Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP).
The mitogenic activity of membrane-associated tyrosine kinases such as Src and the PDGF receptor appear to depend on Ras function. Ras biochemical activity involves regulation of a GTP/GDP cycle and the GTPase activating protein (GAP). Recently, PDGF and v-Src have been shown to stimulate tyrosine phosphorylation of GAP, linking these pathways at the biochemical level. To test whether PDGF and v-Src affect the Ras GTP/GDP cycle, we have measured the guanine nucleotides complexed to Ras in NIH3T3 cells and compared the ratio of GTP to total GTP + GDP detected (percent GTP). In normal quiescent NIH3T3 cells, PDGF stimulated the basal amount of GTP complexed to Ras (7%) by 2.1-fold to 15%. The effect was dependent on PDGF concentration and was observed maximally within 10 min following PDGF challenge. Ras was complexed to 22% GTP in NIH3T3 cells transformed by v-src or v-abl. Overexpression of GAP by 110-fold in NIH3T3 cells reduced the basal level of GTP complexed to Ras to 2.4%; upon challenge with PDGF, Ras was complexed to 6.6% GTP. These results indicate that PDGF receptor activation and tyrosine kinase-encoding oncogene products can stimulate Ras into the GTP complex and that GAP in intact mammalian cells can decrease the amount of GTP complexed to Ras.
Gibbs
J B
JB
Department of Molecular Biology, Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Marshall
M S
MS
Scolnick
E M
EM
Dixon
R A
RA
Vogel
U S
US
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D011905:Genes, ras; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D006150:Guanine Nucleotides; D015345:Oligonucleotide Probes; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D016173:Macrophage Colony-Stimulating Factor; D006160:Guanosine Triphosphate; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins