The ERK1/2 mitogen-activated protein kinase pathway as a master
regulator of the G1- to S-phase transition
S Meloche1and J Pouysse ´ gur2
1Departments of Pharmacology and Molecular Biology, Institut de Recherche en Immunologie et Cance ´rologie, Universite ´ de
Montre ´al, Montreal, Quebec, Canada and2Institute 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
(ERK)1/2 mitogen-activated protein (MAP) kinase path-
way plays a central role in cell proliferation control. In
normal cells, sustained activation of ERK1/ERK2 is
necessary for G1- to S-phase progression and is associated
with induction of positive regulators of the cell cycle and
inactivation of antiproliferative genes. In cells expressing
activated Ras or Raf mutants, hyperactivation of the
ERK1/2 pathway elicits cell cycle arrest by inducing the
accumulation of cyclin-dependent kinase inhibitors. In this
review, we discuss the mechanisms by which activated
ERK1/ERK2 regulate growth and cell cycle progression
of mammalian somatic cells. We also highlight the
findings obtained from gene disruption studies.
Oncogene (2007) 26, 3227–3239. doi:10.1038/sj.onc.1210414
Keywords: signal transduction; MAP kinase; ERK1/2;
cell cycle; G1phase
The cell division cycle is the sequence of events by which
cells faithfully replicate their DNA and then segregate
the duplicated chromosomal DNA equally between two
daughter cells. An intricate network of regulatory
pathways ensures that each cell cycle event is performed
correctly and in proper sequence (Morgan, 2007). The
decision of cells to replicate their genetic material and
divide is made in G1 and is influenced by extracellular
signals (nutrients, mitogens, cytostatic factors, extra-
cellular matrix). Once cells make their decision, they are
irreversibly committed to complete the cycle indepen-
dently of mitogenic factors. One of the key signal
transduction pathways responsible for integrating these
environmental signals and relaying the information to
the cell cycle control system is the Ras-dependent
extracellular signal-regulated kinase 1 (ERK1)/2 mitogen-
activated protein (MAP) kinase pathway.
MAP kinase pathways are evolutionarily conserved
signaling modules by which cells transduce extracellular
signals into intracellular responses (reviewed in Lewis
et al., 1998; Widmann et al., 1999; Pearson et al., 2001).
The prototypical MAP kinase pathway is the ERK1/2
pathway, which is activated preferentially by mitogenic
factors, differentiation stimuli and cytokines. The basic
principles that govern the activation of this pathway
have been established during the 1990s and are relatively
well understood. Binding of ligands to their respective
cell surface receptors induces the activation of the small
GTPase Ras, which recruits the MAP kinase kinase
kinase Raf to the membrane for subsequent activation
by phosphorylation. Activated Raf isoforms phosphory-
late and activate the MAP kinase kinases MEK1/
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-
phorylate a vast array of substrates localized in all
cellular compartments (Lewis et al., 1998; Pearson et al.,
2001; Yoon and Seger, 2006). These include protein
kinases, signaling effectors, receptors, cytoskeletal pro-
teins and nuclear transcriptional regulators. This review
focuses on the importance of the ERK1/2 MAP kinase
pathway in cell proliferation control. We discuss our
current understanding of the mechanisms by which
activated ERK1/ERK2 regulate growth and cell cycle
progression of mammalian somatic cells.
Sustained activation of the ERK1/2 MAP kinase pathway
is necessary for cell proliferation
The first hint about the involvement of ERK1/ERK2
MAP kinases in cell proliferation control came from the
observation that the two kinases are rapidly phosphory-
lated and activated in response to mitogenic stimulation.
Indeed, ERK1 and ERK2 were originally identified as
mitogen-stimulated phosphoproteins of 41–45kDa after
separation by one- or two-dimensional gel electrophor-
esis (Nakamura et al., 1983; Cooper et al., 1984; Kohno,
Correspondence: Dr S Meloche, Departments of Pharmacology and
Molecular Biology, Institut de Recherche en Immunologie et
Cance ´ rologie Universite ´ de Montre ´ al, Montreal, Quebec, Canada
H3C 3J7 or Dr J Pouysse ´ gur, Institute of Signaling, Developmental
Biology and Cancer, CNRS UMR 6543, Universite ´ de Nice-Sofia
Antipolis, Centre A Lacassagne, Nice 06189, France.
E-mails: firstname.lastname@example.org or email@example.com
Oncogene (2007) 26, 3227–3239
& 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00
1985; Kohno and Pouyssegur, 1986). This pair of
tyrosine phosphorylated proteins was later shown to
correspond to the activated forms of two MAP-2/myelin
basic protein (MBP) kinases characterized previously
and referred to as p42mapk(ERK2) and p44mapk(ERK1)
(Rossomando et al., 1989, 1991). Subsequent studies
confirmed that ERK1/ERK2 are activated in response
to virtually all mitogenic factors in diverse cell types.
Detailed kinetic analysis revealed that mitogens induce a
biphasic activation of ERK1 and ERK2 in fibroblasts,
with a rapid and strong burst of kinase activity peaking
at 5–10min followed by a second wave of lower but
sustained activity that persists throughout the G1phase
(Kahan et al., 1992; Meloche et al., 1992; Meloche,
1995). A close correlation was observed between the
ability of extracellular factors to induce sustained
ERK1/2 activation and DNA synthesis (Kahan et al.,
1992; Meloche et al., 1992). For example, the potent
mitogen thrombin was shown to activate ERK1 with
biphasic kinetics in hamster lung fibroblasts. Rapid
inactivation of thrombin with the antagonist hirudin or
pretreatment of cells with pertussis toxin, both of which
abolish late-phase activation while preserving the early
peak of activity, resulted in complete inhibition of DNA
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),
allowing the generalization of the model. In a recent
study that systematically analyzed the impact of the
duration of ERK1/2 activity, it was concluded that
ERK1/2 activation must be sustained until late G1 for
successful S-phase entry (Yamamoto et al., 2006).
