Original Paper

Oncogene (2005) 24, 5207–5217. doi:10.1038/sj.onc.1208707; published online 25 April 2005

A-Raf and Raf-1 work together to influence transient ERK phosphorylation and Gl/S cell cycle progression

Kathryn Mercer1, Susan Giblett1, Anthony Oakden2, Jane Brown2, Richard Marais3 and Catrin Pritchard1

  1. 1Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LEI 7RH, UK
  2. 2Division of Biomedical Services, University of Leicester, University Road, Leicester LEI 7RH, UK
  3. 3Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

Correspondence: C Pritchard, E-mail: cap8@le.ac.uk

Received 11 October 2004; Revised 25 January 2005; Accepted 16 March 2005; Published online 25 April 2005.

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Abstract

The Raf/MEK/ERK (extracellular regulated kinase) signal transduction pathway controls the ability of cells to respond to proliferative, apoptotic, migratory and differentiation signals. We have investigated the combined contribution of A-Raf and Raf-1 isotypes to signalling through this pathway by generating mice with knockout mutations of both A-raf and raf-1 genes. Double knockout (DKO) mice have a more severe phenotype than single null mutations of either gene, dying in embryogenesis at E10.5. The DKO embryos show no changes in apoptosis, but staining for Ki67 indicates a generalized reduction in proliferation. DKO mouse embryonic fibroblasts (MEFs) exhibit a delayed ability to enter S phase of the cell cycle. This is associated with a reduction in levels of transiently induced MEK and ERK phosphorylation and reduced expression of c-Fos and cyclin Dl. Levels of sustained ERK phosphorylation are not significantly altered. Thus, Raf-1 and A-Raf have a combined role in controlling physiological transient ERK activation and in maintenance of cell cycle progression at its usual rate.

Keywords:

A-Raf, Raf-1, knockout, ERK activation, cell cycle, c-Fos, cyclin Dl

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Introduction

Extracellular regulated kinases 1 and 2 (ERK1/2) are highly conserved cellular enzymes that play crucial roles in the control of multiple cellular processes including cell proliferation (Lewis et al., 1998; Chen et al., 2001). Treatment of mammalian cells with mitogens leads to an increase in intracellular levels of Ras.GTP followed by the sequential activation of Raf, MEK1/2 and ultimately ERK1/2 (Marais and Marshall, 1996; Lewis et al., 1998). It has been shown that activation of ERK in this way is an important step in G0–Gl–S phase progression of the cell cycle (Samuels et al., 1993; Kerkhoff and Rapp, 1997, 1998; Woods et al., 1997; Marshall, 1999; Squires et al., 2002). This function is mediated by the ability of ERKs to translocate to the nucleus where they are able to phosphorylate transcription factors, particularly the AP-1 complex that comprises various heterodimers of c-fos (c-fos, FosB, Fra-1, Fra-2) and c-jun (c-Jun, JunB, JunD) family members (Whitmarsh and Davis, 1996; Balmanno and Cook, 1999; Cook et al., 1999; Shaulian and Karin, 2002). This transcription factor complex is involved in promoting D-type cyclin expression, thus allowing the cyclin-dependent kinases CDK4 and CDK6 to phosphorylate and de-repress the retinoblastoma family members (Brown et al., 1998; Kerkhoff and Rapp, 1998; Marshall, 1999; Shaulian and Karin, 2002). The consequent release of the E2F transcription factors from retinoblastoma binding allows expression of genes required for progression through the G0–G1–S transitions of the cell cycle (Stevaux and Dyson, 2002).

There are three Raf family members in mammals; A-Raf, Raf-1 and B-Raf (Mercer and Pritchard, 2003). All three are able to induce MEK/ERK activation (Pritchard et al., 1995), but they differ in their strength of activation of ERK. In kinase cascade assays, immunoprecipitated B-Raf has a far stronger ability to activate MEK/ERK than Raf-1 and Raf-1, in turn, is far stronger than A-Raf (Huser et al., 2001). In B-raf-/- mouse embryonic fibroblasts (MEFs), ERK phosphorylation and activation are reduced to approximately30% that in wild-type cells (Wojnowski et al., 2000; Pritchard et al., 2004) whereas there is no noticeable decrease in ERK phosphorylation or activation in raf-1-/- MEFs or A-raf-/Y MEFs (Huser et al., 2001; Mikula et al., 2001; Mercer et al., 2002). The ability of B-Raf to induce strong ERK activity has been highlighted by the discovery of activating mutations of the BRAF gene in human cancers (Davies et al., 2002; Kimura et al., 2003; Mercer and Pritchard, 2003). The most common mutation, a valine to glutamic acid change at residue 599, is thought to contribute to tumorigenesis by stimulating constitutive ERK phosphorylation. A similar mutation in RAF1 does not induce such high levels of ERK activation and is not detected in human cancers (Davies et al., 2002).

