1993 Fiscal Year Annual Research Report

低分子量GTP結合蛋白質を介する細胞内情報伝達機構とその病態

Research Project

Project/Area Number	03404022
Research Institution	Kode University
Principal Investigator	高井義美神戸大学, 医学部, 教授 (60093514)
Co-Investigator(Kenkyū-buntansha)	貝淵弘三神戸大学, 医学部, 助教授 (00169377)
Keywords	低分子量GTP結合蛋白質 / Ras / MAPキナーゼ / REKS / Rho GDI / Rho / Rab3A / Rabphilin-3A
Research Abstract	私共はこの数年間、Rasに類似した分子量2万余りのGTP結合蛋白質(低分子量G蛋白質)を生体内に多数発見しており、低分子量G蛋白質の役割と作用機構の解析を進めてきた。本年度は、低分子量G蛋白質の機能と標的蛋白質を明らかにすることを目的とした。Rasがどのような標的蛋白質を介して、細胞の増殖や分化を制御しているのかは長い間不明であったが、最近、MAP(Mitogen-Activated Protein)キナーゼ(MAPK;ERK,Extracellular Signal-Regulated Kinaseとも呼ばれる)の活性化の経路の一つにRasが関与していることが明らかにされている。そこで、昨年度、私共はRasの標的蛋白質を同定する目的で、RasがMAPキナーゼキナーゼを活性化してMAPキナーゼを活性化する無細胞系のアッセイ系を確立している。本年度、私共は、このアッセイ系を用いて、Rasによって直接活性化される未知の因子を、高度に精製し、REKS(Ras-dependent ERK Kinase Stimulator)と命名した。REKSはGTP結合型のRasに結合して活性化されるプロテインキナーゼで、MAPキナーゼキナーゼをリン酸化して活性化した。一方、私共は、C3やEDIN、Rho GDIを用いた実験から、Rhoが平滑筋の収縮を制御していることを明らかにした。また、Rhoが細胞形態、細胞膜のラッフリング、細胞運動、細胞質分裂をも制御していることを明らかにした。さらに、ホルボールエステルによる細胞膜のラッフリングと細胞運動をC3とRho GDIが阻害することから、Rhoの上流にCキナーゼが存在することが示唆された。Rab3Aは神経伝達物質の放出に重要であると考えられるが、本年度、私共はウシ大脳のシナプス小胞の膜画分にGTP結合型Rab3Aが特異的に結合する標的蛋白質を見出し、そのcDNAをクローニングして1次構造を決定してRabphilin-3Aと命名した。Rabphilin-3Aはプレシナプスのシナプス小胞に局在しており、シナプスでの神経伝達物質の放出に重要な役割を果たしていると考えられる。これらの私共の研究成果から、低分子量G蛋白質の機能と標的蛋白質について着実な成果を挙げることができ、本年度の研究計画をほぼ達成できたと考えている。

Research Products

(23 results)

All Other

All Publications (23 results)

[Publications] 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)
[Publications] 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)
[Publications] Ullrich,O.: "Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins." J.Biol.Chem.268. 18143-18150 (1993)
[Publications] 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)
[Publications] Kishida,S.: "Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A,a putative Rab3A target protein." J.Biol.Chem.268. 22259-22261 (1993)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] 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)
[Publications] Shirataki,H.: "Rabphilin-3A,a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotaqmin." Mol.Cell.Biol.13. 2061-2068 (1993)
[Publications] 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)
[Publications] 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)
[Publications] Takaishi,K.: "Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility." Oncogene. 9. 273-279 (1993)
[Publications] Kawamura,M.: "Translocation of Ki-ras p21 between membrane and cytoplasm by smg GDS." Biochem.Biophys.Res.Commun.190. 832-841 (1993)
[Publications] 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)
[Publications] Sawai,T.: "Combination of arachidonic acid and guanosine 5'-0-(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)
[Publications] 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)
[Publications] 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)
[Publications] Miki,H.: "Association of Ash/Grb-2 with dynamin through the SH3 domain." J.Biol.Chem.(in press). (1994)
[Publications] 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)
[Publications] 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)
[Publications] Nakanishi,H.: "Kinetic properties of Ash/Grb2-interacting GDP/GTP exchange protein." Biochem.Biophys.Res.Commun.198. 1255-1261 (1994)
[Publications] 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.(in press). (1994)

1993 Fiscal Year Annual Research Report

低分子量GTP結合蛋白質を介する細胞内情報伝達機構とその病態

Principal Investigator

高井 義美 神戸大学, 医学部, 教授 (60093514)

Research Products

[Publications] 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)

[Publications] 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)

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

[Publications] 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)

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

[Publications] 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)

[Publications] 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)

[Publications] 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)

[Publications] 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)

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

[Publications] 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)

[Publications] 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)

[Publications] Takaishi,K.: "Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility." Oncogene. 9. 273-279 (1993)

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

[Publications] 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)

[Publications] Sawai,T.: "Combination of arachidonic acid and guanosine 5'-0-(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)

[Publications] 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)

[Publications] 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)

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

[Publications] 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)

[Publications] 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)

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

[Publications] 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.(in press). (1994)

高井義美神戸大学, 医学部, 教授 (60093514)