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Macrophages exert a wide variety of functions, which necessitate a high level of plasticity on the chromatin level. In the work presented here, we analyzed the role of the polycomb group protein Bmi1 during the acute response of bone marrow derived macrophages (BMDM) to lipopolysaccharide (LPS). Unexpectedly, we observed that Bmi1 was rapidly induced at the protein level and transiently phosphorylated upon LPS treatment. The induction of Bmi1 was dependent on MAP-kinase signaling. LPS treatment of BMDM in the absence of Bmi1 resulted in a pronounced increase in expression of the anti-inflammatory cytokine interleukin-10 (IL-10). Our results identify Bmi1 as a repressor of IL-10 expression during macrophage activation.

Background: We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1., Principal Findings: In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16(INK4a) and p19(ARF)., Significance: The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.

Polycomb group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation. Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG function. We showed previously that PcG complexes are phosphorylated in vivo, which regulates their association with chromatin. The nature of the responsible PcG kinases remained unknown. Here we present the novel finding that the PcG protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways and implicated in differentiation and developmental processes. We identified 3pK as an interaction partner of PcG proteins, in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation, respectively. Activation or overexpression of 3pK resulted in phosphorylation of Bmi1 and other PcG members and their dissociation from chromatin. Phosphorylation and subsequent chromatin dissociation of PcG complexes were expected to result in de-repression of targets. One such reported Bmi1 target is the Cdkn2a/INK4A locus. Cells overexpressing 3pK showed PcG complex/chromatin dissociation and concomitant de-repression of p14(ARF), which was encoded by the Cdkn2a/INK4A locus. Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction. We speculate that phosphorylation may not only affect chromatin association but, in addition, the function of individual complex members. Our findings linked for the first time MAPK signaling pathways to the Polycomb transcriptional memory system. This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

Rap1 is a small GTPase implicated in cell proliferation and differentiation. The mechanisms how endogenous Rap1 is activated by many mitogenic stimuli including the neuropeptide bombesin remained unclear. Here we analyse which signaling pathways are necessary for Rap1 activation. Bombesin-mediated Rap1 activation in Swiss 3T3 and primary mouse embryo fibroblasts requires signaling components similar to those being essential for complex formation between p130Cas and Crk adapter proteins. The Crk/CRKL-binding region of the Rap1-specific exchange factor C3G (CBR) inhibits the bombesin-stimulated Rap1 activity in transfected Swiss 3T3 cells. Further characterization in COS cells showed that the CBR or a c-Crk I SH3 mutant specifically reduces both the basal as well as the stimulated Rap1 activity in a dose-dependent manner, whereas Ras is not affected. The CBR is complexed with endogenous c-Crk II and CRKL and blocks the protein association with catalytically active C3G. Such suppressors of Crk signaling do not affect Erk-phosphorylation induced by bombesin. Embryonic fibroblasts from b-raf knockout mice showed a bombesin-inducible Erk-phosphorylation, providing evidence that B-Raf does not link Rap1 to Erk-activation in bombesin-stimulated fibroblasts. We conclude that cellular Crk/CRKL complexes, recruited to upstream signaling components, contribute to basal and bombesin-induced Rap1 activity, which is independent from the Ras-Raf-Erk pathway under these circumstances.

Mitogenic signals initiated at the plasma membrane are transmitted to the nucleus through an intricate signalling network. We identified the protooncoprotein Cot as a new component of mitogenic signalling cascades, which activates both the classic cytoplasmic cascade and the SAPK stress pathway. Wildtype and activated Cot phosphorylate and activate MEK-1 and SEK-1 in vitro. These findings are consistent with the sequence homology between Cot and the rat gene Tpl-2. Expression of oncogenic Cot in 293, NIH3T3 and PC12 cells leads to in vivo phosphorylation of endogenous c-Jun and Erk-1/2 suggesting that the serine/threonine kinase Cot functions beside c-Raf-1 and Mos as a direct activator of MEK-1. Furthermore, we have examined the biological effects of Cot on the phenotype of fibroblastic and neuronal cells. In order to test a potential c-Raf-1 dependency of Cot transformation, the effect of oncogenic Cot on Raf revertant CHP25 cells was determined. Cot could restore the transformed phenotype indicating that Cot transformation is not dependent on active c-Raf-1 and that Cot is not a target for the putative Raf inhibitor, which is presumably active in the revertant cell line. Expression of oncogenic versions of Raf as well as v-Mos leads to differentiation of PC12 cells. Cot also induces neurite outgrowth of PC12 cells. These data are consistent with the role of Cot in the classic mitogenic cascade and suggest that the simultaneously activated JNK/SAPK stress pathway has no antagonistic effects in this context.

