Title: MEK kinase activity is not necessary for Raf-1 function
Abstract: Article17 April 2001free access MEK kinase activity is not necessary for Raf-1 function Martin Hüser Martin Hüser Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Jeni Luckett Jeni Luckett Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Antonio Chiloeches Antonio Chiloeches Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Kathryn Mercer Kathryn Mercer Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Mabel Iwobi Mabel Iwobi Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Susan Giblett Susan Giblett Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Xiao-Ming Sun Xiao-Ming Sun MRC Toxicology Unit, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Jane Brown Jane Brown Division of Biomedical Services, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Richard Marais Richard Marais Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Catrin Pritchard Corresponding Author Catrin Pritchard Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Martin Hüser Martin Hüser Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Jeni Luckett Jeni Luckett Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Antonio Chiloeches Antonio Chiloeches Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Kathryn Mercer Kathryn Mercer Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Mabel Iwobi Mabel Iwobi Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Susan Giblett Susan Giblett Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Xiao-Ming Sun Xiao-Ming Sun MRC Toxicology Unit, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Jane Brown Jane Brown Division of Biomedical Services, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Richard Marais Richard Marais Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Catrin Pritchard Corresponding Author Catrin Pritchard Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK Search for more papers by this author Author Information Martin Hüser1, Jeni Luckett1, Antonio Chiloeches2, Kathryn Mercer1, Mabel Iwobi1, Susan Giblett1, Xiao-Ming Sun3, Jane Brown4, Richard Marais2 and Catrin Pritchard 1 1Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH UK 2Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK 3MRC Toxicology Unit, University of Leicester, University Road, Leicester, LE1 7RH UK 4Division of Biomedical Services, University of Leicester, University Road, Leicester, LE1 7RH UK ‡M.Hüser, J.Luckett and A.Chiloeches contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1940-1951https://doi.org/10.1093/emboj/20.8.1940 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by Ras.GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1−/− mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1FF/FF mice has no detectable activity towards MEK in vitro, and yet raf-1FF/FF mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1−/− and raf-1FF/FF mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis. Introduction The mammalian raf-1 gene was first identified as the cellular homologue of v-raf, the oncogene responsible for the induction of sarcomas in mice by the MSV3611 virus. Two other genes highly homologous to raf-1 have been cloned: A-raf and B-raf. All three raf genes code for serine/threonine protein kinases and they share a high degree of sequence similarity. The N-terminal region encodes the regulatory domain and binds essential cofactors including Ras while the C-terminal region contains the catalytic kinase domain. Deletion of the N-terminal regulatory regions of all three kinases gives rise to proteins that are constitutively active and are oncogenic in a wide variety of cell types. The kinase domain of B-raf appears to be the most potent of the three in these assays (Pritchard et al., 1995). In the mouse, transcripts for all three raf genes are detectable in all cells (Storm et al., 1990; Barnier et al., 1995). Of the three Raf isotypes, most biochemical studies have focused on Raf-1. Inactive Raf-1 is normally cytosolic, but Raf-1 binds to Ras.GTP in vitro and in vivo and so translocates to the plasma membrane in the presence of active Ras (Marais and Marshall, 1996 and references therein). However, binding to Ras is not sufficient for full Raf-1 activation (Traverse et al., 1993; Marais et al., 1995, 1997; Mason et al., 1999) and additional signals at the plasma membrane including phosphorylation are required (Marais and Marshall, 1996). Our previous studies in COS cells have shown that activation of Raf-1 requires