THE RHOA-DEPENDENT ASSEMBLY OF FOCAL ADHESIONS IN SWISS 3T3 CELLS IS ASSOCIATED WITH INCREASED TYROSINE PHOSPHORYLATION AND THE RECRUITMENTOF BOTH PP125FAK AND PROTEIN-KINASE C-DELTA TO FOCAL ADHESIONS

Mouse Swiss 3T3 fibroblasts cultured in serum-free medium lose their a ctin stress fibres and vinculin-containing focal adhesions, a process that can be reversed by the addition of serum, lysophosphatidic acid ( LPA) or bombesin, and is mediated by rhoA (A. J. Ridley and A. Hall (1 992) Cell 70, 389-399). We have shown that the addition of serum to th ese cells induces the recruitment of the cytoskeletal proteins talin, vinculin and paxillin, and the protein kinases pp125FAK and PKC-delta, to newly formed focal adhesions, and that alpha-actinin is distribute d along the actin stress fibres associated with these structures. The newly formed focal adhesions stained heavily with an antibody to phosp hotyrosine. A similar response was elicited by 100 ng/ml LPA. The effe ct of serum was rapid, with focal staining for paxillin largely restri cted to cell margins seen within 2 minutes of serum addition, and prec eding the assembly of actin filaments. Phosphotyrosine staining differ ed in that it was predominantly punctate and was widely distributed th roughout the cell. By 5 minutes, the paxillin and phosphotyrosine stai ning was concentrated at the ends of actin filaments largely at the ce ll margins. The structures stained ranged from circular to oval, but b y 10 minutes they more closely resembled the elongated focal adhesions found in cultured fibroblasts. Within 10 minutes, the addition of ser um or LPA induced a marked increase in the levels of pp125FAK and paxi llin immune-precipitated by an anti-phosphotyrosine antibody. The resu lts suggest that both pp125FAK and paxillin undergo changes in tyrosin e phosphorylation upon activation of rhoA, and that these changes are associated with the assembly of focal adhesions and actin stress fibre s. The observation that formation of focal adhesions can be induced by the tyrosine phosphatase inhibitor vanadyl hydroperoxide is consisten t with the direct involvement of tyrosine phosphorylation in the assem bly process. The localisation of PKC-delta to newly formed focal adhes ions suggests that serine/threonine phosphorylation may also be import ant in this regard.