GUANOSINE 5'-3-O-(THIO)TRIPHOSPHATE STIMULATES TYROSINE PHOSPHORYLATION OF P125(FAK) AND PAXILLIN IN PERMEABILIZED SWISS 3T3 CELLS - ROLE OF P21(RHO)

Addition of guanosine 5'-3-O-(thio) triphosphate (GTP gamma S) to stre ptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylat ion of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion ki nase (p125(FAK)) and paxillin. GTP gamma S induced tyrosine phosphoryl ation was dose-dependent (EC(50) of 2.5 mu M) and reached maximum leve ls after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M (r) 70,000-80,000 paxillin band. Guanosine 5'-O-(2-thiodiphosphate) in hibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 1 00 mu M. Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation. Varying the Ca2+ concentration from 0 to 6 mu M did not increase tyrosine phosphorylation above basal levels and did not a ffect the ability of GTP gamma S to induce tyrosine phosphorylation. G TP gamma S was able to stimulate tyrosine phosphorylation in the prese nce of nanomolar concentrations of Mg2+. Furthermore, 30 mu M AlF4- on ly weakly induced tyrosine phosphorylation in permeabilized cells. Pre treatment with the Clostridium botulinum C3 exoenzyme which inactivate s p21(rho), markedly reduced the ability of GTP gamma S to stimulate t yrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 band s including p125(FAK) and paxillin in permeabilized Swiss 3T3 cells. F urthermore, a peptide of p21(rho) (p21(rho17-44)) inhibited GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 mu M). This peptide also inhibited tyrosine phosphorylation of p125(FA K) and paxillin. In contrast, 20 mu M p21(ras17-44) peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation. Using permeabili zed cells, our findings demonstrate that GTP gamma S stimulates tyrosi ne phosphorylation of p125(FAK) and paxillin and that a functional p21 (rho) is implicated in this process.