The ERK1/2 Mitogen-Activated Protein Kinase Pathway As a Master Regulator Ofthe G1- to S-Phase Transition

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Oncogene (2007) 26, 3227–3239 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW The ERK1/2 mitogen-activated protein kinase pathway as a master regulator ofthe G1- to S-phase transition

S Meloche1 and J Pouysse´ gur2

1Departments of Pharmacology and Molecular Biology, Institut de Recherche en Immunologie et Cance´rologie, Universite´ de Montre´al, Montreal, Quebec, Canada and 2Institute of Signaling, Developmental Biology and Cancer, CNRS UMR 6543, Universite´ de Nice-Soﬁa Antipolis, Centre A. Lacassagne, Nice, France

The Ras-dependent extracellular signal-regulated kinase extracellular signal-regulated kinase 1 (ERK1)/2 mitogen- (ERK)1/2 mitogen-activated protein (MAP) kinase path- activated protein (MAP) kinase pathway. way plays a central role in cell proliferation control. In MAP kinase pathways are evolutionarily conserved normal cells, sustained activation ofERK1/ERK2 is signaling modules by which cells transduce extracellular necessary for G1- to S-phase progression and is associated signals into intracellular responses (reviewed in Lewis with induction ofpositive regulators ofthe cell cycle and et al., 1998; Widmann et al., 1999; Pearson et al., 2001). inactivation ofantiproliferative genes. In cells expressing The prototypical MAP kinase pathway is the ERK1/2 activated Ras or Rafmutants, hyperactivation ofthe pathway, which is activated preferentially by mitogenic ERK1/2 pathway elicits cell cycle arrest by inducing the factors, differentiation stimuli and cytokines. The basic accumulation ofcyclin-dependent kinase inhibitors. In this principles that govern the activation of this pathway review, we discuss the mechanisms by which activated have been established during the 1990s and are relatively ERK1/ERK2 regulate growth and cell cycle progression well understood. Binding of ligands to their respective ofmammalian somatic cells. We also highlight the cell surface receptors induces the activation of the small findings obtained from gene disruption studies. GTPase Ras, which recruits the MAP kinase kinase Oncogene (2007) 26, 3227–3239. doi:10.1038/sj.onc.1210414 kinase Raf to the membrane for subsequent activation by phosphorylation. Activated Raf isoforms phosphory- Keywords: signal transduction; MAP kinase; ERK1/2; late and activate the MAP kinase kinases MEK1/ cell cycle; G1 phase MEK2, which in turn activate the effector MAP kinases ERK1 and ERK2 by phosphorylation of the Thr and Tyr residues within their activation loop. ERK1/ERK2, which are expressed ubiquitously in mammalian cells, are multifunctional serine/threonine kinases that phos- Introduction phorylate a vast array of substrates localized in all cellular compartments (Lewis et al., 1998; Pearson et al., The cell division cycle is the sequence of events by which 2001; Yoon and Seger, 2006). These include protein cells faithfully replicate their DNA and then segregate kinases, signaling effectors, receptors, cytoskeletal pro- the duplicated chromosomal DNA equally between two teins and nuclear transcriptional regulators. This review daughter cells. An intricate network of regulatory focuses on the importance of the ERK1/2 MAP kinase pathways ensures that each cell cycle event is performed pathway in cell proliferation control. We discuss our correctly and in proper sequence (Morgan, 2007). The current understanding of the mechanisms by which decision of cells to replicate their genetic material and activated ERK1/ERK2 regulate growth and cell cycle divide is made in G1 and is influenced by extracellular progression of mammalian somatic cells. signals (nutrients, mitogens, cytostatic factors, extracellular matrix). Once cells make their decision, they are irreversibly committed to complete the cycle independently of mitogenic factors. One of the key signal Sustained activation ofthe ERK1/2 MAP kinase pathway transduction pathways responsible for integrating these is necessary for cell proliferation environmental signals and relaying the information to the cell cycle control system is the Ras-dependent The first hint about the involvement of ERK1/ERK2 MAP kinases in cell proliferation control came from the Correspondence: Dr S Meloche, Departments of Pharmacology and observation that the two kinases are rapidly phosphory- Molecular Biology, Institut de Recherche en Immunologie et lated and activated in response to mitogenic stimulation. Cance´ rologie Universite´ de Montre´ al, Montreal, Quebec, Canada Indeed, ERK1 and ERK2 were originally identified as H3C 3J7 or Dr J Pouysse´ gur, Institute of Signaling, Developmental Biology and Cancer, CNRS UMR 6543, Universite´ de Nice-Sofia mitogen-stimulated phosphoproteins of 41–45 kDa after Antipolis, Centre A Lacassagne, Nice 06189, France. separation by one- or two-dimensional gel electrophor- E-mails: [email protected] or [email protected] esis (Nakamura et al., 1983; Cooper et al., 1984; Kohno, ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3228 1985; Kohno and Pouyssegur, 1986). This pair of (Brunet et al., 1994; Cowley et al., 1994; Seger et al., tyrosine phosphorylated proteins was later shown to 1994) relaxes growth factor dependency and enhances correspond to the activated forms of two MAP-2/myelin the rate of cell proliferation. basic protein (MBP) kinases characterized previously Although ERK1/2 activation is necessary for G1 and referred to as p42mapk (ERK2) and p44mapk (ERK1) progression, it is nonetheless insufficient to drive cells (Rossomando et al., 1989, 1991). Subsequent studies into S phase (Cheng et al., 1998; Treinies et al., 1999; confirmed that ERK1/ERK2 are activated in response Jones and Kazlauskas, 