ERK1/ERK2 are rapidly inactivated at the G1/S
transition, and their activity is not required for cells to
enter Sphase (Meloche, 1995).
The first demonstration of the direct involvement of
ERK1/2 in the mitogenic response was provided by the
findings that overexpression of ERK1 catalytically
inactive mutants or antisense RNA inhibits the activa-
tion of endogenous ERK1/ERK2 and exerts a domi-
nant-negative effect on fibroblast cell proliferation
(Pages et al., 1993). The suppression of cell proliferation
by antisense ERK1 was fully reverted by co-transfection
of a sense ERK1 construct, confirming the specificity of
the effect. Later on, other studies showed that over-
expression of interfering mutants of MEK1 (Cowley
et al., 1994; Seger et al., 1994), incubation with ERK2
antisense oligonucleotide (Sale et al., 1995) or expression
of active MAP kinase phosphatase 1 (MKP-1/DUSP1)
(Sun et al., 1994; Brondello et al., 1995) inhibits the
induction of DNA synthesis by serum and activated
Ras. Similarly, treatment with synthetic MEK1/2
inhibitors, which prevents activation of ERK1/ERK2,
was reported to inhibit the proliferation of various cell
types, including fibroblasts, T lymphocytes, smooth
muscle cells, hepatocytes and epithelial cell lines (Dudley
et al., 1995; Seufferlein et al., 1996; Karpova et al., 1997;
DeSilva et al., 1998; Williams et al., 1998; Sebolt-
Leopold et al., 1999; Talarmin et al., 1999). Conversely,
expression of constitutively active forms of MEK1
(Brunet et al., 1994; Cowley et al., 1994; Seger et al.,
1994) relaxes growth factor dependency and enhances
the rate of cell proliferation.
Although ERK1/2 activation is necessary for G1
progression, it is nonetheless insufficient to drive cells
into Sphase (Cheng et al., 1998; Treinies et al., 1999;
Jones and Kazlauskas, 2001). Careful temporal dissec-
tion of the signaling events required for cells to move
from G0 through G1 and into Sphase has revealed
that accumulation of phosphatidylinositol-3-OH kinase
(PI(3)K) lipid products is important for late G1
progression (Jones and Kazlauskas, 2001). Some studies
have reported that overexpression of activated MEK1 in
quiescent fibroblasts is sufficient to cause S-phase entry
(Cowley et al., 1994; Gotoh et al., 1999; Treinies et al.,
1999). However, it was later shown that induction of
DNA synthesis can be blocked by incubation of cells
with the PI(3)K inhibitor LY294002, suggesting that
activation of the ERK1/2 pathway induces the synthesis
of autocrine growth factors (Treinies et al., 1999). The
addition of insulin to serum-free culture medium (Gotoh
et al., 1999) may also lead to confounding results, as
insulin is able to substitute for (PI(3)K) lipid products in
late G1 (Jones and Kazlauskas, 2001).
ERK1/2 signaling regulates cell growth
Cell growth is the process by which cells increase their
mass by upregulating the biosynthesis of macromole-
cules, membranes and organelles. In proliferating cells,
cell growth is coordinated with the cell division cycle to
ensure that on average each cell division is accompanied
by a doubling in mass in order to maintain cell size
(Jorgensen and Tyers, 2004). In animals, cell growth is
mainly controlled by extracellular growth factors (Con-
lon and Raff, 1999). The first evidence linking ERK1/2
signaling to cell growth control stemmed from the
finding that PD98059, a synthetic inhibitor of MEK1/
MEK2, blocks the stimulation of global protein syn-
thesis induced by a variety of growth factors acting
through tyrosine kinase receptors or G protein-coupled
receptors in vascular smooth muscle cells (Servant et al.,
1996). At the same time, it was reported that treatment
of cells with PD98059 drastically reduces insulin- and
serum-induced phosphorylation of the translation ini-
tiation factor eIF4E (Flynn and Proud, 1996; Morley
and McKendrick, 1997). These initial observations
suggested that the ERK1/2 pathway might play a role
in translational control. Phosphorylation of eIF4E on
Ser 209 is mediated by the MAP kinase-activated
protein kinase (MK) family members Mnk1/Mnk2,
which are directly phosphorylated and activated by the
MAP kinases ERK1/ERK2 and p38a/b in response to
growth factors or stress stimuli (Fukunaga and Hunter,
1997; Waskiewicz et al., 1997, 1999; Ueda et al., 2004).