The duration and magnitude of ERK activation has a significant impact on whether cells enter the cell cycle or not (Marshall, 1995). In fibroblasts, transient and sustained ERK activation are observed following mitogen treatment (Vouret-Craviari et al., 1993; Cook and McCormick, 1996; Weber et al., 1997; Balmanno and Cook, 1999). The sustained phase of ERK activation correlates with the induction of expression of a subset of AP-1 components (Fra-1, Fra-2, c-Jun and JunB), which in turn are required for sustained cyclin Dl expression and progression through the cell cycle (Kovary and Bravo, 1991, 1992; Lavoie et al., 1996; Balmanno and Cook, 1999; Cook et al., 1999). The transient activation of ERK correlates with transient c-Fos expression and is not associated with the induction of cyclin Dl expression or cell cycle progression in several cell systems (Vouret-Craviari et al., 1993; Cook and McCormick, 1996; Weber et al., 1997). However, other studies have indicated an important role for c-Fos in controlling cyclin Dl expression and cell cycle progression (Kovary and Bravo, 1991; Won et al., 1992; Miao and Curran, 1994; Brown et al., 1998). In particular, mice with knockout mutations of both the c-Fos and FosB genes have defects in proliferation that result at least in part from a failure to induce cyclin Dl expression (Brown et al., 1998).

In this report, we have used gene targeting in mice to investigate the roles of A-Raf and Raf-1 in regulating MEK/ERK activation and downstream cellular responses. Despite the fact that single knockout mutations of either gene do not lead to noticeable changes in ERKl/2 activation (Huser et al., 2001; Mikula et al., 2001; Mercer et al., 2002), the combined knockout mutation of both genes leads to reduced ERKl/2 phosphorylation. These results indicate that A-Raf and Raf-1 play compensatory roles in ERKl/2 activation that cannot be rescued by B-Raf However, only transient ERK phosphorylation is significantly reduced in the double knockout (DKO) MEFs, whereas the sustained phase of ERK phosphorylation is not significantly affected. This reduction in transient ERK phosphorylation is associated with a reduction in transient c-Fos expression and cyclin Dl expression, and delayed progression through the cell cycle.

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Results

Generation of DKO mice

To generate the DKO mice, two rounds of breeding were set up with A-raf-/- and raf-1-/- single mutant mice. In the first round, A-raf+/- female mice were crossed to raf-1+/- male mice. Wild-type (WT), single and double heterozygote mice were obtained at the expected Mendelian frequency, while A-raf/Y and A-rafIYraf-1+l- adult mice were obtained at a reduced frequency and displayed a phenotype typical of that previously described (data not shown; Pritchard et al., 1996). In the second round of breeding, A-raf+/-raf-1+/- female animals were crossed to A-raf/Yraf-1+/- male mice. No DKO animals survived postnatally and so embryos were harvested from matings timed at between E9.5 to birth (Table 1). Genotypes of each embryo were confirmed by PCR analysis of yolk sac DNA (Figure 1a) and Western blot analysis was used to confirm the Raf-1 and/or A-Raf protein deficiencies (Figure 1b). A-raf-/- embryos were obtained at the expected frequency, indicating that loss of A-Raf alone has no detrimental effect on embryogenesis (Table 1). Raf-1-/- embryos were obtained at the expected frequencies up to E13.5 but lethality was observed from this point onwards and very few survived to birth (<3%). DKO embryos were obtained at the expected frequency at E9.5 but they appeared at a reduced frequency at E10.5 onwards and none survived to birth (Table 1). The expression (Figure 1c) and activity (Figure 1d) of the remaining Raf isotype, B-Raf, were not noticeably altered in DKO embryos compared to WT embryos.

Figure 1.
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PCR and protein analysis of embryos. (a) PCR genotyping of embryos. Results are shown for a typical litter arising from an A-raf+/-raf-1+/- intercross. PCR genotyping results are shown for the WT and mutant raf-1 allele (upper panel), WT A-raf allele (middle panel) and mutant A-raf allele (lower panel). DKO embryo is indicated by * in lane 7. (b) Expression of A-Raf and Raf-1. alpha-Raf-1 antibody (upper panel) and an alpha-A-Raf antibody (middle panel) were used to detect the presence/absence of Raf-1 and A-Raf protein in each embryo. As a control for protein loading, the blots were analysed with an antibody for actin (lower panel). DKO embryos are indicated by * in lanes 2 and 7. (c) Expression of B-Raf. An alpha-B-Raf antibody (upper panel) was used to detect the expression level of B-Raf in each embryo. As a control for protein loading, the blots were analysed with an antibody for actin (lower panel). Lanes: 1 – A-raf KO, 2 – A-raf/raf-1 double heterozygote, 3 – DKO, 4 – raf-1 KO. (d) Activity of B-Raf. DKO and WT MEFs were stimulated with EGF over a time course of 0–60 min. B-Raf activity in each sample was determined by performing the immunoprecipitation kinase cascade assay