phosphorylation of Y340 and/or Y341. Substitution of these residues to phenylalanine, creating RafFF, blocks activation of Raf-1 by oncogenic Ras and Src, and by ligand stimulation (Marais et al., 1995, 1997; Diaz et al., 1997; Stokoe and McCormick, 1997; Barnard et al., 1998). Recent data have also suggested that phosphorylation of Raf-1 at serine 338 is required for activation, demonstrating that complex phosphorylation events take place within this region of Raf-1 (Diaz et al., 1997; Barnard et al., 1998; Mason et al., 1999). However, the physiological importance of these phosphorylation events is unclear. The principal functions of the Raf protein kinases appear to be participation in the highly conserved Ras/Raf/MEK/ERK intracellular signalling pathway (Marshall 1994). This pathway is activated by different classes of cell surface receptors including receptor tyrosine kinases (RTKs) and G protein coupled seven transmembrane receptors, all of which confer their biological effects through Ras (Dickson and Hafen, 1994; Marshall, 1994). ERK activation has been associated with many of the downstream consequences of Ras activation and the Raf proteins provide a vital link between activated Ras proteins and the ERKs. A variety of biochemical and genetic data point to the importance of Raf-1 as a MEK activator. Activation of an inducible version of oncogenic Raf-1 induces the rapid activation of MEK and ERK as well as immediate early gene expression in NIH 3T3 cells (Samuels et al., 1993; Kerkhoff and Rapp, 1997). Immunoprecipitated endogenous Raf-1 can phosphorylate MEK1 and -2 in vitro (Howe et al., 1992; Kyriakis et al., 1992; Marais et al., 1998) and the Raf/MEK/ERK cascade can be reconstituted in vitro using proteins expressed in Sf9 cells (Macdonald et al., 1993). Kinase inactive Raf-1 cannot activate MEK in this system. Finally, dominant-negative Raf-1 mutants block growth factor and oncogenic ras-stimulated activation of ERKs in fibroblasts (Schaap et al., 1993; Chao et al., 1994; Troppmair et al., 1994). Intriguingly, a number of observations do not entirely fit with the view that the endogenous Raf-1 protein is a physiologically important MEK activator (Marais and Marshall, 1996). First, only a small proportion (<10%) of the entire cellular Raf-1 is activated upon treatment of cells with growth factors (Dent et al., 1995; Reuter et al., 1995; Jelinek et al., 1996). Secondly, B-Raf has been identified as the major MEK activator in neuronal cell types and NIH 3T3 cells, despite Raf-1 also being expressed in these cells (Moodie et al., 1993, 1994; Catling et al., 1994; Jaiswal et al., 1994; Traverse and Cohen, 1994; Reuter et al., 1995). The kinase domain of B-Raf is a considerably stronger activator of MEK and has a higher affinity for MEK than the kinase domain of Raf-1 (Pritchard et al., 1995; Marais et al., 1997; Papin et al., 1998). Finally, Raf-1 and ERK activation are not coincident in some circumstances (Wood et al., 1992, 1993; Kuo et al., 1996). A pool of Raf-1 is also thought to be located in mitochondria (Wang et al., 1996) and an ERK-independent role for mitochondrial Raf-1 in apoptosis has been postulated (Neshat et al., 2000). Many of the previous studies designed to address the involvement of Raf-1 in mediating signals between Ras and ERKs have used antisense or dominant-negative constructs overexpressed in tissue culture cell lines to inhibit its expression or activity (Kolch et al., 1991; Chao et al., 1994; Troppmair et al., 1994). These approaches have the disadvantage that they may sequester the function/expression of other Raf isotypes (A-Raf and B-Raf) or other Ras effectors. To achieve the required specificity, gene targeting has been used to ablate individual raf genes (Pritchard et al., 1996; Wojnowski et al., 1997, 1998). In this study, we describe the generation of Raf-1 deficient mice (raf-1−/−) as well as mice with a 'knockin' mutation of the endogenous 340/341 tyrosines to phenylalanine (raf-1FF/FF). Although there is no detectable Raf-1 activity in cells derived from either strain of mouse, ERK activation was not compromised in either. However, the raf-1−/− animals die in embryogenesis due to vascularization defects and increased apoptosis, while the raf-1FF/FF animals survive to adulthood and have an apparently normal phenotype. Our results show that the full-length Raf-1 protein is essential for normal mouse development and for protection against apoptosis, but they argue that Raf-1 kinase activity towards MEK is not