Guanosine 5′-3-O-(Thio)triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells: Role of p21rho

Abstract

Addition of guanosine 5′-3-O-(thio)triphosphate (GTPγS) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of Mr 110,000-130,000 and 70,000-80,000 bands. Specifically, GTPγS stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin. GTPγS induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 μM) and reached maximum levels after 1.5 min for the Mr 110,000-130,000 band and 2 min for the Mr 70,000-80,000 paxillin band. Guanosine 5′-O-(2-thiodiphosphate) inhibited GTPγS-induced tyrosine phosphorylation with an IC50 of 100 μM. Protein kinase C did not mediate GTPγS-induced tyrosine phosphorylation. Varying the Ca2+ concentration from O to 6 μM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTPγS to induce tyrosine phosphorylation. GTPγS was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+. Furthermore, 30 μM AIF4- only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTPγS to stimulate tyrosine phosphorylation of Mr 110,000-130,000 and 70,000-80,000 bands including p125FAK and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21rho (p21rho17-44) inhibited GTPγS-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 μM). This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin. In contrast, 20 μM p21ras17-44 peptide failed to inhibit GTPγS-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTPγS stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.