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (deltaRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion mutant-estrogen receptor fusion protein (c-Raf-1-BxB-ER) (N-BxB-ER cells). The transformed morphology of these cells is dependent on the presence of the estrogen antagonist 4-hydroxytamoxifen. Addition of 4-hydroxytamoxifen to N-BxB-ER cells arrested by density or serum starvation causes reentry of these cells into cell proliferation. Increases in the cell number are obvious by 24 h after activation of the oncogenic c-Raf-1 protein in confluent cells. The onset of proliferation in serum-starved cells is further delayed and takes about 48 h. In both cases, the proliferative response of the oncogenic c-Raf-1-induced cell proliferation is weaker than the one mediated by serum and does not lead to exponential growth. This is reflected in a markedly lower expression of the late-S- and G2/M-phase-specific cyclin B protein and a slightly lower expression of the cyclin A protein being induced at the G1/S transition. Oncogenic activation of c-Raf-1 induces the expression of the heparin binding epidermal growth factor. The Jnk1 kinase is putatively activated by the action of the autocrine growth factor. The kinetics of Jnk1 kinase activity is delayed and occurs by a time when we also detect DNA synthesis and the expression of the S-phase-specific cyclin A protein. This finding indicates that oncogenic activation of the c-Raf-1 protein can trigger the entry into the cell cycle without the action of the autocrine growth factor loop. The activation of the c-Raf-1-BxB-ER protein leads to an accumulation of high levels of cyclin D1 protein and a repression of the p27Kip1 cyclin-dependent kinase inhibitor under all culture conditions tested.

In this study we investigated the effect of Listeria monocytogenes infection on the activation of the Raf-MEK-MAP kinase pathway in eukaryotic host cells. HeLa cell infection with L. monocytogenes EGD resulted in a rapid, but transient, phosphorylation of the MAP kinases erk-1 and erk-2, a transient phosphorylation of the MAP kinase kinase MEK-1, and a transient activation of the MAP kinase kinase kinase Raf. In parallel to the transient phosphorylation of the MAP kinases, we detected induced expression of the MAP kinase phosphatase MKP-1. Additionally we present evidence that listeriolysin O is the inducing agent for activation of the Raf-MEK-MAP kinase pathway.

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two-hybrid screen of a mouse-embryo cDNA library we detected an interaction between A-Raf and CK2beta subunit. This binding was specific, as no interaction between CK2beta and B-Raf or c-Raf-1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A-Raf kinase domain. A-Raf kinase activity was enhanced 10-fold upon coexpression with CK2beta in Sf9 cells. The alpha subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf-isoform-specific activation and a regulatory role for CK2beta independent of the CK2alpha subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.

A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.

The Raf family proto-oncogenes encode cytoplasmic protein serine/threonine kinases which play a critical role in cell growth and development. A-raf shares several functional properties with Raf-1 including transforming activity, stimulation of the Raf/MAPK pathway and the ability of dominant negative versions to functionally block Ras signalling. A-raf transcripts are predominantly expressed in the mouse urogenital tissues. Interestingly, the human A-raf promoter region contains three potential glucocorticoid response elements GRE-1, GRE-2 and GRE-3, at positions -17, -34 and -168 respectively from the transcriptional start site. DNA sequence analysis of the mouse A-raf promoter region demonstrated that GRE-1 and -2 were conserved evolutionarily. To determine whether the human A-raf GREs represent functional motifs, an expression vector for the glucocorticoid receptor was cotransfected with A-raf promoter/reporter constructs into HeLa cells. A fivefold dexamethasone-dependent induction of A-raf promoter activity was observed using constructs containing all three GRE motifs whereas point mutations in the GREs either diminished or abolished dexamethasone induction. Electrophoretic mobility shift assays (EMSAs) using purified glucocorticoid receptor DNA binding domain (DBD) demonstrated that both GRE-2 and -3 motifs interact with DBD and oligonucleotide competition experiments established that these have different affinities for DBD. Using nuclear extracts from human and rodent cell lines in EMSAs, a specific protein-DNA complex was observed with GRE-1 which displayed binding properties unlike that of glucocorticoid receptor. These results demonstrate that the A-raf promoter is regulated in part by members of the glucocorticoid family of steroid hormone receptors and suggest a model for the regulation of A-raf expression in urogenital tissues.

