川原 康洋 神戸大学, 医学部附属病院, 講師 (80169755)
SASAKI Takuya Osaka University Medical School, Research Associate, 医学部, 助手 (40241278)
TANAKA Kazuma Osaka University Medical School, Assistant Professor, 医学部, 講師 (60188290)
KAWATA Sumio Osaka University Medical School, Associate Professor, 医学部, 助教授 (90183285)
|Budget Amount *help
¥18,500,000 (Direct Cost : ¥18,500,000)
Fiscal Year 1995 : ¥4,500,000 (Direct Cost : ¥4,500,000)
Fiscal Year 1994 : ¥14,000,000 (Direct Cost : ¥14,000,000)
There is the small GTP-binding protein (G protein) superfamily wich consists of over fifty members. All 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 and tried to prepare their cDNAs and antibodies as tools for diagnosis and treatment of various diseases. Ras is the representative of this superfamily and is well known to requlate 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. Rho regulates reorganization of actin filaments and is implicated in at least maintenance of cell morphology, cell motility, and cytokinesis in mammalian cells. We found t
hat Rho is translocated to a membrane ruffling area when cells are stimulated by HGF and TPA.Rho was also translocated to the cell-cell adhesion area, which was induced by Ca^<2+>. Rho was also observed at the contractile ring produced during cytokinesis. The ERM family (Ezrin, Radixin, and Moesin), which directly interacts with actin filament 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 filament with the plasma membrane. Rho also exists in the yeast Saccharomyces cerevisiae, and we found that RHO1 is essential for budding process. We genetically and biochemically isolated the regulatory proteins (RDI1 and ROM1/ROM2) and target proteins (PKC1, BNI1, glucan synthase) for RHO1.
Rab3A is a member of the Rab family which is particularly implicated in neurotransmitter release from the nerve termini. 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,Rab3A GEP,and Rab3A GAP.The results obtained in this research project suggest that small G proteins play crucial roles in various cell functions and that their cDNAs and antibodies are useful as tools for diagnosis and treatment of various diseases. Less