(The FASEB Journal. 2000;14:2147-2157.)
© 2000 FASEB
Control of the eukaryotic cell cycle by MAP kinase signaling pathways
MARC G. WILKINSON*1 and
JONATHAN B. A. MILLAR
* Department of Molecular Neurobiology, SmithKline Beecham Pharmaceuticals Plc, New Frontiers Science Park, Harlow, Essex, CM19 5AW U.K.; and
Division of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA U.K.
1Correspondence: Department of
Molecular Neurobiology, SmithKline Beecham Pharmaceuticals Plc, New
Frontiers Science Park, Coldharbour Rd., Harlow, Essex CM19 5AW, U.K.
E-mail: Marc_G_Wilkinson{at}sbphrd.com
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ABSTRACT
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In an often rapidly changing environment, cells must adapt by monitoring
and reacting quickly to extracellular stimuli detected by
membrane-bound receptors and proteins. Reversible phosphorylation of
intracellular regulatory proteins has emerged as a crucial mechanism
effecting the transmission and modulation of such signals and is
determined by the relative activities of protein kinases and
phosphatases within the cell. These are often arranged into complex
signaling networks that may function independently or be subject to
cross-regulation. Recently, genetic and biochemical analyses have
identified the universally conserved mitogen-activated protein (MAP)
kinase cascade as one of the most ubiquitous signal transduction
systems. This pathway is activated after a variety of cellular stimuli
and regulates numerous physiological processes, particularly the cell
division cycle. Progression through the cell cycle is critically
dependent on the presence of environmental growth factors and stress
stimuli, and failure to correctly integrate such signals into the cell
cycle machinery can lead to the accumulation of genetic damage and
genomic instability characteristic of cancer cells. Here we focus on
the MAP kinase cascade and discuss the molecular mechanisms by which
these extensively studied signaling pathways influence cell growth and
proliferation.—Wilkinson, M. G., Millar, J. B. A.
Control of the eukaryotic cell cycle by MAP kinase signaling pathways.
Key Words: CDK • proliferation • MAPK • signal transduction
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INTRODUCTION
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THE PAST DECADE has seen an explosion in our
understanding of the molecular mechanisms of extracellular signal
transmission from cell surface receptors to nuclear transcription
factors. Signal transduction pathways relay information from a variety
of different stimuli leading to multiple cellular responses.
Consequently they have attracted a great deal of attention not only as
paradigms for ligand-dependent receptor signaling, but also in disease
pathology and as targets for therapeutic intervention. Work from many
different groups using a variety of model systems has identified the
mitogen-activated protein (MAP) kinase cascade as a key mechanism by
which such signals are transduced by the cell. The core of this cascade
consists of an evolutionarily conserved module of three sequentially
activated protein kinases. Catalytic activation of the MAP kinase
(MAPK) requires phosphorylation on conserved tyrosine and threonine
residues by a dual-specificity MAPK kinase (MAP2K/MAPKK/MEK). The MAP2K
is itself activated by phosphorylation on conserved serine and
threonine residues by a serine/threonine MAP2K kinase
(MAP3K/MAPKKK/MEKK). In addition to phosphorylation of cytoplasmic
targets, activation of MAP kinases promotes their nuclear translocation
and subsequent modulation of transcription factors leading to
stimulus-dependent alterations in gene expression (1
, 2)
.
The most widely characterized MAP kinases are the mammalian
extracellular signal-regulated kinase (ERK) family, discovered in the
search for effector kinases that mediate phosphorylation of the
ribosomal S6 protein in response to insulin stimulation. ERKs were
subsequently identified as targets for MEK1 and 2, which are themselves
linked to Ras-mediated growth factor receptors via the Raf family of
kinases (see Fig. 1
) (3)
. ERK1 and 2 were shown to be highly related to the
previously isolated budding yeast kinase Fus3. This led rapidly to the
genetic and biochemical dissection of a homologous MAP kinase pathway
by which the activation of G protein-coupled receptors by pheromone
leads to cell cycle arrest and alterations in cell morphology
associated with mating. Other distinct MAP kinase pathways have
subsequently been characterized in budding yeast controlling a variety
of disparate cellular processes (4)
.