The serine/threonine protein kinase Raf-1 is a component of a conserved intracellular signaling cascade that controls responses to various extracellular stimuli. Transcription from several promoters, including the oncogene-responsive element in the polyomavirus enhancer, the c-fos promoter, as well as other AP-1- and Ets-dependent promoters, can be induced by Raf-1 kinase. Previously, we have shown that activated Raf-1 kinase transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and have identified the NF-kappaB binding motif as a Raf-1-responsive element (RafRE). We now report that Raf-1 kinase-induced transactivation from the HIV RafRE involves the purine-rich-repeat-binding protein (GABP), which is composed of two distinct subunits (alpha and beta). GABP alpha is an Ets oncogene-related DNA-binding protein, and GABP beta contains four ankyrin-like repeats that have been shown to be essential in protein-protein interactions. In electrophoretic mobility shift assays using nuclear extracts from human Jurkat T cells, a protein-DNA complex which was supershifted with antiserum against GABP alpha and GABP beta was observed. Purified recombinant GABP alpha and beta interact with the HIV RafRE as judged from DNA binding assays. Cotransfection experiments with GABP alpha and beta and Raf-1 kinase demonstrate synergistic transactivation of the HIV-1 promoter. Point mutations in the HIV RafRE abolished the Raf-1 kinase as well as GABP alpha- and beta-induced transactivation. The observed Raf-1-GABP synergism presumably involves phosphorylation of GABP subunits, as treatment of cells with Raf-1 kinase activators serum and 12-O-tetradecanoylphorbol-13-acetate increases phosphorylation of GABP in vivo. However, GABP is not a target of Raf-1 kinase; instead, it is a substrate of mitogen-activated protein kinase (MAPK/ERK), since in vitro phosphorylation of GABP alpha and beta was achieved by the reconstituted protein kinase cascade but not with purified Raf-1 or MEK. These results suggest that Raf-1 kinase- induced activation of the HIV-1 promoter is mediated by the classical cytoplasmic cascade resulting in MAPK/ERK-mediated phosphorylation of GABP alpha and beta. Because the HIV RafRE corresponds to a region within the promoter which is essential for regulation of HIV-1 expression, the data indicate that in addition to NK-kappaB, GABP transcription factors are important for induced expression of HIV.

Synthesis of the protein kinase Mos is required for progesterone-induced activation of MAP kinase, M-phase promoting factor (MPF), and meiotic maturation of Xenopus oocytes. Mos can function as a MAP kinase kinase kinase, leading to activation of MAP kinase; how Mos causes activation of MPF is not yet known. The protein kinase Raf, which acts as a MAP kinase kinase kinase in somatic cells, also appears to be involved in meiotic maturation, but recent work has suggested that the Raf acts downstream of Mos activity during oocyte maturation. Using an oocyte cell-free system, we report here that a dominant negative Raf, which inhibits Ras-induced MAP kinase activation, does not block Mos-induced activation in vitro. These results indicate that, in contrast to previous conclusions, Mos-induced oocyte MAP kinase activation proceeds independently of Raf. Using a dominant-negative MAP kinase construct, we also show that most of the mitogen-induced hyperphosphorylation and dramatic gel retardation of Raf, which is often taken as a marker for the activation of Raf by upstream components, is actually dependent on, and thus downstream of, MAP kinase activation.

Growth factor stimulation of the mitogen-activated protein (MAP) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP kinase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.

Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.

Raf kinases are signal-integrating enzymes that have the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. The connection involves a cascade of protein kinases that is essential for cellular proliferation and differentiation of species ranging from worms to humans. This cascade also mediates transformation by most oncogenes.

