A-Raf and Raf-1 Work Together to Influence Transient ERK Phosphorylation and Gl/S Cell Cycle Progression
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Oncogene (2005) 24, 5207–5217 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc 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 Pritchard*,1 1Department of Biochemistry, University of Leicester, Adrian Building, University Road, Leicester LEI 7RH, UK; 2Division of Biomedical Services, University of Leicester, University Road, Leicester LEI 7RH, UK; 3Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK The Raf/MEK/ERK (extracellular regulated kinase) Kerkhoff and Rapp, 1997, 1998; Woods et al., 1997; signal transduction pathway controls the ability of cells Marshall, 1999; Squires et al., 2002). This function to respond to proliferative, apoptotic, migratory and is mediated by the ability of ERKs to translocate to the differentiation signals. We have investigated the combined nucleus where they are able to phosphorylate transcrip- contribution of A-Raf and Raf-1 isotypes to signalling tion factors, particularly the AP-1 complex that through this pathway by generating mice with knockout comprises various heterodimers of c-fos (c-fos, FosB, mutations of both A-raf and raf-1 genes. Double knockout Fra-1, Fra-2) and c-jun (c-Jun, JunB, JunD) family (DKO) mice have a more severe phenotype than single null members (Whitmarsh and Davis, 1996; Balmanno mutations of either gene, dying in embryogenesis at E10.5. and Cook, 1999; Cook et al., 1999; Shaulian and The DKO embryos show no changes in apoptosis, but Karin, 2002). This transcription factor complex is staining for Ki67 indicates a generalized reduction in involved in promoting D-type cyclin expression, thus proliferation. DKO mouse embryonic fibroblasts (MEFs) allowing the cyclin-dependent kinases CDK4 exhibit a delayed ability to enter S phase of the cell cycle. and CDK6 to phosphorylate and de-repress the This is associated with a reduction in levels of transiently retinoblastoma family members (Brown et al., 1998; induced MEK and ERK phosphorylation and reduced Kerkhoff and Rapp, 1998; Marshall, 1999; Shaulian and expression of c-Fos and cyclin Dl. Levels of sustained Karin, 2002). The consequent release of the E2F ERK phosphorylation are not significantly altered. Thus, transcription factors from retinoblastoma binding al- Raf-1 and A-Raf have a combined role in controlling lows expression of genes required for progression physiological transient ERK activation and in mainte- through the G0–G1–S transitions of the cell cycle nance of cell cycle progression at its usual rate. (Stevaux and Dyson, 2002). Oncogene (2005) 24, 5207–5217. doi:10.1038/sj.onc.1208707; There are three Raf family members in mammals; published online 25 April 2005 A-Raf, Raf-1 and B-Raf (Mercer and Pritchard, 2003). All three are able to induce MEK/ERK activation Keywords: A-Raf; Raf-1; knockout; ERK activation; (Pritchard et al., 1995), but they differ in their strength cell cycle; c-Fos; cyclin Dl 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-rafÀ/À Introduction mouse embryonic fibroblasts (MEFs), ERK phosphor- ylation and activation are reduced to E30% that in Extracellular regulated kinases 1 and 2 (ERK1/2) are wild-type cells (Wojnowski et al., 2000; Pritchard et al., highly conserved cellular enzymes that play crucial roles 2004) whereas there is no noticeable decrease in ERK in the control of multiple cellular processes including cell phosphorylation or activation in raf-1À/À MEFs or proliferation (Lewis et al., 1998; Chen et al., 2001). A-rafÀ/Y MEFs (Huser et al., 2001; Mikula et al., 2001; Treatment of mammalian cells with mitogens leads to an Mercer et al., 2002). The ability of B-Raf to induce increase in intracellular levels of Ras.GTPfollowed by strong ERK activity has been highlighted by the the sequential activation of Raf, MEK1/2 and ulti- discovery of activating mutations of the BRAF gene in mately ERK1/2 (Marais and Marshall, 1996; Lewis human cancers (Davies et al., 2002; Kimura et al., 2003; et al., 1998). It has been shown that activation of ERK Mercer and Pritchard, 2003). The most common in this way is an important step in G0–Gl–S phase mutation, a valine to glutamic acid change at residue progression of the cell cycle (Samuels et al., 1993; 599, is thought to contribute to tumorigenesis by stimulating constitutive ERK phosphorylation. A simi- *Correspondence: C