1993 年度実績報告書

低分子量GTP結合蛋白質の機能修飾物質の開発と有効利用

研究課題

研究課題/領域番号	03558019
研究機関	神戸大学
研究代表者	高井義美神戸大学, 医学部, 教授 (60093514)
研究分担者	佐野公彦神戸大学, 医学部・附属病院, 講師 (40205993) 川原康洋神戸大学, 医学部・附属病院, 講師 (80169755) 佐々木卓也神戸大学, 医学部, 助手 (40241278)
キーワード	低分子量GTP結合蛋白質 / 標的蛋白質 / Rab3A / Rabphilin-3A / シナプス小胞 / REKS / プロテインキナーゼ / Smg GDS
研究概要	本研究では、私共の発見した低分子量G蛋白質とその関連遺伝子のcDNAプローブおよび抗体を作製し、低分子量G蛋白質の活性制御機構や作用機構の解明に有効利用すると共に、種々の病態の理解や病因の解析、診断法、治療法の開発に応用することを目的としている。これまでに低分子量G蛋白質としてRap1およびRab3A、-B、-C活性制御蛋白質としてRab GDIおよびRho GDI、Smg GDSを見出していたが、本年度の本研究でRab3Aの標的蛋白質としてRabphilin-3Aを見出し、そのcDNAを単離している。Rabphilin-3AはN末端側のRab3Aの結合する領域とC末端側のCa^<2+>とセリンリン脂質の結合部位の機能的に異なる部位にわかれ、私共はそれぞれの変異型のcDNAと抗体を作製した。これらの抗体を用いてRabphilin-3Aはシナプス小胞に局在していることが判明した。現在、cDNAや蛋白質、抗体を用いてRab3A、Rabphilin-3Aが神経伝達物質の放出に関与しているか否かを解析中である。一方、私共はRasの標的蛋白質としてREKSを同定していたが、本年度はそれをさらに高度に精製し、その性状を解析した。REKSはプロテインキナーゼでRafやMos,Stellとは異なっていた。現在そのcDNAをクローン化している。他方、Smg GDSはSosがGRB2を介して活性化したRasを細胞膜から細胞質にトランスロケーションする活性をもつことが明らかになった。Smg GDSに関してはアンチセンスを用いて実際に細胞内で作用するか否かを現在検討中である。これらの実験結果から、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、本年度の本研究計画をほぼ達成できたと考えている。

研究成果

(23件)

すべてその他

すべて文献書誌 (23件)

[文献書誌] Miura,Y.: "Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)in Swiss 3T3 cells." J.Biol.Chem.268. 510-515 (1993)
[文献書誌] Itoh,T.: "The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase." J.Biol.Chem.268. 3025-3028 (1993)
[文献書誌] Ullrich,O.: "Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins." J.Biol.Chem.268. 18143-18150 (1993)
[文献書誌] Burstein,E.S.: "Regulation of the GTPase cycle of the neuronally expressed Ras-like GTP-binding protein Rab3A." J.Biol.Chem.268. 22247-22250 (1993)
[文献書誌] Kishida,S.: "Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A,a putative Rab3A target protein." J.Biol.Chem.268. 22259-22261 (1993)
[文献書誌] Sasaki,T.: "Consequences of weak interaction of rho GDI with the GTP-bound forms of rho p21 and rac p21." J.Biol.Chem.268. 23959-23963 (1993)
[文献書誌] Orita,S.: "Comparison of kinetic properties between two mammalian ras p21 GDP/GTP exchange proteins,ras guanine nucleotide-releasing factor and smg GDP dissociation stimulator." J.Biol.Chem.268. 25542-25546 (1993)
[文献書誌] Yamaguchi,T.: "Two functionally different domains of rabphilin-3A,rab3A p25/smg p25A-binding and phospholipid- and Ca^<2+>-binding domains." J.Biol.Chem.268. 27164-27170 (1993)
[文献書誌] Takaishi,K.: "Involvement of rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)in cell motility." Mol.Cell.Biol.13. 72-79 (1993)
[文献書誌] Shirataki,H.: "Rabphilin-3A,a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin." Mol.Cell.Biol.13. 2061-2068 (1993)
[文献書誌] Kishi,K.: "Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)." J.Cell Biol.120. 1187-1195 (1993)
[文献書誌] Itoh,T.: "A protein factor for ras p21-dependent activation of MAP kinase through MAP kinase-kinase." Proc.Natl.Acad.Sci.USA. 90. 975-979 (1993)
[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 (1993)
[文献書誌] Kawamura,M.: "Translocation of Ki-ras p21 between membrane and cytoplasm by smg GDS." Biochem.Biophys.Res.Commun.190. 832-841 (1993)
[文献書誌] Sasaki,T.: "The nucleotide exchange rates of Rho and Rac small GTP-binding proteins are enhanced to different extents by their regulatory protein Smg GDS." Biochem.Biophys.Res.Commun.194. 1188-1193 (1993)
[文献書誌] Sawai,T.: "Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate)induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system." Biochem.Biophys.Res.Commun.195. 264-269 (1993)
[文献書誌] Garrett,M.D.: "Interaction of Sec4 with GDI proteins from bovine brain,drosophila melanogaster and saccharomyces cerevisiae:conservation of GDI membrane dissociation activity." FEBS Lett.331. 233-238 (1993)
[文献書誌] Motoike,T.: "Expression of smg p25A/rab 3A guanine nucleotide dissociation inhibitor(GDI)in neurons and glial cells from rat brain." Neuroscience Letters. 156. 87-90 (1993)
[文献書誌] Miki,H.: "Association of Ash/Grb-2 with dynamin through the SH3 domain." J.Biol.Chem.(in press). (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 Db1 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] 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.(in press). (1994)

