Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.
Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.
Molloy
C J
CJ
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Fleming
T P
TP
Bottaro
D P
DP
Cuadrado
A
A
Aaronson
S A
SA
D016475:3T3 Cells; D000818:Animals; D020690:GTPase-Activating Proteins; D005786:Gene Expression Regulation; D015151:Immunoblotting; D051379:Mice; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D014443:Tyrosine; D011493:Protein Kinase C; D016283:Proto-Oncogene Proteins p21(ras)