The functional impact of eIF4E phosphorylation is not
clear and has been a subject of debate (Scheper and
Proud, 2002). The current view is that phosphorylation
of eIF4E does not lead to a global increase in mRNA
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
translation but rather enhances translation of specific
mRNAs with extensive 50UTR structure (Dever, 2002;
Mamane et al., 2004). In agreement with this idea, no
change in global protein synthesis was observed in
Mnk1/Mnk2-deficient embryonic fibroblasts, despite
complete elimination of eIF4E phosphorylation (Ueda
et al., 2004). So why does inhibition of ERK1/2 activity
has such a profound effect on general protein synthesis
(Servant et al., 1996)? One mechanism by which ERK1/
ERK2 may stimulate global mRNA translation is
through enhancement of mammalian target of rapamy-
cin (mTOR) signaling. mTOR is a master growth
regulator that integrates signals from growth factors,
nutrients and cellular energy status to modulate ribo-
some biogenesis and translation initiation (see Fingar
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
activating protein (GAP) for the small GTPase Rheb
(Manning and Cantley, 2003; Corradetti and Guan,
2006). Rheb binds directly to the mTOR complex 1
(mTORC1) and stimulates mTOR kinase activity in a
GTP-dependent manner. Two groups recently reported
that activation of the ERK1/2 pathway functionally
inactivates the TSC1/TSC2 complex, as a result of the
phosphorylation of TSC2 (Roux et al., 2004; Ma et al.,
2005). One study showed that the serine/threonine
kinase RSK1, a downstream substrate of ERK1/
ERK2, phosphorylates TSC2 in the conserved C-
terminus of the protein (Ser 1798) to impair its
antagonistic action on mTOR signaling (Roux et al.,
2004). The other study proposed that ERK1/ERK2
directly phosphorylate TSC2 on Ser 664 and inhibit its
function by disrupting TSC1/TSC2 complex formation
(Ma et al., 2005). Thus, the ERK1/2 and PI(3)K
pathways cooperate to activate mTOR and promote
Another mechanism by which ERK1/ERK2 may
impact on global protein synthesis is through direct
regulation of ribosomal gene transcription. Activation
of the ERK1/2 pathway by growth factors or con-
ditionally active Raf-1 induces an immediate increase in
ribosomal RNA transcription, whereas inactivation of
the pathway by MEK1/2 inhibitors suppresses basal and
stimulated ribosomal transcription (Stefanovsky et al.,
2001). ERK2 was found to phosphorylate directly the
RNA polymerase I (Pol I) activator upstream binding
factor (UBF) on two conserved threonine residues
located in HMG boxes 1 and 2. Mutation of the two
phosphorylation sites almost completely abolished the
ability of UBF to activate Pol I transcription. ERK1/
ERK2 are also predicted to regulate the synthesis of
ribosomal RNA and other ribosomal components
indirectly via activation of mTOR signaling (Mayer
and Grummt, 2006) and stabilization of c-Myc (see
In addition to proteins, cell growth is associated with
the upregulation of de novo pyrimidine biosynthesis to
meet the increased demand in nucleotides (Evans and
Guy, 2004). Pyrimidine nucleotides serve as essential
precursors for the synthesis of RNA and DNA,
phospholipids, UDP-sugars and glycogen. The rate-
limiting step in the pyrimidine pathway is catalysed by
the carbamoyl-phosphate synthetase (CPSase) enzyme,
which is part of the large multifunctional protein CAD
(Evans and Guy, 2004). Mitogenic stimulation of cells
induces the phosphorylation of CAD and allosterically
activates the CPSase domain by an MEK1/2-dependent
mechanism (Graves et al., 2000). ERK2 directly
phosphorylates CAD on Thr 456 in vitro, and alanine
substitution of this residue abolishes the allosteric
regulation of the enzyme. Furthermore, epidermal
growth factor-stimulated synthesis of UTP is blocked
by incubation with PD98059, consistent with a role of
the ERK1/2 pathway in pyrimidine biosynthesis.
ERK1/2 signaling promotes G1 progression by
G1- to S-phase progression: a brief overview
The regulation of G1- to S-phase transition is governed
by the concerted action of cyclin-dependent kinases
(Cdks) and their regulatory cyclin subunits (reviewed
in Sherr, 2000; Malumbres and Barbacid, 2001). In
response to mitogenic factors, D-type cyclins progres-
sively accumulate during G1phase and assemble with
their catalytic partners Cdk4 and Cdk6. The activity of
Cdk4/6 increases as cells approach the G1/S boundary,
and persist through the first and subsequent cycles as
long as mitogens are present. A major function of cyclin
D-dependent kinases is to phosphorylate members of
the retinoblastoma (Rb) family of proteins. Rb phos-
phorylation disrupts its association with bound E2F
family members, allowing the coordinated transcription
of genes required for DNA replication (Weinberg, 1995;
Harbour and Dean, 2000; Bracken et al., 2004). Among
the E2F-regulated genes are Cyclin E, Cyclin A2 and
Emi1, which are key effectors of the G1/S transition in
normal cells (Hsu et al., 2002; Cobrinik, 2005). Cyclin E
associates with Cdk2 to enforce Rb phosphorylation
and complete its inactivation. A second non-catalytic
function of cyclin D–Cdk4/6 complexes is to sequestrate
the Cdk inhibitors p27Kip1(p27) and p21Cip1(p21) (Geng
et al., 2001; Tong and Pollard, 2001). The binding of
p21/p27 to cyclin D–Cdk4/6 stabilizes the complex
(LaBaer et al., 1997; Cheng et al., 1999) and relieves
cyclin E–Cdk2 from Cdk inhibitor constraint, thereby
G1phase. Once active, Cdk2 also triggers the degrada-
tion of p27 through the SCFSkp2ubiquitin ligase
(Cardozo and Pagano, 2004). Finally, Cdk2 phosphory-
lates and inactivates the anaphase-promoting complex
(APC)-activating subunit Cdh1, which targets a number
of S-phase-promoting factors for degradation (Peters,
2002; Ang and Harper, 2004). The inactivation of Rb-
family proteins, Cdk inhibitors and Cdh1 creates
positive feedback loops that reduce the cell’s depen-
dency on mitogens and contribute to the irreversibility
of S-phase entry.
cyclin E–Cdk2 laterin
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
Induction of D-type cyclins and assembly of cyclin
Various mechanisms have been proposed to explain how
the ERK1/2 MAP kinase pathway regulates G1-phase
progression, although a complete picture is yet to
emerge. One key target of the pathway is D-type cyclins.
