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cDNAs and antibodies for small GTP-binding proteins

小 GTP 结合蛋白的 cDNA 和抗体

基本信息

批准号：
06557012
负责人：
TAKAI Yoshimi
金额：
$ 11.84万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557012/
关键词：
small GTP-binding protein Ras B-Raf Rho The ERM family CD44 Rab3A Rabphilin-3A 低分子量G蛋白質 Rap1 SmgGDS SMAP 14-3-3蛋白質 ERM-CD44系

项目摘要

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

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MRS在20,000到30,000之间的单体蛋白，既显示GDP/GTP结合活性，又显示GTP酶活性。本研究对小G蛋白的功能、激活和作用方式进行了研究，并试图制备小G蛋白的cDNA和抗体，作为诊断和治疗各种疾病的工具。RAS是这一超家族的代表，众所周知，RAS至少通过由MAP、MAP-K和MEK-K组成的MAP-K级联反应来调节基因的表达。我们从牛脑中纯化了一种在无细胞体系中以GTPGammaS-Ki-Ras依赖的方式激活MEK的MEK激酶，并鉴定其为B-Raf与14-3-3蛋白的络合物。Rho调节肌动蛋白细丝的重组，并至少与维持细胞形态、细胞运动和哺乳动物细胞的胞质分裂有关。我们找到了t…当HGF和TPA刺激细胞时，Hat Rho更多地移位到膜皱褶区。Rho也移位到细胞与细胞之间的粘附区，这是由Ca~(2+)和Gt；在胞质分裂过程中产生的收缩环上也观察到Rho。ERM家族(Ezrin、Radixin和Moesin)位于这些区域，通过其C-末端区域与肌动蛋白细丝直接相互作用，并通过其N-末端区域与跨膜蛋白CD44相互作用，因此，Rho可能调节依赖于ERM家族的肌动蛋白细丝与质膜结合的重组。酿酒酵母中也存在RHO，我们发现RHO1在发芽过程中是必不可少的。我们从基因和生物化学上分离了RHO1的调控蛋白(RDI1和ROM1/ROM2)和靶蛋白(PKC1、BNI1、葡聚糖合成酶)。Rab3A是Rab家族的成员，与神经末梢的神经递质释放密切相关。我们分离了其调控蛋白Rab GDI和靶蛋白RabPhilin-3A。在本研究项目中，我们还分离了Rab3A、Rab3A GEP和Rab3A Gap的其他调控蛋白。本研究结果表明，小G蛋白在多种细胞功能中发挥关键作用，其cDNA和抗体可用于各种疾病的诊断和治疗。较少