Involvement of site‐specific FAK phosphorylation in sphingosine‐1 phosphate‐ and thrombin‐induced focal adhesion remodeling: role of Src and GIT

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Abstract
Sphingosine-1 phosphate (S1P) and thrombin are agents with profound but divergent ef- fects on vascular endothelial cell (EC) barrier proper- ties. We have previously reported that S1P-induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G-protein-coupled receptor kinase-interacting proteins GIT1 and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) and site-specific FAK ty- rosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S1P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S1P and thrombin induced dis- tinct patterns of FAK site-specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src-specific in- hibitor PP2 abolished S1P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter throm- bin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phos- phorylation. These observations highlight the differen- tial mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.—Shikata, Y., Birukov, K. G., Birukova, A. A., Verin, A., Garcia, J. G. N. Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion re- modeling: role of Src and GIT. FASEB J. 17, 2240-2249 (2003)
Funding Information
  • National Heart, Lung, and Blood Institute (HL 50533, HL 58064, HL 69340)