ANGIOTENSIN-II STIMULATION OF RAPID PAXILLIN TYROSINE PHOSPHORYLATIONCORRELATES THE FORMATION OF FOCAL ADHESIONS IN RAT AORTIC SMOOTH-MUSCLE CELLS

Angiotensin II is a potent vasoconstrictor that has been also implicat ed in vascular hyperproliferative diseases, including atherosclerosis and restenosis following angioplasty. Treatment of cultured, serum-sta rved rat aortic smooth muscle cells with angiotensin II causes rapid p rotein tyrosine phosphorylation that precedes cell mitogenesis. We hav e identified two of the phosphoproteins as paxillin (75 kilodaltons) a nd the tyrosine kinase pp125(Fak), both components of actin-associated focal adhesion sites. Angiotensin II stimulated a 5-fold increase in the tyrosine phosphorylation of paxillin and a smaller (1.5-fold) incr ease in pp125(Fak) tyrosine phosphorylation. Paxillin tyrosine phospho rylation was evident within 1 minute, and was maximal after 10 minutes . Similar elevated protein tyrosine phosphorylation levels of paxillin were obtained with exposure of the rat aortic smooth muscle cells to peptides endothelin-1 and alpha-thrombin that function, as angiotensin II, through binding to members of the seven transmembrane domain G pr otein coupled receptors. Angiotensin II treatment also stimulated the production of a well-ordered actin-containing stress fiber network and prominent paxillin-containing focal adhesions. The focal adhesions st ained intensely with anti-phosphotyrosine antibody suggesting the tyro sine phosphorylation of paxillin and cytoskeletal reorganization were tightly coupled. Angiotensin II receptor occupancy has been shown prev iously to lead to protein kinase C activation. However, compared to an giotensin II stimulation, a smaller, delayed increase in paxillin tyro sine phosphorylation was observed following direct protein kinase C ac tivation by the phorbol ester phorbol 12-myristate-13-acetate. Paxilli n tyrosine phosphorylation was selective for certain agonists since no increase in tyrosine phosphorylation of this protein was observed fol lowing exposure to the potent mitogen PDGF. Thus, actin-based cytoskel etal changes involving sites of cell adhesion to the extracellular mat rix may play an important role in normal and pathophysiologic smooth m uscle cell growth regulation in response to certain angiotensin II-typ e vasoactive agonists.