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Analysis of physiological function of a focal adhesion protein vinexin

粘着斑蛋白vinexin的生理功能分析

基本信息

批准号：
14560287
负责人：
KIOKA Noriyuki
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Vinexin is a focal adhesion protein belonging to vinexin family (vinexin, CAP (c-cbl associated protein), ArgBP2 (Arg-binding protein 2). Proteins in this family share a sorbin homology (SoHo) domain at their N-terminal half. Functions of SoHo domain are not well known, although it is seemed to be involved in the regulation of actin cytoskeleton. Here we screened the SoHo-binding protein to clarify the function of SoHo domains and found that the fragment of alpha actin could bound to vinexin SoHo domain in two-hybrid system. The interaction of SoHo domain with alpha actin was confirmed by pull down assay and co-immunoprecipitation. Furthermore, the SoHo domain of vinexin was co-precipitated with filamentous actin. Interestingly, SoHo domains of other proteins were also associated. with fliamentous actin. Together, these findings suggest that SoHo domain functions as an actin binding domain.To elucidate the function of vinexin in detail, we analyzed the vinexin knockout mice. Vinexin knockout mice were born, matured and bred as wild type mice. To examine the function in cultured cell, we isolated primary fibroblast from embryo. Unlike to the overexpression of vinexin, knockout of vinexin gene did not affect the activation of ERK or anchorage-dependence of ERK signaling. However, it affected the cell motility. Together these finding suggested that vinexin is involved in cell migration.

Vinexin是一种粘着斑蛋白，属于vinexin家族（vinexin，CAP（c-cbl associated protein），ArgBP 2（Arg-binding protein 2））。该家族中的蛋白质在其N-末端的一半处共享一个sorbin同源性（SoHo）结构域。SoHo结构域的功能尚不清楚，尽管它似乎参与肌动蛋白细胞骨架的调节。为了进一步阐明SoHo结构域的功能，我们筛选了SoHo结合蛋白，并在双杂交系统中发现α肌动蛋白片段可以与vinexin SoHo结构域结合。通过pull down和免疫共沉淀实验证实了SoHo结构域与α肌动蛋白的相互作用。此外，vinexin的SoHo结构域与丝状肌动蛋白共沉淀。有趣的是，其他蛋白质的SoHo结构域也相关。用的是氟胺菌素这些发现表明SoHo结构域作为肌动蛋白结合结构域发挥功能。为了详细阐明vinexin的功能，我们分析了vinexin敲除小鼠。Vinexin敲除小鼠出生、成熟并作为野生型小鼠繁殖。为了检测培养细胞的功能，我们从胚胎中分离了原代成纤维细胞。与vinexin过表达不同，vinexin基因敲除不影响ERK的激活或ERK信号的锚定依赖性。但是，它影响了细胞的运动性。这些发现共同表明，vinexin参与细胞迁移。