Shikata Yasushi, Birukov Konstantin G, Birukova Anna A, Verin Alexander, Garcia Joe G N
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
FASEB J. 2003 Dec;17(15):2240-9. doi: 10.1096/fj.03-0198com.
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.
鞘氨醇-1-磷酸（S1P）和凝血酶是对血管内皮细胞（EC）屏障特性具有深远但不同影响的因子。我们之前报道过，S1P诱导的粘着斑（FA）重塑涉及粘着斑激酶（FAK）、桩蛋白以及G蛋白偶联受体激酶相互作用蛋白GIT1和GIT2之间的相互作用，并提示粘着斑在EC屏障调节中起关键作用。在本研究中，我们检测了凝血酶刺激的人肺动脉内皮细胞中FA蛋白（FAK、桩蛋白、GIT1和GIT2）的重新分布以及FAK位点特异性酪氨酸磷酸化情况。与我们已证明能诱导与皮质肌动蛋白环形成相关的FA蛋白向周边转位的S1P不同，凝血酶导致FA蛋白重新分布到新形成的粗大应力纤维的末端。S1P和凝血酶诱导FAK位点特异性磷酸化的模式不同，S1P刺激靶向FAK Y576磷酸化位点，而凝血酶刺激则使三个FAK位点（Y397、Y576和Y925）发生磷酸化。用Src特异性抑制剂PP2对Src进行药理抑制可消除S1P诱导的FA蛋白转位、皮质肌动蛋白环形成以及FAK [Y576]磷酸化。然而，PP2未能改变凝血酶诱导的形态变化，并且仅部分抑制了FAK位点特异性酪氨酸磷酸化。这些观察结果突出了S1P和凝血酶对粘着斑蛋白复合体重塑和FAK激活的不同机制，并将不同的FA重塑与EC屏障调节联系起来。
