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Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601

2000; Springer Nature; Volume: 19; Issue: 20 Linguagem: Inglês

10.1093/emboj/19.20.5429

1460-2075

Baohong Zhang, Kun‐Liang Guan,

Protein Kinase Regulation and GTPase Signaling

Article16 October 2000free access Activation of B-Raf kinase requires phosphorylation of the conserved residues Thr598 and Ser601 Bao-Hong Zhang Bao-Hong Zhang Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Search for more papers by this author Kun-Liang Guan Corresponding Author Kun-Liang Guan Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Institute of Gerontology, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Search for more papers by this author Bao-Hong Zhang Bao-Hong Zhang Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Search for more papers by this author Kun-Liang Guan Corresponding Author Kun-Liang Guan Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Institute of Gerontology, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA Search for more papers by this author Author Information Bao-Hong Zhang1 and Kun-Liang Guan 1,2 1Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA 2Institute of Gerontology, University of Michigan Medical School, Ann Arbor, MI, 48109-0606 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5429-5439https://doi.org/10.1093/emboj/19.20.5429 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Raf kinase family serves as a central intermediate to relay signals from Ras to ERK. The precise molecular mechanism for Raf activation is still not fully understood. Here we report that phosphorylation of Thr598 and Ser601, which lie between kinase subdomains VII and VIII, is essential for B-Raf activation by Ras. Substitution of these residues by alanine (B-RafAA) abolished Ras-induced B-Raf activation without altering the association of B-Raf with other signaling proteins. Phosphopeptide mapping and immunoblotting with phospho-specific antibodies confirmed that Thr598 and Ser601 are in vivo phosphorylation sites induced by Ras. Furthermore, replacement of these two sites by acidic residues (B-RafED) renders B-Raf constitutively active. Con sistent with these data, B-RafAA and B-RafED exhibited diminished and enhanced ability, respectively, to stimulate ERK activation and Elk-dependent transcription. Moreover, functional studies revealed that B-RafED was able to promote NIH 3T3 cell transformation and PC12 cell differentiation. Since Thr598 and Ser601 are conserved in all Raf family members from Caenorhabditis elegans to mammals, we propose that phosphorylation of these two residues may be a general mechanism for Raf activation. Introduction The Raf proteins are a family of serine/threonine-specific kinases that serve as a central intermediate in transmitting extracellular signals to the mitogen-activated protein kinase (MAPK), also known as the extracellular signal regulated kinase (ERK) cascade, which controls cell growth, differentiation and survival (Marshall, 1995; Morrison and Cutler, 1997; English et al., 1999; Hagemann and Rapp, 1999). Activation of the small GTPase protein Ras is an initial step in the activation of Raf. Activated Raf proteins directly phosphorylate and activate the downstream dual specificity kinase MEK, which in turn phosphorylates ERK on threonine and tyrosine residues and results in a dramatic activation of ERK. Activated ERK is critical for numerous Ras-induced cellular responses, including Elk-dependent transcriptional activation of a number of genes (Hill and Treisman, 1995; Lewis et al., 1998). Three isoforms of Raf proteins have been found in mammalian cells: Raf-1 (or C-Raf), A-Raf and B-Raf (Magnuson et al., 1994). Although the three Raf proteins share high homology in amino acid sequence, it has recently been found that they are differentially regulated and exert different functions (Hagemann and Rapp, 1999). For example, B-Raf has higher affinity and stronger stimulation towards MEK than C-Raf and A-Raf, while A-Raf has the lowest activity due to the weakest binding affinity with Ras (Catling et al., 1994; Reuter et al., 1995; Weber et al., 2000). Mice lacking B-Raf, rather than