A second family of metazoan MAP kinases was recently identified and
shown to be activated by agonists such as inflammatory cytokines,
multiple environmental stresses, DNA damaging agents, and inhibitors of
protein synthesis. These stress-activated MAP kinases (SAPKs) have been
implicated in the control of several diverse processes including cell
proliferation, development, apoptosis, the response to stress, and the
production of inflammatory cytokines. They are defined by two distinct
subfamilies, the c-Jun amino-terminal kinases (JNKs) and the p38 MAP
kinases (5
6
7
8
9
10
11)
. Although SAPK isoforms have been
implicated in signaling events initiated by receptor tyrosine kinases
and G protein-coupled receptors, it is currently unclear exactly how
their catalytic activity is stimulated by upstream regulatory
components, particularly in response to stress. Moreover, considerable
‘cross talk’ has been suggested since many MAP3K enzymes are
promiscuous in their activation of MAPK pathways when transfected into
cells in vitro. Such studies may, however, fail to reflect
adequately the normal in vivo physiological functions of
MAP3Ks and higher order components of the cascade hierarchy. The
structural and functional characterization of the SAPK pathways has
been facilitated by the identification of homologous signaling systems
in more genetically amenable model organisms. In particular, the
fission yeast StyI/Spc1 MAP kinase is highly related to the
p38 enzymes and is activated by a similar range of environmental
stresses (2
, 12
, 13)
. Activation of StyI/Spc1
induces its nuclear translocation and subsequent phosphorylation of the
bZIP transcription factor Atf1, a homologue of ATF2 that is targeted by
the mammalian SAPKs. This indicates a degree of evolutionary
conservation above and beyond the core MAP kinase module itself
(2
, 14
, 15)
. Moreover, genetic dissection of the
StyI/Spc1 pathway in fission yeast is continuing to provide
further insights into the molecular architecture of these ubiquitous
eukaryotic signaling systems (16)
. Such a degree of
structural conservation emphasizes the fact that numerous fundamental
cellular processes are under MAP kinase pathway control. One of the
most important of these is cell growth and proliferation, illustrated
by the identification of mammalian pathway components as
proto-oncoproteins.
In the following sections we document the role of yeast and mammalian
MAP kinase pathways in the modulation of eukaryotic cell cycle
components. We also discuss how mitogenic and antiproliferative signals
can control the cell division cycle by the action of common or opposing
signaling pathways.
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YEAST CELL CYCLE CONTROL BY MAP KINASE PATHWAYS
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The cell division cycle is a complex and dynamic process centrally
driven by a group of enzymes collectively known as the cyclin-dependent
kinases (CDKs). Formation of active CDK holoenzmes requires their
association with cyclin molecules, phosphorylation by CDK-activating
kinase or CAK, and dephosphorylation by members of the Cdc25 family of
phosphatases. Passage between key cell cycle transitions depends on the
periodic activity of CDKs and is governed in part by the abundance of
their cognate cyclin molecules that fluctuate throughout the cell
cycle. In addition, cyclin–CDK complexes can be inhibited, either
sterically or catalytically, by the binding of CDK inhibitors (CKIs)
(17)
. Like the MAP kinase pathway, the molecular
dissection of eukaryotic cell cycle control has been aided considerably
by the study of genetically tractable organisms such as budding and
fission yeast. In the budding yeast Saccharomyces
cerevisiae, the primary CDK involved in cell cycle control is the
Cdc28 kinase. Late G1 progression and passage through START, analogous
to the restriction point in mammalian cells, are controlled by the
association of Cdc28 with three G1 cyclins: Cln1, 2, and 3.
Subsequently, the correct timing of S-phase entry requires the
association of Cdc28 with the B-type cyclins Clb5 and 6 whereas G2
progression and the onset of M-phase requires its association with Clbs
1–4 (18)
. Cell cycle progression mediated by such
cyclin–CDK complexes is often inhibited via signal transduction
pathways as a result of either intracellular stimuli (for example, by
activation of the mitotic spindle assembly checkpoint) or external
factors such as mating pheromone. Cell cycle arrest in response to
mating pheromone provides the best-characterized example of the direct
control of the yeast cell cycle machinery by a MAP kinase pathway.
