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Query: UNIPROT:P06889 (Mol)
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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.
Mol Cell Biol 1994 Dec
PMID: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. 796 25

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.
Mol Cell Biol 1994 Jul
PMID:JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. 800 42

Receptor-bound growth factors elicit intracellular signals that lead to the phosphorylation and activation of numerous intracellular kinases and transcription factors with consequent changes in patterns of gene expression. Several oncogene products are able to mimic these signals, resulting in cell transformation and proliferation. For example, the introduction of oncogenic forms of Raf-1 kinase into fibroblasts induces transformation and leads to the constitutive expression of, among others, the c-fos proto-oncogene. Here it is shown that the elevation of c-fos promoter activity brought about by v-raf is mediated by TCF/Elk-1, which forms a ternary complex with SRF at the serum response element and is a substrate for mitogen-activating protein kinases in vitro. In NIH 3T3 fibroblasts, v-raf activates Erk2, and overexpression of an interfering mutant of Erk2 both blocks the ability of v-raf to activate the c-fos promoter and suppresses transformation. Mutation of individual mitogen-activating protein kinase phosphoacceptor sites in TCF/Elk-1 also compromises v-raf-activated expression of a Gal-Elk/Gal-chloramphenicol acetyltransferase reporter system. However, in at least one instance the introduction of glutamate, but not aspartate, at a phosphoacceptor site is compatible with activation. These results provide compelling evidence that phosphorylation of TCF/Elk-1 by Erk2 is a major link in the Raf-1 kinase-dependent signal transduction pathway that activates c-fos expression.
Mol Cell Biol 1994 Jul
PMID:Inhibition of v-raf-dependent c-fos expression and transformation by a kinase-defective mutant of the mitogen-activated protein kinase Erk2. 800 80

Raf-1 is a serine/threonine kinase that acts downstream of Ras in mitogenic signal transduction pathways, but the mechanism by which Ras transmits signals to Raf-1 is not known. We have examined the interaction between Raf-1 and human H-ras in three different systems that utilize H-ras-induced phenotypes in Saccharomyces cerevisiae. In each system, the effects of H-ras depend on guanosine triphosphate and appear to be mediated through the H-ras effector binding region. H-ras effector function was blocked in each case by expression of the N-terminal regulatory domain of Raf-1. These inhibitory effects did not require the Raf-1 kinase domain. Raf-1 also blocked Rap1A effector function in S. cerevisiae. Raf-1, therefore, appears to interact with H-Ras and Rap1A in these in vivo systems with properties that suggest it is an immediate downstream effector.
Mol Biol Cell 1994 Feb
PMID:Raf-1 interferes with Ras and Rap1A effector functions in yeast. 801 3

Von Hippel Lindau disease (VHL) is a rare autosomal dominant disease associated with tumors and cysts in multiple organ systems. The VHL disease gene is tightly linked to the polymorphic DNA marker 233E2 (D3S720) and flanked by 479H4 (D3S719) on its telomeric and RAF1 on its centromeric side. Two additional markers, D3S1038 and D3S601, have also been identified, and these markers, like D3S720, are very tightly linked to VHL. Previously 93 cosmid clones were mapped to the larger region, 3p24.2-pter, surrounding the VHL disease gene. Using a Southern-based screening strategy on pools of YAC clones we have isolated a contig of overlapping YAC clones that extends about 0.7 megabase centromeric, and about 1.3 megabases telomeric of D3S720 and contains all three tightly linked VHL markers. Individual YACs in this contig were hybridized to grids containing cosmids localized between 3p24.2-pter and to several cosmids localized by fluorescent in situ hybridization (FISH) to 3p25. A total of 28 cosmids were positioned on this contig of overlapping YAC clones. We have also identified homologous YAC clones to many additional cosmid clones localized between 3p24.2-p25, although these have not yet been precisely localized relative to the contig of YAC clones. This contig of YAC clones probably contains the VHL disease gene and should facilitate the isolation and characterization of this gene.
Hum Mol Genet 1993 Aug
PMID:The isolation of a yeast artificial chromosome (YAC) contig extending for 2 megabases in the vicinity of the von Hippel Lindau disease gene. 810 27

Ras, a small GTP-binding protein, is required for functional receptor tyrosine kinase signaling. Ultimately, Ras alters the activity of specific nuclear transcription factors and regulates novel patterns of gene expression. Using a rat prolactin promoter construct in transient transfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolactin promoter in GH4 rat pituitary cells. We also show that the Ras signal is completely blocked by an expression vector encoding a dominant-negative Raf kinase. Additionally, using a molecular genetic approach, we determined that inhibitory forms of p42 mitogen-activated protein kinase and an Ets-2 transcription factor interfere with both the Ras and the Raf activation of the rat prolactin promoter. These findings define a functional requirement for these signaling constituents in the activation of the prolactin gene, a cell-specific gene which marks the lactotroph pituitary cell type. Further, this analysis allowed us to order the components in the Ras signaling pathway as it impinges on regulation of prolactin gene transcription as Ras-->Raf kinase-->mitogen-activated protein kinase-->Ets. In contrast, we show that intact c-Jun expression inhibited the Ras-induced activation of the prolactin promoter, defining it as a negative regulator of this pathway, whereas c-Jun was able to enhance the Ras activation of an AP-1-driven promoter in GH4 cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific prolactin cellular promoter. Thus, we have defined a model system which provides an ideal paradigm for studying Ras/Raf signaling pathways and their effects on neuroendocrine cell-specific gene regulation.
Mol Cell Biol 1994 Mar
PMID:Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression. 811 93

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.
Mol Cell Biol 1993 Dec
PMID:Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction. 824 52

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC) and activation of the Raf-1 protein kinase in Ras-mediated transduction of mitogenic signals. As previously reported, cotransfection of a PC-specific phospholipase C (PC-PLC) expression plasmid bypassed the block to cell proliferation resulting from expression of the dominant inhibitory mutant Ras N-17. In contrast, PC-PLC failed to bypass the inhibitory effect of dominant negative Raf mutants, suggesting that PC-PLC functions downstream of Ras but upstream of Raf. Consistent with this hypothesis, treatment of quiescent cells with exogenous PC-PLC induced Raf activation, even when normal Ras function was blocked by Ras N-17 expression. Further, activation of Raf in response to mitogenic growth factors was blocked by inhibition of endogenous PC-PLC. Taken together, these results indicate that hydrolysis of PC mediates Raf activation in response to mitogenic growth factors.
Mol Cell Biol 1993 Dec
PMID:Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction. 824 81

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.
Mol Cell Biol 1993 Jul
PMID:Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos. 832 Dec 23

Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C.
Mol Cell Biol 1993 May
PMID:Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter. 838 18


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