Early studies showed that expression of activated Ras is
sufficient to induce the accumulation of cyclin D1 in
different cell types (Filmus et al., 1994; Albanese et al.,
1995; Liu et al., 1995; Arber et al., 1996; Winston et al.,
1996). Working downstream of Ras, activation of the
ERK1/2 pathway was found to be both necessary and
sufficient for transcriptional induction of the Cyclin D1
gene (Albanese et al., 1995; Lavoie et al., 1996). As for
entry into Sphase, sustained activation of ERK1/ERK2
is required for the continued expression of cyclin D1 in
G1phase (Weber et al., 1997b; Balmanno and Cook,
1999; Roovers et al., 1999). In fibroblasts, sustained
ERK1/2 activity and the associated expression of cyclin
D1 depend on the synergistic action of mitogenic factors
and cell adhesion to the extracellular matrix (Roovers
et al., 1999). Overexpression of a constitutively active
mutant of MEK1 is sufficient to overcome the adhesion
requirement for expression of cyclin D1. The precise
mechanism and transcriptional regulatory elements that
connect ERK1/2 signaling to Cyclin D1 transcription
remain to be fully defined. The Cyclin D1 promoter
contains a functional AP-1 binding site (Herber et al.,
1994; Albanese et al., 1995) and one probable mechanism
by which the ERK1/2 pathway impinges on Cyclin D1
transcription is by inducing the expression of AP-1
components. Activation of ERK1/ERK2 by mitogens or
conditionally active Raf-1 and MEK1 leads to the
elevated expression of individual Fos and Jun family
proteins (Hill and Treisman, 1995; Whitmarsh and
Davis, 1996; Balmanno and Cook, 1999; Cook et al.,
1999; Treinies et al., 1999). Notably, the repertoire of
Fos and Jun proteins expressed in G1 is determined by
the duration of ERK1/2 activation (Balmanno and
Cook, 1999; Cook et al., 1999). Specifically, sustained
ERK1/2 signaling is required for the induced expression
of Fra-1, Fra-2, c-Jun and JunB. Recent findings have
provided a molecular explanation for how immediate-
early gene products sense the duration of the ERK1/2
signal in fibroblasts (Murphy et al., 2002, 2004). Many
of these gene products including AP-1 family proteins
contain functional docking site for ERK, FXFP (DEF)
domains. Sustained activation of ERK1/ERK2 induces
the phosphorylation of AP-1 proteins at C-terminal
residues, resulting in their stabilization and exposure of
the DEF domain to prime additional phosphorylation
events to further stabilize and activate the proteins.
Thus, a strong correlation exists between sustained
ERK1/2 activity, AP-1 protein expression and accumu-
lation of cyclin D1 in G1phase. ERK1/2 signaling also
regulates Cyclin D1 transcription through phosphoryla-
tion and inactivation of Tob (Maekawa et al., 2002;
Suzuki et al., 2002). Tob is a member of an emerging
family of antiproliferative genes, which acts as a
transcriptional co-repressor and negatively regulates
Cyclin D1 transcription by recruiting histone deacetylase
activity to the promoter (Yoshida et al., 2003).
Stimulation with mitogens induces the rapid phosphory-
lation of Tob by ERK1/ERK2 on three serine residues,
canceling the ability of Tob to suppress cyclin D1
expression by a mechanism that remains to be estab-
lished (Suzuki et al., 2002).
The ERK1/2 pathway may also control the expression
of cyclin D1 at the post-transcriptional level. An
important fraction of cellular eIF4E is found within the
nucleus, where it associates with nuclear bodies (reviewed
in Strudwick and Borden, 2002). In this compartment,
eIF4E functions to promote the nucleo-cytoplasmic
export of a subset of growth-associated mRNAs includ-
ing cyclin D1 (Rousseau et al., 1996; Lai and Borden,
2000; Topisirovic et al., 2002) by a mechanism involving
an 100-nucleotide sequence in the 30UTR of cyclin D1
mRNA (Culjkovic et al., 2005). Mutation of Ser 209 or
treatment with the synthetic inhibitor of Mnk1/Mnk2
CGP57380 markedly reduced the nuclear export of cyclin
D1 mRNA by eIF4E, demonstrating the importance of
phosphorylation in this process (Topisirovic et al., 2004).
Incubation with CGP57380 also decreased cyclin D1
protein levels. On the other hand, overexpression of
eIF4E had no significant effect on the polysomal
distribution of cyclin D1 mRNA (Rosenwald et al.,
1995; Rousseau et al., 1996). These findings suggest a
role for the ERK1/2 pathway in the post-transcriptional
regulation of cyclin D1 mRNA transport.
Although ERK1/2 signaling has been mainly asso-
ciated with the induction of cyclin D1, there are a few
reports implicating this pathway in the regulation of
cyclin D2 (Dey et al., 2000; Piatelli et al., 2002) and
cyclin D3 (Tetsu and McCormick, 2003) expression. The
generality of these observations and the molecular
mechanisms involved remain to be investigated.
