Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains
Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains
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DOI：
10.1091/mbc.9.8.2157
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发表时间：
1998-08-01
影响因子：
3.3
通讯作者：
Hoffmann, FM
中科院分区：
文献类型：
--
作者：
Ahern-Djamali, SM;Conner, AR;Hoffmann, FM
果蝇启用 (Ena) 最初被确定为 Abelson 酪氨酸激酶突变的显性遗传抑制因子，最近被确定为 Ena/人血管舒张刺激磷蛋白 (VASP) 蛋白家族的成员。我们使用遗传、生化和细胞生物学方法来证明 Ena 和人类 VASP 之间的功能关系。此外，我们还定义了 Ena 结构域的作用，这些结构域被认为对其体内活性至关重要。我们已经证明，VASP 可以挽救果蝇中与 Ena 功能丧失相关的胚胎致死性，并且表明，Ena 与 VASP 一样，在培养细胞中表达时与肌动蛋白丝和粘着斑相关。为了定义对 Ena 功能至关重要的序列，我们对影响 Ena/VASP 同源结构域 1 (EVH1) 和 EVH2 的两个致命 ena 突变等位基因中存在的分子损伤进行了表征。导致 EVH1 结构域氨基酸取代的错义突变消除了 Ena 与细胞骨架蛋白 zyxin 的体外结合，zyxin 是之前报道的 VASP 的结合伴侣。导致 C 端截短的 Ena 蛋白缺乏 EVH2 结构域的无义突变无法形成多聚体复合物，并表现出与 zyxin 和 Abelson Src 同源 3 结构域的结合减少。我们的分析表明，Ena 和 VASP 在功能上是同源的，并将保守的 EVH1 和 EVH2 结构域定义为 Ena 生理活性的核心。
Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. Ln addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.