Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP)

Abstract

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.