The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.

We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.

Raf-1 belongs to a family of serine/threonine protein kinases which are highly conserved through evolution in multicellular organisms. Raf-1 kinase has gained much attention due to its function as a critical shuttle enzyme that connects stimulation of growth factor receptors and protein kinase C at the cell membrane to changes in the expression of genes involved in the control of cell growth, differentiation and survival. Regulation of Raf-1 activity is complex and involves Ras, as well as several serine/threonine and tyrosine kinases. Through a series of phosphorylation events, extracellular signals are connected through the Raf-1/MAP kinase pathway to activity-regulation of several oncogene-class transcription factors via phosphorylation of specific serine residues. Under ordinary circumstances, the cascade involving Raf-1 eventually leads to changes in gene expression and protein synthesis. Upon constitutive activation of Raf-1 kinase, as a result of genetic changes, a variety of cell types acquire a transformed phenotype.

Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo coincident with the de novo expression of Ito cell PDGF beta receptor messenger RNA. PDGF-induced mitogenesis was studied in cultured rat hepatic Ito cells which resemble the myofibroblast associated with liver injury. Pretreatment with prostaglandin E markedly suppressed the PDGF response in a dose-dependent fashion. The PDGF-induced cascade was studied with or without PGE to determine the level of regulation which induced the observed suppression. PGE caused no apparent diminution in the abundance of the surface PDGF beta receptor nor its subsequent activation and tyrosine phosphorylation following PDGF stimulation. The cytoplasmic 'secondary messengers' mitogen-activated protein kinase pp42-44 and raf kinase, appeared to be comparably induced and therefore unaffected by PGE. Raf perinuclear translocation was also intact and comparable degrees of nuclear egr, fos, and jun expression occurred. Since other studies have suggested that many of these features of the PDGF cascade may be causally and sequentially linked, the data collectively suggests that the dominant PGE mitogenic suppressive effect resides at a raf-MAP parallel pathway or at a nuclear level distal to the induction of these early growth response genes.

Insulin treatment of untransfected 3T3 L1 cells quickly induced activation of a cytosolic 42-kDa mitogen-activated protein kinase (MAPK) and a 90-kDa S6 kinase (RSK). The activation of these cytosolic kinases was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with inducible ras oncogenes. Furthermore, insulin-induced activation of MAPK and RSK could be blocked by expression of a transfected inducible dominant negative Ras mutant (Asn-17). These results indicate that Ras proteins are obligatory intermediates in the activation of the cytosolic MAPK/RSK cascade by insulin. Insulin treatment of 3T3 L1 cells or expression of transfected ras oncogenes resulted also in hyperphosphorylation of cellular Raf-1. Insulin-induced Raf hyperphosphorylation was inhibited by expression of an inducible dominant negative Ras mutant (Asn-17). We also showed that expression of transfected raf oncogenes induces adipocytic differentiation, as detected by expression of the specific adipocytic marker aP2. In addition, insulin-induced differentiation was significantly blocked by expression of a dominant negative raf mutant. Interestingly, however, the expression of transfected raf oncogenes did not induce MAPK or RSK activation, and the insulin-induced activation of these kinases was not blocked by expression of transfected dominant negative raf mutants. These results are consistent with Raf kinases acting downstream of Ras, but not upstream of MAPK and RSK in insulin-signaling pathways leading to 3T3 L1 differentiation.

Although a pathway that requires sequential activation of Ras, Raf, and MAP kinase kinase has been proposed as the major mechanism for stimulation of mitogen-activated protein kinase (MAP kinase), alternative pathways also exist. A wide variety of extracellular stimuli have been shown to activate MAP kinase; however, the precise mechanisms by which these stimuli mediate the signaling events have not been elucidated. Using a Balb/c-derived cell line expressing a dominant-negative mutant of Raf, we determined whether Raf is required for the activation of MAP kinase by growth factors, phorbol esters, and calcium. Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and phorbol 12,13-dibutyrate activated Ras in both mutant and control cells. However, stimulation of MAP kinase by IGF-I was nearly abolished in the dominant-negative Raf mutant. Stimulation of MAP kinase by the Ca2+ mobilizer thapsigargin was also inhibited in the presence of the Raf mutant. In contrast, EGF and phorbol 12,13-dibutyrate remained potent stimulators of MAP kinase in the dominant-negative Raf cells. The activation of MAP kinase by these stimuli can be further distinguished by differential requirements for Ca2+ and protein kinase C. These results suggest that Raf is required for the activation of MAP kinase by IGF-I and calcium, whereas EGF and possibly phorbol esters may employ alternative Raf-independent pathways for MAP kinase activation.

