細胞内情報伝達系と細胞増殖の制御機構

• 研究課題/領域番号: 02262225
• 研究種目: 重点領域研究
• 配分区分: 補助金
• 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: 河田 正仁 神戸大学, 医学部, 助手 (20224785) ; 菊池 章 神戸大学, 医学部, 助手 (10204827) ; 貝淵 弘三 神戸大学, 医学部, 助教授 (00169377)
• 研究期間 (年度): 1990
• 研究課題ステータス: 完了 (1990年度)
• 配分額: 25,000千円 (直接経費: 25,000千円); 1990年度: 25,000千円 (直接経費: 25,000千円)
• キーワード: ras p21 / smg p21 / ゲラニルゲラニル基 / GDP解離促進蛋白質 / GTPase活性促進蛋白質 / Aキナ-ゼ

研究概要

本年度の研究において、私共はras p21類似低分子量GTP結合蛋白質の中でも、ras p21と同じエフェクタ-領域を有しているsmg p21Bに焦点をあてて解析を進めた。smg p21BのC末端のアミノ酸配列はras p21に類似してCysーAーAーXであるが、このCysにはras p21と異なり、ファルネシル基の代わりにゲラニルゲラニル基が結合していることを明らかにした。また、AーAーXの部分は取り除かれ、Cysのカルボキシル基はメチル化されていた。これらの翻訳後修飾はras p21と同様にsmg p21Bの細胞内局在と機能発現に必須であることを明らかにした。一方、私共はsmg p21のGDP/GTP交換反応を促進する蛋白質であるGDP解離促進蛋白質(GDS)を均一にまで精製し、そのcDNAをクロ-ニングして全一次構造を決定した。smg GDSはsmg p21Bの細胞膜からの解離を促進した。また、Aキナ-ゼはsmg p21Bの179番目のセリンをリン酸化するが、リン酸化されたsmg p21Bはsmg GDSとの親和性が高まることが明らかになった。私共は昨年度、smg p21のGTPase活性促進蛋白質(GAP)を部分精製した。したがって、smg p21はAキナ-ゼ、smg GDS、smg p21 GAPによりその活性が制御され、さらに細胞膜への結合も制御されることが明らかとなった。最近、ras p21がチロシンキナ-ゼの下流に位置していると報告されている。私共はsmg p21がras p21GAPを抑制することを明らかにした。したがって、smg p21の標的蛋白質の1つはras p21 GAPであり、smg p21はras p21GAPの活性を抑制することにより、チロシンキナ-ゼ系の作用を促進すると考えられる。このように、smg p21は種々の細胞内情報伝達系のクロスト-クに重要であると考えられる。以上の成績を得たことにより本年度の研究計画はほぼ達成することができた。

