cDNAs and antibodies for small GTP-binding proteins

有一个小的GTP结合蛋白（G蛋白）超家族，由50多个成员组成。所有成员都是单体蛋白，其Mrs在20，000和30，000之间，表现出GDP/GTP结合和GTP酶活性。在本研究项目中，我们研究了小G蛋白的功能和激活和作用模式，并试图制备其cDNA和抗体，作为诊断和治疗各种疾病的工具。Ras是该超家族的代表，并且众所周知至少通过由MAP激酶、MAP激酶（MEK）和MEK激酶组成的MAP激酶级联来调节基因表达。我们从牛脑中纯化了一种MEK激酶，该激酶在无细胞系统中以GTP γ S-Ki-Ras依赖的方式激活MEK，并将其鉴定为与14-3-3蛋白复合的B-Raf。Rho调节肌动蛋白丝的重组，并且至少涉及哺乳动物细胞中细胞形态、细胞运动和胞质分裂的维持。我们找到了 关于我们 当细胞被HGF和TPA刺激时，Rho移位到细胞膜皱褶区，同时，Rho也移位到细胞间粘附区，这是由Ca^2+诱导的。在胞质分裂期间产生的收缩环处也观察到Rho。ERM家族（Ezrin、Radixin和Moesin）通过C端区域与肌动蛋白丝直接相互作用，通过N端区域与跨膜蛋白CD 44直接相互作用，因此Rho可能调节ERM家族依赖的肌动蛋白丝与质膜的结合的重组。Rho也存在于酿酒酵母中，并且我们发现Rho 1是芽殖过程所必需的。我们从基因和生化上分离了RHO 1的调节蛋白（RDI 1和ROM 1/ROM 2）和靶蛋白（PKC 1、BNI 1、葡聚糖合成酶）。Rab 3A是Rab家族的成员，特别是参与神经末梢的神经递质释放。本研究还分离了Rab 3A、Rab 3A GEP和Rab 3A GAP的调控蛋白，结果表明，Rab 3A小G蛋白在多种细胞功能中发挥重要作用，其cDNA和抗体可作为诊断和治疗多种疾病的工具。少

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 … More 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