The protein kinase cascade Raf-MAPKK/MEK-MAPK/ERK connects protein tyrosine kinase receptors in the membrane with control of transcription factor activity in the nucleus. We have examined whether Raf is obligatory for activation of this cascade and whether this signaling pathway is relevant to transformation. By use of transient assays with epitope-tagged ERK-1 cDNA and a dominant inhibitory mutant of Raf-1 we found that serum and 12-O-tetradecanoylphorbol-13-acetate as well as representatives of three classes of oncogenes (protein tyrosine kinases abl/src, Ras, and protein serine/threonine kinases mos/cot) were all Raf-dependent for stimulation of MAPK. All of the MAPK stimulating oncogenes were also activators of Raf kinase as judged by shift induction. It thus appears that there is little or no redundancy in pathways used by growth regulators for activation of MAPK/ERK. Furthermore, the ability to stimulate MAPK/ERK appears to be critical for transformation by oncogenic Raf-1 and ERK-1 and -2 synergized with v-raf in a focus induction assay on NIH3T3 cells and kinase dead mutants of ERK-2 were inhibitory. Raf/ERK synergism was also observed in transcriptional transactivation of the oncogene-response element in the polyoma enhancer. We conclude that this Raf signaling pathway, which connects to many upstream activators and downstream effectors, is essential for transformation by most oncogenes.

The transfection of several oncogenes, particularly c-raf-1, into mammalian in vitro cell lines has been reported to be associated with increased radioresistance. We have thus investigated (by scanning photodensitometry of western blots) the phenotypic expression of the c-raf-1, c-myc and c-ras protein products in 19 human in vitro cell lines, whose intrinsic cellular sensitivity to 4 MeV photon irradiation has also been determined. High levels of c-raf-1 proto-oncogene product expression did not correlate with increased cellular radioresistance, but rather showed a significant correlation with intrinsic cellular radiosensitivity to photon irradiation for alpha (r = 0.664, P = 0.002), and SF2 (r = -0.655, P = 0.002). There was no significant correlation for the ras family, c-myc or actin. These results conflict with those of previous studies in which transfection of the activated forms of the c-raf-1 oncogene were associated with increased radioresistance, and suggest the possibility that the full length proto-oncogene may influence cellular radiosensitivity in a different manner from that of the activated oncogene.

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.

The raf protooncogenes are the cellular counterparts of the v-raf oncogene expressed by a murine sarcoma virus. The raf protooncogenes encode cytoplasmic serine/threonine-specific protein kinases which can be activated from different growth factor receptors by phosphorylation. The mRNAs of raf protooncogenes are found in a large variety of normal adult tissues, including the central nervous system. As concerns the distribution and localization of their protein products (the raf kinases), very few data are to be found in the literature. This is the first detailed description of their light microscopic localization in neocortical and allocortical areas of rodents. Preembedding immunohistochemical studies were performed on vibratome sections from the brains of adult guinea pigs and albino rats. The localizations of two isoenzymes, raf-1 kinase and B-raf kinase, were studied with the help of isoenzyme-specific polyclonal antibodies. Both of the antibodies detected raf protein-like immunoreactivity in many neurons and scattered glial cells of the sensory neocortex, and the cingular, pyriform, perirhinal and entorhinal allocortical areas. Pyramidal and non-pyramidal cells of Ammon's horn, granule cells of the dentate fascia and the large neurons in the hilar region were immunoreactive, too. The findings indicated that B-raf protein kinase and raf-1 kinase are present almost ubiquitously in the neurons of the investigated cortical structures. The intensity of staining obtained with serial dilutions of the antibodies indicated that the cytoplasmic concentration of B-raf kinase is tended to be higher than that of raf-1 kinase. The present findings suggested that the raf kinases are localized in postsynaptic structures, mainly in dendrites and cell bodies. Their cytosolic localization and their ability to undergo intracellular translocation during activation and phosphorylation raise the possibility that they play a pivotal role in the intracellular signaling of neurons.