研究成果

• [文献書誌] Fukumot,Y.: "Activation of the cーfos serumーresponse element by the activated cーHaーras protein in a manner independent of protein kinase C and cAMPーdependent protein kinase." J.Biol.Chem.265. 774-780 (1990)
• [文献書誌] Sasaki,T.: "Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A,a ras p21ーlike GTPーbinding protein." J.Biol.Chem.265. 2333-2337 (1990)
• [文献書誌] Hata,Y.: "Inhibition of the ras p21 GTPaseーactivating proteinーstimulated GTPase activity of cーHaーras p21 by smg p21 having the same putative effector domain as ras p21s." J.Biol.Chem.265. 7104-7107 (1990)
• [文献書誌] Ueda,T.: "Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20,a ras p21ーlike GTPーbinding protein." J.Biol.Chem.265. 9373-9380 (1990)
• [文献書誌] Mizoguch,A.: "Localization and subcellular distribution of smg p25A,a ras p21ーlike GTPーbinding protein,in rat brain." J.Biol.Chem.265. 11872-11879 (1990)
• [文献書誌] Araki,S.: "Regulation of reversible binding of smg p25A,a ras p21ーlike GTPーbinding protein,to synaptic plasma membranes and vesicles by its specific regulatory protein,GDP dissociation inhibitor." J.Biol.Chem.265. 13007-13015 (1990)
• [文献書誌] Yamamoto,T.: "Purification and characterization from bovine brain cytosol of proteins that regulate the GDP/GTP exchange reaction of smg p21s,ras p21ーlike GTPーbinding proteins." J.Biol.Chem.265. 16626-16634 (1990)
• [文献書誌] Kim,S.: "Tissue and subcellular distributions of the smg 21/rapl/ Krevー1 proteins which are partly distinct from those of cーras p21s" Mol.Cell.Biol.10. 2645-2652 (1990)
• [文献書誌] Matsiu,Y.: "Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A,a ras p21ーlike GTPーbinding protein." Mol.Cell.Biol.10. 4116-4122 (1990)
• [文献書誌] Hoshijima,M.: "Purification and characterization from bovine brain membranes of a GTPーbinding protein with a Mr of 21,000 ADPーribosylated by an ADPーribosyltransferase contaminated in botulinum toxin type C1ーidentification as the rhoA gene product." Mol.Brain.Res.7. 9-16 (1990)
• [文献書誌] Yamaomo,J.: "A GTPaseーactivating protein for rhoB p20,a ras p21ーlike GTPーbinding proteinーpartial purification,characterization and subcellular distribution in rat brain." Mol.Brain.Res.8. 105-111 (1990)
• [文献書誌] Sano,K.: "Differential expression of the Nーmyc,cーfos,and smg p25A genes in human neuroblastoma cells during neuronal and Schwannian differentiation." Mol.Brain.Res.9. 149-152 (1990)
• [文献書誌] Kawata,M.: "Posttranslationally processed structure of the human platelet protein smg p21B:Evidence for geranylgeranylation and carboxyl methylation of the Cーterminal cysteine." Proc.Natl.Acad.Sci.USA. 87. 8960-8964 (1990)
• [文献書誌] Sano,K.: "Expression of the smg p25A (a ras p21ーlike GTPーbinding protein) gene in human neuroblastoma cell lines and tumor tissues." Cancer Res.50. 7242-7245 (1990)
• [文献書誌] Hori,Y.: "Activation of the serumーresponse and TPAーresponse elements by expression of the vーabl protein:comparison of the mode of action of the vーabl protein with those of protein kinase C,cyclic AMPーdependent protein kinase,and the activated cーraf protein." Oncogene. 5. 1201-1206 (1990)
• [文献書誌] Fukumoto,Y.: "Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins,ras p21ーlike small GTPーbinding proteins." Oncogene. 5. 1321-1328 (1990)
• [文献書誌] Matsui,Y.: "Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B." Biochem.Biophys.Res.Commun.166. 1010-1016 (1990)
• [文献書誌] Fujioka,H.: "A small GTPーbinding protein (G protein) recognized by smg p25A GDP dissociation inhibitor (GDI) in human platelet membranes and GDI for this small G protein in human platelet cytosol." Biochem.Biophys.Res.Commun.168. 1244-1252 (1990)
• [文献書誌] Isomura,M.: "Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol." Biochem.Biophys.Res.Commun.169. 652-659 (1990)
• [文献書誌] Ohmori,T.: "Purification and characterization of a novel GTPーbinding protein with a M_r value of 24,000 from rat liver." Biochem.Biophys.Res.Commun.169. 816-823 (1990)
• [文献書誌] Kawahara,Y.: "Identification of a major GTPーbinding protein in bovine aortic smooth muscle cytosol as the rhoA gene product." Biochem.Biophys.Res.Commun.170. 673-683 (1990)
• [文献書誌] Oku,N.: "Comparison of the mode of action of ras p21 with those of protein kinases A and C in the stimulation of gene expression in NIH/3T3 cells." FEBS.Lett.269. 148-152 (1990)
• [文献書誌] Hiroyoshi,M.: "Role of the Cーterminal region of smg p21,a ras p21ーlike small GTPーbinding protein,in membrane and smg p21 GDP/GTP exchange protein interactions." J.Biol.Chem.266. 2962-2969 (1991)
• [文献書誌] Hata,Y.: "Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase Aーcatalyzed phosphorylation of smg p21." J.Biol.Chem.(1991)
• [文献書誌] Katayama,M.: "The postーtranslationally modified Cーterminal structure of bovine aortic smooth muscle rhoA p21." J.Biol.Chem.(1991)
• [文献書誌] Araki,S.: "Role of the Cーterminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein." Mol.Cell.Biol.(1991)
• [文献書誌] Sasaki,T.: "A mammalian inhibitory GDP/GTP exchange protein (GDI) for smg p25A is active on the yeast SEC4 protein." Mol.Cell.Biol.(1991)
• [文献書誌] Kaibuchi,K.: "Molecular cloning and characterization of the stimulatory GDP/GTP exchange protein (GDS) for smg p21s,ras p21ーlike small GTPーbinding proteins." Mol.Cell.Biol.(1991)
• [文献書誌] Isomura,M.: "Regulation of binding of rhoB p20 to membranes by its specific regulatory protein,GDP dissociation inhibitor." Oncogene. (1991)
• [文献書誌] Hori,Y.: "Postーtranslational modifications of the Cーterminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins." Oncogene. (1991)
• [文献書誌] Kawata,M.: "The molecular heterogeneity of the smgー21/Krevー1/rapl proteins,a GTPーbinding protein having the same effector domain as ras p21s,in bovine aortic smooth muscle membranes." Oncogene. (1991)
• [文献書誌] Ueda,T.: "Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24KG,a ras p21ーlike GTPーbinding protein,with properties similar to those of smg p25A GDI." Biochemistry. (1991)
• [文献書誌] Motoike,T.: "Expression of smg p25A,a ras p21ーlike small GTPーbinding protein,during postnatal development of rat cerebellum." Dev.Brain Res.(1991)
• [文献書誌] Kawamura,S.: "Stoichiometric interaction of smg p21 with its GDP/GTP exchange protein and its novel action to regulate the translocation of smg p21 between membrane and cytoplasm." Biochem.Biophys.Res.Commun.(1991)
• [文献書誌] Shimizu,K.: "Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for the rho protein by use of its specific antibody." Biochem.Biophys.Res.Commun.(1991)
• [文献書誌] Yoshida,Y.: "A geranylgeranyltransferase for rhoA p21 distinct from the farnesyltransferase for ras p21s." Biochem.Biophys.Res.Commun.(1991)