title: Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion remodeling: role of Src and GIT.
abstract: 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.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shikata
authors.forename: Konstantin G
authors.initials: KG
authors.lastname: Birukov
authors.forename: Anna A
authors.initials: AA
authors.lastname: Birukova
authors.forename: Alexander
authors.initials: A
authors.lastname: Verin
authors.forename: Joe G N
authors.initials: JG
authors.lastname: Garcia
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000032:analysis / Q000502:physiology*; D006801:Humans; D008246:Lysophospholipids; D051419:Paxillin; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000502:physiology*; D011743:Pyrimidines / Q000494:pharmacology; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: C412373:AG 1879; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; C485647:GIT1 protein, human; C430213:GIT2 protein, human; D020690:GTPase-Activating Proteins; D008246:Lysophospholipids; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011743:Pyrimidines; C060506:sphingosine 1-phosphate; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D013917:Thrombin; D013110:Sphingosine