ADHESION-INDUCED TYROSINE PHOSPHORYLATION OF THE P130 SRC SUBSTRATE

Adhesion of cells to the extracellular matrix leads to an increase in the tyrosine phosphorylation of a specific set of proteins, three of w hich have now been identified as the focal adhesion proteins pp125(FAK ), paxillin and tensin. In addition, we have previously noted the adhe sion-induced tyrosine phosphorylation of a fourth protein, with an app arent molecular mass of 130. As in the case of FAK, paxillin and tensi n, a 130 kDa protein is also found to be highly tyrosine phosphorylate d in Rous sarcoma virus (RSV)-transformed cells, This protein forms a stable complex with pp60(src) and is directly phosphorylated by activa ted forms of c-svc, Using a monoclonal antibody (mAb 4F4) specific for the sue-associated p130 we show that p130 is also phosphorylated in r esponse to cell adhesion, Immunoprecipitation of p130 followed by an a nti-phosphotyrosine immunoblot revealed that adhesion of rat embryo fi broblasts (REF52) to fibronectin (FN) led to a significant increase in the phosphotyrosine content of p130. Furthermore, a comparison of cel l lysates before and after immunoprecipitation confirmed the absence o f tyrosine phosphorylated p130 from lysates immunoprecipitated with mA b 4F4. Immunofluorescence staining of REFS2s revealed that p130 is fou nd in focal adhesions as well as along stress fibers in a pattern remi niscent of that exhibited by a-actinin, In addition, in many cells, we found significant staining in the nucleus, but evidence is presented that the nuclear staining is not due to tyrosine phosphorylated p130, Finally, unlike pp125(FAK), p130 does not appear to be itself a kinase as evidenced by immune-complex kinase assays carried out in the prese nce or absence of exogenous substrates.