Activation of the intrinsic tyrosine kinase activity of certain growth factor receptors and related oncogenes, resulting in the phosphorylation of important intracellular target proteins, has been postulated to be a critical event in mitogenic signal transduction as well as malignancy. Ras proteins (ras p2l) have been implicated in the PDGF-stimulated mitogenic signalling pathway in fibroblasts, yet the function of the endogenous ras p2l in normal mitogenic signal transduction remains to be elucidated. It is well established that the GTP-bound forms of ras p2l are active and the GDP-bound forms are inactive. Recently, a cytosolic GTPase activating protein (GAP) has been shown to catalyze, in vivo, the conversion of p2l-GTP to p2l-GDP so that this recycling proceeds at a rate greater than 100-fold in excess of the intrinsic p2l hydrolytic rate. GAP has no effect on oncogenic ras p2l proteins, which allows them to remain in their active GTP-bound states. Using intact quiescent NIH/3T3 cells, we observed that PDGF stimulation induced rapid and sustained tyrosine phosphorylation of GAP which was found to be associated with cell membranes, the known site of ras p2l biologic activity. Tyrosine-phosphorylated GAP coimmunoprecipitated with activated PDGF receptors as well as a 62-kd tyrosine phosphorylated protein. In experiments using NIH/3T3 cells transformed by certain oncogenes encoding activated tyrosine kinases, including abl, fgr, and src, tyrosine phosphorylated GAP was also readily detected. These data provide the first direct biochemical evidence linking the ras p2l/GAP mitogenic signalling system to the biochemical cascade activated by receptor tyrosine kinases and related oncogenes.
