Tsubouchi Asako, Sakakura Junko, Yagi Ryohei, Mazaki Yuichi, Schaefer Erik, Yano Hajime, Sabe Hisataka
Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.
J Cell Biol. 2002 Nov 25;159(4):673-83. doi: 10.1083/jcb.200202117.
RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.
在整合素结合的初始阶段，RhoA活性会被短暂抑制，此时主要发生由Cdc42和/或Rac1介导的膜伸展和褶皱。桩蛋白是一种整合素组装蛋白，有四个主要的酪氨酸磷酸化位点，酪氨酸31和酪氨酸118的磷酸化与细胞黏附和迁移相关。我们发现，酪氨酸31/118突变导致RhoA激活增强，应力纤维过早形成，在NMuMG细胞黏附和迁移过程中，有效膜伸展和褶皱大量丧失。这些表型与亲本细胞中由RhoA(G14V)诱导的表型相似，并且可以通过在表达突变体的细胞中表达RhoA(T19N)、Rac1(G12V)或p190RhoGAP来消除。发现磷酸化的酪氨酸31/118与p120RasGAP的两个src同源（SH）2结构域结合，内源性桩蛋白与p120RasGAP共沉淀。已知p190RhoGAP是p120RasGAP SH2结构域的主要细胞内结合伴侣。我们发现酪氨酸31/118磷酸化的桩蛋白与p190RhoGAP竞争结合p120RasGAP，并提供证据表明从p120RasGAP释放的p190RhoGAP在细胞黏附过程中有效抑制RhoA活性。我们得出结论，酪氨酸31/118磷酸化的桩蛋白作为局部抑制RhoA活性的模板，是NMuMG细胞黏附和迁移过程中有效膜伸展和褶皱所必需的。
