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Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration
Asako Tsubouchi1, Junko Sakakura, Ryohei Yagi
1Department of Molecular Biology, Osaka Bioscience Institute, Osaka 565-0874, Japan.
Abstract:
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.
Insights
Phosphorylated paxillin (Tyr31/118) suppresses RhoA activity, enabling cell membrane spreading and ruffling during adhesion and migration. This mechanism is crucial for efficient cell movement.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Integrin engagement initiates cell adhesion and migration processes.
- RhoA, Cdc42, and Rac1 are key regulators of the actin cytoskeleton and cell morphology.
- Paxillin is an integrin-associated protein involved in cell adhesion and migration.
Purpose of the Study:
- To elucidate the role of paxillin phosphorylation at Tyr31 and Tyr118 in regulating RhoA activity during cell adhesion and migration.
- To investigate the molecular mechanism by which paxillin phosphorylation influences cell spreading and ruffling.
Main Methods:
- Site-directed mutagenesis of paxillin phosphorylation sites (Tyr31/118).
- Expression of dominant-negative and constitutively active Rho GTPases and RhoGAP.
- Co-immunoprecipitation assays to study protein-protein interactions.
Main Results:
- Mutation of paxillin Tyr31/118 led to enhanced RhoA activation, premature stress fiber formation, and impaired membrane spreading/ruffling.
- These phenotypes were rescued by inhibiting RhoA, activating Rac1, or expressing p190RhoGAP.
- Phosphorylated paxillin (Tyr31/118) binds to p120RasGAP, competing with p190RhoGAP binding.
Conclusions:
- Tyr31/118-phosphorylated paxillin acts as a scaffold to recruit p120RasGAP, facilitating the release and activation of p190RhoGAP.
- Released p190RhoGAP suppresses RhoA activity, which is essential for efficient membrane spreading and ruffling during cell adhesion and migration.
- Paxillin phosphorylation is a critical regulatory mechanism for controlling cell adhesion and migration dynamics.