ABSTRACT

Addition of guanosine 5` - 3 - O - (thio) triphosphate (GTPS) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M 110,000-130,000 and 70,000-80,000 bands. Specifically, GTPS stimulated tyrosine phosphorylation of both focal adhesion kinase (p125) and paxillin. GTPS induced tyrosine phosphorylation was dose-dependent (EC of 2.5 µM) and reached maximum levels after 1.5 min for the M 110,000-130,000 band and 2 min for the M 70,000-80,000 paxillin band. Guanosine 5`-O-(2-thiodiphosphate) inhibited GTPS-induced tyrosine phosphorylation with an IC of 100 µM. Protein kinase C did not mediate GTPS-induced tyrosine phosphorylation. Varying the Ca concentration from 0 to 6 µM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTPS to induce tyrosine phosphorylation. GTPS was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg. Furthermore, 30 µM AlF only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21, markedly reduced the ability of GTPS to stimulate tyrosine phosphorylation of M 110,000-130,000 and 70,000-80,000 bands including p125 and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21(p21) inhibited GTPS-induced tyrosine phosphorylation in a dose-dependent manner (IC 1 µM). This peptide also inhibited tyrosine phosphorylation of p125 and paxillin. In contrast, 20 µM p21 peptide failed to inhibit GTPS-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTPS stimulates tyrosine phosphorylation of p125 and paxillin and that a functional p21 is implicated in this process.

FOOTNOTES

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The abbreviations used are: FAK, focal adhesion kinase; anti-Tyr(P), anti-phosphotyrosine; C3 toxin, Clostridium botulinum C3 exoenzyme; DMEM, Dulbecco's modified Eagle's medium; GDPS, guanosine 5`-3-O-(thio)diphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate; LPA, lysophosphatidic acid; mAb, monoclonal antibody; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PAGE, polyacrylamide gel electrophoresis; PIP-PLC, phosphatidylinositol 4,5-bisphosphate-specific phospholipase C; SLO, streptolysin O; Py72, phosphotyrosine; PIPES, 1,4-piperazineethanesulfonic acid.

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