Guanosine 5'-3-O-(thio)triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells. Role of p21rho.
Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin. GTP gamma S induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 microM) and reached maximum levels 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) inhibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 100 microM. Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation. Varying the Ca2+ concentration from 0 to 6 microM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTP gamma S to induce tyrosine phosphorylation. GTP gamma S was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+. Furthermore, 30 microM AlF4- only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTP gamma S to stimulate tyrosine phosphorylation of M(r) 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 GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 microM). This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin. In contrast, 20 microM p21ras17-44 peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTP gamma S stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.
Seckl
M J
MJ
Imperial Cancer Research Fund, London, United Kingdom.
Morii
N
N
Narumiya
S
S
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051419:Paxillin; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D020741:rho GTP-Binding Proteins
D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D008274:Magnesium; D002118:Calcium