1993 年度 実績報告書

低分子量GTP結合蛋白質の機能修飾物質の開発と有効利用

研究代表者

高井 義美 神戸大学, 医学部, 教授 (60093514)

研究成果

[文献書誌] Miura,Y.: "Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)in Swiss 3T3 cells." J.Biol.Chem.268. 510-515 (1993)

[文献書誌] Itoh,T.: "The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase." J.Biol.Chem.268. 3025-3028 (1993)

[文献書誌] Ullrich,O.: "Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins." J.Biol.Chem.268. 18143-18150 (1993)

[文献書誌] Burstein,E.S.: "Regulation of the GTPase cycle of the neuronally expressed Ras-like GTP-binding protein Rab3A." J.Biol.Chem.268. 22247-22250 (1993)

[文献書誌] Kishida,S.: "Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A,a putative Rab3A target protein." J.Biol.Chem.268. 22259-22261 (1993)

[文献書誌] Sasaki,T.: "Consequences of weak interaction of rho GDI with the GTP-bound forms of rho p21 and rac p21." J.Biol.Chem.268. 23959-23963 (1993)

[文献書誌] Orita,S.: "Comparison of kinetic properties between two mammalian ras p21 GDP/GTP exchange proteins,ras guanine nucleotide-releasing factor and smg GDP dissociation stimulator." J.Biol.Chem.268. 25542-25546 (1993)

[文献書誌] Yamaguchi,T.: "Two functionally different domains of rabphilin-3A,rab3A p25/smg p25A-binding and phospholipid- and Ca^<2+>-binding domains." J.Biol.Chem.268. 27164-27170 (1993)

[文献書誌] Takaishi,K.: "Involvement of rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)in cell motility." Mol.Cell.Biol.13. 72-79 (1993)

[文献書誌] Shirataki,H.: "Rabphilin-3A,a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin." Mol.Cell.Biol.13. 2061-2068 (1993)

[文献書誌] Kishi,K.: "Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein(rho GDI)." J.Cell Biol.120. 1187-1195 (1993)

[文献書誌] Itoh,T.: "A protein factor for ras p21-dependent activation of MAP kinase through MAP kinase-kinase." Proc.Natl.Acad.Sci.USA. 90. 975-979 (1993)

[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 (1993)

[文献書誌] Kawamura,M.: "Translocation of Ki-ras p21 between membrane and cytoplasm by smg GDS." Biochem.Biophys.Res.Commun.190. 832-841 (1993)

[文献書誌] Sasaki,T.: "The nucleotide exchange rates of Rho and Rac small GTP-binding proteins are enhanced to different extents by their regulatory protein Smg GDS." Biochem.Biophys.Res.Commun.194. 1188-1193 (1993)

[文献書誌] Sawai,T.: "Combination of arachidonic acid and guanosine 5'-O-(3-thiotriphosphate)induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system." Biochem.Biophys.Res.Commun.195. 264-269 (1993)

[文献書誌] Garrett,M.D.: "Interaction of Sec4 with GDI proteins from bovine brain,drosophila melanogaster and saccharomyces cerevisiae:conservation of GDI membrane dissociation activity." FEBS Lett.331. 233-238 (1993)

[文献書誌] Motoike,T.: "Expression of smg p25A/rab 3A guanine nucleotide dissociation inhibitor(GDI)in neurons and glial cells from rat brain." Neuroscience Letters. 156. 87-90 (1993)

[文献書誌] Miki,H.: "Association of Ash/Grb-2 with dynamin through the SH3 domain." J.Biol.Chem.(in press). (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 Db1 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] 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.(in press). (1994)

1993 年度実績報告書

高井義美神戸大学, 医学部, 教授 (60093514)