2001). Careful temporal dissec- to virtually all mitogenic factors in diverse cell types. tion of the signaling events required for cells to move Detailed kinetic analysis revealed that mitogens induce a from G0 through G1 and into S phase has revealed biphasic activation of ERK1 and ERK2 in fibroblasts, that accumulation of phosphatidylinositol-3-OH kinase with a rapid and strong burst of kinase activity peaking (PI(3)K) lipid products is important for late G1 at 5–10 min followed by a second wave of lower but progression (Jones and Kazlauskas, 2001). Some studies sustained activity that persists throughout the G1 phase have reported that overexpression of activated MEK1 in (Kahan et al., 1992; Meloche et al., 1992; Meloche, quiescent fibroblasts is sufficient to cause S-phase entry 1995). A close correlation was observed between the (Cowley et al., 1994; Gotoh et al., 1999; Treinies et al., ability of extracellular factors to induce sustained 1999). However, it was later shown that induction of ERK1/2 activation and DNA synthesis (Kahan et al., DNA synthesis can be blocked by incubation of cells 1992; Meloche et al., 1992). For example, the potent with the PI(3)K inhibitor LY294002, suggesting that mitogen thrombin was shown to activate ERK1 with activation of the ERK1/2 pathway induces the synthesis biphasic kinetics in hamster lung fibroblasts. Rapid of autocrine growth factors (Treinies et al., 1999). The inactivation of thrombin with the antagonist hirudin or addition of insulin to serum-free culture medium (Gotoh pretreatment of cells with pertussis toxin, both of which et al., 1999) may also lead to confounding results, as abolish late-phase activation while preserving the early insulin is able to substitute for (PI(3)K) lipid products in peak of activity, resulted in complete inhibition of DNA late G1 (Jones and Kazlauskas, 2001). synthesis. These early observations were confirmed by several laboratories using different cellular models (Cook and McCormick, 1996; Weber et al., 1997b; Talarmin et al., 1999; Jones and Kazlauskas, 2001), ERK1/2 signaling regulates cell growth allowing the generalization of the model. In a recent study that systematically analyzed the impact of the Cell growth is the process by which cells increase their duration of ERK1/2 activity, it was concluded that mass by upregulating the biosynthesis of macromole- ERK1/2 activation must be sustained until late G1 for cules, membranes and organelles. In proliferating cells, successful S-phase entry (Yamamoto et al., 2006). cell growth is coordinated with the cell division cycle to ERK1/ERK2 are rapidly inactivated at the G1/S ensure that on average each cell division is accompanied transition, and their activity is not required for cells to by a doubling in mass in order to maintain cell size enter S phase (Meloche, 1995). (Jorgensen and Tyers, 2004). In animals, cell growth is The first demonstration of the direct involvement of mainly controlled by extracellular growth factors (Con- ERK1/2 in the mitogenic response was provided by the lon and Raff, 1999). The first evidence linking ERK1/2 findings that overexpression of ERK1 catalytically signaling to cell growth control stemmed from the inactive mutants or antisense RNA inhibits the activa- finding that PD98059, a synthetic inhibitor of MEK1/ tion of endogenous ERK1/ERK2 and exerts a domi- MEK2, blocks the stimulation of global protein syn- nant-negative effect on fibroblast cell proliferation thesis induced by a variety of growth factors acting (Pages et al., 1993). The suppression of cell proliferation through tyrosine kinase receptors or G protein-coupled by antisense ERK1 was fully reverted by co-transfection receptors in vascular smooth muscle cells (Servant et al., of a sense ERK1 construct, confirming the specificity of 1996). At the same time, it was reported that treatment the effect. Later on, other studies showed that over- of cells with PD98059 drastically reduces insulin- and expression of interfering mutants of MEK1 (Cowley serum-induced phosphorylation of the translation ini- et al., 1994; Seger et al., 1994), incubation with ERK2 tiation factor eIF4E (Flynn and Proud, 1996; Morley antisense oligonucleotide (Sale et al., 1995) or expression and McKendrick, 1997). These initial observations of active MAP kinase phosphatase 1 (MKP-1/DUSP1) suggested that the ERK1/2 pathway might play a role (Sun et al., 1994; Brondello et al., 1995) inhibits the in translational control. Phosphorylation of eIF4E on induction of DNA synthesis by serum and activated Ser 209 is mediated by the MAP kinase-activated Ras. Similarly, treatment with synthetic MEK1/2 protein kinase (MK) family members Mnk1/Mnk2, inhibitors, which prevents activation of ERK1/ERK2, which are directly phosphorylated and activated by the was reported to inhibit the proliferation of various cell MAP kinases ERK1/ERK2 and p38a/b in response to types, including fibroblasts, T lymphocytes, smooth growth factors or stress stimuli (Fukunaga and Hunter, muscle cells, hepatocytes and epithelial cell lines (Dudley 1997; Waskiewicz et al., 1997, 1999; Ueda et al., 2004). et al., 1995; Seufferlein et al., 1996; Karpova et al., 1997; The functional impact of eIF4E phosphorylation is not DeSilva et al., 1998; Williams et al., 1998; Sebolt- clear and has been a subject of debate (Scheper and Leopold et al., 1999; Talarmin et al., 1999). Conversely, Proud, 2002). The current view is that phosphorylation expression of constitutively active forms of MEK1 of eIF4E does not lead to a global increase in mRNA

