1995 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が、RasファミリーのRap1の作用機構については不明である。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、Rabは細胞内小胞輸送系を介して、細胞の形態や極性を制御していることが明らかにされており、RhoもRabもがん細胞の浸潤や転移において重要な役割を果たしている可能性がでてきている。しかし、RhoとRabの作用機構についても十分に明らかにされていない。Rap1の作用機構としては、これまでに、Rasに対して阻害的に働く機構と促進的に働く機構とが提唱されていたが、本研究において、私共は、Rap1が、Rasの標的蛋白質として私共が見い出している、B-Rafを活性化することを明らかにした。その結果、Rap1がRasに対して促進的に作用する機構が存在することが明らかとなった。また、私共が見い出しているSmgGDSは、RasとRhoに作用してGDP/GTP交換反応を促進して活性化するが、本年度、私共は、SmgGDSに結合する新しい蛋白質を見い出し、SMAP(SmgGDS-Associated Protein)と命名した。一方、本研究において、私共は、Rhoが細胞外Ca^<2+>によって誘起されるadherens junctionとtight junctionの形成にも関与していることを明らかにすると共に、Rhoがアクチン細胞骨格系と細胞膜との結合を制御するERM-CD44系を介してアクチン細胞骨格系の再編成を制御していることを明らかにした。また、酵母をモデル系に用いて、酵母RHO1の標的蛋白質がPKC1(Cキナーゼホモログ)、BNI1(細胞質分裂関連蛋白質)、およびグルカン合成酵素であることを明らかにした。他方、本研究において、私共は、Rab3Aの標的蛋白質として私共が見い出しているRabphilin3Aに類似してC2様ドメインを二つ持つ蛋白質を見い出してDoc2(Double C2)と命名した。さらに、Rabphilin-3AもDoc2も共にエクソサイトーシス(開口分泌)を制御していることを明らかにした。以上の結果を得たことにより、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] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase" J.Biol.Chem.270. 2460-2465 (1995)
[文献書誌] Bourgoin,S.: "Low molecular weight GTP-binding Proteins in HL-60 granulocytes." J.Biol.Chem.270. 3172-3178 (1995)
[文献書誌] Yamamori,B.: "Purification of a Ras-dependent mitogen-activated Protein kimase kinase kinase from bovine brain cytosol and its identification as a complex of B-raf and 14-3-3 Proteins." J.Biol.Chem.270. 11723-11726 (1995)
[文献書誌] Chung,S-H.: "Evidence that the Rab3A-binding Protein,Rabphilin-3A,enhances requlated secretion-Studies in adrenal Chromaffin cells." J.Biol.Chem.270. 16714-16718 (1995)
[文献書誌] Kuribara,H.: "Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21,and its inhibition by clostridium botulinum C3 exoenzyme." J.Biol.Chem.270. 25667-25671 (1995)
[文献書誌] McGeady,P.: "The farnesyl group of H-ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase." J.Biol.Chem.270. 26347-26351 (1995)
[文献書誌] Gotoh,T.: "Identification of Rapl as a target for the Crk SH3 Domain-binding guanine nucleotide-releasing factor C3G." Mol.Cell.Biol.15. 6746-6753 (1995)
[文献書誌] Nonaka,H.: "A downstream target of RHO1 small GTP-binding protein is PKC1,a homolog of protein kinase C,which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae." EMBO J.14. 5931-5938 (1995)
[文献書誌] Takaishi,K.: "Translocation of activated Rho from the cytoplasm to membrane ruffling area,cell-cell adhesion sites and cleavage furrows." Oncogene. 11. 39-48 (1995)
[文献書誌] Nishimura,N.: "Cloning of a brain-type isoform of human rab GDI and its expression in human neroblastoma cell lines and tumor specimens." Cancer Res.55. 5445-5450 (1995)
[文献書誌] Orita,S.: "Doc2: a novel brain protein having two repeated C2-like domains." Biochem.Biophys.Res.Commun.206. 439-448 (1995)
[文献書誌] Araki,K.: "purif:cation and characterization of Rab GDIβ from rat brain." Biochem.Biophys.Res.Commun.211. 296-305 (1995)
[文献書誌] Takahashi,K.: "Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules." Biochem.Biophys.Res.Commun.217. 979-986 (1995)
[文献書誌] Sakaguchi,G.: "Molecular cloning of an isoform of Doc2 having two C2-like domains." Biochem.Biophys.Res.Commun.217. 1053-1061 (1995)
[文献書誌] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine-and Ca2+-dependent manner." Mol.Brain.Res.28. 29-36 (1995)
[文献書誌] Regazzi,R.: "Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells-comparison with the effect of Rab3AL peptide." Biochim.Biophys.Acta. 1268. 269-278 (1995)
[文献書誌] Takai,Y.: "Rho as a regulator of the cytoskeleton." Trends in Biochemical Sciences. 20. 227-231 (1995)
[文献書誌] Ohtsuka,T.: "Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein." J.Biol.Chem.271. 1258-1261 (1996)
[文献書誌] Orita,S.: "Doc2 enhances Ca2+-dependent exocytosis from PC12 cells." J.Biol.Chem.(in press).
[文献書誌] Fujita,Y.: "phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C-Its implication in requlating the interaction of Munc-18/n-Sec1/rbSec1 with Syntaxin." J.Biol.Chem.(in press).
[文献書誌] Drgonova,J.: "Rholp a yeast protein at the juncture between cell polarization and morphogenesis." Science. (in press).
[文献書誌] Ozaki,K.: "Romlp,and Rom2p are GDP/GTP exchange proteins(GEPs) for the Rholp small GTP-binding protein in Saccharomyces cerevisiae." EMBO J.(in press).
[文献書誌] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein-partial purification and characterization from rat brain." Oncogene. (in press).
[文献書誌] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. (in press).
[文献書誌] Komuro,R.: "Involment of rabphilin-3A in Ca2+-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.(in press).