C-Raf or A-Raf, show disturbances in cell survival, indicating that B-Raf may possess specific functions in cell death regulation (Pritchard et al., 1996; Wojnowski et al., 1997). Indeed, B-Raf specifically promotes cell survival by activating the MAPK pathway (Erhardt et al., 1999). B-Raf is also differentially regulated by cAMP-dependent protein kinase A (PKA) activation through Rap1 GTPase, which specifically activates B-Raf but not C-Raf (Ohtsuka et al., 1996; Vossler et al., 1997; MacNicol and MacNicol, 1999). Structurally, the Raf proteins can be divided into two functional regions: the N-terminal regulatory domains (CR1 and CR2) and the C-terminal kinase domain (CR3) (Daum et al., 1994). The CR1 region contains Ras binding domains, which are essential for Raf activation by Ras (Vojtek et al., 1993; Nassar et al., 1995; Gorman et al., 1996; Mott et al., 1996). When Ras is activated, Raf translocates from the cytosol to the plasma membrane where it binds to the active GTP-bound Ras and becomes activated. Although multiple steps have been involved in Raf activation, the molecular mechanism of Raf activation is not fully understood. Many of the studies were performed on C-Raf. It is postulated that phosphorylation of Raf is one of the critical steps for Raf activation. However, the effect of phosphorylation on Raf activity is complex because both inhibitory and stimulatory phosphorylation sites have been characterized (Morrison and Cutler, 1997). For example, phosphorylation of Ser43 (possibly by PKA) (Cook and McCormick, 1993; Morrison et al., 1993; Wu et al., 1993; Schramm et al., 1994) or Ser259 (possibly by PKB/AKT) (Michaud et al., 1995; Muslin et al., 1996; Rommel et al., 1999; Zimmermann and Moelling, 1999) exerts an inhibitory effect on C-Raf activity. In contrast, phosphorylation of Thr269 (possibly by CAP/KSR) (Yao et al., 1995; Zhang et al., 1997), Ser338 (possibly by Pak3) (King et al., 1998; Mason et al., 1999), Ser497/Ser499 (possibly by PKC) (Kolch et al., 1993; Carroll and May, 1994; Cai et al., 1997) or Ser621 (Michaud et al., 1995) has a positive effect on C-Raf activity. The effect of Ser621 phosphorylation is more complex in that it may also be involved in negative regulation of B-Raf by PKA (Mischak et al., 1996). Furthermore, tyrosine phosphorylation of Tyr341 activates C-Raf (Fabian et al., 1993; Jelinek et al., 1996; Mason et al., 1999). Using phospho-specific antisera, Mason et al. (1999) have found that active oncogenic Ras induces predominantly Ser338 phosphorylation, whereas activated Src gives predominantly Tyr341 phosphorylation. Moreover, a synergistic activation was observed when both sites are phosphorylated (Mason et al., 1999). However, the current model for C-Raf activation cannot explain the activation of other Raf family members due to the fact that Ser338 and Tyr341 of C-Raf are not completely conserved in other Raf proteins. For example, Caenorhabditis elegans lin-45 Raf contains an aspartate at the position corresponding to Ser338 of C-Raf, whereas Drosophila Raf contains an asparagine residue at the corresponding site of Tyr341. Thus, receptor tyrosine kinases (RTKs) and Ras may induce phosphorylation of other residues to activate C.elegans lin-45 and Drosophila Raf proteins. Similarly, B-Raf is strongly activated by oncogenic Ras but not by Src, probably due to the replacement of the tyrosine residue by an aspartate at the site equivalent to Tyr341 of C-Raf. Moreover, Ser445 of B-Raf, corresponding to Ser338 of C-Raf, is not involved in Ras-induced B-Raf activation in that it is constitutively phosphorylated and not stimulated by Ras (Mason et al., 1999). Thus, mutation of B-Raf Ser445 to alanine has little effect on Ras-induced B-Raf activation, whereas the corresponding mutation in C-Raf abolishes Ras-dependent activation (Mason et al., 1999). These results demonstrate that the mechanism for regulation of B-Raf activation is different from that for C-Raf. However, a common unidentified biochemical activation event may be shared by all Raf family kinases because all Raf proteins can be activated by Ras and RTKs. Thus, genetic data have unequivocally demonstrated that the C.elegans and Drosophila Raf are activated