Inhibition of platelet-derived growth factor type-beta receptor autophosphorylation in cells transformed by activated ras

The regulation of events triggered by activation of receptor tyrosine kinases, and the subversion of these pathways by oncogenes is of great importance in unraveling the biochemical mechanisms of cellular transformation. Autophosphorylation of the platelet-derived growth factor type-beta receptor (PDGFbetaR) and subsequent activation of the PDGFbetaR pathway is blocked in cells expressing activated ras genes. In Balb/c-3T3 fibroblasts transformed by Kirsten v-ras (KBalb), a factor has been identified that dominantly inhibits autophosphorylation of the PDGFbetaR.;We have further characterized this factor and have demonstrated that it is a protein that associates with cellular membranes. Partial purification by anion-exchange chromatography has demonstrated that this inhibitor can be physically separated from the PDGFbetaR, resulting in recovery of kinase activity. Column fractions containing the PDGFbetaR inhibitor co-purify with the protein tyrosine phosphatase Syp, the adaptor molecule Grb2 and activated p21ras.;The presence of Syp in the inhibitory fraction and the general phosphatase activity present in the KBalb membrane preparation, prompted investigation into the role of phosphatases in the inhibition of PDGFbetaR activation. Sodium orthovanadate treatment of KBalb membranes in vitro had no affect on PDGFbetaR autophosphorylation. However, in vivo treatment of whole cells resulted in recovery of PDGFbetaR kinase activity. Accompanying the apparent suppression of inhibitory activity in the KBalb cells was a morphological change that reverted the transformed cells to a more normal fibroblastic appearance. These experiments suggest the following: (1) the ability of the PDGFbetaR in KBalb cells to phosphorylate requires inhibition of a vanadate-sensitive step, possibly implicating a phosphatase in the mechanism of inhibition. (2) The recovery of PDGFbetaR kinase activity may be linked to cell shape or changes in cytoskeletal elements. (3) The alterations in cell morphology may involve vanadate-regulatory mechanisms that in turn affect the ability of the PDGFbetaR to phosphorylate.