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Phenotype analysis of single knockout and DKO mice

Embryos were harvested at E10.5. and photographed (Figure 2a–d). A-raf-/- embryos exhibited no abnormalities and were identical in size and developmental age to WT embryos (Figure 2a, b). Raf-1-/- embryos appeared relatively normal although they were consistently small in size and were developmentally retarded by approximately 0.5 days of gestation (Figure 2c). By contrast, the DKO embryos were grossly abnormal (Figure 2d). They were extremely small, demonstrated tail truncations and the vast majority died before they reached E10.5. Embryos at E10.5 were fixed, embedded in paraffin and sectioned for histological analysis. Sections were stained either with haematoxylin and eosin (Figure 2e–h), subjected to TUNEL analysis to detect apoptotic cells (Figure 2i–l) or stained with an antibody for the S phase marker Ki67 (Figure 2m–t). Consistent with previous observations, the A-raf-/- embryos showed no differences in levels of apoptosis or proliferation compared to WT embryos (compare Figure 2j, n, r with Figure 2i, m, q). The raf-1-/- embryos also showed no increase in apoptosis (Figure 2k) or changes in proliferation (Figure 2o, s). The DKO embryos showed no increase in TUNEL staining (Figure 2l). However, the number of cells staining with Ki67 was noticeably reduced in these embryos (Figure 2p, t), indicating a generalized reduction in the percentage of cells in S phase.

Figure 2.
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Phenotype analysis of embryos. WT embryos are shown in (a,e,i,m,q); A-raf-/- embryos are shown in (b,f,j,n,r); raf-1-/- embryos are shown in (c,g,k,o,s); DKO embryos are shown in (d,h,l,p,t). In (ad), embryos were extracted from yolk sacs and photographed. In (et), embryos were fixed, sectioned and stained with haematoxylin and eosin (eh), subjected to TUNEL analysis (il) or stained with an antibody for Ki67 (mt). qt shows higher magnification of Ki67 staining of the somites and adjacent mesenchymal tissue. Scale bars: ad, 200 mum. qt, 25 mum

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Growth, apoptosis and proliferation analysis of single knockout and DKO MEFs

Primary MEFs were derived from the DKO, A-raf-/-, raf-1-/- and sibling WT embryos by standard procedures. Growth rates were determined by counting cells in triplicate over 9 days in culture following immediate isolation from the embryo (Figure 3a). A-raf-/- MEFs had growth profiles similar to that of the control MEFs. By day 7, the growth of raf-1-/- MEFs was slightly reduced compared to the WT cells. However, the growth of the DKO MEFs was significantly compromised compared to MEFs of all other genotypes (Figure 3a).

Figure 3.
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Growth, apoptosis and proliferation analysis of primary MEFs. (a) Growth analysis of DKO MEFs (closed circles) compared to raf-1-/- (closed squares), A-raf-/- (open squares) and WT MEFs (open circles) over 8 days in culture. (b) Apoptosis analysis of primary MEFs. Cells were either untreated or treated with alpha-CD95 antibody for 20 h. The percentage of cells undergoing apoptosis was quantified by FACS analysis of annexin V staining. Each experiment was performed three times and the data show mean valuesplusminusstandard deviation. (c) Analysis of progression through the cell cycle. Propidium iodide staining followed by FACS analysis was performed to assess DNA content of asynchronously growing MEFs. The percentages of cells in the Gl (left panel), S (middle panel) and G2/M (right panel) phases of the cell cycle were determined. Each experiment was performed eight times and the data show mean valuesplusminusstandard deviation. (d) Analysis of entry into S phase. BrdU proliferation assays were performed to compare the percentage of DKO and WT cells entering S phase over a time course of stimulation with serum from 2 to 16 h

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Apoptosis was induced by treatment of the primary MEFs with alpha-CD95 antibody and cell death was assessed by annexin V staining (Huser et al., 2001; Mercer et al., 2002). Treatment of cells with alpha-CD95 antibody led to a small but significant increase in apoptosis in all samples. No significant difference was observed in the level of apoptosis induced in the A-raf-/- cells compared to WT cells, while the raf-1-/- and DKO cells appeared to be more protected from alpha-CD95 antibody-induced apoptosis than WT cells (Figure 3c). These results show that increases in apoptosis cannot account for the greatly reduced growth of the DKO cells or the slightly reduced growth of the raf-1-/- cells (Figure 3a).