by RTKs and Ras pathways (Sternberg and Han, 1998). Similarly, biochemical studies in mammalian cells have shown that oncogenic mutant Ras activates A-Raf, B-Raf and C-Raf. We investigate Raf activation using B-Raf as a model because the C.elegans and Drosophila Raf are closer to B-Raf than C-Raf. Furthermore, B-Raf activation has been studied much less. In this report, we examined the potential phosphorylation sites in B-Raf responsible for Ras-induced activation. Using mutational analysis and phosphopeptide mapping, we demonstrate that Thr598 and Ser601 are the major phosphorylation sites in response to oncogenic Ras, and phosphorylation of these two residues is required for full activation of B-Raf. These two residues, which are located within the kinase activation loop between kinase subdomains VII and VIII of B-Raf, are conserved in C-Raf, but no data have been reported about the importance of these residues in Raf activation. Furthermore, functional studies reveal that phosphorylation of Thr598 and Ser601 is important for B-Raf to induce ERK activation, Elk-1-dependent transcription, NIH 3T3 transformation and PC12 differentiation. These results provide the first evidence that phosphorylation of Thr598 and Ser601 of B-Raf is responsible, at least in part, for oncogenic Ras-induced B-Raf activation. Since Thr598 and Ser601 are completely conserved in all Raf members including C.elegans lin-45, Drosophila Raf and mammalian C-Raf and A-Raf, our data suggest that phosphorylation of these two sites may be a general mechanism for activation of the Raf family kinases. Results Phosphorylation of both Thr598 and Ser601 is required for full activation of B-Raf induced by oncogenic Ras Phosphorylation of serine and/or threonine residue(s) within kinase subdomains VII and VIII has been found to be responsible for the activation of a number of kinases including ERK (Payne et al., 1991), MEK (Alessi et al., 1994; Zheng and Guan, 1994; Resing et al., 1995), PKA (Shoji et al., 1983), PDK1 (Casamayor et al., 1999), glycogen synthase kinase (Hughes et al., 1993) and PKB/Akt (Alessi and Cohen, 1998). We wanted to identify the common activating phosphorylation sites in Raf in response to Ras. Amino acid sequences between kinase subdomains VII and VIII of Raf family members were aligned (Figure 1A). In B-Raf, there are five putative phosphorylation sites (Thr598, Ser601, Ser604, Ser606 and Ser613) within this region, while Ser615 is highly conserved in almost all of the kinases and is unlikely to be a target for regulation. It has been reported that Ser497 and Ser499 in C-Raf, which correspond to Ser604 and Ser606 in B-Raf, are the PKC phosphorylation sites. Therefore, we examined, using mutational analysis, whether other serine and threonine residues within this region in B-Raf are potential phosphorylation sites in response to Ras, and whether the phosphorylation contributes to B-Raf activation. The following N-terminal hemagglutinin (HA)-tagged B-Raf mutations were created: B-RafT598A, B-RafS601A and B-RafS613A (Figure 1A). COS1 cells were transfected with HA-tagged wild-type B-Raf (B-Raf) or the above individual mutants in the presence or absence of an active version of Ras, HRasV12. HA-B-Raf was immunoprecipitated and its activity was assayed using the coupled kinase assay method. As expected, HRasV12 greatly increased the B-Raf activity reflected by Elk phosphorylation by ∼6-fold compared with non-stimulated cells expressing B-Raf only (Figure 1B, compare lanes 2 and 3). This increase in Elk1 phosphorylation by oncogenic Ras is substrate MEK dependent, indicating that our kinase assay is B-Raf specific (Figure 1B, lane 4). When the mutants were tested, we observed that B-RafT598A and S601A decreased the oncogenic Ras-induced B-Raf activity to ∼10 and 60% of the wild type, respectively (Figure 1B, compare lanes 6, 8 and 3), whereas S613A did not alter the kinase activity (Figure 1B, lane 10). These results suggest that both T598 and S601 are essential for full activation of B-Raf. To examine this, we substituted both T598 and S601 by alanine (B-RafAA). Interestingly, compared with the wild type, basal B-Raf activity of this double mutant was reduced by ∼40% and