Expression of activated MEK1 not only drives the
accumulation of D-type cyclins but also facilitates the
assembly of cyclin D1–Cdk4 complexes in fibroblasts
(Cheng et al., 1998). This effect might be mediated by
downstream phosphorylation of the chaperone Hsc70,
which stabilizes newly synthesized cyclin D1 and helps
assemble active cyclin D1–Cdk4 holoenzymes (Diehl
et al., 2003). Activation of MEK1 appears to be
sufficient to trigger the association of cyclin D1 with
Hsc70 (Diehl et al., 2003).
Stabilization of c-Myc
c-Myc is a member of the Myc family of transcription
factors that plays a central role in regulating cell growth,
cell cycle progression and apoptosis (reviewed in
Pelengaris et al., 2002). c-Myc heterodimerizes with its
obligate partner Max to activate or repress transcription
of a vast array of target genes (Zeller et al., 2003;
Adhikary and Eilers, 2005). Among the best-defined
c-Myc targets that function directly in cell cycle control
are cyclin D2 (Bouchard et al., 1999; Coller et al., 2000),
Cdk4 (Hermeking et al., 2000), p21 (Claassen and Hann,
2000; Coller et al., 2000) and Cdc25A (Galaktionov
et al., 1996; Zeller et al., 2003). In addition, c-Myc also
impacts profoundly on cell growth by activating genes
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
encoding ribosomal proteins and translation factors
(Adhikary and Eilers, 2005), and by stimulating
transcription of ribosomal RNA genes by Pol I (Arabi
et al., 2005; Grandori et al., 2005). Activation of ERK1/
ERK2 markedly enhances c-Myc protein stability as a
result of direct phosphorylation of Ser 62 (Sears et al.,
2000). c-Myc contains a DEF domain that is required
for the full phosphorylation of Ser 62 in response to
growth factors (Murphy et al., 2004). It is noteworthy
that ERK1/2 signaling and induction of c-Myc are both
necessary to drive cells from G0 to late G1phase (Jones
and Kazlauskas, 2001).
Regulation of p21 and p27 expression
Stimulation of cells with mitogenic factors induces a
transient accumulation of the Cdk inhibitor p21 in early
G1phase by a p53-independent mechanism (Li et al.,
1994; Michieli et al., 1994; Noda et al., 1994; Sheikh
et al., 1994; Macleod et al., 1995). This induction of p21
is dependent on ERK1/2 signaling and is exerted, at least
in part, at the level of transcription (Liu et al., 1996;
Zezula et al., 1997). In contrast to the induction of cyclin
D1, a transient activation of ERK1/ERK2 is sufficient to
induce the expression of p21 (Bottazzi et al., 1999). The
transient accumulation of p21 likely contributes to the
stabilization of cyclin D–Cdk4 complexes in G1 (LaBaer
et al., 1997; Cheng et al., 1999; Bagui et al., 2003). Thus
the ERK1/2 pathway may assist in both the assembly
and stabilization of cyclin D1–Cdk4/6 complexes.
The Cdk inhibitor p27 acts as a primary negative
regulator of cell proliferation in a variety of cell types
(Nakayama and Nakayama, 1998). The levels of p27 are
high in quiescent cells and decline in response to
mitogenic factor stimulation. Several studies have
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.,
1999; Treinies et al., 1999; Lenferink et al., 2000; Yang
et al., 2000; Delmas et al., 2001; Mirza et al., 2004; Gysin
et al., 2005; Sakakibara et al., 2005). In contrast, other
studies failed to document any significant change in
p27 levels following inhibition of ERK1/2 signaling
by synthetic MEK1/2 inhibitors or dominant-negative
ERK2, or after conditional activation of the pathway by
activated Raf-1 or MEK1 (Sewing et al., 1997; Takuwa
and Takuwa, 1997; Weber et al., 1997a; Cheng et al.,
1998; Ladha et al., 1998; Chen et al., 1999; Tetsu and
McCormick, 2003). It was even reported that activated
Raf-1 markedly increases p27 expression in small cell
lung cancer cell lines (Ravi et al., 1998). These contra-
dictory observations are difficult to reconcile with a
direct and general role of the ERK1/2 pathway in the
regulation of p27 by mitogenic factors. It cannot be
excluded, however, that this signaling pathway contri-
butes to p27 inactivation in specific cell types or contexts.
It is also unclear whether the involvement of ERK1/
ERK2 is direct and if such regulation occurs in normal
cells. One of the key mechanisms that regulates p27
abundance is proteolysis by the ubiquitin–proteasome
pathway (Pagano et al., 1995). p27 is phosphorylated on
Thr 187 by cyclin E–Cdk2 in late G1 (Sheaff et al., 1997;
Vlach et al., 1997) and is then recognized and targeted
for ubiquitination by the SCFSkp2E3 ligase in complex
with the cofactor Cks1 (Carrano et al., 1999; Sutterluty
et al., 1999; Tsvetkov et al., 1999; Ganoth et al., 2001;
Spruck et al., 2001). The degradation of p27 at the G1/S
transition depends on the accumulation of cyclin E and
concomitant activation of Cdk2, events that are condi-
tional on earlier activation of cyclin D–Cdk4/6 com-
plexes by the ERK1/2 pathway. However, there is
evidence that ERK1/2 signaling can trigger downregula-
tion of p27 expression by a Cdk2- and Skp2-independent
mechanism (Delmas et al., 2001; Mirza et al., 2004).
Activation of the ERK1/2 pathway may also contribute
indirectly to p27 regulation through the synthesis of
autocrine growth factors.