FACS analysis of propidium iodide-stained cells showed that A-raf-/- and raf-1-/- MEFs had a similar proportion of cells in each phase of the cell cycle compared to control cells (Figure 3c). Our recent analysis has shown that the raf-1-/- cells consistently grow to a lower saturation density (KM, unpublished data) that may account for their slightly reduced ability to grow (Figure 3a). However, the DKO cells had a significantly reduced percentage of cells in S phase (Figure 3c); 5.7% of the DKO MEFs compared with 8.3% of the WT MEFs were in S phase (n=8; 95% CI for difference 0.89% to 4.48%, P=0.006). Bromodeoxyuridine (BrdU) incorporation assays showed that, following serum stimulation for 8 h, only 12% of DKO cells had entered S phase compared to 23% of WT cells (Figure 3d). However, by 16 h, the proportion of cells in S phase was approximately the same for the DKO (38%) and WT (42%) cells. These results suggest that the reduction in growth of the DKO cells is associated with a delayed ability to pass through the G0–G1–S transitions of the cell cycle.

Assessment of MEK/ERK phosphorylation in DKO MEFs

MEFs of each genotype were serum starved and treated with serum for various lengths of time, protein lysates were prepared and Western blots were incubated with antibodies specific for phosphoMEKl/2 or phosphoERKl/2. In the A-raf-/-, raf-1-/- and WT control MEFs, the levels of phosphoMEK increased following 2 min of serum treatment and continued to increase up to 10 min of treatment but started to decline after this point (Figure 4a). For the DKO MEFs, the levels of phosphoMEK increased slightly following 2–10 min of stimulation but they were significantly reduced in comparison to control MEFs (Figure 4a). Similar profiles of phosphorylation were observed for phosphoERK (Figure 4b). Levels of phosphoERK in cells stimulated with serum for 2–5 min were significantly lower in the DKO cells compared to the control cells (Figure 4b). At later time points from 10 min onwards, the levels of phosphoERK reached similar levels to those in control MEFs. The profiles of phosphoERK stimulation over a time course of EGF treatment were similar to that observed with serum (Figure 4c). The sustained activation of phosphoERK following serum stimulation was also assessed (Figure 4d). At time points over 1 h of stimulation, there was no significant difference in the levels of phosphoERK observed in DKO cells compared to WT cells (Figure 4d). Total protein lysates were also generated from DKO embryos as well as from littermate control embryos. The total level of phosphoERK in the DKO embryo lysates was reduced compared to that in control embryo lysates (Figure 4e).

Figure 4.
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MEK and ERK phosphorylation (a) MEK phosphorylation following serum stimulation. Primary MEFs of each genotype were treated with serum over the indicated time course and protein cell lysates were harvested. Western blots were prepared and analysed with an antibody against phosphoMEK (upper panels) and actin (lower panels). (bd) ERK phosphorylation following serum (b, d) and EGF (c) stimulation. Western blots were prepared and analysed with an antibody against phosphoERKl/2 (upper panels) and total ERK (lower panels). (e) ERK phosphorylation in embryo lysates. Protein lysates were harvested from individual embryos of the genotypes indicated and Western blots were analysed with an antibody against phosphoERKl/2 (upper panel) or against actin (lower panel). (f) Quantitation of ERKl/2 phosphorylation following serum stimulation. Quantitation was achieved by scanning Western blots from multiple experiments using NIH Image Software. Fold changes in ERKl/2 phosphorylation over basal in WT samples were calculated and pooled at each time point. Each experiment was performed six times and the data show mean valuesplusminusstandard error

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Throughout these experiments, some variations were observed in the profiles of ERK phosphorylation between cells of the same genotype. For this reason, the experiments were repeated multiple times. The levels of phosphoERK induced at each time point in each experiment were quantified using NIH Image software, pooled and analysed using a paired t-test (Figure 4f). The results of this analysis confirm that at 2 min (n=6; P=0.015) and at 5 min (n=6; P=0.043) of serum treatment the levels of phosphoERK are significantly reduced in the DKO cells compared to WT cells. However, at all other time points, although there is a trend towards a lower level of phosphoERK in the DKO samples, this is not statistically significant at any time point.