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3229 translation but rather enhances translation of specific precursors for the synthesis of RNA and DNA, mRNAs with extensive 50UTR structure (Dever, 2002; phospholipids, UDP-sugars and glycogen. The rate- Mamane et al., 2004). In agreement with this idea, no limiting step in the pyrimidine pathway is catalysed by change in global protein synthesis was observed in the carbamoyl-phosphate synthetase (CPSase) enzyme, Mnk1/Mnk2-deficient embryonic fibroblasts, despite which is part of the large multifunctional protein CAD complete elimination of eIF4E phosphorylation (Ueda (Evans and Guy, 2004). Mitogenic stimulation of cells et al., 2004). So why does inhibition of ERK1/2 activity induces the phosphorylation of CAD and allosterically has such a profound effect on general protein synthesis activates the CPSase domain by an MEK1/2-dependent (Servant et al., 1996)? One mechanism by which ERK1/ mechanism (Graves et al., 2000). ERK2 directly ERK2 may stimulate global mRNA translation is phosphorylates CAD on Thr 456 in vitro, and alanine through enhancement of mammalian target of rapamy- substitution of this residue abolishes the allosteric cin (mTOR) signaling. mTOR is a master growth regulation of the enzyme. Furthermore, epidermal regulator that integrates signals from growth factors, growth factor-stimulated synthesis of UTP is blocked nutrients and cellular energy status to modulate ribo- by incubation with PD98059, consistent with a role of some biogenesis and translation initiation (see Fingar the ERK1/2 pathway in pyrimidine biosynthesis. and Blenis, 2004; Hay and Sonenberg, 2004; Wulls- chleger et al., 2006 for reviews). mTOR activity is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 heterodimer, which acts as a GTPase ERK1/2 signaling promotes G1 progression by activating protein (GAP) for the small GTPase Rheb multiple mechanisms (Manning and Cantley, 2003; Corradetti and Guan, 2006). Rheb binds directly to the mTOR complex 1 G1- to S-phase progression: a brief overview (mTORC1) and stimulates mTOR kinase activity in a The regulation of G1- to S-phase transition is governed GTP-dependent manner. Two groups recently reported by the concerted action of cyclin-dependent kinases that activation of the ERK1/2 pathway functionally (Cdks) and their regulatory cyclin subunits (reviewed inactivates the TSC1/TSC2 complex, as a result of the in Sherr, 2000; Malumbres and Barbacid, 2001). In phosphorylation of TSC2 (Roux et al., 2004; Ma et al., response to mitogenic factors, D-type cyclins progres- 2005). One study showed that the serine/threonine sively accumulate during G1 phase and assemble with kinase RSK1, a downstream substrate of ERK1/ their catalytic partners Cdk4 and Cdk6. The activity of ERK2, phosphorylates TSC2 in the conserved C- Cdk4/6 increases as cells approach the G1/S boundary, terminus of the protein (Ser 1798) to impair its and persist through the first and subsequent cycles as antagonistic action on mTOR signaling (Roux et al., long as mitogens are present. A major function of cyclin 2004). The other study proposed that ERK1/ERK2 D-dependent kinases is to phosphorylate members of directly phosphorylate TSC2 on Ser 664 and inhibit its the retinoblastoma (Rb) family of proteins. Rb phos- function by disrupting TSC1/TSC2 complex formation phorylation disrupts its association with bound E2F (Ma et al., 2005). Thus, the ERK1/2 and PI(3)K family members, allowing the coordinated transcription pathways cooperate to activate mTOR and promote of genes required for DNA replication (Weinberg, 1995; cell growth. Harbour and Dean, 2000; Bracken et al., 2004). Among Another mechanism by which ERK1/ERK2 may the E2F-regulated genes are Cyclin E, Cyclin A2 and impact on global protein synthesis is through direct Emi1, which are key effectors of the G1/S transition in regulation of ribosomal gene transcription. Activation normal cells (Hsu et al., 2002; Cobrinik, 2005). Cyclin E of the ERK1/2 pathway by growth factors or con- associates with Cdk2 to enforce Rb phosphorylation ditionally active Raf-1 induces an immediate increase in and complete its inactivation. A second non-catalytic ribosomal RNA transcription, whereas inactivation of function of cyclin D–Cdk4/6 complexes is to sequestrate the pathway by MEK1/2 inhibitors suppresses basal and the Cdk inhibitors p27Kip1 (p27) and p21Cip1 (p21) (Geng stimulated ribosomal transcription (Stefanovsky et al., et al., 2001; Tong and Pollard, 2001). The binding of 2001). ERK2 was found to phosphorylate directly the p21/p27 to cyclin D–Cdk4/6 stabilizes the complex RNA polymerase I (Pol I) activator upstream binding (LaBaer et al., 1997; Cheng et al., 1999) and relieves factor (UBF) on two conserved threonine residues cyclin E–Cdk2 from Cdk inhibitor constraint, thereby located in HMG boxes 1 and 2. Mutation of the two facilitating activation of cyclin E–Cdk2 later in phosphorylation sites almost completely abolished the G1 phase. Once active, Cdk2 also triggers the degrada- ability of UBF to activate Pol I transcription. ERK1/ tion of p27 through the SCFSkp2 ubiquitin ligase ERK2 are also predicted to regulate the synthesis of (Cardozo and Pagano, 2004). Finally, Cdk2 phosphory- ribosomal RNA and other ribosomal components lates and inactivates the anaphase-promoting complex indirectly via activation of mTOR signaling (Mayer (APC)-activating subunit Cdh1, which targets a number and Grummt, 2006) and stabilization of c-Myc (see of S-phase-promoting factors for degradation (Peters, below). 2002; Ang and Harper, 2004). The inactivation of Rb- In addition to proteins, cell growth is associated with family proteins, Cdk inhibitors and Cdh1 creates the upregulation of de novo pyrimidine biosynthesis to positive feedback loops that reduce the cell’s depen- meet the increased demand in nucleotides (Evans and dency on mitogens and contribute to the irreversibility Guy, 2004). Pyrimidine nucleotides serve as essential of S-phase entry.