the oncogenic Ras-induced kinase activation was abolished (Figure 1B, compare lanes 11, 12 and 2, 3). Figure 1.Activation of B-Raf by oncogenic Ras requires both Thr598 and Ser601 phosphorylation. (A) Sequence alignment of Raf kinase activation loop (residues 593–622 for human B-Raf). Residues subjected to site-directed mutagenesis are indicated by arrows. An asterisk denotes the protein kinase C phosphorylation sites. (B) Oncogenic Ras-induced kinase activation of B-Raf and its mutants. Fifty nanograms of HA-tagged pcDNA3 (vector control, lane 1), HA-tagged B-Raf (lanes 2--4), B-RafT598A (lane 5 and 6), B-RafS601A (lanes 7 and 8), B-RafS613A (lanes 9 and 10), B-RafAA (lanes 11 and 12) or B-RafED (lanes 13 and 14) were transiently transfected in COS cells alone (open bars) or with 100 ng of oncogenic Ras (HRasV12, solid bars), as indicated at the top of the panel. The kinase was immunoprecipitated and its activity, reflected by phosphorylation of GST–Elk1 (pElk1), was measured using the coupled assay (see Materials and methods). The upper panel shows autoradiographs of pElk1 that are representative of five independent experiments. The intensity of pElk1 bands was quantified by PhosphorImager (lower panel); results were subtracted from the reading of lane 1 (background) and expressed as fold increase with respect to the cells transfected with wild-type B-Raf without stimulation (lane 2). Lane 4* denotes that GST–MEK was omitted in the assay as a control. Results are mean ± SD from three independent experiments. Western blot of the immunoprecipitated kinase is shown in the middle panel, indicating equivalent protein loadings. (C) Activation of B-RafED by oncogenic Ras. COS cells were transfected with vector (lane 1), 50 ng of wild-type B-Raf (lanes 2 and 3), 5 ng of B-RafED (lanes 4 and 5), 25 ng of B-RafED (lanes 6 and 7) or 50 ng of B-RafED (lanes 8 and 9) in the presence or absence of HRasV12, as indicated at the top of the panel. Results of kinase assay and western blotting of B-Raf using α-HA were representative of two separate experiments. (D) Carbachol-induced kinase activation of B-Raf and its mutants. Cells were co-transfected with the above DNA constructs and hM3. After 24 h, cells were starved in FBS-free medium for 5 h followed by stimulation with carbachol for 5 min. Kinase immunoprecipitation, kinase assay and quantitation of results are as described in (B). Download figure Download PowerPoint To test further that T598 and S601 are the potential Ras phosphorylation sites that contribute to B-Raf activity, we made substitutions of T598 and S601 with acidic residues (B-RafED, T598 to glutamic acid and S601 to aspartic acid) and found that B-RafED became constitutively active with a kinase activity comparable to that of the wild-type B-Raf induced by oncogenic Ras (Figure 1B, compare lanes 13 and 3). The kinase activity of B-RafED was further slightly increased by oncogenic Ras. To test whether an additional phosphorylation event is involved in B-Raf activaiton, we reduced the amount of B-RafED in the transfection to avoid potential saturation of the substrates for the in vitro coupled kinase assay. Cells were transfected with 5 ng (Figure 1C, lanes 4 and 5), 25 ng (Figure 1C, lanes 6 and 7) or 50 ng (Figure 1C, lanes 8 and 9) of B-RafED in the presence or absence of HRasV12. Indeed, results in Figure 1C clearly demonstrated that B-RafED could be further stimulated by oncogenic Ras, suggesting that there may be an additional phosphorylation mechanism for Ras-induced B-Raf activation. However, the magnitute of Ras stimulation on B-RafED was significantly reduced. Western blotting results demonstrated that equal amounts of protein were used for the kinase assays (Figure 1B and C, middle panel). Phosphorylation of Elk1 was quantified and is presented in the bottom panel of Figure 1B and C; all results were substracted from the background (lane 1) and are expressed as the fold increase compared with cells transfected with wild-type B-Raf only (lane 2). The above results demonstrate that Thr598 and Ser601 are putative phosphorylation sites important for B-Raf activation in response to active Ras. Next we asked whether the phosphorylation of T598 and S601 