Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src

Thrombin and angiotensin II (angII) have trophic properties as mediators of vascular remodeling. Focal adhesions and actin cytoskeleton are involved i n cell growth, shape, and movement and may be important in vascular remodel ing. To characterize mechanisms by which thrombin and angII modulate vessel structure, we studied the effects of these G protein-coupled receptor liga nds on focal adhesions in vascular smooth muscle cells (VSMCs). Both thromb in and angII stimulated bundling of actin filaments to form stress fibers, assembly of focal adhesions, and protein tyrosine phosphorylation at focal adhesions, such as p130Cas, paxillin, and tensin. To test whether c-Src pla ys a critical role in focal. adhesion rearrangement, we analyzed cells with altered c-Src activity by retroviral transduction of wild-type (WT) and ki nase-inactive (KI) c-Src into rat VSMCs, and by use of VSMCs from WT (src(/+)) and Src-deficient (src(-/-)) mice. Tyrosine phosphorylation of Gas, pa xillin, and tensin were markedly decreased in VSMCs expressing KI-Src and i n src(-/-) VSMCs. Expression of KI-Src did not inhibit stress fiber formati on by thrombin. Surprisingly, actin bundling was markedly decreased in VSMC s from src(-/-) mice both basally and after thrombin stimulation, compared with src(+/+) mice. We also studied the effect of KI-Src and WT-Src on VSMC spreading. Expression of KI-Src reduced the rate of VSMC spreading on coll agen, whereas WT-Src enhanced cell spreading. In conclusion, c-Src plays a critical role in agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in VSMCs. c-Src kinase activity is required for the cytoskeletal turnover that occurs in cell spreading, whereas c-Src appears to regulate actin bundling via a kinase-independent mechanism.