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3230 Induction of D-type cyclins and assembly of cyclin activity to the promoter (Yoshida et al., 2003). D–Cdk4 complexes Stimulation with mitogens induces the rapid phosphory- Various mechanisms have been proposed to explain how lation of Tob by ERK1/ERK2 on three serine residues, the ERK1/2 MAP kinase pathway regulates G1-phase canceling the ability of Tob to suppress cyclin D1 progression, although a complete picture is yet to expression by a mechanism that remains to be estab- emerge. One key target of the pathway is D-type cyclins. lished (Suzuki et al., 2002). Early studies showed that expression of activated Ras is The ERK1/2 pathway may also control the expression sufficient to induce the accumulation of cyclin D1 in of cyclin D1 at the post-transcriptional level. An different cell types (Filmus et al., 1994; Albanese et al., important fraction of cellular eIF4E is found within the 1995; Liu et al., 1995; Arber et al., 1996; Winston et al., nucleus, where it associates with nuclear bodies (reviewed 1996). Working downstream of Ras, activation of the in Strudwick and Borden, 2002). In this compartment, ERK1/2 pathway was found to be both necessary and eIF4E functions to promote the nucleo-cytoplasmic sufficient for transcriptional induction of the Cyclin D1 export of a subset of growth-associated mRNAs includ- gene (Albanese et al., 1995; Lavoie et al., 1996). As for ing cyclin D1 (Rousseau et al., 1996; Lai and Borden, entry into S phase, sustained activation of ERK1/ERK2 2000; Topisirovic et al., 2002) by a mechanism involving is required for the continued expression of cyclin D1 in an 100-nucleotide sequence in the 30 UTR of cyclin D1 G1 phase (Weber et al., 1997b; Balmanno and Cook, mRNA (Culjkovic et al., 2005). Mutation of Ser 209 or 1999; Roovers et al., 1999). In fibroblasts, sustained treatment with the synthetic inhibitor of Mnk1/Mnk2 ERK1/2 activity and the associated expression of cyclin CGP57380 markedly reduced the nuclear export of cyclin D1 depend on the synergistic action of mitogenic factors D1 mRNA by eIF4E, demonstrating the importance of and cell adhesion to the extracellular matrix (Roovers phosphorylation in this process (Topisirovic et al., 2004). et al., 1999). Overexpression of a constitutively active Incubation with CGP57380 also decreased cyclin D1 mutant of MEK1 is sufficient to overcome the adhesion protein levels. On the other hand, overexpression of requirement for expression of cyclin D1. The precise eIF4E had no significant effect on the polysomal mechanism and transcriptional regulatory elements that distribution of cyclin D1 mRNA (Rosenwald et al., connect ERK1/2 signaling to Cyclin D1 transcription 1995; Rousseau et al., 1996). These findings suggest a remain to be fully defined. The Cyclin D1 promoter role for the ERK1/2 pathway in the post-transcriptional contains a functional AP-1 binding site (Herber et al., regulation of cyclin D1 mRNA transport. 1994; Albanese et al., 1995) and one probable mechanism Although ERK1/2 signaling has been mainly asso- by which the ERK1/2 pathway impinges on Cyclin D1 ciated with the induction of cyclin D1, there are a few transcription is by inducing the expression of AP-1 reports implicating this pathway in the regulation of components. Activation of ERK1/ERK2 by mitogens or cyclin D2 (Dey et al., 2000; Piatelli et al., 2002) and conditionally active Raf-1 and MEK1 leads to the cyclin D3 (Tetsu and McCormick, 2003) expression. The elevated expression of individual Fos and Jun family generality of these observations and the molecular proteins (Hill and Treisman, 1995; Whitmarsh and mechanisms involved remain to be investigated. Davis, 1996; Balmanno and Cook, 1999; Cook et al., Expression of activated MEK1 not only drives the 1999; Treinies et al., 1999). Notably, the repertoire of accumulation of D-type cyclins but also facilitates the Fos and Jun proteins expressed in G1 is determined by assembly of cyclin D1–Cdk4 complexes in fibroblasts the duration of ERK1/2 activation (Balmanno and (Cheng et al., 1998). This effect might be mediated by Cook, 1999; Cook et al., 1999). Specifically, sustained downstream phosphorylation of the chaperone Hsc70, ERK1/2 signaling is required for the induced expression which stabilizes newly synthesized cyclin D1 and helps of Fra-1, Fra-2, c-Jun and JunB. Recent findings have assemble active cyclin D1–Cdk4 holoenzymes (Diehl provided a molecular explanation for how immediate- et al., 2003). Activation of MEK1 appears to be early gene products sense the duration of the ERK1/2 sufficient to trigger the association of cyclin D1 with signal in fibroblasts (Murphy et al., 2002, 2004). Many Hsc70 (Diehl et al., 2003). of these gene products including AP-1 family proteins contain functional docking site for ERK, FXFP (DEF) domains. Sustained activation of ERK1/ERK2 induces Stabilization of c-Myc the phosphorylation of AP-1 proteins at C-terminal c-Myc is a member of the Myc family of transcription residues, resulting in their stabilization and exposure of factors that plays a central role in regulating cell growth, the DEF domain to prime additional phosphorylation cell cycle progression and apoptosis (reviewed in events to further stabilize and activate the proteins. Pelengaris et al., 2002). c-Myc heterodimerizes with its Thus, a strong correlation exists between sustained obligate partner Max to activate or repress transcription ERK1/2 activity, AP-1 protein expression and accumu- of a vast array of target genes (Zeller et al., 2003; lation of cyclin D1 in G1 phase. ERK1/2 signaling also Adhikary and Eilers, 2005). Among the best-defined regulates Cyclin D1 transcription through phosphoryla- c-Myc targets that function directly in cell cycle control tion and inactivation of Tob (Maekawa et al., 2002; are cyclin D2 (Bouchard et al., 1999; Coller et al., 2000), Suzuki et al., 2002). Tob is a member of an emerging Cdk4 (Hermeking et al., 2000), p21 (Claassen and Hann, family of antiproliferative genes, which acts as a 2000; Coller et al., 2000) and Cdc25A (Galaktionov transcriptional co-repressor and negatively regulates et al., 1996; Zeller et al., 2003). In addition, c-Myc also Cyclin D1 transcription by recruiting histone deacetylase impacts profoundly on cell growth by activating genes