is also responsible for B-Raf activation induced by G-protein-coupled receptors. COS1 cells were co-transfected with B-Raf or B-Raf mutants and human muscarinic receptor, hM3, followed by stimulation with 500 μM carbachol for 5 min. Similar to the results induced by oncogenic Ras, phosphorylation of T598 and S601 is also essential for G-protein-coupled receptor-induced B-Raf activation (Figure 1D). Phosphopeptide mapping of B-Raf To provide further evidence that T598 and S601 of B-Raf are the Ras-dependent phosphorylation sites, in vivo metabolic labeling with [32P]orthophosphate and two-dimensional phosphopeptide mapping were performed. Figure 2A shows the signals of 32P-labeled B-Raf immunoprecipitates resolved by SDS–PAGE. The phosphorylation of B-Raf was increased after stimulation with oncogenic Ras, as compared with the basal phosphorylation without Ras (Figure 2A, lanes 1 and 2). The hyperphosphorylation induced by Ras resulted in a mobility shift of B-Raf. However, this increase in kinase phosphorylation was reduced in B-RafAA, and a lesser degree of the mobility shift was seen under the same condition of Ras stimulation (Figure 2A, lane 3). These results suggest that phosphorylation of T598 and S601 represents a significant portion of the total B-Raf phosphorylation induced by oncogenic Ras. The changes in B-Raf phosphorylation were not due to the protein loading, since western blotting indicated equal loading of each sample (Figure 2A, bottom). Figure 2.Tryptic phosphopeptide mapping of B-Raf. (A) Metabolic labeling and immunoprecipitation of B-Raf and B-RafAA. COS1 cells grown on a 60 mm plate were transfected with wild-type B-Raf (lane 1), B-Raf + HRasV12 (lane 2) or B-RafAA + HRasV12 (lane 3), labeled with [32P]orthophosphate, and immunoprecipitated with anti-HA antibody. Immunocomplexes were separated by SDS–PAGE and visualized by antoradiography. Positions of size markers are indicated in kilodaltons (KD) on the right. An immunoblot of the above immunoprecipitation using anti-HA antibody is shown at the bottom. (B, C and D) Phosphopeptide mappings for wild-type B-Raf, B-Raf + HRasV12 and B-RafAA + HRasV12, respectively. The 32P-labeled bands were excised from the membrane, digested with trypsin and analyzed by two-dimensional thin-layer electrophoresis. The directions for electrophoresis (E, from cathode to anode) and TLC (T) are indicated in the lower left corner of each panel. Arrows point to positions of phosphopeptides for references between different panels. Dashed circles indicate phosphopeptides present in (C) that are missing in (D). Open arrowheads indicate the Ras-induced phosphopeptide not affected in B-RafAA. (E) Phosphorylation of Thr598 and Ser601 determined by specific anti-phospho Thr598 and Ser601 antibodies. COS cells were transfected with pcDNA (vector, lane 1), wild-type B-Raf (lane 2), co-transfected with wild-type B-Raf and HRasV12 (lane3), B-RafAA (lane 4) or co-transfected B-RafAA and HRasV12 (lane 5). Immunoprecipitates of B-Raf and B-RafAA were isolated and resolved by SDS–PAGE, transferred to membrane, and blotted with anti-phospho Thr598 (top panel) or anti-phospho Ser601 antibody (middle panel). Protein loading was examined by blotting using HA antibody (bottom panel). Download figure Download PowerPoint The labeled bands were excised, digested with trypsin and analyzed by two-dimensional phosphopeptide mapping. Compared with the phosphopeptide pattern of unstimulated wild-type B-Raf (Figure 2B), wild-type B-Raf stimulated with oncogenic Ras displayed two additional phosphopeptides, as circled in Figure 2C. However, these two phosphopeptides were missing in the oncogenic Ras-stimulated B-RafAA sample (Figure 2D), suggesting that these two phosphopeptides are derived from phosphorylation of T598 and S601. In contrast, another Ras-induced phosphopeptide (indicated by open arrowheads) was not affected by the B-RafAA mutation (Figure 2C and D). Reference phosphopeptides, which were not altered by Ras stimulation, are indicated by arrows. These results confirmed that phosphorylation of Thr598 and Ser601 is stimulated by Ras. To examine directly that Thr598 and Ser601 are phos phorylated by oncogenic Ras, we generated