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 previous work has demonstrated that fibroblasts adherent to multimeric FN within the context of a fibrin matrix (FN-fibrin) exhibit clear phenotypic 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 activation.


METHODS
NIH 3T3 cells were plated in the presence or absence of pervanadate (PV), a phosphotyrosine phosphatase inhibitor, on wells coated with FN or FN-fibrin matrix. Spread cell areas were measured after increasing incubation times and are recorded as mean cell area (mm2) +/- SEM. Alternatively, cells were lysed and equal amounts of protein were analyzed by immunoblot using a monoclonal antibody specific for phosphotyrosine.


RESULTS
PV significantly inhibited cell spreading on FN-fibrin matrices. In contrast, PV treatment had little effect on cell area on FN alone. Analysis of cell lysates revealed that protein tyrosine phosphorylation events differ in a substrate-dependent manner.


CONCLUSIONS
Cell attachment to a FN-fibrin matrix induces distinct cell shape and cytoskeletal organization. Inactivation of tyrosine-specific phosphatases enhances this distinction and inhibits the spreading of cells attached to this substrate. The phosphotyrosyl protein content 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 cytoskeletal organization and related signaling events.
Corbett
S A
SA
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick 08903, USA.
Schwarzbauer
J E
JE
D016475:3T3 Cells / Q000187:drug effects / Q000502:physiology; D000818:Animals; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000502:physiology*; D005337:Fibrin / Q000494:pharmacology; D005353:Fibronectins / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D004338:Drug Combinations; D005353:Fibronectins; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; D005337:Fibrin