Analysis of expression of c-Fos in DKO MEFs

The AP1 transcription factor complex is an important element in mediating the effects of the ERK pathway on cell proliferation (Herber et al., 1994; Albanese et al., 1995; Shaulian and Karin, 2002). Of the AP1 components, only the induction of c-Fos expression has been specifically associated with the transient stimulation of ERK activation (Balmanno and Cook, 1999; Cook et al., 1999). Therefore, we examined whether the expression of c-Fos was disrupted in the DKO cells (Figure 5a). In WT cells, c-Fos expression was induced following 45 min of treatment with serum and phosphorylated forms of c-Fos were observed at later time points, particularly after 4 h. By contrast, the level of expression of c-Fos in the DKO cells was reduced at 45 min of serum treatment compared to the levels in WT cells. Although phosphorylated c-Fos isoforms were observed at the later time points in the DKO cells, their level of expression was reduced (Figure 5a). MEFs with a single knockout mutation of Raf-1 or A-Raf did not have noticeable changes in c-Fos expression or phosphorylation (data not shown).

Figure 5.
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Analysis of the expression of cell cycle control proteins. (a) Expression of c-Fos in MEFs. WT and DKO MEFs were serum starved for 20 h and then stimulated with serum for up to 8 h. Cell lysates were harvested, Western blots were prepared and analysed with an antibody against c-Fos (upper panel). A nonspecific band detected by the c-Fos antibody that remains unchanged during the time course provided a control for protein loading (lower panel). (b) Expression of cyclin Dl in embryos. Cell lysates were harvested from E10.5 embryos and Western blots were analysed with an antibody against cyclin Dl. An antibody against actin was used to confirm protein loading. (c) Expression of cyclin Dl, cdk4 and cyclin D3 in primary MEFs. Cell lysates were harvested and Western blots were analysed with antibodies against cyclin Dl, cyclin D3 and Cdk4. An antibody against actin was used to confirm protein loading. (d) Expression of cyclin Dl in MEFs over a time course of stimulation with serum. WT and DKO MEFs were serum starved for 20 h and then stimulated with serum for 0–20 h as indicated. Cell lysates were harvested, Western blots were prepared and analysed with an antibody against cyclin Dl (upper panel). An antibody against actin was used to confirm protein loading (lower panel)

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Analysis of expression of cell cycle proteins in DKO MEFs

Consistent with the reduction in G0–G1–S cell cycle progression, levels of expression of cyclin Dl were reduced in cycling DKO cells and in whole embryo lysates (Figure 5b and c). Levels of Cdk4 were slightly reduced in the DKO MEFs, whereas levels of cyclin D3 expression were unchanged (Figure 5b). Levels of cyclin Dl were assessed over a time course of stimulation with serum (Figure 5d). In WT cells, serum stimulation gave rise to an induction of expression of cyclin Dl at 1 h and this continued to rise up to 20 h. In the DKO cells, there was no detectable cyclin Dl expression until after 4 h of serum stimulation. Therefore, consistent with the proliferation data, induction of cyclin Dl expression is delayed in the DKO cells compared to the WT cells

Rescue of DKO phenotype by overexpression of Raf-1

We transfected the DKO cells with vectors expressing Raf isotypes in an attempt to rescue the phenotype. Coexpression of A-Raf and Raf-1 together proved technically difficult. Therefore, we overexpressed Raf-1 alone. DKO cells were transfected with vectors expressing either myc-tagged human Raf-1 or with a control vector expressing GFP using the Amaxa 'Nucleofector'. Transfection of the DKO cells with the Raf-1 vector clearly led to the overexpression of Raf-1 (Figure 6a). This was associated with a significant increase in the levels of phospho ERK (Figure 6a) and cyclin Dl (Figure 6b) as well rescue of the percentage of cells entering S phase (Figure 6c).

Figure 6.
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Rescue of the DKO phenotype by overexpression of Raf-1. (a) Rescue of phosphoERK levels. DKO cells were transiently transfected with vectors expressing either GFP or human Raf-1. Cell lysates were prepared from WT and DKO cells, as well as transfected cells that were either unstimulated cells or stimulated with serum for 10 min. Western blots were analysed with antibodies for Raf-1 (top), phosphoERK (middle) and actin as a loading control (bottom). (b) Rescue of cyclin Dl levels. Cell lysates were prepared from WT and DKO cells, as well as transfected cells grown in complete medium. Western blots were analysed with antibodies for cyclin Dl (top) and actin as a loading control (bottom). (c) Rescue of proliferation. WT, DKO and transfected cells were serum starved for 20 h and then stimulated with serum for 4 h. The percentage of cells entering S phase was determined by performing a BrdU proliferation assay (see Materials and methods)

