低分子量G蛋白質による細胞の増殖、運動の制御機構

• 研究課題/領域番号: 09254103
• 研究種目: 特定領域研究(A)
• 配分区分: 補助金
• 研究機関: 広島大学
• 研究代表者: 菊池 章 広島大学, 医学部, 教授 (10204827)
• 研究分担者: 松田 道行 国立国際医療センター研究所, 臨床病理研究部, 部長 (10199812) ; 服部 成介 国立精神神経センター, 神経研究所・診断研究部, 室長 (50143508) ; 佐藤 孝哉 東京工業大学, 生命理工学部, 寄附講座教員 (20251655) ; 大矢 禎一 東京大学, 大学院・新領域創成科学研究科, 教授 (20183767) ; 松井 泰 東京大学, 大学院・理学系研究科, 助手 (50229407) ; 金保 安則 東京工業大学, 生命理工学部, 助教授 (00214437) ; 東江 昭夫 東京大学, 大学院・理学系研究科, 教授 (90029249)
• 研究期間 (年度): 1997 – 1999
• 研究課題ステータス: 完了 (1999年度)
• 配分額: 92,000千円 (直接経費: 92,000千円); 1999年度: 32,000千円 (直接経費: 32,000千円); 1998年度: 32,000千円 (直接経費: 32,000千円); 1997年度: 28,000千円 (直接経費: 28,000千円)
• キーワード: 発がん / 転移 / 低分子量G蛋白質 / Ras / Rho / 酵母 / Ral / RasGRF1 / GAP1m / CalDAGI / cAMP-GEF / PLD / 細胞増殖 / 細胞運動

研究概要

低分子量G蛋白質RasとRhoの細胞増殖や運動における役割を解析した。Ras/RalGDS/Ral/RalBP1/POB1の下流にEps15とEpsinが存在して、インスリンとEGF依存性の受容体のエンドサイトーシスを制御した。RasのGDP/GTP交換反応促進蛋白質Ras-GRF1が、G蛋白質共役型受容体刺激によりチロシンリン酸化され、DHドメインを介してRacのGDP/GTP交換反応を促進した。SH2領域およびRasGRF相同領域をもつ新規因子ChatはCasに結合し、MAPキナーゼによりリン酸化された。Rap1の不活性化因子Rap1GAPのファミリーである新規蛋白質Rap1GAPIIのN末端側のGolocoモチーフが三量体G蛋白質Giのαサブユニットに刺激依存性に結合し、Rap1を不活化した。Rac1依存性のラッフル膜形成にARF6とPI(4)P5Kαが関与した。酵母Rho3の不活性型と結合する蛋白質として単離したRab型G蛋白質Ypt11は、Rho3の標的蛋白質Myo2とも結合し、Myo2の活性がYpt11により制御された。翻訳後修飾を受けている酵母Rho1はその標的蛋白質であるグルカン合成酵素を活性化すると共にFks1とも効率よく結合した。酵母Rho1の標的タンパク質でフォルミンファミリーに属するBNI1が細胞膜表面の極性マーカーとして働き、細胞質微小管と相互作用して核分裂の方向を制御した。
これらの結果から、低分子量G蛋白質のGDP/GTP交換反応促進蛋白質(RasGRF,C3G,Dbl)がチロシンキナーゼにより活性化されることが明らかになった。また、低分子量G蛋白質間の制御機構が酵母から哺乳動物細胞まで普遍的に存在すると考えられた。さらに、Rho1がアクチン系と微小管系の両者を制御する可能性が示唆された。

