細胞増殖シグナル伝達の機構と癌遺伝子産物の作用

• Project/Area Number: 01015067
• Research Category: Grant-in-Aid for Cancer Research
• Allocation Type: Single-year Grants
• Research Institution: Kobe University
• Principal Investigator: 高井 義美 神戸大学, 医学部, 教授 (60093514)
• Co-Investigator(Kenkyū-buntansha): 溝口 明 神戸大学, 医学部, 講師 (90181916) ; 菊池 章 神戸大学, 医学部, 助手 (10204827) ; 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, 講師 (00169377)
• Project Period (FY): 1989
• Project Status: Completed (Fiscal Year 1989)
• Budget Amount: ¥13,000,000 (Direct Cost: ¥13,000,000); Fiscal Year 1989: ¥13,000,000 (Direct Cost: ¥13,000,000)
• Keywords: cーfos / ras p21 / cーrafー1蛋白質 / Cキナーゼ / Aキナーゼ / GAP

Research Abstract

本年度は当初の目的に従い、まず細胞性癌遺伝子による遺伝子の転写制御と細胞内伝達系の関係を解明するため、cーfos遺伝子の5′側上流領域、血清反応性エレメント、cAMP反応性エレメント、TPA反応性エレメントをクロラムフェニコール・アセチルトランスフェラーゼやルシフェラーゼ発現ベクターに組み込み、癌遺伝子産物であるras p21や活性型のcーrafー1蛋白質による転写制御について検討した。その結果、ras p21、cーrafー1蛋白質はいずれもCキナーゼ、Aキナーゼとは独立に血清反応性エレメントを介してcーfos遺伝子の転写を誘導することが明らかになった。また、私共はこれまでウシ大脳、ヒト血小板から10数種類のras p21類似低分子量GTP結合蛋白質を分離し5種類(rho AP21・rhoB p20、Kiーras p21、smg p21、smg P25A)を均一蛋白質にまで精製しているが、本年度はこれらのraf P21類似低分子量GTP結合蛋白質の作用機構を解明するため、これらの蛋白質と直接作用する蛋白質の同定を試みた。すでにras p21のGTPase活性を促進する蛋白質GTPaseーactivating protein(GAP)が精製されているが、私共はsmg p21に特異的に作用するGAPをウシ大脳・ヒト血小板可溶性画分から、rhoB p20に特異的に作用するGAPをウシ大脳可溶性画分から分離することに成功した。さらにsmg p25Aに対してはGDP結合型に作用してGDPの解離を抑制し、GDP結合型からGTP結合型への転換を阻害する全く新しいタイプの調節蛋白質をウシ大脳から精製し、GDP dissociation inhibitor(GDI)と命名した。rhoB p20に対しても同じタイプの調節蛋白質をウシ大脳から精製した。このように、ras p21類似低分子量GTP結合蛋白質にはそれぞれ特異的なGAPやGDI等の調節蛋白質が存在し、細胞増殖、癌化機構に関わる巨大な情報伝達系を形成していることが明らかになった。以上、本年度は予想以上の成果をあげた。

Research Products

• [Publications] Ohmori,T.: "Small molecular weight GTP-binding proteins in human platelet membranesーーーPurification and characterization of a novel GTPーbinding protein with a molecular weight of 22,000." J.Biol.Chem.264. 1877-1881 (1989)
• [Publications] Kikuchi,A.: "Purification and characterization from bovine brain cytosol of two GTPaseーactivating proteins specific for smg p21,a GTPーbinding protein having the same effector domain as cーras p21s." J.Biol.Chem.264. 9133-9136 (1989)
• [Publications] Kaibuchi,K.: "Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C." J.Biol.Chem.264. 13489-13496 (1989)
• [Publications] Kawata,M.: "Phosphorylation of smg p21,a ras p21ーlike GTPーbinding protein,by cyclic AMP-dependent protein kinase in a cellーfree system and in response to prostaglandin E_1 in intact human platelets." J.Biol.Chem.264. 15688-15695 (1989)
• [Publications] Kaibuchi,K.: "Activation of the serum-response element and TPA-response element by the activated c-raf-1 protein in a manner independent of protein kinase C." J.Biol.Chem.264. 20855-20858 (1989)
• [Publications] Adachi,M.: "Elevation of intracellular calcium ion by prostaglandin E_1 and its inhibition by protein kinase C in human megakaryocyte leukemia cell line." Cancer Res.49. 3805-3808 (1989)
• [Publications] Mizoguchi,A.: "Localization and subcellular distribution of cellular ras gene products in rat brain." Mol.Brain Res.5. 31-44 (1989)
• [Publications] Kim,S.: "Intrasynaptosomal distribution of the ras,rho and smg-25A GTP-binding proteins in bovine brain." Mol.Brain Res.6. 167-176 (1989)
• [Publications] Ueda,T.: "GTPase activating proteins for the smg-21 GTP-binding protein having the same effector domain as the ras proteins in human platelets." Biochem.Biophys.Res.Commun.159. 1411-1419 (1989)
• [Publications] Shoji,I.: "Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A." Biochem.Biophys.Res.Cmmun.162. 273-281 (1989)
• [Publications] Mizoguchi,A.: "Tissue distribution of smg p25A,A ras p21ーlike GTPーbinding protein,studied by use of a specific monoclonal antibody." Biochem.Biophys.Res.Commun.162. 1438-1445 (1989)
• [Publications] Kuroda,S.: "Kinetic analysis of the binding of guanine nucleotides to bovine brain rhoB p20,a ras p21-like GTP-binding protein." Biochem.Biophys.Res.Commun.163. 674-681 (1989)
• [Publications] Kawata,M.: "Identification of a major GTP-binding protein in bovine aortic smooth muscle membranes as smg p21,a GTP-binding protein having the same effector domain as ras p21s." Biochem.Biophys.Res.Commun.163. 1418-1427 (1989)
• [Publications] Ohga,N.: "Rabbit intestine contains a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to rhoB p20,a ras p21-like GTP-binding protein." Biochem.Biophys.Res.Commun.163. 1523-1533 (1989)
• [Publications] Yamamoto,K.: "Small molecular weight GTP-binding proteins and signal transduction." Clinica.Chimica.Acta.185. 347-356 (1989)
• [Publications] Fukumoto,Y.: "Activation of the c-fos serum-response element by the activated c-Ha-ras protein in a manner independent of protein kinase C and cAMP-dependent protein kinase." J.Biol.Chem.265. 774-780 (1990)
• [Publications] 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-like GTP-binding protein." J.Biol.Chem.265. 2333-2337 (1990)
• [Publications] Hoshijima,M.: "Purification and characterization from bovine brain membranes of a GTP-binding protein with a Mr of 21,000 ADP-ribosylated by an ADP-ribosyltransferase contained in botulinum toxin type C1ーーーIdentification as the rhoA gene product." Mol.Brain Res.7. 9-16 (1990)
• [Publications] 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)
• [Publications] Hata,Y.: "Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s." J.Biol.Chem.(1990)
• [Publications] Yamamoto,J.: "A GTPase-activating protein(GAP)for rhoB p20,a ras p21-like GTP-binding proteinーーーPartial purification,characterization and subcellular distribution in rat brain." Mol.Brain Res.(1990)
• [Publications] Kim,S.: "Tissue and subcellular distributions of smg p21s,ras p21-like GTP-binding proteins having the same putative effector domain as ras p21s,partly distinct from those of c-ras p21s." Mol.Cell.Biol.(1990)