Interactions of tensin with actin and identification of its three distinct actin-binding domains.
Interactions of tensin with actin and identification of its three distinct actin-binding domains.
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DOI：
10.1083/jcb.125.5.1067
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发表时间：
1994-06
期刊：
影响因子：
--
通讯作者：
Chen LB
中科院分区：
文献类型：
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作者：
Lo SH;Janmey PA;Hartwig JH;Chen LB
Tensin是一种200 kD的焦接触磷蛋白，与Src（SH 2结构域）和几种肌动蛋白结合蛋白具有序列同源性。这些特征表明，张力蛋白可能连接细胞膜的细胞骨架和直接响应酪氨酸激酶信号通路。在这里，我们确定了三个不同的肌动蛋白结合结构域内张力蛋白。在通过杆状病毒系统过表达后纯化的重组张力蛋白以Kd = 0.1 μ M与肌动蛋白丝结合，以1：10（张力蛋白/肌动蛋白）的摩尔比交联肌动蛋白丝，并通过以Kd = 20 nM的倒钩末端加帽延迟肌动蛋白组装。构建张力蛋白片段并表达为融合蛋白以映射具有这些活性的结构域。张力蛋白的三个区域与肌动蛋白相互作用：两个区域由氨基酸1至263和263至463组成，与F-肌动蛋白共沉积，但不改变肌动蛋白组装的动力学;一个区域由氨基酸888-989组成，与插入蛋白序列同源，阻碍肌动蛋白聚合。一个爪形张力蛋白二聚体有六个潜在的肌动蛋白结合位点，并能在焦点接触处包围两个肌动蛋白丝的末端。
Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross- links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.