necessary for these processes. Results Generation and phenotype analysis of raf-1−/− and raf-1FF/FF mutant mice The generation of the mutant mice is described in the Supplementary data (available at The EMBO Journal Online.) Both mutations were established on the mixed 129Ola/C57BL6 and 129Ola/MF-1 backgrounds. For the raf-1 knockout, raf-1+/− animals were intercrossed and PCR genotype analysis was performed on tail DNAs from surviving offspring. No surviving animals with the raf-1−/− genotype were obtained (n = 14 matings) but raf-1+/− animals were born at the expected Mendelian frequency and were indistinguishable from raf-1+/+ littermates. Therefore, raf-1+/− intercrosses were set up and embryos were genotyped. On the 129Ola/C57BL6 background, at E9.5, a number of abnormal embryos were observed and these all PCR genotyped as raf-1−/− (Table I). On the 129Ola/MF-1 background, in early backcross generations, a phenotype similar to that on the 129Ola/C57BL6 background was observed (Table II). However, a number of normal embryos at E9.5–E10.5 also typed as raf-1−/− (Table II). When the raf-1 mutation was further backcrossed to the MF-1 strain, raf-1−/− embryos were observed at E12.5 up to birth but these were small and morphologically abnormal. Table 1. Genotyping data from raf-1+/− intercrosses: C57BL6 background Age raf-1+/+ raf-1+/− raf-1−/− normal raf-1−/− abnormal Resorbeda Untypedb E3.5 2 5 3 0 – 3 E8.5 2 4 3 0 0 0 E9.5 17 26 0 12 1 0 E10.5 7 16 0 10 9 0 E11.5 14 20 0 6 14 0 E12.5 4 2 0 0 0 0 E15.5 0 2 0 0 2 0 Total 46 75 6 28 26 3 a Resorbed tissue had degenerated too much to dissect cleanly. b DNA samples did not PCR amplify. Table 2. Genotyping data from raf-1+/− intercrosses: MF-1 background Age raf-1+/+ raf-1+/− raf-1+/− normal raf-1−/− abnormal Resorbeda Untypedb E8.5 2 6 1 0 0 0 E9.5 3 14 1 1 0 0 E10.5 3 1 3 2 0 1 E12.5 3 4 0 1 3 3 E13.5 7 9 0 1 1 1 E14.5 6 6 0 3 2 7 E18.5 16 20 0 3 0 2 Birth (P1) 3 10 0 5c – 2 Total 43 70 5 16 6 16 a Resorbed tissue had degenerated too much to dissect cleanly. b DNA samples did not PCR amplify. c Animals died a few hours after birth. On the 129Ola/C57BL6 background, at E9.5, raf-1−/− embryos were morphologically smaller and were reduced in size by approximately one-third (Figure 1A and B). They were developmentally arrested, had fewer somites and were anaemic, but were still alive as judged by the presence of a regular heartbeat. Strikingly, all of the mutants lacked small and large blood vessels in the yolk sac (Figure 1C and D), as visualized by staining with an antibody to platelet endothelial cell adhesion molecule-1 (PECAM-1; Figure 1E). PECAM-1 staining of the raf-1−/− embryos also revealed abnormal vascular network formation (Figure 1F–I). In the head region, there was a reduction in the number of large and small blood vessels, the vessels were disorganized and there were no capillary sprouts into the neuroectoderm (Figure 1H and I). Haemorrhaging was observed in approximately one- quarter of the mutant embryos (data not shown). There was a significant reduction in the number and density of cells throughout the mutant embryos but cell size appeared larger (Figure 1J and K). Staining of the embryonic brain with the Ki67 antibody, a marker for cells in S phase, showed that there was no significant reduction in the numbers of proliferating cells in the raf-1−/− embryos (Figure 1L and M). However, the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay indicated that there was an increase in the numbers of apoptotic cells throughout the mutant embryos (Figure 1N and O). Figure 1.Phenotype of the 129Ola/C57BL6 raf-1−/− embryos. (A) raf-1−/− embryo at E9.5; (B) raf-1+/+ littermate embryo at E9.5; (C) raf-1−/− embryo at E9.5 in yolk sac; (D) raf-1+/+ littermate embryo at E9.5 in yolk sac; (E) PECAM-1 immunostaining of yolk sacs from wild-type embryo at E9.5 (left) and from mutant embryo (right). (F–I) PECAM-1 staining of raf-1+/+ embryo (F and H) and raf-1−/− embryo (G and I) at E9.5. (H) and (I) are magnifications of the embryonic brain regions. (J–O) Longitudinal sections through forebrain of raf-1+/+ (J, L and N) and raf-1−/− (K, M and O) embryos at E9.5. (J) and (K) were stained with haematoxylin and eosin, (L) and (M) were immunostained with a Ki67 antibody, and (N) and (O) were processed for TUNEL staining. Scale bars: A–E, 250 μm; F and G, 100 μm; H and I, 25 μm, J–M, 50 μm; N and O, 100 μm. NE, neural ectoderm; SE, surface ectoderm; Lu, lumen of prospective forebrain; MT, cephalic mesenchyme tissue. Download