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3231 encoding ribosomal proteins and translation factors pathway (Pagano et al., 1995). p27 is phosphorylated on (Adhikary and Eilers, 2005), and by stimulating Thr 187 by cyclin E–Cdk2 in late G1 (Sheaff et al., 1997; transcription of ribosomal RNA genes by Pol I (Arabi Vlach et al., 1997) and is then recognized and targeted et al., 2005; Grandori et al., 2005). Activation of ERK1/ for ubiquitination by the SCFSkp2 E3 ligase in complex ERK2 markedly enhances c-Myc protein stability as a with the cofactor Cks1 (Carrano et al., 1999; Sutterluty result of direct phosphorylation of Ser 62 (Sears et al., et al., 1999; Tsvetkov et al., 1999; Ganoth et al., 2001; 2000). c-Myc contains a DEF domain that is required Spruck et al., 2001). The degradation of p27 at the G1/S for the full phosphorylation of Ser 62 in response to transition depends on the accumulation of cyclin E and growth factors (Murphy et al., 2004). It is noteworthy concomitant activation of Cdk2, events that are condi- that ERK1/2 signaling and induction of c-Myc are both tional on earlier activation of cyclin D–Cdk4/6 com- necessary to drive cells from G0 to late G1 phase (Jones plexes by the ERK1/2 pathway. However, there is and Kazlauskas, 2001). evidence that ERK1/2 signaling can trigger downregulation of p27 expression by a Cdk2- and Skp2-independent mechanism (Delmas et al., 2001; Mirza et al., 2004). Regulation of p21 and p27 expression Activation of the ERK1/2 pathway may also contribute Stimulation of cells with mitogenic factors induces a indirectly to p27 regulation through the synthesis of transient accumulation of the Cdk inhibitor p21 in early autocrine growth factors. G1 phase by a p53-independent mechanism (Li et al., et al et al 1994; Michieli ., 1994; Noda ., 1994; Sheikh Downregulation of antiproliferative genes et al., 1994; Macleod et al., 1995). This induction of p21 In addition to upregulating proliferation-associated is dependent on ERK1/2 signaling and is exerted, at least genes, activation of the ERK1/2 pathway also leads to in part, at the level of transcription (Liu et al., 1996; downregulation of antiproliferative genes. A recent gene Zezula et al., 1997). In contrast to the induction of cyclin profiling analysis has identified a set of 173 genes that are D1, a transient activation of ERK1/ERK2 is sufficient to downregulated by an ERK1/2-dependent mechanism induce the expression of p21 (Bottazzi et al., 1999). The during G1-phase progression (Yamamoto et al., 2006). transient accumulation of p21 likely contributes to the Importantly, overexpression experiments revealed that stabilization of cyclin D–Cdk4 complexes in G1 (LaBaer some of these genes clearly have the ability to inhibit et al., 1997; Cheng et al., 1999; Bagui et al., 2003). Thus fibroblast growth factor-stimulated DNA synthesis. The the ERK1/2 pathway may assist in both the assembly list of genes includes Tob1, Ddit3 and JunD, all of which and stabilization of cyclin D1–Cdk4/6 complexes. have been associated with the inhibition of cell prolifera- The Cdk inhibitor p27 acts as a primary negative tion. Continuous activation of ERK1/ERK2 throughout regulator of cell proliferation in a variety of cell types G1 is required to maintain decreased expression levels of (Nakayama and Nakayama, 1998). The levels of p27 are antiproliferative genes, identifying another mechanism high in quiescent cells and decline in response to that link the duration of ERK1/2 signaling to the mitogenic factor stimulation. Several studies have progression from G1 to S phase. suggested that the Ras–ERK1/2 signaling pathway is involved in the mitogen-induced downregulation of p27 (Kawada et al., 1997; Kerkhoff and Rapp, 1997; Woods et al., 1997; Greulich and Erikson, 1998; Rivard et al., Is ERK1/2 signaling important for G2/M progression and 1999; Treinies et al., 1999; Lenferink et al., 2000; Yang mitosis ofsomatic cells? et al., 2000; Delmas et al., 2001; Mirza et al., 2004; Gysin et al., 2005; Sakakibara et al., 2005). In contrast, other The ERK1/2 signaling pathway has also been implicated studies failed to document any significant change in in the regulation of the G2/M transition and mitosis in p27 levels following inhibition of ERK1/2 signaling somatic cells. However, the evidence in support of such a by synthetic MEK1/2 inhibitors or dominant-negative role has been controversial. Although some studies have ERK2, or after conditional activation of the pathway by reported that ERK1/ERK2 are phosphorylated/acti- activated Raf-1 or MEK1 (Sewing et al., 1997; Takuwa vated during G2/M (Tamemoto et al., 1992; Edelmann and Takuwa, 1997; Weber et al., 1997a; Cheng et al., et al., 1996; Wright et al., 1999; Roberts et al., 2002), 1998; Ladha et al., 