MODULATION OF PROTEIN-TYROSINE PHOSPHORYLATION BY THE EXTRACELLULAR-MATRIX

Fibronectin (FN) cross-linked to fibrin following injury provides the provisional matrix required for cells to begin tissue repair. Our prev ious work has demonstrated that fibroblasts adherent to multimeric FN within the context of a fibrin matrix (FN-fibrin) exhibit clear phenot ypic differences from those adherent to a dimeric FN-coated, surface. We hypothesize that this response to multimeric FN may be mediated by altered protein tyrosine phosphatase activity following integrin activ ation, 1Methods: NIH 3T3 cells were plated in the presence or absence of per vanadate (PV), a phosphotyrosine phosphatase inhibitor, on well s coated with FN or FN-fibrin matrix. Spread cell areas were measured after increasing incubation times and are recorded as mean cell area ( mm(2)) +/- SEM. Alternatively, cells were lysed and equal amounts of p rotein were analyzed by immunoblot using a monoclonal antibody specifi c for phosphotyrosine, Results: PV significantly inhibited cell spread ing on FN-fibrin matrices. In contrast, PV treatment had little effect on cell area on PN alone. Analysis of cell lysates revealed that prot ein tyrosine phosphorylation events differ in a substrate-dependent ma nner. Conclusion: Cell attachment to a FN-fibrin matrix induces distin ct cell shape and cytoskeletal organization. Inactivation of tyrosine- specific phosphatases enhances this distinction and inhibits the sprea ding of cells attached to this substrate, The phosphotyrosyl protein c ontent of treated cells on FN-fibrin matrix is also diminished. These results suggest that cell-extracellular matrix interactions affect the tyrosine phosphorylation balance of the cell, thus modifying cytoskel etal organization and related signaling events. (C) 1997 Academic Pres s.