For cells to undergo successful sexual conjugation with a member
of the opposite mating type, they must first synchronize their cell
cycles by arresting in G1. This allows efficient morphogenesis and
formation of a diploid zygote. The signal transduction pathway at the
core of the mating response comprises the MAPK Fus3, the MAP2K Ste7,
and the MAP3K Ste11, which are tethered into a high molecular weight
complex by the scaffold protein Ste5 and activated by the Ste20 kinase
(19)
. After binding of mating pheromone to its cognate
seven transmembrane receptor, this complex is recruited to the plasma
membrane. Ste5 is responsible for targeting the complex to the membrane
and is regulated by a novel nuclear shuttling mechanism. In the absence
of pheromone, Ste5 shuttles constitutively through the nucleus.
Treatment with pheromone, however, increases nuclear export of Ste5 and
only this pool is competent to localize to the cell periphery, bind to
the G protein-associated ß
subunit (Ste4/Ste18), and activate the
mating MAPK pathway (20)
. The exact modification that
nuclear transit imparts in order to facilitate this interaction is
unclear, but this mechanism is thought to prevent inappropriate
activation of the mating pathway in the absence of pheromone
(20)
. After activation, the MAP kinase Fus3 then
translocates to the nucleus, where it phosphorylates the CDK inhibitor
Far1 (21
22
23)
. The action of this CKI is restricted to the
G1 phase of the cell cycle by two mechanisms. First, Far1 mRNA is under
cell cycle regulation, peaking between M and G1 phases
(24)
; second, Far1 protein is stable only in G1 and is
rapidly degraded in other phases of the cell cycle via the SCF
ubiquitin ligase complex (25)
. Activation of the pheromone
response pathway provokes a cell cycle arrest in G1 by increasing the
stoichiometry of Far1 relative to Cln1/2–Cdc28 complexes. This occurs
in two ways. In response to pheromone, the levels of Far1 are increased
in a Fus3-dependent manner by increasing mRNA expression and possibly
by inhibiting protein degradation (25
26
27)
. Second, Fus3
represses the expression of the G1/S-specific cyclins CLN1,
CLN2, and CLB5 (28)
. Consequently,
in response to mating pheromone in G1, the levels of Far1 increase
relative to Cln1/2-Cdc28 and Fus3-mediated phosphorylation of Far1
promotes its association with and inhibition of these kinase complexes
leading to cell cycle arrest (21
22
23)
. The exact mechanism
of Far1-mediated inhibition is currently unclear and may not involve
catalytic repression of Cln1/2-Cdc28 kinase, but rather inhibition of
substrate binding or kinase sequestration (29)
. In the
absence of mating, prolonged exposure to pheromone eventually leads to
adaptation and the resumption of mitotic proliferation. This is thought
to occur via two mechanisms. First, the levels of Cln1 and 2 increase
relative to Far1 as a consequence of their transcriptional derepression
after inactivation of the Fus3 MAP kinase. Second, phosphorylation of
Far1 by Cln1/2-Cdc28 leads to its ubiquitin-dependent degradation. This
forms a positive feedback mechanism by which Cln1/2–Cdc28 complexes
are reactivated leading to the resumption of vegetative proliferation
(see Fig. 2A
) (25
, 28)
. In addition, Fus3 may directly
promote proliferation by the activation of genes under the control of
the MADS box transcription factor Mcm1 (28)
.
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Figure 2. A) Mating pheromone-induced cell cycle arrest in budding
yeast is mediated by the Fus3 MAP kinase pathway. Pheromone binding to
its cognate seven transmembrane receptor promotes the activation of the
mating response MAP kinase cascade via the Ste20 protein kinase.
Subsequently, nuclear translocation of the MAP kinase Fus3 facilitates
the expression of the CDK inhibitor Far1 and inhibits its
ubiquitin-mediated degradation. Fus3 also represses transcription of
the G1 cyclins CLN1 and CLN2, which are
critical for the activity of the Cdc28 kinase. In addition, Fus3
phosphorylates Far1, which promotes its association with and inhibition
of the remaining Cln1/2–Cdc28 kinase complexes leading to G1 arrest
prior to START. B) Cell cycle control in fission yeast.
The activity of the central CDK Cdc2 requires its association with the
B-type cyclin Cdc13. This complex is inhibited after phosphorylation of
Cdc2 by the Weel kinase and is activated by dephosphorylation of
tyrosine-15 by the Cdc25 phosphatase immediately prior to the onset of
M-phase. The StyI/Spc1 MAPK pathway comprises the MAP3Ks Wak1 and Win1,
the MAP2K Wis1 and the MAPK StyI/Spc1, which is homologous to the
metazoan p38 MAP kinases. This pathway impinges on cell cycle control
and may indirectly modulate the activity of Cdc2, although the
mechanism by which this occurs is currently unclear.
