-3A on the synaptic vesicle." Biochem.:Biophys.Res.Commun.202. 1235-1243 (1994)
[文献書誌] 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)
[文献書誌] Miyazaki,M.: "Identification as β-adducin of a protein interacting with Rabphilin-3A in the Presence of Ca_<1+> and Phosphatidylserine." Biochem.Biophys.Res.Commun.205. 406-466 (1994)
[文献書誌] Imazumi,K.: "Identification of a Rabphilin-3A-interacting protein as GTP cyclohydorase I in PC12 cells." Biochem.Biophys.Res.Commun.205. 1409-1416 (1994)
[文献書誌] Kato,M.: "phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II." Biochem.Biophys.Res.Commun.205. 1776-1784 (1994)
[文献書誌] Miyazaki,A.: "Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins." FEBS Lett.350. 333-336 (1994)
[文献書誌] Fujita,Y.: "GDP/GTP exchange reaction-stimulating activity of Rabphilin-3A for Rab3A small GTP-binding protein." FEBS Lett.353. 67-70 (1994)
[文献書誌] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs__Identification of REKS as a Ras-dependent MEK kinase." J.Biol.Chem.270. 2460-2465 (1995)
[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinase kinase knase from bovine brain cytosol ans its identification as a complex of B-Raf and 14-3-3 proteins." J.Biol.Chem.in press (1995)
[文献書誌] Orita,S.: "Doc2:a novel brain protein having two repeated C_2-like domains." Biochem.Biophy.Res.Commun.206. 439-448 (1995)
[文献書誌] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115′000 polypeptide in a phosphatidylserine-and Ca_<1+>-dependent manner." Mol.Brain.Res.28. 29-36 (1995)

1994 年度 実績報告書

低分子量G蛋白質による細胞機能の制御機構

研究代表者

高井 義美 大阪大学, 医学部, 教授 (60093514)

研究成果

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

[文献書誌] 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)

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

[文献書誌] Masuda,T.: "Molecular cloning and characterization of yeast rho GDP dissociation inhibitor." J.Biol.Chem.269. 19713-19718 (1994)

[文献書誌] 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. 2917-22920 (1994)

[文献書誌] Shirataki,H.: "Rabphilin-3A is associated with synaptic vesicle through a vesicle protein in a manner independent of Red3A." J.Biol.Chem.269. 32717-32720 (1994)

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

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

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

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

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

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

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

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

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

[文献書誌] 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)

[文献書誌] Miyazaki,M.: "Identification as β-adducin of a protein interacting with Rabphilin-3A in the Presence of Ca_<1+> and Phosphatidylserine." Biochem.Biophys.Res.Commun.205. 406-466 (1994)

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

[文献書誌] Kato,M.: "phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II." Biochem.Biophys.Res.Commun.205. 1776-1784 (1994)

[文献書誌] Miyazaki,A.: "Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins." FEBS Lett.350. 333-336 (1994)

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

[文献書誌] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs__Identification of REKS as a Ras-dependent MEK kinase." J.Biol.Chem.270. 2460-2465 (1995)

[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinase kinase knase from bovine brain cytosol ans its identification as a complex of B-Raf and 14-3-3 proteins." J.Biol.Chem.in press (1995)

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

[文献書誌] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115′000 polypeptide in a phosphatidylserine-and Ca_<1+>-dependent manner." Mol.Brain.Res.28. 29-36 (1995)

1994 年度実績報告書

高井義美大阪大学, 医学部, 教授 (60093514)