| Co-Investigator(Kenkyū-buntansha) |
SHIRATAKI Hiromichi Osaka University, Medical School, Assistant Professor, 医学部, 助手 (90249962)
SASAKI Takuya Osaka University, Medical School, Assistant Professor, 医学部, 助手 (40241278)
TANAKA Kazuma Osaka University, Medical School, Associate Professor, 医学部, 助教授 (60188290)
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| Budget Amount *help |
¥30,600,000 (Direct Cost: ¥30,600,000)
Fiscal Year 1996: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1995: ¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1994: ¥20,100,000 (Direct Cost: ¥20,100,000)
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| Research Abstract |
There is the small GTP-binding protein (G protein) superfamily which consists of over sixty members. All the members are monomeric proteins with Mrs between 20,000 and 30,000 which exhibit both GDP/GTP-binding and GTPase activities. In the present research project, we studied the functions and modes of activation and action of small G proteins. Ras is the representative of this superfamily and is well known to regulate gene expression at least through the MAP kinase cascade consisting of MAP kinase, MAP kinase kinase (MEK), and MEK kinase. We purified from bovine brain a MEK kinase which activated MEK in a GTPgammaS-Ki-Ras-dependent manner in a cell-free system and identified it as B-Raf complexed with 14-3-3 proteins. We also found that Rapl, which has the same amino acid sequence at its effector domain as that of Ras, is also involved in the activation of the B-Raf-dependent MAP kinase cascade. Rho regulates reorganization of actin filaments and is implicated in at least maintenance
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of cell morphology, cell motility, and cytokinesis in mammalian cells. We found that Rho is translocated to a membrane ruffling area when cells are stimulated by HGF and TPA.Rho was also observed at the contractile ring produced during cytokinesis. The ERM family (Ezrin, Radixin, and Moesin), which directly interacts with actin filaments through its C-terminal region and with a transmembrane protein, CD44, through its N-terminal region, is located at these areas. Therefore, Rho may regulate reorganization of the ERM family-dependent association of actin filaments with the plasma membrane. We also found that Rho GDI directly interacts with ERM,initiating the activation of Rho by reducing the Rho GDI activity. Rho also exists in the yeast Saccharomyces cerevisiae, and we found that Rho is essential for budding process. We genetically and biochemically isolated the regulatory proteins (RDI1 and ROM1/ROM2) and target proteins (PKC1, BNI1/BNR1, glucan synthase) for Rho. BNI1 and BNR1 directly interact with profilin at the FH1 domains, suggesting that Rho regulates the reorganization of actin filaments through the BNI1/BNR1-profilin system. Rab3A is a member of the Rab family which is particularly implicated in neurotransmitter release from the nerve terminal. We had isolated its regulatory protein, named Rab GDI,and its target protein, named Rabphilin-3A.In this research project, we isolated other regulatory proteins for Rab3A,Rab3 GEP and Rab3 GAP. Less
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