Uncoupling Crk signal transduction by Pseudomonas exoenzyme T
Uncoupling Crk signal transduction by Pseudomonas exoenzyme T
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DOI：
10.1074/jbc.m504901200
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发表时间：
2005-10-28
影响因子：
4.8
通讯作者：
Barbieri, JT
中科院分区：
文献类型：
--
作者：
Deng, Q;Sun, JJ;Barbieri, JT
外泌酶T （ExoT）是铜绿假单胞菌的一种双功能III型细胞毒素，具有Rho gtpase激活蛋白和adp核糖基转移酶活性。ExoT的adp -核糖基转移酶活性刺激肌动蛋白细胞骨架的解聚，而不依赖于Rho gtpase激活蛋白的功能，并且ExoT随后被证明可以adp -核糖基化Crk（激酶的CT10调节因子）-I和Crk- ii。Crk蛋白是包含SH2和SH3结构域的真核衔接蛋白，是整合素信号通路中导致Rac1和Rap1功能的组成部分。质谱分析鉴定出Arg（20）为adp -核糖基化位点。Arg（20）是位于SH2结构域中的一个保守残基，它是与上游信号分子如paxillin和p130（cas）相互作用所必需的。谷胱甘肽s -转移酶下拉和远Western实验显示，adp核糖化的Crk-I或Crk-I（R20K）不能结合p130cas或paxillin。这表明adp核糖基化抑制了Crk与这些黏附蛋白的直接相互作用。野生型Crk-I的过表达减少了ExoT对细胞的圆切作用，而显性活性Rac1的表达则干扰了ExoT对细胞的圆切作用。因此，通过直接抑制Crk与局灶黏附蛋白的结合，Crk的adp核糖基化解除了整合素信号通路。
Exoenzyme T (ExoT) is a bifunctional type III cytotoxin of Pseudomonas aeruginosa that possesses both Rho GTPase-activating protein and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity of ExoT stimulated depolymerization of the actin cytoskeleton independent of Rho GTPase-activating protein function, and ExoT was subsequently shown to ADP-ribosylate Crk (CT10 regulator of kinase)-I and Crk-II. Crk proteins are eukaryotic adaptor proteins comprising SH2 and SH3 domains that are components of the integrin signaling pathway leading to Rac1 and Rap1 functions. Mass spectroscopic analysis identified Arg(20) as the site of ADP-ribosylation by ExoT. Arg(20) is a conserved residue located within the SH2 domain that is required for interactions with upstream signaling molecules such as paxillin and p130(cas). Glutathione S-transferase pull-down and far Western assays showed that ADP-ribosylated Crk-I or Crk-I(R20K) failed to bind p130cas or paxillin. This indicates that ADP-ribosylation inhibited the direct interaction of Crk with these focal adhesion proteins. Overexpression of wild-type Crk-I reduced cell rounding by ExoT, whereas expression of dominant-active Rac1 interfered with the ability of ExoT to round cells. Thus, the ADP-ribosylation of Crk uncouples integrin signaling by direct inhibition of the binding of Crk to focal adhesion proteins.