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In the fission yeast Schizosaccharomyces pombe, the
major point of control over the cell division cycle occurs not at the
G1/S transition but immediately prior to the onset of M-phase. A signal
transduction network regulating the activity of a homologous cyclin-CDK
pair, the Cdc13–Cdc2 kinase complex, governs the timing of mitotic
initiation. Moreover, since the components of this network exist in
humans, G2/M control in fission yeast has served as a paradigm for this
evolutionarily conserved process (30
, 31)
. Phosphorylation
of Cdc2 on tyrosine-15, critical for the inhibition of the complex, is
catalyzed by the Wee1 kinase, whereas dephosphorylation of this residue
by the Cdc25 phosphatase is the key event governing the initiation of
mitosis (32
33
34)
. Consequently, the balance between the
levels of Wee1 and Cdc25 sets a ‘threshold’ for the activity of the
Cdc13–Cdc2 complex that determines the correct timing of M-phase
initiation (see Fig. 2B
). A variety of different mutants
have been isolated that affect cell cycle control in fission yeast and
are defined based on their size at division, being either delayed or
advanced in the onset of M-phase. These can be broadly grouped into two
classes: those that affect the activity of Cdc2 and those that define
components of the stress-activated StyI/Spc1 MAP kinase pathway. The
central elements of this pathway are the MAPK StyI/Spc1, the MAP2K
Wis1, and the MAP3Ks Wak1 and Win1 (2)
. Cells deleted for
sty1 or wis1 are highly elongated as a
consequence of a delay in the timing of mitotic initiation, which is
exacerbated in response to stress. Although such mutants still undergo
cell cycle arrest in response to stress, they are unable to resume
proliferation and die (12
, 35)
. This suggests that the
StyI/Spc1 MAP kinase pathway is required for recovery from a
stress-induced cell cycle arrest. It is currently unclear exactly how
StyI/Spc1 influences the basal cell cycle machinery. One possibility is
that StyI/Spc1 may promote the expression of the B-type cyclin Cdc13.
Since cells deleted for atf1, which encodes a transcription
factor target for the MAP kinase, have no apparent cell cycle defect,
other transcriptional targets may be required. Alternatively, StyI/Spc1
may be required for the assembly or stability of mitotic cyclin–CDK
complexes or other cell cycle components (see Fig. 2B
).
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MAMMALIAN CELL CYCLE CONTROL BY MAP KINASE PATHWAYS
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In contrast to yeast, the mammalian cell cycle is subject to
control by numerous cyclin–CDK complexes. Early in the G1 phase of the
cell cycle, cyclinD–CDK4/6 kinase complexes are active and are subject
to growth factor regulation. Subsequent entry into and progression
through S-phase is regulated respectively by cyclinE-CDK2 and
cyclinA-CDK2, and the onset of mitosis is governed by cyclinB-CDK1/2
(see Fig. 3
). In addition, the activities of CDKs are inhibited by numerous CKIs,
of which there are two families: Ink4 and p21. The Ink4 family consists
of Ink4a-d and is selective for Cdk4 and Cdk6, binding through
conserved ankyrin repeat domains. The p21 family is composed of
p21CIP1/WAF1, p27KIP1, and
p57KIP2 and shows a much broader range of
specificity, being able to inhibit all G1 cyclin–CDK complexes and to
a limited extent cyclin B-associated CDKs. CKIs bind to and inhibit the
action of CDKs either physically or catalytically and also function as
assembly factors for cyclin–CDK complexes (17)
. In
mammalian cells the ability of extracellular signals to influence the
cell cycle machinery is restricted to either G0 or G1 phases. After
passage through the restriction point, cells are refractory to external
cues and become committed to completing a full mitotic cycle, even in
the presence of anti-proliferative molecules such as transforming
growth factor ß. Cyclin D-dependent kinases appear to be the primary
cell cycle targets for extracellular stimuli since cell types that
normally proliferate independently of mitogens (for example, during
embryogenesis) have little or no cyclinD-dependent kinase activity. The
effect of growth factor-dependent signal transduction pathways on the
mammalian cell cycle consequently centers largely on the control of
these kinase complexes at the G1/S transition and is one of the major
functions of the MAP kinase pathway.