figure Download PowerPoint On the 129Ola/MF-1 background, the raf-1−/− embryos died within a few hours of birth. They were 50–60% lighter in weight than the raf-1+/+ embryos and they were anaemic (Figure 2A). The placenta was considerably smaller than the raf-1+/+ placenta and was disorganized (Figure 2B and C). The spongiotrophoblast layer was considerably reduced in size, a number of the mesenchymal cells of the labyrinthine layer were undifferentiated and there were fewer blood vessels (Figure 2D and E). As with the 129Ola/C57BL6 raf-1−/− embryos, there were fewer cells in the mutant liver than the wild-type liver, but the mutant cells appeared to be greater in size (Figure 2F–K). In addition, there were notably fewer areas of haemopoiesis (Figure 2F and G). There did not appear to be any difference in the number of proliferating cells in the raf-1−/− liver compared with the raf-1+/+ liver (Figure 2H and I). There was also no significant difference in the number of TUNEL positive cells in the raf-1−/− liver sections compared with the raf-1+/+ liver sections (Figure 2J and K), indicating no significant difference in levels of spontaneous apoptosis. The fact that the raf-1−/− livers are hypocellular is likely to have been caused by increased susceptibility to apoptosis at earlier developmental stages (see below). Figure 2.Phenotype of the 129Ola/MF-1 raf-1−/− embryos. (A) Photograph of raf-1−/− embryo (left) and raf-1+/+ littermate embryo (right) at E15.5. (B and C) Cross-sections of placentas from raf-1−/− (B) and raf-1+/+ (C) embryos showing the labyrinthine and spongiotrophoblast (Sp) layers. (D and E) Cross-sections of labyrinthine layer of placentas from raf-1−/− (D) and raf-1+/+ (E) embryos. (F–K) Cross-sections of liver from littermate raf-1−/− (G, I and K) and raf-1+/+ embryos at E15.5. These sections were either stained with haematoxylin and eosin (F and G), immunostained with the Ki67 antibody (H and I) or processed for TUNEL staining (J and K). Scale bars: B and C, 400 μm; D–K, 50 μm. Download figure Download PowerPoint To assess the raf-1FF/FF phenotype, raf-1+/FF animals on the 129Ola/C57BL6 and 129Ola/MF-1 backgrounds were intercrossed and PCR genotype analysis was performed on tail DNAs from surviving offspring. Of 148 animals assessed, 45 typed as raf-1+/+, 67 as raf-1+/FF and 36 as raf-1FF/FF (n = 19 matings). Therefore, there was no significant difference in the ratio of raf-1+/+ to raf-1FF/FF animals surviving to adulthood (P = 0.15). The raf-1FF/FF animals on both genetic backgrounds survived for >1 year, were normal in weight and had no behavioural abnormalities. T-cell development was normal in these animals as judged by CD4 and CD8 staining of T cells (data not shown). Analysis of proliferation and apoptosis in raf-1−/− and raf-1FF/FF MEFs Mouse embryonic fibroblasts (MEFs) were derived from the raf-1−/− and raf-1FF/FF embryos and characterized for their ability to proliferate and undergo apoptosis in vitro. We consistently failed to culture raf-1−/− MEFs from E10.5 on the 129Ola/C57BL6 background as most of the cells were dead following embryo homogenization. This may be because of the apparent spontaneous apoptosis of the raf-1−/− embryos observed on this genetic background (Figure 1N and O). However, we successfully obtained raf-1−/− MEFs from E14.5 embryos on the 129Ola/MF-1 background. Raf-1FF/FF MEFs were isolated from E14.5 embryos resulting from raf-1+/FF intercrosses on both genetic backgrounds. Sibling raf-1+/+ MEFs were also isolated in each case. There was no detectable difference between the growth rates of the raf-1−/− cells compared with raf-1+/+ cells over 8 days in culture (Figure 3A), or the raf-1FF/FF cells compared with the raf-1+/+ cells (data not shown). Cell proliferation was measured by assessing bromodeoxyuridine (BrdU) incorporation in MEFs that had been made quiescent and then stimulated with 10% fetal calf serum (FCS). Again, the raf-1−/− and raf-1FF/FF MEFs showed no difference in their ability to undergo DNA synthesis as measured by this assay compared with raf-1+/+ MEFs (Figure 3B). Figure 3.Proliferation and apoptosis analysis of MEFs. (A) Growth curves of raf-1+/+ MEFs (open circles) compared with raf-1−/− MEFs (closed circles) over 8 days in culture are shown. (B) DNA synthesis of raf-1−/− and raf-1FF/FF primary MEFs compared with raf-1+/+ cells induced by 10% serum. The data represent pooled data from four experiments of cells with each genotype. (C and D) Levels of apoptosis in raf-1FF/FF