1998; Chen et al., 1999; Tetsu and others concluded that the kinases are inactivated in McCormick, 2003). It was even reported that activated mitosis (Takenaka et al., 1998; Gomez-Cambronero, Raf-1 markedly increases p27 expression in small cell 1999; Hayne et al., 2000; Harding et al., 2003). These lung cancer cell lines (Ravi et al., 1998). These contra- results should, however, be interpreted with caution: dictory observations are difficult to reconcile with a first, in long-term cellular synchronization studies, the direct and general role of the ERK1/2 pathway in the synchrony of cells is not sufficient to measure accurately regulation of p27 by mitogenic factors. It cannot be the level of activation of ERK1/ERK2 in each stage of excluded, however, that this signaling pathway contri- the cell cycle; second, the use of microtubule-disrupting butes to p27 inactivation in specific cell types or contexts. agents to isolate mitotic cells does not reflect normal It is also unclear whether the involvement of ERK1/ progression into M phase. Immunofluorescence studies ERK2 is direct and if such regulation occurs in normal with phospho-specific antibodies also suggested that cells. One of the key mechanisms that regulates p27 ERK1/ERK2 and MEK1/MEK2 are active in mitosis abundance is proteolysis by the ubiquitin–proteasome and localized at kinetochores, spindle poles and midbody

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3232 (Shapiro et al., 1998; Zecevic et al., 1998; Willard and cellular outcome of this pathway (reviewed in Roovers Crouch, 2001; Liu et al., 2004). However, more recent and Assoian, 2000; Ebisuya et al., 2005). Several studies studies have shown that phospho-specific antibodies to have shown that strong activation of ERK1/ERK2 by MEK1/MEK2 strongly cross-react with phosphorylated conditionally active Ras or Raf causes cell cycle arrest in nucleophosmin, a multifunctional nuclear phosphopro- established cell lines by inducing the expression of the tein involved in centrosome duplication (Cha et al., 2004; Cdk inhibitor p21 (Pumiglia and Decker, 1997; Sewing Hayne et al., 2004). Also, the labeling of centromeric et al., 1997; Woods et al., 1997; Kerkhoff and Rapp, regions, centrosomes and midbodies observed with the 1998; Tombes et al., 1998). The persistent accumulation phospho-ERK1/2 antibody could not be blocked by of p21 has been associated with inhibition of Cdk4 and pretreatment with the MEK1/2 inhibitor U0126, Cdk2 enzymatic activities and the resulting arrest in G1. although the drug completely suppressed the weak Titration of activated Raf expression levels demon- phospho-ERK1/2 signal observed by immunoblot ana- strated that cells display bell-shaped dose–response lysis in mitotic cells (Shinohara et al., 2006). Thus, there curves; low Raf signal promotes cell cycle progression is no conclusive evidence that ERK1/ERK2 MAP and S-phase entry, whereas high Raf activity inhibits kinases are active in mitosis. DNA synthesis (Sewing et al., 1997; Woods et al., 1997). Inhibition of the ERK1/2 pathway by pharmacologi- These studies have led to a model that integrates the cal or genetic approaches was reported to delay the G2/ amplitude and duration of ERK1/2 activity with cell M progression of cells (Wright et al., 1999; Hayne et al., cycle progression (Roovers and Assoian, 2000). A 2000; Roberts et al., 2002; Liu et al., 2004; Knauf et al., robust early phase of ERK1/2 signaling followed by a 2006). However, the interpretation of these studies is moderate sustained phase leads to transient induction complicated by the fact that MEK1/MEK2 inhibitors of p21 and accumulation of cyclin D1, allowing G1 were added for long periods of time and it is therefore progression. However, a robust and prolonged activa- difficult to distinguish whether the delay in mitosis entry tion of ERK1/ERK2 causes G1 arrest due to long-term results from a direct effect of ERK1/ERK2 in late G2 or p21 induction and Cdk2 inhibition. It should be noted from an indirect consequence of earlier cell cycle defects. that high-intensity ERK1/2 signaling induces the The role of ERK1/2 signaling in the G2/M transition expression of the Cdk inhibitors p16Ink4a and p15Ink4b in and mitotic progression was revisited in a recent study certain cell lines, which can also contribute to cell cycle using time-lapse video microscopy in live cells to arrest (Malumbres et al., 2000; Han et al., 2005). precisely monitor the timing of these transitions The induction of cell cycle arrest by hyperactivation (Shinohara et al., 2006). The inhibitors of MEK1/ of the ERK1/2 pathway is not restricted to cell lines but MEK2 were added to cell cultures 15 min before is also observed in non-immortalized primary cells. recording the observations, which is enough to sup- Expression of activated forms of Ras, Raf or MEK1 was press ERK1/ERK2 activity completely. These studies shown to elicit cell cycle arrest in primary fibroblasts, revealed that ERK1/2 signaling is not required in late Schwann cells, hepatocytes, T lymphocytes, keratino- G2 for the timely entry into or exit from mitosis in both cytes, astrocytes and epithelial intestinal cells (Lloyd normal and transformed cells. No evidence for defect in et al., 1997; Serrano et al., 1997; Lin et al., 1998; Tombes centrosome duplication, bipolar spindle assembly, chro- et al., 1998; Zhu et al., 1998; Chen et al., 1999; Fanton mosome segregation or cytokinesis was observed when et al., 2001; Roper et al., 2001; Boucher et al., 2004). the ERK1/2 pathway was inactivated during late G2. Notably, the proliferation arrest observed in primary Using CENP-F staining as a marker for G2 progression, fibroblasts, astrocytes and epithelial intestinal cells is it was found that inhibition of ERK1/ERK2 activity permanent and phenotypically related to cellular senes- causes a transient delay in early–mid-G2, which cence (Serrano et al., 1997; Lin et al., 1998; Zhu et al., translates into a retardation of the mitotic index. 