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Discussion

Mice containing knockout mutations of the raf-1 and A-raf genes have been previously documented (Pritchard et al., 1996; Huser et al., 2001; Mercer et al., 2002). In contrast to the present study, the analysis of these single mutant mice provided no evidence for a role of A-Raf and Raf-1 in ERK1/2 activation or cell proliferation, but suggested an MEK kinase-independent role of Raf-1 in the prevention of apoptosis (Huser et al., 2001; Jesenberger et al., 2001; Mikula et al., 2001; Hindley and Kolch, 2002). In this report, we intercrossed the A-raf/Y and raf-T' mice on the MF1 background to generate mice carrying knockout mutations of both genes and show that A-Raf and Raf-1 work together to influence ERK1/2 activation, c-Fos expression, cyclin Dl expression and G0–G1–S cell cycle progression. Thus, the role of Raf-1 as an MEK kinase involved in proliferation is dependent on A-Raf. Our results show that A-Raf and Raf-1 compensate for one another in the single mutant mice and that B-Raf cannot compensate for the combined loss of A-Raf and Raf-1. These data are consistent with recent results from one of our laboratories showing that the downregulation of A-Raf and Raf-1 by siRNA in B-Raf-transformed melanoma cells is associated with a reduction in DNA synthesis in both cases (Karasarides et al., 2004). The downregulation of B-Raf in these cells by siRNA also led to a reduction in DNA synthesis. Therefore, both A-Raf/Raf-1 and B-Raf are involved in activating pools of MEK/ERK, both of which have an important influence on cell proliferation. To further explore the role of Raf-1 and B-Raf in regulating cell proliferation via MEK/ERK activation, we are currently generating knockin mice with kinase inactive versions of Raf-1 and B-Raf.

Previous studies of raf-1-/- mice showed that the lack of Raf-1 is associated with an increase in apoptosis (Huser et al., 2001; Jesenberger et al., 2001; Mikula et al., 2001; Hindley and Kolch, 2002) whereas the raf-1-/- embryos and MEFs analysed here do not show an apoptotic phenotype (Figures 2 and 3). The difference between these studies is the genetic background of the mice investigated. The previous studies analysed mice on either the 129Ola/C57BL6 or 129Ola/129Sv mixed inbred genetic backgrounds, whereas mice on the outbred MF-1 background were studied here. This would suggest that the role of Raf-1 in apoptosis is complex and is highly dependent on the genetic origin of the cells under study and indeed may explain some conflicting data in the literature that has defined Raf-1 both as a promoter (Blagosklonny et al., 1996, 1997; Kauffmann-Zeh et al., 1997; Basu et al., 1998) and inhibitor of apoptosis (Baccarini, 2002).

The combined disruption of A-Raf and Raf-1 leads to a significant decrease in transient ERK activation following growth factor stimulation (Figure 4). The fact that transient MEK phosphorylation is also disrupted in the DKO cells indicates that the changes observed in phosphoERK are more likely to be due to changes in the activities of Raf/MEK rather than changes in the activities of MEK or MAPK phosphatases.

Previous data indicated that the three Rafs have tissue-specific patterns of gene expression (Storm et al., 1990). However, through the availability of good antibodies for each Raf protein, it has now become clear that all three have ubiquitous patterns of gene expression (Mercer and Pritchard, 2003). Therefore, the different role of each Raf isotype in regulating endogenous MEK/ERK activation is more likely to be due to different mechanisms in the regulation of their kinase activities rather than differences in their expression patterns. Indeed, the profiles of activation of endogenous Raf-1 and A-Raf in MEFs following growth factor stimulation, as measured by the immunoprecipitation MEK/ERK kinase cascade assay, correlate well with the transient peak of MEK/ERK activation they are involved in stimulating (Huser et al., 2001). Raf-1 and A-Raf activities are stimulated five-fold and 1.5-fold, respectively, following growth factor treatment and maximal activities are observed after 5 min of treatment. Their activities drop significantly after this peak and return to basal levels by 60 min. By contrast, the profile of B-Raf activation in MEFs correlates with sustained ERK activation. B-Raf has a high level of basal activity in unstimulated cells and its activity is further stimulated 1.5-fold by growth factors and reaches a maximum after 5 min of treatment. However, its activity is sustained for far longer than that of Raf-1 or A-Raf as it remains active at later time points (Huser et al., 2001).