cells compared with raf-1+/+ cells (C) and in raf-1−/− cells compared with raf-1+/+ cells (D). Cells were either not treated (NT) or treated with serum-free media (SFM), etoposide or anti-Fas antibody for 20 h. The percentage of cells undergoing apoptosis was quantified by flow cytometric analysis of annexin V staining. Each experiment was performed seven times and the data show mean values ± standard deviation. (E) An example of flow cytometric analysis of annexin V staining of cells for data presented in (C) and (D). The percentage of annexin V positive cells is indicated. (F) Hoechst 33258 staining of apoptotic cells. raf-1+/+ cells (left panels) and raf-1−/− cells (right panels) were either untreated (top panels) or treated with anti-Fas antibody (bottom panels). Download figure Download PowerPoint Except for the C57BL6 raf-1−/− MEFs, the primary MF-1 raf-1−/− and raf-1FF/FF MEFs did not show evidence of spontaneous apoptosis under normal growth conditions (Figure 3C–E). Apoptosis was induced by treatment of raf-1+/+, raf-1−/− and raf-1FF/FF MEFs with etoposide, anti-Fas antibody or by serum withdrawal, and cell death was assessed by annexin V or Hoechst 33258 staining. The raf-1FF/FF cells showed no increase or decrease in programmed cell death (PCD) upon treatment with these apoptotic agents compared with raf-1+/+ cells (Figure 3C). In contrast, raf-1−/− cells showed a significant increase in PCD (Figure 3D–F). Upon treatment with etoposide, the raf-1+/+ cells showed 21.1% PCD whereas the raf-1−/− cells showed 30.0% PCD (n = 7; 95% CI for difference 0.5–17.2%, P = 0.04; Figure 3D and E). Upon treatment with anti-Fas antibody, the raf-1+/+ cells showed 29.9% PCD whereas the raf-1−/− cells showed 48.3% PCD (n = 7; 95% CI for difference 9.5–27.4%, P = 0.0007; Figure 3D and E). The raf-1−/− cells did not show a significant increase in PCD upon serum withdrawal as this treatment induced 26.0% PCD for the raf-1−/− cells and 18.7% for the raf-1+/+ cells (n = 7; 95% CI for difference −6.8 to 21.4%, P = 0.284; Figure 3D and E). Hoechst 33258 staining confirmed that the raf-1−/− cells were more susceptible to PCD induced by the anti-Fas antibody than the raf-1+/+ cells, as assessed by nuclear morphology (Figure 3F). Raf kinase activities in the mutant cells For measuring Raf kinase activities, primary MEFs were immortalized with the SV40 large T antigen and permanent cell lines were derived. Only cells expressing similar levels of the T antigen and with similar growth rates were analysed and compared (data not shown). We first compared the activities of the endogenous Raf proteins in raf-1+/+ MEFs using the immunoprecipitation kinase cascade assay (Marais et al., 1997). For these initial studies, the conditions were optimized for B-Raf activity, since B-Raf is the most active isotype under our assay conditions (Mason et al., 1999). In agreement with our previous studies, B-Raf had elevated basal activity in unstimulated cells, whereas both Raf-1 and A-Raf were inactive (Figure 4A). When stimulated with epidermal growth factor (EGF), B-Raf activity in raf-1+/+ cells was increased by ∼2-fold and returned to basal levels by 60 min (Figure 4A). However, we did not detect any activation of Raf-1 or A-Raf in this assay (Figure 4A), despite numerous reports previously describing the activation of Raf-1 in growth factor stimulated cells (Marshall, 1994 and references therein). We obtained similar results using two other B-Raf-specific antibodies, generated to peptides from different regions of B-Raf, but did not detect B-Raf activity with these antibodies in MEFs derived from B-raf−/− embryos (Wojnowski et al., 1997; our unpublished data). However, despite this extremely strong kinase activity of B-Raf, the protein could not be detected in these cells with these antibodies even by 35S-labelling or by immunoprecipitation combined with western analysis (data not shown). By contrast, A-Raf and Raf-1 are present at high levels in MEFs as detected by western analysis. Figure 4.Raf kinase activities. (A) Time course of activation of immunoprecipitated A-Raf, B-Raf and Raf-1 in the kinase cascade assay in raf-1+/+ cells following stimulation with EGF. The conditions for the assay were the same as those used for routinely measuring B-Raf activity. (B) Time course showing the fold Raf-1 activation in raf-1+/+, raf-1−/− and raf-1FF/FF MEFs following EGF stimulation. Standard conditions for measuring Raf-1 activity were utilized. (C) Time course showing the fold A-Raf activation