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Figure 3. Modulation of the mammalian cell cycle machinery by MAP kinase
pathways. DNA synthesis (S-phase) and mitosis (M-phase) are the key
events in the cell cycle where the chromosomes are duplicated and
segregated between mother and daughter cells. These events are
separated by two intervening gap phases, G1 and G2, depicted on the
bottom panel (not to scale). The restriction point (R) denotes the
point at which cells become committed to completing a full mitotic
cycle. Activation of the Ras/ERK pathway by growth factors and adhesive
signals in early G1 promotes the expression of cyclinD by
phosphorylation of AP1 and ETS transcription factors that bind to
defined elements within the cyclinD promoter. In addition, through
up-regulation of p21CIP1/WAF1, ERK promotes assembly of
cyclinD–CDK4/6 kinase complexes. These complexes then subsequently
phosphorylate pRB leading to displacement of pRB and histone
deacetylases (HDAC), which results in activation of genes regulated by
the transcription factor E2F at the restriction point. E2F promotes the
expression of cyclinA and cyclinE in late G1, which associate with
CDK2. CyclinE–CDK2, the rate-limiting kinase complex required for
S-phase entry, is inhibited in late G1 by the CDK inhibitor
p27KIP1. ERK phosphorylation of p27KIP1
promotes its degradation, release of active cyclinE-CDK2 and therefore
entry into S-phase. The p38 MAPK causes cell cycle arrest by inhibiting
phosphorylation of AP1 and ETS transcription factors required for
cyclinD synthesis. In addition, the JNK pathway may inhibit cell cycle
progression by attenuating E2F-dependent transcription.
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Expression of oncogenic Ras or constitutively active MEK in primary
fibroblasts causes cell cycle arrest and a terminal phenotype similar
to cellular senescence, with high levels of the CKIs INK4a and
p21CIP1/WAF1. The latter is dependent on the
transcription factor p53, which is also significantly elevated in these
cells (36
, 37)
. Sustained Ras/ERK pathway activity as a
result of artificially high constitutively active Raf kinase has also
been shown to arrest the cell cycle and cause increased levels of
p21CIP1/WAF1 (38
, 39)
. These studies
reveal an inherent fail-safe mechanism to arrest the cell cycle and
therefore prevent transformation in the presence of abnormally
sustained Ras/ERK pathway activity. How then does the ERK pathway
influence normal proliferation? A key step governing progression into
S-phase is the activation of the E2F family of transcription factors
that directly regulate genes required for cell proliferation and DNA
synthesis. During early G1, E2F is inhibited by the action of the tumor
suppressor pRB, the product of the retinoblastoma susceptibility gene.
By binding to E2F, pRB recruits histone deacetylases to the promoters
of E2F-responsive genes and thereby represses their expression
(40)
. This is relieved in late G1 by the release of E2F as
a consequence of pRB phosphorylation by cyclinD-CDK4/6. Several studies
have demonstrated an involvement of the classical Ras/ERK pathway in G1
progression by the direct control of cyclinD1 expression. This appears
to be primarily mediated by ERK-dependent modulation of AP-1 and ETS
transcription factors that bind to defined elements in the cyclinD1
promoter (41
42
43)
. The Ras/ERK pathway is further
implicated in the regulation of pRB by the demonstration that cells
lacking functional pRB do not require cyclinD1 and are unable to arrest
the cell cycle after expression of a dominant negative Ras mutant or
microinjection of neutralizing anti-Ras antibodies
(44
45
46
47)
. Further studies showed that overexpression of
direct inhibitors of cyclinD1–CDK4/6 complexes, such as
p21CIP1/WAF1 and p16INK4a,
inhibits Ras-induced proliferation (48)
. Thus, stimulation
of synthesis and assembly of cyclinD1-dependent kinase complexes during
early G1 appears to be a bona fide role for the ERK pathway
in the control of the cell cycle.
