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Sphingosine-1 phosphate (S1P) and thrombin are agents with profound but divergent effects on vascular endothelial cell (EC) barrier properties. 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 tyrosine 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 distinct 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 inhibitor PP2 abolished S1P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter thrombin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phosphorylation. These observations highlight the differential mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.

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Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.