in MEFs following EGF stimulation. The conditions were the same as for the Raf-1 assay. (D) Time course showing the fold B-Raf activation in MEFs following EGF stimulation. The B-Raf assay conditions were utilized. Each experiment was performed in triplicate and error bars show standard deviations. Download figure Download PowerPoint The above results suggest that the specific activity of B-Raf towards MEK as measured by this kinase cascade assay is far greater than either Raf-1 or A-Raf. To examine Raf-1 and A-Raf activation, we increased the sensitivity of the assay by using 10-fold more lysate (1 mg) and by increasing the first incubation period from 15 to 30 min (Materials and methods). These have been developed as standard conditions for measuring Raf-1 activity (Marais et al., 1997). Immortalized raf-1+/+, raf-1−/− and raf-1FF/FF MEFs were made quiescent and stimulated with EGF, platelet-derived growth factor (PDGF), phorbol 12-myristate 13-acetate (PMA) or lysophosphatidic acid (LPA) over a time course of up to 60 min. Raf-1 kinase activity was stimulated 4- to 6-fold in raf-1+/+ cells following 2–5 min treatment with EGF and returned to basal levels within 60 min (Figure 4B). As expected, no Raf-1 kinase activity was detected in the raf-1−/− cells (Figure 4B). In the raf-1FF/FF MEFs, no activation of Raf-1 was detected in response to EGF; the level of activity observed was comparable to that in the raf-1−/− cells and to background levels in assays performed without substrate. The same Raf-1 activation results were obtained upon stimulation with PDGF, PMA and LPA (data not shown). In raf-1+/+ cells, A-Raf kinase activity was much lower than Raf-1 activity and was stimulated only by 1.3-fold upon 2 min treatment with EGF. Similar levels of A-Raf activity were detected in the raf-1−/− and raf-1FF/FF cells compared with raf-1+/+ cells (Figure 4C). We compared B-Raf activation in the raf-1+/+, raf-1−/− and raf-1FF/FF cells using the assay conditions described above for B-Raf. The levels of basal B-Raf kinase activity in the raf-1−/− and raf-1FF/FF cells were similar to that observed in raf-1+/+ cells (Figure 4D). The time course of activation by EGF was similar in the three cell lines, although the level of induction of B-Raf in raf-1−/− and raf-1FF/FF was elevated slightly compared with raf-1+/+ cells. ERK activation in the mutant cells To measure ERK activation, cells were made quiescent and stimulated with EGF and levels of phospho-ERK were assessed. In the raf-1+/+ cells, phospho-ERK increased following 2 min of EGF treatment and continued to increase up to 5 min of treatment but reached basal levels after 60 min (Figure 5A). For the raf-1−/− and raf-1FF/FF cells, the level of phospho-ERK was similar to that in the raf-1+/+ cells (Figure 5A). ERK phosphorylation was assessed in cells that were made quiescent and then stimulated with EGF, PDGF, serum, LPA and PMA for 10 min (Figure 5B). In the raf-1+/+ cells, ERK phosphorylation was induced following treatment with all stimuli. A similar level of ERK phosphorylation was observed in the raf-1−/− and raf-1FF/FF cells with all stimuli (Figure 5B). ERK activation was also measured by using myelin basic protein (MBP) as a substrate for immunoprecipitated p42ERK. As with the ERK phosphorylation data, ERK activation increased in all cells following 2 min EGF treatment and reached maximum at 10 min (Figure 5C). There was no difference observed in either the time course or level of ERK activity between the raf-1+/+, raf-1−/− or raf-1FF/FF cells following EGF treatment (Figure 5C). ERK activation was also assessed upon stimulation of quiescent cells with sub-saturating amounts of EGF. MEFs of each genotype were made quiescent and then stimulated with varying concentrations of EGF from 0 to 10 ng/ml for 10 min. With increasing doses of EGF, a greater level of ERK activation was observed in all cell lines (Figure 5D). However, no difference was observed in the level of MBP phosphorylation upon treatment with any given dose of EGF between the raf-1+/+, raf-1−/− and raf-1FF/FF cells (Figure 5D). Figure 5.ERK activation. (A) Stimulation of ERK phosphorylation in raf-1+/+, raf-1−/− and raf-1FF/FF MEFs over a time course of EGF treatment. (B) Stimulation of ERK phosphorylation in raf-1+/+, raf-1−/− and raf-1FF/FF MEFs following treatment with different stimuli for 10 min. The blots in (A) and (B) were incubated with an anti-phosphoERK antibody (top panels) and an anti-ERK2 antibody (bottom panels) to control f