1998; Chen et al., 1999; Fanton et al., 2001; Boucher However, this delay is transient, and after 4–5 h the et al., 2004). ERK1/2-induced cell cycle arrest of normal cells resume cycling into mitosis. The molecular basis of cells is generally associated with upregulation of p53, this requirement in early- to mid-G2 is not known. p21 and p16Ink4a, although the repertoire of Cdk Together, these observations suggest that ERK1/2 inhibitors solicited varies depending on cell type and signaling exerts a modulatory role in early G2 for context. Interestingly, certain cells use two distinct and timely progression towards mitosis, but is dispensable redundant cell cycle inhibitory pathways. In normal after this time for mitosis to proceed. The ERK1/2 human astrocytes with an intact p16Ink4a/Rb pathway, pathway may also be involved in the G2 DNA damage activated Raf-1 induces an irreversible senescence-like checkpoint response (Abbott and Holt, 1999; Tang proliferation arrest associated with upregulation of et al., 2002; Yan et al., 2005). p16Ink4a expression (Fanton et al., 2001). However, when the p16Ink4a/Rb pathway is disrupted by expression of E7 oncoprotein in normal astrocytes or by genetic inactivation in high-grade glioma cell lines, Raf-1 activation Too strong ERK1/2 signaling leads to reversible or leads to reversible cell cycle arrest as a consequence of permanent cell cycle arrest p21 induction. It has been proposed that induction of Cdk inhibitors in response to abnormal mitogenic Not only the duration, but also the magnitude of the signaling and the resulting cell cycle arrest is a crucial ERK1/2 signal plays a key role in determining the final mechanism for protection against cancer development

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3233 (Serrano et al., 1997; Lloyd, 1998). The recent demon- A similar phenotype was observed in mice with deletion stration that ‘oncogene-induced senescence’ occurs of both Erk1 and Erk2 genes, although the penetrance in vivo and can be documented in pre-cancerous human of the defect was highly variable; some mice had very melanocytic naevi (expressing B-RafV600E) or mouse few DN4 cells, whereas others were only moderately lung adenomas (induced by K-RasV12) has provided affected. Immunoblot analysis of ERK2 levels in DN3 strong support for the physiological importance of this and DN4 populations revealed that ERK2 expression tumor suppression mechanism (Collado et al., 2005; was reduced but still detectable at the DN4 stage, Michaloglou et al., 2005). indicating that some cells have escaped Erk2 excision. These results suggest that ERK2 signaling is required for proliferation of thymocytes through the DN4 stage of development (Fischer et al., 2005). Role ofERK1 and ERK2: lessons fromknockout mice One important question that remains unanswered is whether ERK1 and ERK2 isoforms have unique roles in Further demonstration of the critical role of ERK1/ cell proliferation control or if they are used interchange- ERK2 MAP kinases in the regulation of cell prolifera- ably to reach a necessary threshold of ERK activity that tion was provided by gene disruption experiments. is specific to each cell type. ERK1 and ERK2 display Analysis of Erk1À/À mice revealed that ERK1 is not 83% amino-acid identity overall and it is generally essential for embryonic development and for normal assumed, although not rigorously tested, that the two growth or fertility (Pages et al., 1999). No difference in kinases phosphorylate common substrates. Interest- proliferation rate, mitogenic factor requirement, magni- ingly, a global analysis of protein phosphorylation in tude of DNA synthesis stimulation or timing of re-entry yeast using proteome chips has revealed that closely into the cell cycle from G0 could be documented in related kinases can recognize distinct sets of substrates ERK1-deficient embryonic fibroblasts as compared to (Ptacek et al., 2005). For example, the yeast protein wild-type cells. While the expression level of ERK2 was kinase A homologues Tpk1, Tpk2 and Tpk3, which are unchanged in ERK1À/À fibroblasts, the mitogen-stimu- highly related in sequence (from 67 to 84% amino-acid lated phosphorylation of ERK2 was more sustained in identity between isoforms), have unique substrate these cells, suggesting a possible compensatory effect. specificity and share only a few substrates. Analysis of On the other hand, loss of ERK1 resulted in a marked mice bearing inactivating mutations of Erk1 and Erk2 inhibition of DNA synthesis in thymocytes stimulated genes suggests that ERK1 and ERK2 have both by T-cell receptor crosslinking, despite the expression overlapping and unique cellular functions. The pheno- and activation of ERK2. Disruption of the Erk2 gene type of Erk2À/À mice observed in trophoblast cells and leads to early embryonic lethality (Hatano et al., 2003; thymocytes cannot be simply explained by the lack of Saba-El-Leil et al., 2003; Yao et al., 2003). Erk2À/À mice redundant expression of ERK1 (Saba-El-Leil et al., die in utero around day e7.5 likely owing to a defect in 2003; Fischer et al., 2005). In a mouse