A common requirement for p21c-ras function in the mitogenic signaling pathways of Swiss 3T3 fibroblasts

The role of p21c-ras in the activation of DNA synthesis in quiescent Swiss 3T3 fibroblasts was examined by scrape loading or microinjecting the neutralizing monoclonal antibody Y13-259 into the cells. Y13-259 delayed but did not block both the serum-stimulated entry of the cells into S phase and the accumulation of cdc2 mRNA and protein at the G1-S boundary. Introduction of Y13-259 also stimulated expression of sufficient p21c-ras to neutralize the loaded antibody; this finding suggests that the delay of S phase is attributable to the time taken to synthesize an excess of p21c-ras over the antibody and implies autoregulation of c-ras expression. Y13-259 had no effect upon phosphoinositide-mediated responses ([Ca2+]i increase and activation of protein kinase C) to platelet-derived growth factor or bombesin, demonstrating that p21c-ras is not required for mitogen activation of protein kinase C. Y13-259 inhibited 5-bromo-2'-deoxyuridine incorporation in response to all of the combinations of mitogens used to stimulate quiescent cells (fetal calf serum, platelet-derived growth factor, and the combination of insulin with 12-O-tetradecanoylphorbol-13-acetate, bombesin, epidermal growth factor, or prostaglandin E1), indicating that p21c-ras functions on pathways common to all of the effective combinations of mitogens examined.