| Project/Area Number |
07044248
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KAKIZUKA Akira (1996) Faculty of Medicine, Kyoto university, 医学研究科, 助教授 (80204329)
成宮 周 (1995) 京都大学, 医学研究科, 教授 (70144350)
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| Co-Investigator(Kenkyū-buntansha) |
SYMONDS Marc ONYX Pharmaceutics, 研究員
VUORI Kristiina La Jolla Cancer Foundation, 研究員
WITTINGHOFER Alfred Max-Planck Institute, 部長
RIDLEY Anne Ludwing Institute for Cancer Research, 室長
CULOTTI Joseph University of Toronto, 準教授
BIRGE Raymond The Rockfeller University, 助教授
MOOLENAAR Wouter The Netherlands Cancer Institute, 室長
NAKAGAWA Osamu Faculty of Medicine, Kumamoto University, 医学部, 助手 (40283593)
USHIKUBI Humitaka Faculty of Medicine, Kyoto university, 医学研究科, 講師 (50243035)
HIRATA Masakazu Faculty of Medicine, Kyoto university, 医学研究科, 助手 (40261143)
WITTINGHOFER アルフレッド マックスプランク分子生物学研究所, 部長
RIDLEY Anne ルードヴィッヒ研究所, 室長
LULOTTI Jose トロント大学, 準教授
斎藤 雄二 京都大学, 医学研究科, 助手 (40263090)
垣塚 彰 京都大学, 医学研究科, 助教授 (80204329)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥11,000,000 (Direct Cost: ¥11,000,000)
Fiscal Year 1996: ¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1995: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | rho p21 / p160ROCK / focal adhesion / intergrin / p140mDia / actin polymerization / Neural plasticity / cell adhesion / rho p21 / ボツリヌスC_3酵素 / PKN / P160^<ROCK> / 細胞形態変化 / SH_3 |
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| Research Abstract |
We have isolated new Rho effector molecules, p160ROCK,ROCK-II,Rhotekin and p140mDia using ligand overlay blotting and yeast two hybrid system. We are now characterizing functions of these effector molecules.
p160ROCK induces formation of stress fibers and focal adhesions, as observed in transfection experiments. A dominant negative mutant of p160ROCK was constructed, and co-transfected in HeLa cells together with Val14-RhoA,a dominant active mutant RhoA.This dominant negative mutant of p160ROCK suppressed the Val14-RhoA phenotype, namely the Rho induced stress fibers and focal adhesion formation. These results demonstrate that p160ROCK works downstream of Rho, mediating the formation of stress fibers and focal adhesions. We have also isolated Rhotekin with yeast two hybrid system. The Rho binding domain of Rhotekin shows homology to the Rho binding domains in PKN and Rhophilin, defining a new consensus sequence for Rho binding. Rhotekin binds to Rho and Rho GTPase activity is decreased
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by this binding. Recently, we also isolated p140mDia with yeast two hybrid system. p140mDia has a Rho-binding domain in its N-terminus and polyproline regions in the middle of the protein. p140mDia binds to profilin (which regulates actin polymerization)through its poly-proline regions. In immunofluorescence analysis, p140mDia was localized at the periphery near the membrane, and at the cleavage furrow during cytokinesis.
We participated in two international meeting, presenting these results and exchanging new informations. Our cDNA of Rho effector molecules have been sent to numerous researchers in the world and several collaborations have been undertaken. For example Dr.Wittenghofer's group (Max-Planck institute) is determining the 3D-structure of our Rho effectors using X-ray diffraction. Dr.Symonds (ONYX pharmaceutics) helped us with cytological techniques such as microinjection, and Dr.Vuori (La Jolla Cancer Foundation) is analyzing cell adhesion through intergrin, a function that involves p160ROCK.We are also collaborating with Dr.Moolenaar on neurite retraction in response to extra cellular stimulation. Less
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