The reasons for the differences in profiles of activation/deactivation of the three Rafs are not entirely clear but must reflect biochemical similarities between A-Raf and Raf-1 and important biochemical differences between these two Raf kinases and B-Raf. All three Raf kinases are located in the cytosol bound to 14-3-3 in inactive states and translocate to the plasma membrane in the presence of active Ras (Kolch, 2000; Marais et al., 1995; Marais et al., 1997; Avruch et al., 2001; Dhillon and Kolch, 2002; Ory et al., 2003). All three Rafs require phosphorylation/dephosphorylation for full activation at the membrane but the patterns of phosphorylation differ somewhat (Dhillon and Kolch, 2002; Chong et al., 2003). Both Raf-1 and B-Raf require dephosphorylation of Akt consensus sequences at serine 259 in Raf-1 and at the equivalent serine 364 in B-Raf (Chong et al., 2001; Zhang et al., 2001; Dhillon et al., 2002) as well as phosphorylation of threonine and serine residues in the activation loop for full activation (Zhang and Guan, 2000; Chong et al., 2001). In B-Raf, the purpose of these activation loop phosphorylations appear to be to disrupt the P-loop–DFG interaction stabilizing the inactive conformation of the kinase (Wan et al., 2004). However, Raf-1 also requires phosphorylation of tyrosine 341 and serine 338 residues in the N region of the kinase domain to achieve full activation, whereas B-Raf does not require phosphorylation in this region as it is constitutively phosphorylated at serine 445, the equivalent residue to serine 338 in Raf-1, and possesses a phosphomimetic aspartic acid residue at 448, the equivalent residue to tyrosine 341 in Raf-1 (Marais et al., 1995, 1997; Mason et al., 1999; Dhillon and Kolch, 2002; Dhillon et al., 2002). A-Raf appears to be regulated in a similar way to Raf-1 (Marais et al., 1997). The process of Raf deactivation has been little studied to date but must involve loss of Ras binding due to the conversion of Ras.GTP to Ras. GDP by RasGAPs, phosphorylation/dephosphorylation events leading to the reformation of the P-loop–DFG interaction, and the formation of new protein–protein interactions in the cytosol, particularly with 14-3-3. The sustained activation of B-Raf following growth factor treatment would suggest that this deactivation process is more prolonged for B-Raf than it is for Raf-1 or A-Raf.

An important step for cell cycle re-entry and progression through Gl is expression of the D-type cyclins and consequent activation of cyclinD : cdk4/6 complexes (Jiang et al., 1993; Resnitzky et al., 1994; Stacey, 2003). A key mechanism by which signalling through the Raf/MEK/ERK can induce cell cycle progression is through stimulation of cyclin Dl mRNA expression (Kerkhoff and Rapp, 1997; Woods et al., 1997; Marshall, 1999). This is achieved by the ability of ERKs to translocate to the nucleus and phosphorylate Ets and AP-1 family members that transactivate the cyclin Dl promoter (Herber et al., 1994; Albanese et al., 1995; Lavoie et al., 1996; Whitmarsh and Davis, 1996; Balmanno and Cook, 1999; Cook et al., 1999; Shaulian and Karin, 2002). In fibroblasts, several studies have shown that the sustained activation of ERKs induced by various mitogens is associated with cell cycle progression, whereas transient peaks of ERK activation are not (Vouret-Craviari et al., 1993; Cook and McCormick, 1996; Weber et al., 1997; Balmanno and Cook, 1999). An extensive study of the AP-1 components expressed during these different phases showed that while many AP-1 components (c-Fos, Fra-1, Fra-1, c-Jun and JunB) are expressed at immediate early time points following mitogen treatment of cells, the expression of c-Fos is not observed at sustained time points whereas the expression of the other AP-1 components persists at later time points (Kovary and Bravo, 1992; Balmanno and Cook, 1999; Cook et al., 1999). The role of the immediate early induction of c-Fos expression in cell cycle re-entry was therefore brought into question (Balmanno and Cook, 1999; Cook et al., 1999).

In this report, our results show that transiently induced ERK phosphorylation is significantly reduced in the DKO cells and the cell cycle is not maintained at its usual rate. This may be mediated in part by transient phosphoERK induction of c-Fos expression and consequent induction of cyclin Dl mRNA expression (Figure 5), although transient phosphoERK may also influence other parts of the cell cycle machinery that have yet been subjected to investigation. A recent study has shown that, when ERK activation is transient, c-Fos expression is induced but the protein is unstable and is degraded (Murphy et al., 2002). However, under conditions when ERK activation is sustained, the c-Fos induced during the immediate early phase is phosphorylated on multiple sites by ERK and p90RSK and is thus stabilized and contributes to the induction of expression of cyclin Dl. Our data support this important functional role of c-Fos as a sensor for the duration of Raf/ERK signalling.

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Materials and methods

Derivation of mice and culture ofMEFs

Mice containing a homozygous knockout mutation of the A-raf gene and the raf-1 gene have been described previously (Pritchard et al., 1996; Huser et al., 2001). These mice were backcrossed onto the MF1 genetic background. Embryos were collected at E10.5–E14.5, homogenized and fibroblasts were derived by standard procedures (Huser et al., 2001). PCR genotyping was performed using the primers and conditions described previously (Huser et al., 2001; Mercer et al., 2002). Each primary MEF culture was isolated from a single embryo. Fibroblasts were cultured in high glucose (4.5 g/1) Dulbecco's modified Eagle's medium (DMEM; Life Technologies) containing 10% (v/v) foetal calf serum (FCS; SeraQ) and 100 U/ml penicillin/streptomycin (Life Technologies) in a 10% CO2 humidified incubator at 37°C.