The Raf-1 proto-oncogene product is a highly regulated serine/threonine kinase that functions in signal transduction downstream from growth factor receptors and upstream from nuclear proto-oncogene products. Using a transient cotransfection assay we have found that activated Raf-1 activates expression from the HIV-LTR. Analysis of a series of 5' deletion and point mutations revealed the NF-kappa B motifs as the Raf-responsive element in the HIV-LTR. Moreover, Raf-BXB activated expression from heterologous promoters driven by the HIV NF-kappa B binding sites. In addition to Raf, we show that v-Src, v-H-Ras and v-Mos activate HIV-LTR expression through the NF-kappa B binding sites and v-H-Ras-induced HIV-LTR expression is mediated by Raf-1. These findings may have implications for the involvement of the cellular homologues of these oncogenes in the switch from latent to productive infection by HIV in response to T-cell activation.

Engagement of the T cell receptor/CD3 complex activates the serine/threonine kinase, Raf-1, but the physiologic consequences of its activation have not been determined. The effects of Raf-1 on interleukin 2 (IL2) production in T cells were examined using activated and inhibitory forms of Raf-1. A truncated active form of Raf-1 was expressed constitutively from the metallothionein promoter in a malignant T cell line, Jurkat. Treatment of the cells with zinc and cadmium greatly increased active Raf-1 expression. This increase in Raf-1 expression allowed antibodies to CD3 and to CD28 to stimulate IL2 production in the absence of phorbol myristate acetate (PMA) and enhanced IL2 production stimulated by these antibodies in the presence of PMA. The action of active Raf-1 was to increase IL2 gene transcription as it enhanced transcription of a reporter gene linked to IL2 promoter. Finally, the dominant negative form of Raf-1 inhibited transcription directed by the IL2 promoter that was induced by the mitogen phytohemagglutinin (PHA) and PMA. We conclude that Raf-1 activity is necessary for IL2 gene transcription and secretion. These data indicate a role for Raf-1 in the immune response.

The c-Raf-1 serine/threonine kinase is an important component of signal transduction pathways mediating the effects of a variety of growth factors. In activated T cells, IL-2 has been shown to induce activation of c-Raf-1, but c-Raf-1 has not previously been shown to be activated through the T-cell receptor (TCR) in resting G0 T cells. Using a sensitive immune complex kinase reaction, we show that cross-linking of the stimulatory and costimulatory receptors CD3, CD4, or CD28 induces c-Raf-1 activation in highly purified resting peripheral blood human T cells. In contrast, cross-linking the nonstimulatory receptor CD45 did not induce c-Raf-1. Surprisingly, although earlier studies had shown delayed kinetics in response to Thy-1 stimulation in murine cells, c-Raf-1 activation in response to CD3 cross-linking was one of the earliest measurable events. In spite of its early kinetics, c-Raf-1 activation was found to be downstream of several other early signal transduction events, including activation of a tyrosine kinase and a tyrosine phosphatase. Several lines of evidence suggest that activation of c-Raf-1 in response to TCR stimulation may be PKC-dependent: first, phorbol esters are extremely potent activators of c-Raf-1 in human T cells; second, the kinetics of accumulation of products of phosphatidylinositol hydrolysis coincides with the kinetics of c-Raf-1 activation; and third, physiologic activation of the PLC/PKC pathway through a transfected, G-protein-coupled receptor HM1 induced similar levels of c-Raf-1 activation with a similar time course. We conclude that c-Raf-1 activation is tightly coupled to TCR stimulation and may participate in signal transduction pathways in resting, G0 T cells. The observation that the HM1 receptor can also activate c-Raf-1 suggests that T cells have the capability to utilize both tyrosine kinase-dependent and tyrosine kinase-independent mechanisms of c-Raf-1 activation.