model of trophoblast development. Analysis of chimeric embryos myocardial infarction, loss of a single Erk2 allele was demonstrated that Erk2 functions cell autonomously in shown to increase cardiac cell death, providing genetic extra-embryonic lineages for the development of the evidence for a role of ERK1/2 signaling in cell survival ectoplacental cone and extra-embryonic ectoderm (Lips et al., 2004). Strikingly, complete inactivation of (Saba-El-Leil et al., 2003). No proliferation of polar Erk1 did not enhance infarction susceptibility despite trophectoderm cells is observed in Erk2 homozygous similar impact on total cardiac ERK1/2 enzymatic mutants. Given the primordial role of fibroblast growth activity. On the other hand, ERK1 deficiency was found factor 4 (FGF4) in promoting the proliferation of to decrease the incidence and progression of skin trophectoderm cells (Rossant and Cross, 2001) and the papillomas in a two-stage mouse skin carcinogenesis importance of ERK1/ERK2 MAP kinases in transdu- model using 7,12-dimethylbenz(a)anthracene as initiator cing signals from growth factor receptors, it has been and 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) as suggested that ERK2 functions downstream of FGF promoter (Bourcier et al., 2006). Keratinocytes isolated receptor 2 and is required to transduce the mitogenic from Erk1À/À mice showed a reduced proliferative signal of FGF4 in polar trophectoderm cells (Saba- response to mitogens as compared to wild-type cells. El-Leil et al., 2003). Interestingly, ERK1 is widely Intriguingly, it has been recently suggested that ERK1 expressed in early mouse embryos. The lack of acts as a negative regulator of cell proliferation in compensation of ERK2 deficiency by ERK1 raises the fibroblasts by restraining ERK2-dependent signaling intriguing possibility that ERK2 may play a specific role (Vantaggiato et al., 2006). This idea of a competition in the developing embryo. In a recent study, the role of model is based on the observations that inactivation of ERK1 and ERK2 in T-cell development was analysed ERK1 by either genetic disruption or RNAi silencing by crossing Erk1À/À mice to mice bearing a conditional increases the proliferation rate of fibroblasts, whereas allele of Erk2 (Fischer et al., 2005). No difference in knockdown of ERK2 expression dramatically inhibits thymic subpopulations was observed in mice deficient cell proliferation. Reciprocally, overexpression of only for ERK1. However, conditional deletion of Erk2 ERK1, but not ERK2, was found to attenuate the in the T-cell lineage resulted in a substantial increase in ability of activated Ras to transform NIH 3T3 cells the number of DN3 thymocytes, suggesting a partial (Vantaggiato et al., 2006). However, these findings are block in the maturation from DN3 to DN4 stage. discrepant with observations made by others. No

Oncogene ERK1/2 MAP kinases in cell cycle control S Meloche and J Pouysse´gur 3234 difference in the proliferation rate of ERK1-deficient addressed. Knock-in gene replacement strategies with embryonic fibroblasts was observed in a previous study complementation studies and chemical genetic ap- (Pages et al., 1999), whereas RNAi silencing of ERK1 proaches should help to determine unequivocally expression inhibited the proliferation of HeLa cells to whether ERK1 and ERK2 have evolved specific cellular the same extent as ERK2 silencing (Liu et al., 2004). functions. The systematic characterization of the phy- Physiologically, Erk1À/À mice do not grow faster than siological substrates of ERK1 and ERK2 will be critical their littermate controls in three different genetic back- to understanding how these kinases work at the cellular grounds (Pages et al., 1999; S Meloche, unpublished level. Historically, phosphorylation has been studied at observations). Additional work using unbiased genetic the level of single molecules, but new proteomic approaches will be required to clarify the individual strategies now allow the global characterization of the roles of ERK1 and ERK2. cell phosphoproteome. The Ras–ERK1/2 MAP kinase pathway is viewed as an attractive signaling pathway for the development of targeted cancer therapies (Sebolt- Concluding remarks Leopold and Herrera, 2004). A better understanding of this pathway is likely to have enormous repercussions The ERK1/2 MAP kinase pathway has emerged as a on drug development. central regulator of cell proliferation by controlling both cell growth and cell cycle progression. Major advances Acknowledgements have been made over the last 10 years in identifying the targets of this signaling pathway that are linked to the S Meloche holds a Canada Research Chair in Cellular cell-cycle control machinery. The importance of the Signaling. Work in the author’s laboratory was supported by amplitude and duration of the ERK1/2 signal in normal grants from the National Cancer Institute of Canada, Canadian Institutes for Health Research and Cancer Research cell proliferation and oncogene-induced senescence has Society to SM, and by the Centre National de la Recherche been highlighted in a series of elegant studies. However, Scientifique, Centre A Lacassagne, Ministe` re de l’Education, several important issues still remain unresolved. The de la Recherche et de la Technologie, Ligue Nationale Contre individual contribution of ERK1 and ERK2 isoforms le Cancer (Equipe labellise´ e) and Association pour la to these cellular processes has not been adequately Recherche sur le Cancer to JP.

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