INHIBITION OF PLATELET-DERIVED GROWTH FACTOR-MEDIATED AND EPIDERMAL GROWTH FACTOR-MEDIATED MITOGENESIS AND SIGNALING IN 3T3 CELLS EXPRESSING DELTA-RAF-1-ER, AN ESTRADIOL-REGULATED FORM OF RAF-1

We have recently described the properties of Delta Raf-1:ER, a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the human estrogen receptor. In this study, we demo nstrate that activation of Delta Raf-1:ER in quiescent 3T3 cells (C2 c ells), while sufficient to promote morphological oncogenic transformat ion, was insufficient to promote the entry of cells into DNA synthesis . Indeed, activation of Delta Raf-1:ER potently inhibited the mitogeni c response of cells to platelet-derived growth factor (PDGF) and epide rmal growth factor (EGF) treatment. Addition of beta-estradiol to quie scent C2 cells led to rapid, sustained activation of Delta Raf-1:ER an d MEK but only two- to threefold activation of p42 mitogen-activating protein (MAP) kinase activity. Addition of PDGF or EGF to quiescent C2 cells in which Delta Raf-1:ER was inactive led to rapid activation of Raf-1, MEK, and p42 MAP kinase activities, and entry of the cells int o DNA synthesis. In contrast, when Delta Raf-1:ER was activated in qui escent C2 cells prior to factor addition, there was a significant inhi bition of certain aspects of the signaling response to subsequent trea tment with PDGF or EGF. The expression and activation of PDGF receptor s and the phosphorylation of p70(S6K) in response to PDGF treatment we re unaffected by prior activation of Delta Raf-1:ER In contrast, PDGF- mediated activation of Raf-1 and p42 MAP kinases was significantly inh ibited compared with that of controls. Interestingly, the mitogenic an d signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior act ivation of Delta Raf-1:ER It seems likely that at least two mechanisms contribute to the effects of Delta Raf-1:ER in these cells. First, ac tivation of Delta Raf-1:ER appeared to uncouple the activation of Raf- 1 from the activation of the PDGF receptor at the cell surface. This m ay be due to the fact that mSOS1 is constitutively phosphorylated as a consequence of the activation of Delta Raf-1:ER Second, quiescent C2 cells expressing activated Delta Raf-1:ER appear to contain an inhibit or of the MAP kinase pathway that, because of its apparent sensitivity to sodium orthovanadate, may be a phosphotyrosine phosphatase. It is likely that the inhibitory effects of Delta Raf-1:ER observed in these cells are a manifestation of the activation of some of the feedback i nhibition path ways that normally modulate a cell's response to growth factors. 3T3 cells expressing Delta Raf-1:ER will be a useful tool in unraveling the role of Raf-1 kinase activity in the regulation of suc h pathways.