1994 年度実績報告書

低分子量G蛋白質とその関連蛋白質の機能修飾物質の開発およびその有効利用

研究課題

研究課題/領域番号	06557012
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	川原康洋神戸大学, 医学部附属病院, 講師 (80169755) 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 / B-Raf / 14-3-3蛋白質 / Rho / ERM-CD44系 / Rab3A / Rabphilin-3A
研究概要	本研究では、私共の発見した低分子量G蛋白質とその関連蛋白質の変異型cDNAやそのプローブ、特異抗体を作製し、低分子量G蛋白質の機能と作用機能の解明に有効利用すると共に、これらを用いて低分子量G蛋白質の制御異常による病態の解析や新しい診断法、治療法の開発に応用することを目的としている。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、すで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までに精製し、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。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、C3やRhoGDIを用いた実験から、RhoがHGFと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、epitiope tagをつけたRho遺伝子を導入した細胞を用いて、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、細胞真接着部位、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、アクチン細胞骨格と細胞膜との結合を制御している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Rabphilin-3Aの抗体を作製し、その抗体を用いてRabphilin-3AがRab3Aと共にシナプス小胞に局在していることを明らかにした。さらに、Rabphilin-3AはRad3A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。以上の結果から、低分子量G蛋白質の機能と作用機構の解明に着実な成果をあげることができ、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 soecies 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 atimulator 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 Rad3A." J.Biol.Chem.269. 32717-32720 (1994)
[文献書誌] Nishiyama,T.: "Rac p21 is involved in insulin-induced mambrane 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)
[文献書誌] Yamochi,W.: "Growth site-localization of Rhol small GTP-binding protein and its involvement in but 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 Rad3A,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 Db1 oncogene product as a GDP/GTP exchange protein for the Rho family:Its properties in compartison 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 hippocamous." 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 Ce^<2+> and phosphatidylserine." 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 MEK kinase." J.Biol.Chem.270. 2460-2465 (1994)
[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinese kinase kinase from bovine brain cytosol and 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.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

低分子量G蛋白質とその関連蛋白質の機能修飾物質の開発およびその有効利用

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 soecies 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 atimulator 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 Rad3A." J.Biol.Chem.269. 32717-32720 (1994)

[文献書誌] Nishiyama,T.: "Rac p21 is involved in insulin-induced mambrane 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)

[文献書誌] Yamochi,W.: "Growth site-localization of Rhol small GTP-binding protein and its involvement in but 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 Rad3A,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 Db1 oncogene product as a GDP/GTP exchange protein for the Rho family:Its properties in compartison 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 hippocamous." 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 Ce^<2+> and phosphatidylserine." 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 MEK kinase." J.Biol.Chem.270. 2460-2465 (1994)

[文献書誌] Yamamori,B.: "Purification of a ras-dependent mitogen-activated protein kinese kinase kinase from bovine brain cytosol and 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.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)