anti-phospho Thr598 (pThr598) and Ser601 (pSer601) antibodies, which specifically recognize phosphorylated Thr598 and Ser601 in B-Raf, respectively. Cells were transfected with vector (pcDNA3), wild-type B-Raf or B-RafAA in the presence or absence of HRasV12. Immunoprecipitates of B-Raf and B-RafAA were botted with pThr598 or pSer601 and the western blotting results are shown in Figure 2E. Only wild-type B-Raf but not B-RafAA was phosphorylated at both Thr598 and Ser601 after stimulation of HRasV12, as determined by pThr598 and pSer601 (compare lanes 3 and 5 in the top and middle panels in Figure 2E). These results support the view that the pThr598 and pSer601 antibodies selectively recognize the phosphorylated B-Raf. We have also performed experiments with B-RafT598A or B-RafS601A single mutants, and similar results were obtained. Thus, B-RafT598A eliminated the recognition by pThr598 but not by pSer601 antibody in response to HRasV12 stimulation (data not shown). Conversely, the B-RafS601A mutant eliminated the recognition by pSer601 but not by pThr598 antibody (data not shown). These data provided the direct evidence that B-Raf was indeed phosphorylated at Thr598 and Ser601 in response to oncogenic Ras stimulation. Furthermore, our results indicate that phosphorylation of Thr598 and Ser601 can occur independently. B-RafT598A, B-RafS601A or B-RafAA does not alter the association of B-Raf with 14-3-3, HSP90 or MEK Raf kinase activation is also regulated by interaction with other signaling components including 14-3-3 proteins and heat shock protein 90 (HSP90) (Morrison and Cutler, 1997; Roy et al., 1998; Tzivion et al., 1998; Grammatikakis et al., 1999). B-Raf also directly binds to and activates MEK. To exclude the possibility that the decreased kinase activity in the mutants is due to global conformational changes and disruption of protein–protein interactions, we conducted experiments examining the interactions of B-Raf, B-RafT598A, B-RafS601 or B-RafAA with endogenous 14-3-3 protein, HSP90 or MEK. Immunoprecipitates of overexpressed B-Raf and mutants were isolated and probed with antibodies against 14-3-3, HSP90 or MEK. The results in Figure 3 clearly showed that either wild-type B-Raf or mutants were able to bind to all three endogenous signaling proteins, suggesting that the decreased kinase activity is not a result of disrupted protein–protein interactions. Figure 3.B-RafT598A, B-RafS601A or B-RafAA does not alter the association with 14-3-3, HSP90 or MEK. COS cells were transiently transfected with pcDNA3 (vector, lane 1), HA-B-Raf (lane 2), HA-B-RafT598A (lane 3), HA-B-RafS601A (lane 4) or HA-B-RafAA (lane 5). Cells were lysed and the HA-tagged B-Raf was immuno precipitated (IP) with anti-HA and western blotted with anti-HA (B-Raf), anti-14-3-3, anti-HSP90 or anti-MEK antibodies. Download figure Download PowerPoint Modulation of kinase activity by B-RafAA or B-RafED affects ERK activation and Elk-dependent transcription Next, we wished to test the effects of B-RafAA and B-RafED on activation of ERK in vivo. Cells were co-transfected with HA-tagged B-Raf, B-RafAA or B-RafED and Myc-tagged ERK. Myc-ERK was immunoprecipitated by anti-Myc antibody and its kinase activity was determined. As expected and consistent with the in vitro B-Raf kinase assay results, mutant B-RafAA reduced the ability to activate ERK kinase by ∼40% compared with wild-type B-Raf (Figure 4A, compare lanes 4 and 3), whereas constitutively active B-RafED induced a large increase in ERK activity, at a level similar to oncogenic Ras-induced ERK activity (Figure 4A, compare lanes 5 and 2). Immunoblotting demonstrated equivalent levels of protein loading detected by anti-Myc or anti-HA (Figure 4A, middle or bottom panel). It is noteworthy that wild-type B-Raf exerts high basal activity even under the serum-starved condition, leading to a significant activation of ERK (Figure 4A, compare lanes 3 and 1). Figure 4.Effects of B-Raf, B-RafAA or B-RafED on ERK activity and Elk-dependent transcription. (A) Activation of ERK by B-Raf, B-RafAA or B-RafED. Myc-ERK was co-transfected with pcDNA3, HA-tagged B-Raf, B-RafAA or B-RafED. Co-transfection of Myc-ERK and HRasV12 serves as a positive