Histology

For sectioning, embryos were fixed in methacarn (10% (v/v) glacial acetic acid, 30% (v/v) chloroform, 60% (v/v) methanol), and paraffin embedded. Paraffin sections at 5 mum thickness were mounted onto microscope slides pretreated with silane. For Ki67 staining, after blocking in 6% (v/v) hydrogen peroxide in methanol for 30 min and 1 : 25 dilution of swine serum for 15 min, sections were incubated with a 1 : 200 dilution of a rabbit polyclonal Ki67 antibody (Vector Laboratories, Burlingame, CA, USA) at room temperature overnight. Antigen–antibody complexes were visualized with biotinylated secondary antibody and with the avidin–biotin–peroxidase complex (Vector Laboratories, Burlingame, CA, USA). For TUNEL assays, the ApoAlert™ DNA fragmentation assay kit (BD Biosciences) was used following the manufacturer's instructions.

Cell stimulations, B-Raf kinase assays and immunoblotting

Primary MEFs were placed in serum-free media for 20 h and then stimulated with either 10 ng/ml EGF or with 10% (v/v) FCS over a time course of up to 20 h. Protein lysates and Western blots were carried out as described previously (Luckett et al., 2000). B-Raf assays were performed using the immunoprecipitation kinase cascade assay (Marais et al., 1997). Primary antibodies were a 1 : 1000 dilution of a rabbit polyclonal antibody for A-Raf (Santa Cruz Biotechnology Inc.), a 1 : 1000 dilution of a mouse monoclonal antibody for Raf-1 (Transduction Laboratories), a 1 : 1000 dilution of a mouse monoclonal antibody for B-Raf (Santa Cruz Biotechnology Inc.), a 1 : 1000 dilution of an antibody for actin (Sigma), a 1 : 1000 dilution of a mouse monoclonal antibody against Thr202/Tyr204 phospho-p44/42 ERK1/2 (Cell Signalling Tech.), a 1 : 1000 dilution of a rabbit polyclonal antibody for ERK2 (Zymed Laboratories Inc.), a 1 : 1000 dilution of a rabbit polyclonal antibody against Ser217/Ser221 phospho-MEKl/2 (Cell Signalling Tech.), a 1 : 2000 dilution of a mouse monoclonal antibody against cyclin Dl, a 1 : 2000 dilution of a mouse monoclonal antibody against cdk4, a 1 : 2000 dilution of a mouse monoclonal antibody against cdk6, a 1 : 2000 dilution of a mouse monoclonal antibody against cyclin D3 (all from Cell Signalling Tech.) and a 1 : 1000 dilution of a rabbit polyclonal antibody for c-Fos (Santa Cruz Biotechnologies).

Transfection of MEFs

DKO MEFs were transfected with vectors expressing either myc-tagged full-length human Raf-1 or GFP using a Nucleofector under the MEF conditions recommended by the manufacturer (Amaxa Biosystems, Germany). At 24 h following transfection, cells were placed in serum-free media for 20 h and then either left unstimulated or stimulated with 10% (v/v) FCS for 10 min. Protein lysates and western blots were carried out as described above.

Proliferation and apoptosis assays

For growth curves, 2 times 104 cells were plated and counted at 24 h intervals in triplicate using a haemocytometer. For cell cycle analysis, primary cells at 80% confluency on 6 cm dishes were collected and fixed in 70% (v/v) ethanol at 4°C for 30 min. Fixed cells were then resuspended in PBS containing 10 mug/ml propidium iodide and 100 mug/ml RNase at room temp for 1 h. FACS analysis was performed using a Becton Dickinson flow cytometer. BrdU incorporation assays for proliferation were performed by using the BrdU labelling and detection kit provided by Roche. The percentage of BrdU-positive cells was visualized by fluorescence microscopy using a Zeiss Axiophot microscope. To induce apoptosis, primary cells at 80% confluency on 6 cm dishes were treated with 50 ng/ml anti-CD95 antibody with 0.5 muM cycloheximide for 20 h in a 37°C humidifying incubator. Annexin V staining and FACS analysis were performed as described previously (Huser et al., 2001).

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Acknowledgements

We are extremely grateful to The Wellcome Trust and Cancer Research UK for providing financial support for this project. We thank Mabel Iwobi and Vicky Aldridge for help during the initial stages of this project and Simon Cook for advice on detecting c-Fos.

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