Downregulation of antiproliferative genes
In addition to upregulating proliferation-associated
genes, activation of the ERK1/2 pathway also leads to
downregulation of antiproliferative genes. A recent gene
profiling analysis has identified a set of 173 genes that are
downregulated by an ERK1/2-dependent mechanism
during G1-phase progression (Yamamoto et al., 2006).
Importantly, overexpression experiments revealed that
some of these genes clearly have the ability to inhibit
fibroblast growth factor-stimulated DNA synthesis. The
list of genes includes Tob1, Ddit3 and JunD, all of which
have been associated with the inhibition of cell prolifera-
tion. Continuous activation of ERK1/ERK2 throughout
G1 is required to maintain decreased expression levels of
antiproliferative genes, identifying another mechanism
that link the duration of ERK1/2 signaling to the
progression from G1 to Sphase.
Is ERK1/2 signaling important for G2/M progression and
mitosis of somatic cells?
The ERK1/2 signaling pathway has also been implicated
in the regulation of the G2/M transition and mitosis in
somatic cells. However, the evidence in support of such a
role has been controversial. Although some studies have
reported that ERK1/ERK2 are phosphorylated/acti-
vated during G2/M (Tamemoto et al., 1992; Edelmann
et al., 1996; Wright et al., 1999; Roberts et al., 2002),
others concluded that the kinases are inactivated in
mitosis (Takenaka et al., 1998; Gomez-Cambronero,
1999; Hayne et al., 2000; Harding et al., 2003). These
results should, however, be interpreted with caution:
first, in long-term cellular synchronization studies, the
synchrony of cells is not sufficient to measure accurately
the level of activation of ERK1/ERK2 in each stage of
the cell cycle; second, the use of microtubule-disrupting
agents to isolate mitotic cells does not reflect normal
progression into Mphase. Immunofluorescence studies
with phospho-specific antibodies also suggested that
ERK1/ERK2 and MEK1/MEK2 are active in mitosis
and localized at kinetochores, spindle poles and midbody
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
(Shapiro et al., 1998; Zecevic et al., 1998; Willard and
Crouch, 2001; Liu et al., 2004). However, more recent
studies have shown that phospho-specific antibodies to
MEK1/MEK2 strongly cross-react with phosphorylated
nucleophosmin, a multifunctional nuclear phosphopro-
tein involved in centrosome duplication (Cha et al., 2004;
Hayne et al., 2004). Also, the labeling of centromeric
regions, centrosomes and midbodies observed with the
phospho-ERK1/2 antibody could not be blocked by
although the drug completely suppressed the weak
phospho-ERK1/2 signal observed by immunoblot ana-
lysis in mitotic cells (Shinohara et al., 2006). Thus, there
is no conclusive evidence that ERK1/ERK2 MAP
kinases are active in mitosis.
Inhibition of the ERK1/2 pathway by pharmacologi-
cal or genetic approaches was reported to delay the G2/
M progression of cells (Wright et al., 1999; Hayne et al.,
2000; Roberts et al., 2002; Liu et al., 2004; Knauf et al.,
2006). However, the interpretation of these studies is
complicated by the fact that MEK1/MEK2 inhibitors
were added for long periods of time and it is therefore
difficult to distinguish whether the delay in mitosis entry
results from a direct effect of ERK1/ERK2 in late G2 or
from an indirect consequence of earlier cell cycle defects.
The role of ERK1/2 signaling in the G2/M transition
and mitotic progression was revisited in a recent study
using time-lapse video microscopy in live cells to
precisely monitor the timing of these transitions
(Shinohara et al., 2006). The inhibitors of MEK1/
MEK2 were added to cell cultures 15min before
recording the observations, which is enough to sup-
press ERK1/ERK2 activity completely. These studies
revealed that ERK1/2 signaling is not required in late
G2 for the timely entry into or exit from mitosis in both
normal and transformed cells. No evidence for defect in
centrosome duplication, bipolar spindle assembly, chro-
mosome segregation or cytokinesis was observed when
the ERK1/2 pathway was inactivated during late G2.
Using CENP-F staining as a marker for G2 progression,
it was found that inhibition of ERK1/ERK2 activity
causes a transient delay in early–mid-G2, which
translates into a retardation of the mitotic index.
However, this delay is transient, and after 4–5h the
cells resume cycling into mitosis. The molecular basis of
this requirement in early- to mid-G2 is not known.
Together, these observations suggest that ERK1/2
signaling exerts a modulatory role in early G2 for
timely progression towards mitosis, but is dispensable
after this time for mitosis to proceed. The ERK1/2
pathway may also be involved in the G2 DNA damage
checkpoint response (Abbott and Holt, 1999; Tang
et al., 2002; Yan et al., 2005).
Too strong ERK1/2 signaling leads to reversible or
permanent cell cycle arrest
Not only the duration, but also the magnitude of the
ERK1/2 signal plays a key role in determining the final
cellular outcome of this pathway (reviewed in Roovers
and Assoian, 2000; Ebisuya et al., 2005). Several studies
have shown that strong activation of ERK1/ERK2 by
conditionally active Ras or Raf causes cell cycle arrest in
established cell lines by inducing the expression of the
Cdk inhibitor p21 (Pumiglia and Decker, 1997; Sewing
et al., 1997; Woods et al., 1997; Kerkhoff and Rapp,
1998; Tombes et al., 1998). The persistent accumulation
of p21 has been associated with inhibition of Cdk4 and
Cdk2 enzymatic activities and the resulting arrest in G1.