1995 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] Kuroda,S.: "Purification and characterization of REKS from Xenopus eggs Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase" J.Biol.Chem.270. 2460-2465 (1995)

[文献書誌] Bourgoin,S.: "Low molecular weight GTP-binding Proteins in HL-60 granulocytes." J.Biol.Chem.270. 3172-3178 (1995)

[文献書誌] Yamamori,B.: "Purification of a Ras-dependent mitogen-activated Protein kimase kinase kinase from bovine brain cytosol and its identification as a complex of B-raf and 14-3-3 Proteins." J.Biol.Chem.270. 11723-11726 (1995)

[文献書誌] Chung,S-H.: "Evidence that the Rab3A-binding Protein,Rabphilin-3A,enhances requlated secretion-Studies in adrenal Chromaffin cells." J.Biol.Chem.270. 16714-16718 (1995)

[文献書誌] Kuribara,H.: "Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21,and its inhibition by clostridium botulinum C3 exoenzyme." J.Biol.Chem.270. 25667-25671 (1995)

[文献書誌] McGeady,P.: "The farnesyl group of H-ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase." J.Biol.Chem.270. 26347-26351 (1995)

[文献書誌] Gotoh,T.: "Identification of Rapl as a target for the Crk SH3 Domain-binding guanine nucleotide-releasing factor C3G." Mol.Cell.Biol.15. 6746-6753 (1995)

[文献書誌] Nonaka,H.: "A downstream target of RHO1 small GTP-binding protein is PKC1,a homolog of protein kinase C,which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae." EMBO J.14. 5931-5938 (1995)

[文献書誌] Takaishi,K.: "Translocation of activated Rho from the cytoplasm to membrane ruffling area,cell-cell adhesion sites and cleavage furrows." Oncogene. 11. 39-48 (1995)

[文献書誌] Nishimura,N.: "Cloning of a brain-type isoform of human rab GDI and its expression in human neroblastoma cell lines and tumor specimens." Cancer Res.55. 5445-5450 (1995)

[文献書誌] Orita,S.: "Doc2: a novel brain protein having two repeated C2-like domains." Biochem.Biophys.Res.Commun.206. 439-448 (1995)

[文献書誌] Araki,K.: "purif:cation and characterization of Rab GDIβ from rat brain." Biochem.Biophys.Res.Commun.211. 296-305 (1995)

[文献書誌] Takahashi,K.: "Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules." Biochem.Biophys.Res.Commun.217. 979-986 (1995)

[文献書誌] Sakaguchi,G.: "Molecular cloning of an isoform of Doc2 having two C2-like domains." Biochem.Biophys.Res.Commun.217. 1053-1061 (1995)

[文献書誌] Miyazaki,M.: "Rabphilin-3A binds to a Mr 115,000 polypeptide in a phosphatidylserine-and Ca2+-dependent manner." Mol.Brain.Res.28. 29-36 (1995)

[文献書誌] Regazzi,R.: "Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells-comparison with the effect of Rab3AL peptide." Biochim.Biophys.Acta. 1268. 269-278 (1995)

[文献書誌] Takai,Y.: "Rho as a regulator of the cytoskeleton." Trends in Biochemical Sciences. 20. 227-231 (1995)

[文献書誌] Ohtsuka,T.: "Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein." J.Biol.Chem.271. 1258-1261 (1996)

[文献書誌] Orita,S.: "Doc2 enhances Ca2+-dependent exocytosis from PC12 cells." J.Biol.Chem.(in press).

[文献書誌] Fujita,Y.: "phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C-Its implication in requlating the interaction of Munc-18/n-Sec1/rbSec1 with Syntaxin." J.Biol.Chem.(in press).

[文献書誌] Drgonova,J.: "Rholp a yeast protein at the juncture between cell polarization and morphogenesis." Science. (in press).

[文献書誌] Ozaki,K.: "Romlp,and Rom2p are GDP/GTP exchange proteins(GEPs) for the Rholp small GTP-binding protein in Saccharomyces cerevisiae." EMBO J.(in press).

[文献書誌] Matsuda,S.: "A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein-partial purification and characterization from rat brain." Oncogene. (in press).

[文献書誌] Taniguchi,T.: "Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers." Oncogene. (in press).

[文献書誌] Komuro,R.: "Involment of rabphilin-3A in Ca2+-dependent exocytosis from PC12 cells." Biochem.Biophys.Res.Commun.(in press).

1995 年度実績報告書

高井義美大阪大学, 医学部, 教授 (60093514)