The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation
The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation
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DOI：
10.1074/jbc.274.33.23549
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发表时间：
1999-08-13
影响因子：
4.8
通讯作者：
Reinhard, M
中科院分区：
文献类型：
--
作者：
Bachmann, C;Fischer, L;Reinhard, M
血管扩张剂刺激的磷酸蛋白(Vasp)是Ena/Vasp家族中的一员，参与肌动蛋白细胞骨架的调控，所有家族成员都有一个三方结构，包括一个N端的Ena/Vasp同源(EVH)1结构域，一个更发散的富含Pro的中心部分，以及一个共同的C端约160-190个氨基酸的EVH2区。通过对不同截短的Vasp结构进行化学交联、蔗糖梯度沉淀和凝胶过滤分析，我们证明了Vasp EVH2区域是四聚的必要条件和充分条件。此外，共沉淀和荧光鬼臼蛋白染色显示，EVH2区在体外结合和捆绑F-肌动蛋白，并定位于转基因细胞中的应力纤维。对该区域内高度保守的片段的功能贡献分析表明，人Vasp的259-276残基是与F-肌动蛋白相互作用所必需的，而343-380残基是四聚所必需的，可能是通过卷曲形成的，与F-肌动蛋白的相互作用被Vasp四聚增强。结果表明，C-末端EVH2片段不仅在序列上是保守的，而且形成了一个独特的功能实体。这些数据表明，EVH2片段代表了一个新的寡聚和F-肌动蛋白结合域。
Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton, All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160-190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259-276 of human VASP are essential for the interaction with F-actin, whereas residues 343-380 are required for tetramerization, probably via coiled-coil formation, Interactions with F-actin are enhanced by VASP tetramerization, The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.