Titration of activated Raf expression levels demon-
strated that cells display bell-shaped dose–response
curves; low Raf signal promotes cell cycle progression
and S-phase entry, whereas high Raf activity inhibits
DNA synthesis (Sewing et al., 1997; Woods et al., 1997).
These studies have led to a model that integrates the
amplitude and duration of ERK1/2 activity with cell
cycle progression (Roovers and Assoian, 2000). A
robust early phase of ERK1/2 signaling followed by a
moderate sustained phase leads to transient induction
of p21 and accumulation of cyclin D1, allowing G1
progression. However, a robust and prolonged activa-
tion of ERK1/ERK2 causes G1 arrest due to long-term
p21 induction and Cdk2 inhibition. It should be noted
that high-intensityERK1/2 signaling
expression of the Cdk inhibitors p16Ink4aand p15Ink4bin
certain cell lines, which can also contribute to cell cycle
arrest (Malumbres et al., 2000; Han et al., 2005).
The induction of cell cycle arrest by hyperactivation
of the ERK1/2 pathway is not restricted to cell lines but
is also observed in non-immortalized primary cells.
Expression of activated forms of Ras, Raf or MEK1 was
shown to elicit cell cycle arrest in primary fibroblasts,
Schwann cells, hepatocytes, T lymphocytes, keratino-
cytes, astrocytes and epithelial intestinal cells (Lloyd
et al., 1997; Serrano et al., 1997; Lin et al., 1998; Tombes
et al., 1998; Zhu et al., 1998; Chen et al., 1999; Fanton
et al., 2001; Roper et al., 2001; Boucher et al., 2004).
Notably, the proliferation arrest observed in primary
fibroblasts, astrocytes and epithelial intestinal cells is
permanent and phenotypically related to cellular senes-
cence (Serrano et al., 1997; Lin et al., 1998; Zhu et al.,
1998; Chen et al., 1999; Fanton et al., 2001; Boucher
et al., 2004). ERK1/2-induced cell cycle arrest of normal
cells is generally associated with upregulation of p53,
p21 and p16Ink4a, although the repertoire of Cdk
inhibitors solicited varies depending on cell type and
context. Interestingly, certain cells use two distinct and
redundant cell cycle inhibitory pathways. In normal
human astrocytes with an intact p16Ink4a/Rb pathway,
activated Raf-1 induces an irreversible senescence-like
proliferation arrest associated with upregulation of
p16Ink4aexpression (Fanton et al., 2001). However, when
the p16Ink4a/Rb pathway is disrupted by expression of E7
oncoprotein in normal astrocytes or by genetic inactiva-
tion in high-grade glioma cell lines, Raf-1 activation
leads to reversible cell cycle arrest as a consequence of
p21 induction. It has been proposed that induction of
Cdk inhibitors in response to abnormal mitogenic
signaling and the resulting cell cycle arrest is a crucial
mechanism for protection against cancer development
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
(Serrano et al., 1997; Lloyd, 1998). The recent demon-
stration that ‘oncogene-induced senescence’ occurs
in vivo and can be documented in pre-cancerous human
melanocytic naevi (expressing B-RafV600E) or mouse
lung adenomas (induced by K-RasV12) has provided
strong support for the physiological importance of this
tumor suppression mechanism (Collado et al., 2005;
Michaloglou et al., 2005).
Role of ERK1 and ERK2: lessons from knockout mice
Further demonstration of the critical role of ERK1/
ERK2 MAP kinases in the regulation of cell prolifera-
tion was provided by gene disruption experiments.
Analysis of Erk1?/?mice revealed that ERK1 is not
essential for embryonic development and for normal
growth or fertility (Pages et al., 1999). No difference in
proliferation rate, mitogenic factor requirement, magni-
tude of DNA synthesis stimulation or timing of re-entry
into the cell cycle from G0 could be documented in
ERK1-deficient embryonic fibroblasts as compared to
wild-type cells. While the expression level of ERK2 was
unchanged in ERK1?/?fibroblasts, the mitogen-stimu-
lated phosphorylation of ERK2 was more sustained in
these cells, suggesting a possible compensatory effect.
On the other hand, loss of ERK1 resulted in a marked
inhibition of DNA synthesis in thymocytes stimulated
by T-cell receptor crosslinking, despite the expression
and activation of ERK2. Disruption of the Erk2 gene
leads to early embryonic lethality (Hatano et al., 2003;
Saba-El-Leil et al., 2003; Yao et al., 2003). Erk2?/?mice
die in utero around day e7.5 likely owing to a defect in
trophoblast development. Analysis of chimeric embryos
demonstrated that Erk2 functions cell autonomously in
extra-embryonic lineages for the development of the
(Saba-El-Leil et al., 2003). No proliferation of polar
trophectoderm cells is observed in Erk2 homozygous
mutants. Given the primordial role of fibroblast growth
factor 4 (FGF4) in promoting the proliferation of
trophectoderm cells (Rossant and Cross, 2001) and the
importance of ERK1/ERK2 MAP kinases in transdu-
cing signals from growth factor receptors, it has been
suggested that ERK2 functions downstream of FGF
receptor 2 and is required to transduce the mitogenic
signal of FGF4 in polar trophectoderm cells (Saba-
El-Leil et al., 2003). Interestingly, ERK1 is widely
expressed in early mouse embryos. The lack of
compensation of ERK2 deficiency by ERK1 raises the
intriguing possibility that ERK2 may play a specific role
in the developing embryo. In a recent study, the role of
ERK1 and ERK2 in T-cell development was analysed
by crossing Erk1?/?mice to mice bearing a conditional
allele of Erk2 (Fischer et al., 2005). No difference in
thymic subpopulations was observed in mice deficient
only for ERK1. However, conditional deletion of Erk2
in the T-cell lineage resulted in a substantial increase in
the number of DN3 thymocytes, suggesting a partial
block in the maturation from DN3 to DN4 stage.