control (lane 2). Cells were cultured for 24 h followed by starvation for 15 h. Myc-ERK was immuno precipitated with anti-Myc monoclonal antibody 9E10 and assayed for kinase activity using GST–Elk1 as a substrate. ERK kinase activity is shown in the top panel and fold activation was determined by PhosphorImager analysis. Immunoblot detection of Myc-ERK or HA-B-Raf is shown in the middle or bottom panel. (B) Elk1-dependent transcription activation by B-Raf, B-RafAA or B-RafED. COS cells were transfected with Gal4-Elk1 and Gal4-LUC and B-Raf, B-RafAA or B-RafED. A pCMV-LacZ plasmid was co-transfected as an internal control for variations in transfection efficiency. Cells were cultured for 24 h followed by 15 h starvation. Luciferase activity was measured and normalized against the co-transfected β-galactosidase activity and results are mean ± SD from three independent experiments. Download figure Download PowerPoint The concerted activation of the Ras/Raf/MEK/ERK pathway is known to be involved in many cell functions, including proliferation, differentiation and survival. Activated ERK is able to phosphorylate numerous transcription factors and thus regulate gene expression. One of the best characterized substrates for ERK is Elk1, a member of the ternary complex factor family. To examine whether the manipulation of B-Raf phosphorylation (mutants B-RafAA and B-RafED) affects Elk-dependent gene transcription, we measured the transcriptional activity of Elk using a luciferase reporter assay. The activity of a Gal4–Elk1 reporter (Sugimoto et al., 1998), which contains the C-terminal ERK-responsive domain of Elk1 fused to the DNA binding domain of Gal4, was tested. Consistent with the data on ERK activation shown in Figure 4A, Gal4–Elk1 activation in B-RafAA-expressing cells was reduced by 40%, whereas it was increased ∼4-fold in B-RafED-expressing cells as compared with the cells expressing wild-type B-Raf (Figure 4B). B-RafED induces NIH 3T3 cell transformation and PC12 cell differentiation Activation of Raf has been shown to be involved in diverse biological functions such as cell transformation and cell differentiation. To examine the effect of phosphorylation of Thr598 and Ser601 on cell transformation, NIH 3T3 cells were co-transfected with wild-type B-Raf or B-RafED, and pCMV-lacZ. Transfection efficiency was monitored by β-galactosidase activity. We observed that cells transfected with B-RafED reproducibly induced cell transformation, although the numbers of foci were low compared with those induced by oncogenic Ras. In contrast, at the same transfection efficiency, there was no focus formed in cells transfected with wild-type B-Raf. Three individual foci were cloned in medium containing G418 to establish stably transfected cells expressing B-RafED. The morphology of cells stably expressing wild-type HA-B-Raf or HA-B-RafED is shown in Figure 5A. The B-RafED cells showed morphological changes typical of transformed cells: cells were more refractile and less flattened. In addition, the B-RafED clones lost cell contact inhibition of growth. Western blotting using anti-HA antibody confirmed the expression of wild-type B-Raf and B-RafED in the above stably transfected cells. Figure 5.B-RafED induces NIH 3T3 cell transformation and PC12 cell differentiation. (A) Morphology of NIH 3T3 cells stably expressing wild-type B-Raf or B-RafED. NIH 3T3 cells were transfected with wild-type B-Raf or B-RafED. Stably transfected cells were selected as described in Materials and methods. Note that morphology for cells expressing B-RafED is from three individual cell colonies. The western blotting for B-Raf expression in stably transfected cells is shown in the lower panel. (B) PC12 cell differentiation induced by B-Raf3A, B-Raf-ED and B-Raf3AED. PC12 cells were co-transfected with pEGFP vector and pcDNA3 (vector control), wild-type B-Raf (WT-B-Raf), B-Raf3A, B-RafED or B-Raf3AED. Two days after transfection, cells were shifted to NGF-minus differentiation medium (DMEM supplemented with 2% horse serum and 1% FBS medium) for 3 days. Cells were examined under the fluorescence microscope for visualization of transfected cells (by GFP) and differentiation (by the presence of neur