G(i)-mediated Cas tyrosine phosphorylation in vascular endothelial cells stimulated with sphingosine 1-phosphate - Possible involvement in cell motility enhancement in cooperation with Rho-mediated pathways

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activa tion, it is important to examine the effects of this bioactive lipid on vas cular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated si gnaling pathways related to human umbilical vein endothelial cell (HUVEC) m otility, with a special emphasis on the cytoskeletal docking protein Crk-as sociated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Ga s, which was inhibited by the Gi inactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn cons titutively associated with and phosphorylated Gas, suggesting that Gas tyro sine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimu lation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine- phosphorylated Gas, and the Cas-Crk complex translocated to the cell periph ery (membrane ruffles), through mediation of Gi (Fyn) but not Rho. In contr ast, tyrosine phosphorylation of focal adhesion kinase, and formation of st ress fibers and focal adhesion were mediated by Rho but not G(i) (Fyn). Fin ally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay usi ng gold sol-coated plates and a Boyden's chamber assay, was markedly inhibi ted not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into G (i)-mediated signaling pathways (involving Gas) and Rho-mediated ones (invo lving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the S ph-1-P receptors EDG-1 (coupled with G(i)) and EDG-3 (coupled with G(13) an d G(q)) and the EDG-3 antagonist suramin was found to block specifically Rh o-mediated responses, it is likely that Gas-related responses following G(i ) activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.