A similar phenotype was observed in mice with deletion
of both Erk1 and Erk2 genes, although the penetrance
of the defect was highly variable; some mice had very
few DN4 cells, whereas others were only moderately
affected. Immunoblot analysis of ERK2 levels in DN3
and DN4 populations revealed that ERK2 expression
was reduced but still detectable at the DN4 stage,
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).
One important question that remains unanswered is
whether ERK1 and ERK2 isoforms have unique roles in
cell proliferation control or if they are used interchange-
ably to reach a necessary threshold of ERK activity that
is specific to each cell type. ERK1 and ERK2 display
83% amino-acid identity overall and it is generally
assumed, although not rigorously tested, that the two
kinases phosphorylate common substrates. Interest-
ingly, a global analysis of protein phosphorylation in
yeast using proteome chips has revealed that closely
related kinases can recognize distinct sets of substrates
(Ptacek et al., 2005). For example, the yeast protein
kinase A homologues Tpk1, Tpk2 and Tpk3, which are
highly related in sequence (from 67 to 84% amino-acid
identity between isoforms), have unique substrate
specificity and share only a few substrates. Analysis of
mice bearing inactivating mutations of Erk1 and Erk2
genes suggests that ERK1 and ERK2 have both
overlapping and unique cellular functions. The pheno-
type of Erk2?/?mice observed in trophoblast cells and
thymocytes cannot be simply explained by the lack of
redundant expression of ERK1 (Saba-El-Leil et al.,
2003; Fischer et al., 2005). In a mouse model of
myocardial infarction, loss of a single Erk2 allele was
shown to increase cardiac cell death, providing genetic
evidence for a role of ERK1/2 signaling in cell survival
(Lips et al., 2004). Strikingly, complete inactivation of
Erk1 did not enhance infarction susceptibility despite
similar impact on total cardiac ERK1/2 enzymatic
activity. On the other hand, ERK1 deficiency was found
to decrease the incidence and progression of skin
papillomas in a two-stage mouse skin carcinogenesis
model using 7,12-dimethylbenz(a)anthracene as initiator
and 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) as
promoter (Bourcier et al., 2006). Keratinocytes isolated
from Erk1?/?mice showed a reduced proliferative
response to mitogens as compared to wild-type cells.
Intriguingly, it has been recently suggested that ERK1
acts as a negative regulator of cell proliferation in
fibroblasts by restraining ERK2-dependent signaling
(Vantaggiato et al., 2006). This idea of a competition
model is based on the observations that inactivation of
ERK1 by either genetic disruption or RNAi silencing
increases the proliferation rate of fibroblasts, whereas
knockdown of ERK2 expression dramatically inhibits
ERK1, but not ERK2, was found to attenuate the
ability of activated Ras to transform NIH 3T3 cells
(Vantaggiato et al., 2006). However, these findings are
discrepant with observations made by others. No
ERK1/2 MAP kinases in cell cycle control
S Meloche and J Pouysse ´gur
difference in the proliferation rate of ERK1-deficient
embryonic fibroblasts was observed in a previous study
(Pages et al., 1999), whereas RNAi silencing of ERK1
expression inhibited the proliferation of HeLa cells to
the same extent as ERK2 silencing (Liu et al., 2004).
Physiologically, Erk1?/?mice do not grow faster than
their littermate controls in three different genetic back-
grounds (Pages et al., 1999; S Meloche, unpublished
observations). Additional work using unbiased genetic
approaches will be required to clarify the individual
roles of ERK1 and ERK2.
The ERK1/2 MAP kinase pathway has emerged as a
central regulator of cell proliferation by controlling both
cell growth and cell cycle progression. Major advances
have been made over the last 10 years in identifying the
targets of this signaling pathway that are linked to the
cell-cycle control machinery. The importance of the
amplitude and duration of the ERK1/2 signal in normal
cell proliferation and oncogene-induced senescence has
been highlighted in a series of elegant studies. However,
several important issues still remain unresolved. The
individual contribution of ERK1 and ERK2 isoforms
to these cellular processes has not been adequately
addressed. Knock-in gene replacement strategies with
complementation studies and chemical genetic ap-
proaches should help to determine unequivocally
whether ERK1 and ERK2 have evolved specific cellular
functions. The systematic characterization of the phy-
siological substrates of ERK1 and ERK2 will be critical
to understanding how these kinases work at the cellular
level. Historically, phosphorylation has been studied at
the level of single molecules, but new proteomic
strategies now allow the global characterization of the
cell phosphoproteome. The Ras–ERK1/2 MAP kinase
pathway is viewed as an attractive signaling pathway for
the development of targeted cancer therapies (Sebolt-
Leopold and Herrera, 2004). A better understanding of
this pathway is likely to have enormous repercussions
on drug development.
S Meloche holds a Canada Research Chair in Cellular
Signaling. Work in the author’s laboratory was supported by
grants from the National Cancer Institute of Canada,
Canadian Institutes for Health Research and Cancer Research
Society to SM, and by the Centre National de la Recherche
Scientifique, Centre A Lacassagne, Ministe ` re de l’Education,
de la Recherche et de la Technologie, Ligue Nationale Contre
le Cancer (Equipe labellise ´ e) and Association pour la
Recherche sur le Cancer to JP.
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