Signal Transduction and Cellular Functions of the Small GTPase Rho and its Effectors

• Project/Area Number: 08102007
• Research Category: Grant-in-Aid for Specially Promoted Research
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: NARUMIYA Shuh Kyoto University, Department of Pharmacology, Professor, 医学研究科, 教授 (70144350)
• Co-Investigator(Kenkyū-buntansha): 石崎 敏理 京都大学, 医学研究科, 助手 (70293876) ; 斎藤 雄二 京都大学, 医学研究科, 助手 (40263090)
• Project Period (FY): 1996 – 2000
• Project Status: Completed (Fiscal Year 2000)
• Budget Amount: ¥305,000,000 (Direct Cost: ¥305,000,000); Fiscal Year 2000: ¥42,000,000 (Direct Cost: ¥42,000,000); Fiscal Year 1999: ¥49,000,000 (Direct Cost: ¥49,000,000); Fiscal Year 1998: ¥47,000,000 (Direct Cost: ¥47,000,000); Fiscal Year 1997: ¥68,000,000 (Direct Cost: ¥68,000,000); Fiscal Year 1996: ¥99,000,000 (Direct Cost: ¥99,000,000)
• Keywords: Small GTPase Rho / cell adhesion / cell motility / cytokinesis / ROCK kinase / mDia / actin / microtubules / Rho / アクチン細胞骨格 / ECT-2 / アクリン突起 / Pho / ストレス・ファイバー / LIMキナーゼ / コフィリン / 精子 / citronキナーゼ / 神経突起 / がん浸潤 / がん化 / シナプス / 低分子量GTP結合蛋白質Rho / p160ROCK / ROCK阻害薬 / Na-H交換体 / 蛋白質リン酸化酵素 / 低分子量G蛋白質 Rho / 細胞接着斑 / ストレス ファイバー / アクチン重合 / p140mDia / インテグリン

Research Abstract

Effectors for the small GTPase Rho were determined, and signal tranuction pathways from Rho to the reorganization of the actin cytoskeleton were identified. (1) We identified that ROCK-kinase, that is activated downstream of Rho, moblizes at least three pathways to regulate the actin cytoskeleton, one being phsporylaiton and inacitvation of myosin phosphatas to increase the myosin-based contractility, the second phosphorylaiton and acivaton of LIM-linase leading to inactivation of actin depolymerizing activity of cofilin, and the third phosphorylation of type I NaH exchanger. We further showed that these Rho-ROCK pathways are impliated in elicitation of hypertension and invasion and metastasis of tumor cells. (2) We identified p140mDia as a novel Rho effector protein. This protein binds profiin through its FH1 region and regulate alignment of microtubules through its FH2 region, and thereby integrate the organization of the actin cytoskeleton and microtubules. We showed that this action of mDia is essential in Rho-induced inducion and alignment of stress fibers. (3) We identified Citron-kinase, and showed that it works as a Rho effector in cytokinesis. We heve also developed a pull-down assay for GTP-Rho, and showed that ECT2 is a Rho exchanger working in cytokinesis. (4) We identified ropporin as a binding protein of rhophilin, and showed that the two proteins together localize in the sperm tail. We also showed that ropporin makes a homo dimer and binds to another sperm protein, AKAP-110.

