Inhibition of platelet-derived growth factor- and epidermal growth factor-mediated mitogenesis and signaling in 3T3 cells expressing delta Raf-1:ER, an estradiol-regulated form of Raf-1.

Abstract

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 demonstrate that activation of delta Raf-1:ER in quiescent 3T3 cells (C2 cells), while sufficient to promote morphological oncogenic transformation, was insufficient to promote the entry of cells into DNA synthesis. Indeed, activation of delta Raf-1:ER potently inhibited the mitogenic response of cells to platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) treatment. Addition of beta-estradiol to quiescent C2 cells led to rapid, sustained activation of delta Raf-1:ER and 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 into DNA synthesis. In contrast, when delta Raf-1:ER was activated in quiescent C2 cells prior to factor addition, there was a significant inhibition of certain aspects of the signaling response to subsequent treatment with PDGF or EGF. The expression and activation of PDGF receptors and the phosphorylation of p70S6K in response to PDGF treatment were unaffected by prior activation of delta Raf-1:ER. In contrast, PDGF-mediated activation of Raf-1 and p42 MAP kinases was significantly inhibited compared with that of controls. Interestingly, the mitogenic and signaling responses of quiescent C2 cells to stimulation with fetal bovine serum or phorbol myristate acetate were unaffected by prior activation 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, activation 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 may 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 inhibitor 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 inhibition pathways 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 such pathways.

Inhibition of a signaling pathway in cardiac muscle cells by active mitogen-activated protein kinase kinase.

“…Because we obtain high levels of ERK2 activity in the MKK (AN3/S218E/S222D)-transfected cells, the effects that we observe are not likely to result from ERK inhibition by phosphatases. The mechanism of inhibition in our experiments therefore seems to be quite different from that occurring during ARaf-l:ERmediated inhibition of 3T3 cell growth (Samuels and McMahon, 1994).…”

“…Similarly, estrogen-induced ARaf-1 :ER prevents NIH 3T3 cell proliferation induced by tyrosine kinase-linked growth factors. This inhibition is correlated with phosphorylation of Sos and the activation of a vanadate-sensitive phosphatase that prevents ERK activation (Samuels and McMahon, 1994). Feedback inhibition of a molecule like Sos that functions early in the signaling pathway would be expected to switch off the entire pathway.…”

Cell cycle targets of Ras/Raf signalling

“…In NIH3T3 ®broblasts the activation of an inducible oncogenic version of the cRaf-1 protein (c-Raf-1-BxB-ER TM ) has been shown to be su cient to drive cells arrested by serum starvation or cell density back into the cell cycle (Kerkho and Rapp, 1997). In the same cellular background a similar Raf construct was shown to block cell proliferation when activated (Samuels and McMahon, 1994;Pumiglia and Decker, 1997;Woods et al, 1997;Sewing et al, 1997). An explanation for these obvious di erences was recently found, when three groups showed that the activation of constitutive low Raf signals leads to cell proliferation and that very high constitutive Raf signals lead to a cell cycle arrest Woods et al, 1997;.…”

Phospholipase D overcomes cell cycle arrest induced by high-intensity Raf signaling

“…As noted for activated Ras (Franza et al, 1986;Hirakawa and Ruley, 1988), too much Raf signaling leads to cell cycle arrest rather than proliferation (Samuels and McMahon, 1994;Kerkho and Rapp, 1998;Woods et al, 1997;Zhu et al, 1998;Sewing et al, 1997). These observations suggest that high intensity Raf signals are recognized as inappropriate, leading to a block in cell cycle progression.…”