We examined the effect of overexpression of growth factor-regulated second messenger enzymes, alone and in combination, on transformation of NIH3T3 cells. Signal transducers included phospholipase C-gamma (PLC-gamma), protein kinase C-gamma (PKC-gamma), and two proto-oncogenes, c-H-ras and c-raf-1. Three of these proteins, PLC-gamma, PKC-gamma and Raf-1, did not transform NIH3T3 cells alone or in combination. c-H-ras, which under its own promoter control has low transforming activity, also did not cooperate with PLC-gamma or PKC-gamma. In contrast, the combination of normal or oncogenic p21 H-Ras with the Raf-1 kinase dramatically increased transformation efficiency. The level of Ras protein required for transformation was reduced in Raf-1 co-transfectants, implying that, at low levels of p21 Ras, p74 Raf-1 is rate limiting. As transformation by Ras depends on jun-mediated transcriptional events, we also examined H-ras and c-raf-1 cooperation in transcriptional transactivation of TPA-responsive element (TRE)-dependent reporters. Like the H-ras/c-raf-1 cooperation in transformation, we observed this synergistic stimulation of TRE-dependent transcription. This pathway for transformation and transcriptional activation by increased levels of normal Ras and Raf may be important in tumors that show overexpression but lack mutationally activated forms of these two proto-oncogenes.

Treatment of cells with various growth factors and mitogens results in the rapid hyperphosphorylation and activation of the Raf-1 kinase. To determine if phosphorylation events affect Raf-1 activity, we have initiated experiments to identify the phosphorylation sites of Raf-1. In this report, we find that Ser43, Ser259, and Ser621 are the major sites of Raf-1 which are phosphorylated in mammalian cells and in Sf9 insect cells infected with a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase activity are also phosphorylated on these sites in vivo, indicating that these phosphorylation events are not a consequence of autophosphorylation. Furthermore, we find that Thr268 is the predominant Raf-1 residue phosphorylated in in vitro autokinase assays. In addition, we have examined the biochemical activity of baculovirus-expressed Raf-1 proteins containing mutations at these phosphorylation sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold more active than wild-type Raf-1 and Ser621 mutant proteins were inactive as kinases. Analysis of the residues surrounding Ser259 and Ser621 indicates that RSXSXP may be a consensus sequence for the kinase responsible for phosphorylation of Raf-1 at these sites. Interestingly, these RSXSXP sequences are completely conserved throughout evolution in all Raf family members.

The c-raf-1 protooncogene encodes a Ser/Thr protein kinase. A mitogen-activated protein kinase-kinase (MAPKK) purified from bovine brain is phosphorylated and activated 4-9-fold in vitro by c-Raf-1 from mitogen-treated cells. c-Raf-1 protein kinase activity, measured by the phosphorylation of brain MAPKK substrate, is detectably activated within 1 min after addition of platelet-derived growth factor (PDGF) to 3T3 cells, increasing more rapidly than the endogenous NIH 3T3 cell MAPKK activity. c-Raf-1 activation is also induced by insulin, phorbol ester, thrombin, and endothelin. PDGF-, epidermal groth factor-, and insulin-stimulated 32P-c-Raf-1 yield very similar, complex tryptic 32P-peptide maps, wherein only 2 of 10 32P-peptides appear entirely de novo after growth factor addition. Mitogen-activated protein kinase/extracellular signal-regulated kinase-2 can phosphorylate c-Raf-1 in vitro on 4-6 tryptic 32P-peptides, all of which comigrate with tryptic 32P-peptides derived from c-Raf-1 labeled in situ. Mitogen-activated protein kinase phosphorylation of c-Raf-1 in vitro, however, does not 1) generate 32P-peptides that comigrate with those that appear de novo after PDGF or insulin treatment in situ; 2) does not convert c-Raf-1 polypeptides to a slower mobility on SDS-polyacrylamide gel electrophoresis as is seen after PDGF or insulin; 3) does not alter c-Raf-1 kinase activity toward MAPKK. Thus, based on overlapping site specificity, Erk-2 is a viable candidate to be among the PDGF-stimulated c-Raf-1 kinases. Although PDGF/insulin-stimulated c-Raf-1 Ser/Thr phosphorylation may be necessary to sustain the active state, a role for mitogen-activated protein kinase/extracellular signal-regulated kinase-2 phosphorylation in the initiation of c-Raf-1 activation is unlikely.

