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首页> 外文期刊>Molecular and Cellular Biology >Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation
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Phosphotyrosine (p-Tyr)-Dependent and -Independent Mechanisms of p190 RhoGAP-p120 RasGAP Interaction: Tyr 1105 of p190, a Substrate for c-Src, Is the Sole p-Tyr Mediator of Complex Formation

机译：p190 RhoGAP-p120 RasGAP相互作用的磷酸酪氨酸（p-Tyr）依赖性和非依赖性机制：p190的Tyr 1105（c-Src的底物）是复合物形成的唯一p-Tyr介体

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p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.
机译：p190 RhoGAP是一种190 kDa蛋白，可与p120 RasGAP稳定结合，并通过小型GTPases Rho家族成员调节肌动蛋白动态。先前的研究表明，p190酪氨酸磷酸化水平，表皮生长因子（EGF）诱导的肌动蛋白重排的程度和动力学与EGF诱导的细胞周期进程之间存在直接关系，这表明p190将Ras介导的有丝分裂信号与通过肌动蛋白细胞骨架。确定p190中的哪些酪氨酸残基被磷酸化，哪些因素调节这些位点的磷酸化以及酪氨酸磷酸化对p190功能的影响是了解p190在这些过程中可能发挥的作用的关键。为了开始研究这些问题，我们使用了生物化学方法来表征C3H10T1 / 2鼠成纤维细胞内源性p190上体内磷酸化酪氨酸残基的数量和相对水平。仅检测到两个含有磷酸酪氨酸（p-Tyr）的胰蛋白酶磷酸肽，一个主要位点为Y1105，一个次要位点（未识别）。与EGF受体相比，c1-Src的过表达在体内对Y1105的磷酸化（但不是次要位点）进行了更大程度的体内调节，并在体外被c-Src有效地催化，表明Y1105是C1-Src的选择性和优先靶标。体外和体内的c-Src。使用谷胱甘肽 S -转移酶（GST）融合蛋白进行体外和体内共沉淀分析，该融合蛋白包含p190中间域的野生型和Y1105F变体，以及在COS-7细胞中异位表达的全长p190变体，并且C3H10T1 / 2细胞中的内源性p190和p120显示p190可以在存在和不存在p190酪氨酸磷酸化的情况下与p120结合。不依赖p-Tyr的复合物占在p-Tyr存在下形成的复合物的10％至20％。 Y1105从Tyr突变为Phe导致完全失去p-Tyr依赖的复合物形成，表明p-Y1105是介导与p120结合的唯一p-Tyr残基。这些研究描述了c-Src可以调节p190-p120缔合的特定机制，并且还证明了p190-p120结合的p-Tyr非依赖性手段的重要作用。

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