Calyculin‐A induces focal adhesion assembly and tyrosine phosphorylation of p125^Fak, p130^Cas, and paxillin in Swiss 3T3 cells

Abstract

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) 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); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) 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 p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) 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 p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

Rapamycin inhibits F-actin reorganization and phosphorylation of focal adhesion proteins

“…Inhibition of mTOR suppresses phosphorylation of the focal adhesion proteins A close relationship between F-actin reorganization and tyrosine phosphorylation of focal adhesion proteins has been described (Leventhal et al, 1997;Guvakova and Surmacz, 1999;Leopoldt et al, 2001). As IGF-I stimulation very quickly induced F-actin reorganization, next we examined whether IGF-I impacts tyrosine phosphorylation of the focal adhesion proteins in Rh1 and Rh30 cells.…”

Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption

“…Active ROCK inhibits myosin light chain phosphatase (Kimura et al, 1996), thus causing the stimulation of actomyosin contractility. In turn, application of local forces by actomyosin contractility stimulates FA and stress fiber formation (Chrzanowska-Wodnicka and Burridge, 1996;Leopoldt et al, 2001), whereas inhibitors of actomyosin contractility induce disruption of these structures (Volberg et al, 1994;Helfman et al, 1999;Zamir et al, 1999;). Growth of FAs and focal complexes can also be 975 Tyrosine phosphorylation of focal adhesion components is involved in the regulation of focal adhesion formation and turnover, yet the underlying molecular mechanisms are still poorly defined.…”

RNA Interference Elucidates the Role of Focal Adhesion Kinase in HLA Class I-Mediated Focal Adhesion Complex Formation and Proliferation in Human Endothelial Cells

“…In earlier studies, we observed that class I-induced activation of the PI3K/Akt cell survival pathway and fibroblast growth factor (FGF) receptor (FGFR) expression was inhibited by cytochalasin D and latrunculin A, suggesting that FAK regulates both class I-mediated activation of the PI3K/Akt survival pathway and cell proliferation (10). However, much of what we conjecture about FAK interactions with effectors in the class I signal transduction pathway is based upon the pharmacological inhibitors cytochalasin D and latrunculin A that prevent actin polymerization (20). This approach, although informative, is limited by the challenges inherent in establishing the specificity of these agents.…”