Research Products

• [Publications] Uehata,M.et al: "Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension"Nature. 389・6654. 990-994 (1997)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Madaule,P. et al.: "Role of citron kinase as a target of the small GTPase Rho in cytokinesis"Nature. 394・6692. 491-494 (1998)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Itoh,K. et al: "An essential part for Rho-associated kinase in the transcellullar invasion of tumor cells"Nature Medicine. 5・2. 221-225 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Maekawa,M. et al.: "Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase"Science. 285. 895-898 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Watanabe N., et al: "Cooperation between mDia 1 and ROCK in Rho-induced actin reorganization"Nature Cell Biology. 1・3. 136-143 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Ishizaki T., et al: "Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia 1"Nature Cell Biology. 3-1. 8-14 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Uehata, M.et al.: "Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension"Nature. 389(-6654). 990-994 (1997)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Madaule, P.et al.: "Role of citron kinase as a target of the small GTPase Rho in cytokinesis"Nature. 394(-6692). 491-494 (1998)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Itoh, K.et al.: "An essential part for Rho-associated kinase in the transcellular invasion of tumor cells"Nature Medicine. 5(-2). 221-225 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Maekawa, M.et al.: "Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase"Science. 285. 895-898 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Watanabe, N.et al: "Cooperation between mDia 1 and POCK in Rho-induced actin reorganization"Nature Call Biology. 1(-3). 136-143 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ishizaki, T.et al: "Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia 1"Nature Cell Biology. 3(-1). 8-14 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Ishizaki,T. et al.: "Coordination of microtubules and the actin cytoskeleton by the Rho effector mDia 1."Nature Cell Biology. 3-1. 8-14 (2001)
• [Publications] Kato,T. et al.: "Localization of a mammalian homolog of diaphanous, mDia 1, to the mitotic spindle in HeLa cells."Journal of Cell Science. 114. 775-784 (2001)
• [Publications] Bito,H. et al.: "A critical role for a Rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons."Neuron. 26-2. 431-441 (2000)
• [Publications] Kimura,K. et al.: "Accumulation of GTP-bound RhoA during cytokinesis and a critical role of ECT2 in this accumulation."The Journal of Biological Chemistry. 275-23. 17233-6 (2000)
• [Publications] Kosako,H. et al.: "Rho-kinase/ROCK is in volvedin cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow."Oncogene. 19-52. 6059-64 (2000)
• [Publications] Amano,T. et al: "LIM-kinase 2 induces formation of stress fibers, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505."Biochemical Journal. 354. 149-159 (2001)
• [Publications] Watanabe, N. et al: "Molecular basis of the activation of mDial (p140mDia) and its cooperation with ROCK in Rho induced actin reorganization"Nature Cell Biology. 1. 130-135 (1999)
• [Publications] Maekawa M. et al: "Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase"Science. 285. 895-898 (1999)
• [Publications] Ohashi K. et al: "Rho-associated kinase ROCK activities LIM-kinase 1 by phosphorylation at Threonine 508 within the activation loop"The Journal of Biological Chemistry. 275. 3577-3582 (2000)
• [Publications] Nakamura K. et al: "Rhophilin, a small GTPase Rho-binding protein, is abundantly expressed in the mouse testis and localized in the principal piece of the sperm tail"FEBS Lett.. 445. 9-13 (1999)
• [Publications] Fujita A. et al: "Ropporin, a sperm-specific binding protein of rhophilin, that is localized in the fibrous sheath of sperm flagella"Journal of Cell Science. 113. 103-112 (2000)
• [Publications] Ishizaki T. et al: "Pharmacological properties of Y-27632, a specific inhibitor of Rho-associated kinases"Molecular Pharmacology. (in press).
• [Publications] Madaule,P.et al.: "Role of citron kinase as a target of the small GTPase Rho in cytokinesis" Nature. 394. 491-494 (1998)
• [Publications] Tominaga,T.et al.: "p160ROCK mediates RhoA activation of Na-H exchange." EMBOJ.17. 4712-4722 (1998)
• [Publications] Hirose,M.et al.: "Molecular dissection of the Rho-associated protein kinase(p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells." J.Cell Biol.141. 1625-1636 (1998)
• [Publications] Furuyashiki,T.et al.: "Citron,a Rho-target,interacts with PSD-95/SAP-90 at glutamatergic synapses in the thalamus." J.Neuroscience. 19. 109-118 (1999)
• [Publications] Itoh,K.et al.: "An essential part for Rho-associated kinase in the transcellular invasion of tumor cells." Nature Med.5. 221-225 (1999)
• [Publications] Sahai,E.et al.: "Transformation mediated by RhoA requires ROCK family kinase activity." Curr.Biol.9. 136-145 (1999)
• [Publications] Uehata,M.et al: "Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension" Nature. 389. 990-994 (1997)
• [Publications] Watanabe,N.et al: "p140mDia,a mammalian homologue of Drosophila diaphanous,is target protein for Rho small GTPase and is a ligand for prof 1" EMBO Journal. 16. 3044-3056 (1997)
• [Publications] Fujita,A.et al: "Integrin-dependent translocation of p160ROCK to cytoskeleta complex in thrombin-stimulated human platelets" Biochemical Journal. 328. 769-775 (1997)
• [Publications] Ishizaki,T.et al: "p160ROCK,a Rho associated coiled-coil forming protein kinase works downstream of Rho and induces focal addesions" FEBS Letters. 404. 118-124 (1997)
• [Publications] Hirai,A.et al: "Geranylgeranylated rho small GTPase (s) are essential for the degradation of p27kip 1 and facilitate the progression from G1 to S phase in growth-stimulated rat FRTL -5 cells" Journal of Biological Chemistry. 272. 13-16 (1997)
• [Publications] Altun-Gultekin,Z.F.et al: "Activation of rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein" Molecular Cellular Biology. (in press). (1998)
• [Publications] Reid,T.,et al.: "Rhotekin,a new putative target for Rho bearing homology to a serine/threonine kinase,PKN,and rhophilin in the Rho-binding domai" J.Biol.Chem.271. 13556-13560 (1996)
• [Publications] Ishizaki,T.,et al.: "The small GTP-binding protein Rho binds to and activates a160 Da Ser/Thr protein kinase homologous to myotonic dystrophy kinase." EMBO J.15. 1885-1893 (1996)
• [Publications] Nakagawa,O.,et al.: "Characterization of the interaction netween RhoA and the amino-terminal region of RKN." FEBS Lett.392. 189-193 (1996)
• [Publications] Fujisawa,K.,et al.: "Identification of the Rho binding domain of p160ROCK,a Rho-associated coiled coil-containing protein kinase." J.Biol.Chem.271. 23022-23028 (1996)
• [Publications] Watanabe,N.,et al.: "p140mDia,a mammalian homologue of Drosophila diaphanous,is a target protein for Rho small GTPase amid is a ligand for profilin." EMBO.J.(in press).
• [Publications] Ishizaki,T.,et al.: "p160ROCK,a Rho-associated coiled-coil forming protein kinase,works downstream of Rho and induces focal adhesions." FEBS.Lett.(in press).
• [Publications] Narumiya,S.: "J.Biochem." The small GTPase Rho : cellular functions and signal transduction., 215-228 (1996)
• [Publications] Narumiya,S.,et al.: "FEBS.Lett." Rho effectors and reorganization of actin cytoakeleton.(in press),