1994 年度実績報告書

細胞の増殖・分化のシグナル伝達機構

研究課題

研究課題/領域番号	06283221
研究種目	重点領域研究
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	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 / MAPキナーゼ / B-Raf / 14-3-3蛋白質 / Smg GDS / ERM-CD44系
研究概要	低分子量G蛋白質のうち、RasはMAPキナーゼ系を介して種々の遺伝子の発現を促進することにより細胞の増殖や分化を制御していることが明らかとなっている。一方、私共を含めたいくつかのグループによって、Rhoはアクチン細胞骨格系を介して細胞の形態や運動を制御していることが明らかにされている。しかし、RasとRhoの活性化機構と作用機構については十分に明らかにされていない。本年度、Rasについては、私共が開発した無細胞系によって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にまで精製し、それがB-Rafプロテインキナーゼと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蛋白質はB-Rafに直接作用し、Ras存在下でB-Rafを相乗的に活性化することを明らかにした。また、私共が見出しているSmg GDSが、Rasを活性型であるGTP結合型に変換する活性だけではなく、GTP結合型のRasと安定な三者複合体を形成してRasを細胞膜から解離する活性を有することを明らかにした。一方、Rhoに関しては、Dbl発がん遺伝子産物がRhoのGDP解離促進蛋白質としての活性を有することを明らかにし、Dblが細胞内でRhoを活性化している可能性を示した。また、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明らかにした。さらに、Rhoを静止時の細胞では細胞質に存在するが、種々の細胞刺激により、細胞質から細胞膜ラッフリング領域、細胞間接着部位、細胞質分裂時の収縮環等の細胞膜部位へトランスロケーションすることを明らかにした。これらの部位には、アクチン細胞骨格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細胞骨格系の再編成に関与している可能性を示した。以上、RasとRhoの活性化機構と作用機構の詳細がかなり明らかになり、本年度の研究は順調に進んだと考えられる。

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 smg GDP dissociation stimulator and mammalian counterpart 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. 22917-22920 (1994)
[文献書誌] 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)
[文献書誌] Nishiyama,T.: "Rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-0-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 involvement 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 regulator 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 sites 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^<2+> and phosphatidylseirne." Biochem.Biophys.Res,Commun.205. 460-466 (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 MEX kinase." J.Biol.Chem.270. 2460-2465 (1995)
[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain sytosol and its identificatio 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.Biophys.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^<2+>-dependent manner." Mol.Brain.Res.28. 29-36 (1995)

1994 年度 実績報告書

細胞の増殖・分化のシグナル伝達機構

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 smg GDP dissociation stimulator and mammalian counterpart 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. 22917-22920 (1994)

[文献書誌] 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)

[文献書誌] Nishiyama,T.: "Rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-0-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 involvement 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 regulator 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 sites 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^<2+> and phosphatidylseirne." Biochem.Biophys.Res,Commun.205. 460-466 (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 MEX kinase." J.Biol.Chem.270. 2460-2465 (1995)

[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain sytosol and its identificatio 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.Biophys.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^<2+>-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)