1990 年度実績報告書

<ras>___ー p21類似GTP結合蛋白質のcDNAと抗体の有効利用,特に病態、診断への応用

研究課題

研究課題/領域番号	01870017
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) 佐野公彦神戸大学, 医学部, 助手 (40205993) 川原康洋神戸大学, 医学部, 助手 (80169755) 貝淵弘三神戸大学, 医学部, 助教授 (00169377)
キーワード	<ras>___ー p21 / 低分子量GTP結合蛋白質 / プレニル化 / GDP / GTP交換反応調節蛋白質 / GTPase活性化蛋白質 / 腫瘍マ-カ-
研究概要	私共は分子量2万余りの低分子量GTP結合蛋白質(G蛋白質)(cーKiー<ras>___ー p21,<rho>___ー A p21,<rho>___- B p21,<smg>___ー p21A,<smg>___ー p21B,<smg>___ー p25A)やその活性調節蛋白質を精整している。昨年度の本研究において、私共はこれらの活性調節蛋白質として、<smg>___ー p21に対するGTP ase活性促進蛋白質(GAP)および<smg>___ー p25Aに対するGDP解離抑制蛋白質(GDI)を見出した。本年度の研究において、私共は新しい種類の活性調節蛋白質であるGDP解離促進蛋白質(GDS)を<smg>___ー p21と<rho>___ー p21に対して見出した。さらに、<smg>___ー p21 GDSのcDNAを単離してその一次構造を明らかにするとともに特異抗体を作整した。今後、<smg>___ー p21 GDSのcDNAや特異抗体を用いて種々の病態と<smg>___ー p21 GDSとの関係を解析する必要がある。一方、<ras>___ー p21のC末端のCysーAーAーXのCys残基にファルネシル基が結合し、次いでAーAーX部分が除かれ、さらにCys残基がメチル化を受けることが明らかになっている。そこで、私共はC末端のアミノ酸配列がCysーAーAーXである<smg>___ー p21Bと<rho>___ー A p21の翻訳後修飾を解析して、両低分子量G蛋白質のC末端側のCys残基にはファルネシル基とは異なるゲラニルゲラニル基が結合し、AーAーX部分は取り除かれ、Cys残基がメチル化を受けることを明らかにした。また、<smg>___ー p25AのC末端側はCysーXーCysと<smg>___ー p21とは異なっているが、少なくとも一方のcys残基にはゲラニルゲラニル基が結合していることも明らかにした。さらに、これらの翻訳後修飾が低分子量G蛋白質の細胞内局在や、GDIやGDSなどの調節蛋白質との結合に重要な役割を果たすことを示した。また、種々の組織中のファルネシルトランスフェラ-ゼやゲラニルゲラニルトランスフェラ-ゼ活性を測定するアッセイ方法を開発した。これらの酵素活性の変動が種々の病態と関連している可能性があり、今後、この点について解析を進めていく予定である。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
(36件)

すべてその他

すべて文献書誌 (36件)

[文献書誌] Fukumoto,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] Mizoguchi,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/rap1/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] Matsui,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] Yamamoto,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/rap1 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 farnesylーtransferase for ras p21s." Biochem.Biophys.Res.Commun.(1991)

1990 年度 実績報告書

<ras>___ー p21類似GTP結合蛋白質のcDNAと抗体の有効利用,特に病態、診断への応用

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] Fukumoto,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] Mizoguchi,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/rap1/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] Matsui,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] Yamamoto,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] 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/rap1 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 farnesylーtransferase for ras p21s." Biochem.Biophys.Res.Commun.(1991)

1990 年度実績報告書

高井義美神戸大学, 医学部, 教授 (60093514)