In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

Previously we found that interleukin 2 (IL-2) induces tyrosine phosphorylation and activation of the serine/threonine-specific kinase encoded by the raf-1 protooncogene in a T-cell line, CTLL-2. Here we extended these findings by exploring the effects of selective removal of phosphate from tyrosines in p72-74-Raf-1 kinase that had been immunoprecipitated from IL-2-stimulated CTLL-2 cells. Treatment in vitro of IL-2-activated Raf-1 with the tyrosine-specific phosphatases CD45 and TCPTP (formerly called T-cell protein tyrosine phosphatase) reduced Raf kinase activity to nearly baseline levels. This effect was completely inhibited by the phosphatase inhibitor sodium orthovanadate. In contrast, treatment of Raf-1 with a serine/threonine-specific phosphatase, protein phosphatase 1 (PP-1), resulted in a more modest decrease in Raf in vitro kinase activity, and this effect was prevented by okadaic acid. Two-dimensional phosphoamino acid analysis confirmed the selective removal of phosphate from tyrosine by CD45 and from serine and threonine by PP-1. The immunoreactivity of p72-74-Raf-1 with anti-phosphotyrosine antibodies was also completely abolished by treatment with CD45 in the absence but not in the presence of sodium orthovanadate. These findings provide evidence that the IL-2-stimulated phosphorylation of Raf-1 on tyrosines plays an important role in upregulating the activity of this serine/threonine-specific kinase in CTLL-2 cells and, as such, provides a model system for studying the transfer of growth factor-initiated signals from protein tyrosine kinases to serine/threonine-specific kinases.

The cellular raf oncogenes encode cytoplasmic serine/threonine specific protein kinases. Since there are some indications that they are involved in neuronal plasticity, we investigated the localization of raf protein kinases immunohistochemically. The spinal cord of adult guinea pigs was fixed by means of transcardial perfusion, transverse sections were cut from each segment and stained with monoclonal and polyclonal antibodies, raised against synthetic raf peptide and recombinant raf protein, respectively. The motor neurons of the ventral horn and the large- and medium sized neurons of laminae I, IV, V, VI, VII and VII were stained in every segment of the spinal cord. The widespread occurrence of raf kinases in the spinal cord and the literature data indicating their participation in growth factor mediated processes, raise the possibility that the raf protooncogenes play a fundamental role in the regulation of transmembrane signalling of these neurons.

A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.

The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.

The B-raf gene was localized to human chromosome region 7p11-7qter by Southern blot analysis of its segregation in a panel of rodent-human hybrids, using a B-raf cDNA clone. Chromosomal in situ hybridization refined the localization to 7q33-36. Analysis of genomic B-raf clones identified a B-raf pseudogene in addition to the active gene. Based on the chromosomal mapping data we conclude that the pseudogene is located near the active gene.

Raf-1 serine-threonine protein kinase has the hallmarks of a critical switch that connects growth factor receptor activation at the cell membrane with transcriptional events in the nucleus. We show by use of Raf-1 dominant-negative mutants that Raf-1 is required for serum-, TPA-, and Ras-induced expression from the oncogene-responsive element in the polyomavirus enhancer. The minimal region of Raf-1 that displays this dominant-negative phenotype (Raf-C4) contains a cysteine finger motif. Raf-C4 appears to function by titrating out a Raf-1-activating factor that is induced by Ras following serum or TPA treatment of NIH-3T3 cells. In addition, we show that Raf-1 and Ras cooperate in trans-activation through the oncogene-responsive element and that the cysteine-rich region is necessary for this effect.

Treatment of PC12 cells with nerve growth factor does not alter the levels of B-raf mRNA, but does induce rapid phosphorylation of B-raf proteins. Phosphorylation was observed after 1.5 min and reached a maximum by 10-15 min. B-raf protein was phosphorylated almost exclusively on serine residues; no tyrosine phosphorylation was detected. Nerve growth factor-induced phosphorylation was not affected by depletion of protein kinase C or by removal of extracellular calcium but was inhibited by K-252a. Concomitant with the increase in serine phosphorylation, nerve growth factor treatment also increased the serine/threonine kinase activity of B-raf protein within 1-2 min.