研究成果

• [文献書誌] Nakashima,S.: "Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors"EMBO J.. 18. 3629-3642 (1999)
• [文献書誌] Sawamoto,K.: "The Drosophila Ral GTPase regulates developmental cell shape changes through the Jun NH_2-terminal kinase pathway"J.Cell Biol.. 146. 361-372 (1999)
• [文献書誌] Fukata,M.: "Cdc42 and Racl regulate the interaction of IQGAP1 with β-catenin"J.Biol.Chem.. 274. 26044-26050 (1999)
• [文献書誌] Matsubara,K.: "Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation"Oncogene. 18. 1303-1312 (1999)
• [文献書誌] Kiyono,M.: "G protein βγsubunit-dependent Rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25Mm"Proc.Natl.Acad.Sci.USA. 96. 4826-4831 (1999)
• [文献書誌] Nishida,K.: "Anti-apoptotic function of Rac in hematopoietic cells"Oncogene. 18. 407-415 (1999)
• [文献書誌] Sakai,T.: "Mouse semaphorin H induces PC12 cell neurite outgrowth activating Ras-mitogen-activated protein kinase signaling pathway via Ca^<2+> influx"J.Biol.Chem.. 274. 29666-29671 (1999)
• [文献書誌] Sakakibara,A.: "Chat, a Cas/HEF1-associated adaptor protein that integrates multiple signaling pathways"J.Biol.Chem.. 275. 6404-6410 (2000)
• [文献書誌] Mochizuki,N.: "Activation of ERK/MAPK pathway by an isoform of rap1GAP associated with Gαi"Nature. 400. 891-894 (1999)
• [文献書誌] Ichiba,T.: "Activation of C3G guanine nucleotide exchange factor for Rap1 by phosphorylation of tyrosine 504"J.Biol.Chem.. 274. 14376-14381 (1999)
• [文献書誌] Inoue,S.B.: "Prenylation of Rholp is required for activation of yeast 1, 3-β-glucan synthase"J.Biol.Chem.. 274. 38119-38124 (1999)
• [文献書誌] Kikyo,M.: "An FH domain-containing Bnrlp is a multifunctional protein interacting with a variety of cytoskeletal proteins in Saccharomyces cerevisiae"Oncogene. 18. 7046-7054 (1999)
• [文献書誌] Robinson,N.G.: "Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70"Mol.Cell.Biol.. 19. 3580-3587 (1999)
• [文献書誌] Fujiwara,T.: "The formin Bnilp regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae"Mol.Cell.Biol.. 19. 8016-8027 (1999)
• [文献書誌] Tanaka,K.: "Formin family proteins in cytoskeletal control"Biochem.Biophys.Res.Commun.. 267. 479-481 (2000)
• [文献書誌] Honda,A.: "Phosphatidylinositol 4-phosphate 5-kinase a is a downstream effector of the small G protein ARF6 in membrane ruffle formation"Cell. 99. 521-532 (1999)
• [文献書誌] Yamazaki,M.: "Interaction of the small G protein RhoA with the C terminus of human phospholipase D1"J.Biol.Chem.. 274. 6035-6038 (1999)
• [文献書誌] Ikeda,M.: "Identification and characterization of a novel protein interacting with Ral-binding proteinl,a putative effector protein of Ral." J.Biol.Chem.273・2. 814-821 (1998)
• [文献書誌] Shirouzu,M.: "Interactions of the amino acid residues at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS." J.Biol.Chem.273・13. 7737-7742 (1998)
• [文献書誌] Deng,J.: "Involvement of Ras in Bruton's tyrosine kinase-mediated JNK activation." J.Biol.Chem.273・27. 16787-16791 (1998)
• [文献書誌] Jiang,Y.: "The G protein G12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain" Nature. 395・6704. 808-813 (1998)
• [文献書誌] Matsumura,I.: "Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiantion of a human factor-dependent hematopoietic cell line." Mol.Cell.Biol. 18・7. 4282-4290 (1998)
• [文献書誌] Hashimoto,Y.: "Phosphorylation of CrkII adaptor protein at Tyrosine 221 by epidermal growth factor receptor." J.Biol.Chem.273・27. 17186-17191 (1998)
• [文献書誌] Okada,S.: "Insulin regulates the dynamic balance batween Ras and Rapl signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes." EMBO J.17・9. 2554-2565 (1998)
• [文献書誌] Kawasaki,H.: "A family of cAMP-binding proteins that directly activate Rapl." Science. 282・5397. 2275-2279 (1998)
• [文献書誌] Caplin B.E.: "Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase.creating the perfect farnesyltransferase" J.Biol.Chem.273・16. 9472-9479 (1998)
• [文献書誌] Homma,K.: "Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis." J.Biol.Chem.273・25. 15779-15786 (1998)
• [文献書誌] Okano,H.: "Importance of phenylalanine residues of yeast calmodulin for target binding and activation." J.Biol.Chem.273・41. 26375-26382 (1998)