Pritchard; E-mail: [email protected] lar mutation in RAF1 does not induce such high levels of Received 11 October 2004; revised 25 January 2005; accepted 16 March ERK activation and is not detected in human cancers 2005; published online 25 April 2005 (Davies et al., 2002). Influence of A-Raf and Raf-1 on ERK phosphorylation and Gl/S cell cycle progression K Mercer et al 5208 Table 1 Genotyping data of embryos obtained from A-rafÀ/Àraf-1+/À Â A-raf/Y raf-1+/À intercrosses Genotype Age P1 Total Expected E9.5E10.5Ell.5E12.5E13.5E14.5 A-raf+/+raf-1+/+ a 3235 1 562522 A-raf+/Àraf-1+/+ 6576 1 383622 A-rafÀ/Àraf-1+/+ a 7 6 13 9 5 2 6 48 44 A-raf+/+raf-1+/À a 5 2 20 7 1 3 12 50 44 A-raf+/Àraf-1+/À 6 6 11 16 6 1 17 63 44 A-rafÀ/Àraf-1+/À a 18 5 26 22 5 1 9 86 88 A-raf+/+raf-1À/À a 1044 1 011122 A-raf+/Àraf-1À/À 4235 0 001422 A-rafÀ/Àraf-1À/À a 5543 1 001844 aMale and female embryos indistinguishable The duration and magnitude of ERK activation has a Results significant impact on whether cells enter the cell cycle or not (Marshall, 1995). In fibroblasts, transient and Generation of DKO mice sustained ERK activation are observed following mito- gen treatment (Vouret-Craviari et al., 1993; Cook and To generate the DKO mice, two rounds of breeding À/À À/À McCormick, 1996; Weber et al., 1997; Balmanno and were set up with A-raf and raf-1 single mutant þ /À Cook, 1999). The sustained phase of ERK activation mice. In the first round, A-raf female mice were þ /À correlates with the induction of expression of a subset of crossed to raf-1 male mice. Wild-type (WT), single and double heterozygote mice were obtained at the AP-1 components (Fra-1, Fra-2, c-Jun and JunB), which /Y in turn are required for sustained cyclin Dl expression expected Mendelian frequency, while A-raf and A-raf IY þ lÀ and progression through the cell cycle (Kovary and raf-1 adult mice were obtained at a reduced Bravo, 1991, 1992; Lavoie et al., 1996; Balmanno and frequency and displayed a phenotype typical of that Cook, 1999; Cook et al., 1999). The transient activation previously described (data not shown; Pritchard et al., þ /À þ /À of ERK correlates with transient c-Fos expression and is 1996). In the second round of breeding, A-raf raf-1 /Y þ /À not associated with the induction of cyclin Dl expression female animals were crossed to A-raf raf-1 male or cell cycle progression in several cell systems (Vouret- mice. No DKO animals survived postnatally and so Craviari et al., 1993; Cook and McCormick, 1996; embryos were harvested from matings timed at between Weber et al., 1997). However, other studies have E9.5 to birth (Table 1). Genotypes of each embryo were indicated an important role for c-Fos in controlling confirmed by PCR analysis of yolk sac DNA (Figure 1a) cyclin Dl expression and cell cycle progression (Kovary and Western blot analysis was used to confirm the Raf-1 À/À and Bravo, 1991; Won et al., 1992; Miao and Curran, and/or A-Raf protein deficiencies (Figure 1b). A-raf 1994; Brown et al., 1998). In particular, mice with embryos were obtained at the expected frequency, knockout mutations of both the c-Fos and FosB genes indicating that loss of A-Raf alone has no detrimental À/À have defects in proliferation that result at least in part effect on embryogenesis (Table 1). Raf-1 embryos from a failure to induce cyclin Dl expression (Brown were obtained at the expected frequencies up to E13.5 et al., 1998). but lethality was observed from this point onwards and In this report, we have used gene targeting in mice very few survived to birth (o3%). DKO embryos were to investigate the roles of A-Raf and Raf-1 in regulating obtained at the expected frequency at E9.5 but they MEK/ERK activation and downstream cellular re- appeared at a reduced frequency at E10.5 onwards and sponses. Despite the fact that single knockout mutations none survived to birth (Table 1). The expression of either gene do not lead to noticeable changes (Figure 1c) and activity (Figure 1d) of the remaining in ERKl/2 activation (Huser et al., 2001; Mikula et al., Raf isotype, B-Raf, were not noticeably altered in DKO 2001; Mercer et al., 2002), the combined knockout embryos compared to WT embryos. mutation of both genes leads to reduced ERKl/2 phosphorylation. These results indicate that A-Raf Phenotype analysis of single knockout and DKO mice and Raf-1 play compensatory roles in ERKl/2 activa- tion that cannot be rescued by B-Raf However, Embryos were harvested at E10.5.