Calyculin‐A induces focal adhesion assembly and tyrosine phosphorylation of p125Fak, p130Cas, and paxillin in Swiss 3T3 cells

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Abstract
Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125Fak in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 ± 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (10 nM) tyrosine phosphorylation of p125Fak was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125Fak through a protein kinase C- and Ca2+-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125Fak in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125Fak in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130Cas and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125Fak, p130Cas, and paxillin, acting downstream of ROK.