• [文献書誌] Kagami,M.: "Sro7p,a Saccharomyces cerevisiae counterpart of the tumor suppressor l (2) g1,is functionally realted to myosines." Genetics. 149・4. 1717-1727 (1998)
• [文献書誌] Yokozeki,T.: "Purification and characterization of phosphatidic acid-regulated protein kinase and its substrate,p29." J.Neurochem.71・1. 410-417 (1998)
• [文献書誌] Matsubara,K.: "Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral Activation." Oncogene. 18・6. 1303-1312 (1999)
• [文献書誌] Nishida,K.: "Anti-apoptotic function of Rac in hematopoietic cells." Oncogene. 18・2. 407-415 (1999)
• [文献書誌] Sawamoto,K.: "Ectopic expression of mutationally-activated RalGTPase inhibits cellshape changes during Drosophila eye development." Oncogene. (in press). (1999)
• [文献書誌] Robinson,N.: "Rho3 of Saccharomyces cerevisiae,which regulates the actin cytoskeleton and exocytosis,is a GTPase which interacts with Myo2 and Exo70." Mol.Cell.Biol.(in press). (1999)
• [文献書誌] Dohke,Y.: "Translocation of Arfl to the secretory granules in rat parotid acinar cells." Arch.Biochem.Biophys.(in press). (1999)
• [文献書誌] Murai,H.: "Characteriation of Ral GDP dissociation stimulator like(RGL)activities to regulate c-fos promoter and the GDP/GTP exchange of Ral." J.Biol.Chem.272・16. 10483-10490 (1997)
• [文献書誌] Fukata,M.: "Regulation of cross-linking of actin filament by IQGAP1,a target for CDC42." J.Biol.Chem.272・47. 29579-29583 (1997)
• [文献書誌] Okazaki,M.: "Synergistic activation of c-fos promoter activity by Ral GDP dissociation stimulator and Raf." Oncogene. 14・5. 515-521 (1997)
• [文献書誌] Kishida,S.: "Colocalization of Ras and Ral on the membrane is required for Ras-dependent Ral activation through Ral GDP dissociation stimulator." Oncogene. 15・24. 2899-2907 (1997)
• [文献書誌] Matsubara,K.: "The post-translational modifications of Ral and Rac are important for the action of Ral-binding protein 1,a putative effector protein of Ral." FEBS Lett.410・2,3. 169-174 (1997)
• [文献書誌] Ikeda,M.: "Identification and Characterization of a novel protein which interacts with RalBP1,a putative effector protein of Ral." J.Biol.Chem.273・2. 814-821 (1998)
• [文献書誌] Ikeda,S.: "Axin,a negative regulator of the Wnt signaling pathway,makes a complex with GSK-β and β-catenin and promotes GSK-3β-dependentβ-catenin phosphorylations." EMBO J.17・5. 1371-1384 (1998)
• [文献書誌] Shirouzu,M.: "Interactions of the amino acid residues at position 31 on the c-Ha-Ras Protein with Raf-1 and RalGDS." J.Biol.Chem.(in press). (1998)
• [文献書誌] Yamamoto,H.: "Axil,a member of Axin family,interacts both GSK-3β and β-catenin and inhibits axis formatio of Xenopus embryo." Mol.Cell.Biol.(in press). (1998)
• [文献書誌] Kishida,S.: "Axin,a negative regulator of the Wnt signaling pathway pathway,directly interacts with APC and regulates the stabilization of β-catenin." J.Biol.Chem.(in press). (1998)
• [文献書誌] Terada,K.: "Ras-dependent activation of c-Jun N-terminal kinase/stress-activated protein kinase in response to interleukin-3 stimulation in hematopoietic BaF3 cells." J.Biol.Chem.272・7. 4544-4548 (1997)
• [文献書誌] Gotoh,T.: "Activation of R-Ras by Ras-guanine nucleotide-releasing factor." J.Biol.Chem.272・30. 18602-18607 (1997)
• [文献書誌] Li,S.: "Activation of R-Ras GTPase by GTPase-activation proteins for Ras,Gap1m and p120 GAP." J.Biol.Chem.272・31. 19328-19332 (1997)
• [文献書誌] Kominami,K.: "Yeast counterparts of subunits S5a and p58(S3)of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1." Mol.Biol.Cell.8・1. 171-187 (1997)
• [文献書誌] Uesono,Y.: "Ssd1 of Saccharomyces cerevisiae associates with RNA." J.Biol.Chem.272・26. 16103-16109 (1997)
• [文献書誌] Kawasaki,M.: "Identification of three core regions essential for protein splicing of the yeast Vma1 protopzyme." J.Biol.Chem.272・25. 15668-15674 (1997)
• [文献書誌] Takita,Y.: "Applications of the long and accurate polymerase chain reaction method in yeast molecular biology : Direct sequencing of the amplified DNA and its introduction into yeast." Yeast. 13・8. 763-768 (1997)
• [文献書誌] Tanaka,K.: "A target of phosphatidylinositol 3,4,5-trisphosphate with a Zinc finger motif similar to that of the ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains." Eur.J.Biochem.245・2. 512-519 (1997)