REQUIREMENT OF PHOSPHOLIPASE C-GAMMA, THE TYROSINE PHOSPHATASE SYP AND THE ADAPTER PROTEINS SHC AND NCK FOR PDGF-INDUCED DNA-SYNTHESIS - EVIDENCE FOR THE EXISTENCE OF RAS-DEPENDENT AND RAS-INDEPENDENT PATHWAYS

We have investigated the roles of the phosphotyrosine phosphatase Syp (also called SH-PTP2), phospholipase C (PLC) gamma 1, rasGTPase Activa ting Protein (rasGAP) and the adapter molecules Nck and She in the mit ogenic response induced by PDGF in fibroblasts, Two separate approache s were used to inhibit the biological activity of these signalling pro teins in vivo. Either glutathione S-transferase (GST) fusion proteins containing the SH2 domains of these proteins, or antibodies specific f or these polypeptides, were microinjected into cells. GST-SH2 fusion p roteins are expected to act as dominant inhibitors by competing for ph ysiological SH2-mediated interactions, while microinjected antibodies can directly block protein functions, Inhibition of PLC gamma, Syp, Sh e and Nck signals blocked PDGF-stimulated cells in G(1) showing a requ irement for these proteins for S-phase entry. Inhibition of rasGAP, in contrast, had no effect on S-phase entry, We next examined which of t hese signals were required for PDGF-induced cFos expression, a Ras-dep endent event important for signalling, By using the same approaches wi th cells expressing beta-galactosidase under the control of a c-fos pr omoter, we showed that PLC gamma Syp and She were necessary for ligand -induced cFos expression whereas Nck and phosphatidylinositol 3-kinase were not. From these results we concluded that PDGF generates Ras-dep endent and Ras-independent pathways important for DNA synthesis.