The Ras/ERK pathway also acts post-translationally on cyclinD- and
E-dependent kinase assembly and catalytic activity. This was first
alluded to by the observation that ectopically expressed cyclinD can
only be incorporated into functional kinase complexes in the presence
of exogenous growth factors (49)
. Subsequently, the
synthesis of the p21 family CKIs has been shown to be directly
modulated by the Ras/ERK pathway. In previously quiescent cells, growth
factor stimulation causes cell cycle re-entry from G0 and transient
p21CIP1/WAF1 expression, in a manner dependent on
ERK activity (50)
. Since primary cells lacking both
p21CIP1/WAF1 and p27KIP1
genes have significantly depleted cyclinD–CDK4/6 complexes in G1, the
functional consequence of p21CIP1/WAF1 expression
at this stage of the cell cycle is to contribute, in addition to
p27KIP1, to the assembly of cyclinD–CDK4/6
complexes and hence promote G1 progression (17
, 50
51
52)
.
In addition, entry into S-phase is promoted by proteolytic degradation
of p27KIP1 in late G1, contributing to the
release from inhibition of the rate-limiting cyclinE–CDK2 complex
(17)
. Although cyclinE-CDK2 itself contributes to this
degradation, a significant component may be dependent on ERK activity
(53)
. ERK is able to phosphorylate
p27KIP1 in vitro, preventing the CKI from
interacting with and inhibiting CDK2. Moreover, at this stage in the
cell cycle, expression of a dominant negative Ras mutant or
pharmacological inhibition of MEK leads to maintenance of
p27KIP1 protein levels and results in a G1/S
arrest, suggesting that ERK modulates p27KIP1 in
vivo (54
55
56)
. Although degradation of
p27KIP1 has been linked to the
ubiquitin-proteasome pathway, after constitutive ERK pathway activation
specific proteasome inhibitors are unable to stabilize
p27KIP1 protein levels, suggesting that
degradation occurs via a ubiquitin-independent pathway (55
, 57)
.
The key molecular events that contribute to the irreversible decision
to enter S-phase at the restriction point can therefore all be
modulated by ERK pathway activity: 1) the initiation of
cyclinA and cyclinE mRNA synthesis as a consequence of
cyclinD1-CDK4/6-mediated phosphorylation of pRB and liberation of E2F;
2) the assembly of cyclinA- and cyclinE–CDK2 kinase
complexes by the up-regulation of the pool of CKIs acting as assembly
factors; and 3) the release of catalytically active CDK2
complexes by the degradation of the inhibitory CKI
p27KIP1 that is bound tightly to these complexes
in late G1 (see Fig. 3
).
In addition to mitogenic growth factors, the majority of
untransformed cells also require attachment to an extracellular matrix
(ECM) for normal cell cycle progression. Cells become anchored to the
ECM through an interaction involving surface receptors called
integrins, composed of heterodimers between 
and ß chains.
Integrin engagement has been demonstrated to activate the ERK pathway
both directly (58
59
60)
and indirectly in association with
growth factor stimulation (61
62
63)
. Increases in cyclinD1
mRNA and protein by many anchorage-dependent cell types require both
attachment to the ECM and stimulation by growth factors. Similar
requirements exist for pRB phosphorylation and hence expression of
E2F-dependent genes such as cyclinE (64
65
66
67)
. In growth
factor-treated cells, attachment to fibronectin or ligation of the
5ß1 integrin using a specific antibody results in potent ERK
activation and concomitant expression of cyclinD1. Furthermore, the
adhesion requirement for cyclinD1 expression can be suppressed by the
expression of a constitutively active form of ERK (68)
.
Exactly how integrins are coupled to the ERK pathway is currently
unclear. Several studies indicate an involvement of the cytoplasmic
tyrosine kinases Src and FAK in ERK activation (59
60
61
62
63
64
65
66
67
68
69
70)
while another study suggests a FAK-independent mechanism. In this case,
a subset of integrins, composed of 1 and vß3, are required, in
addition to the adaptor protein Shc, for signaling to the Ras/ERK
pathway and consequently for G1 progression. Ligation of other
integrins or expression of dominant negative Shc results in cell cycle
arrest and apoptosis under the same conditions (71)
. These
studies demonstrate a clear role for the ERK pathway in
integrin-mediated ECM-dependent cell survival and proliferation. This
requirement, however, may be confined to early G1, since control of
certain cell cycle components—for example,
p21CIP1/WAF1, which initially requires
ERK—becomes refractory to this pathway in late G1 (50)
.
Additional parallel pathways may consequently mediate ECM-dependent
proliferation at this stage. In addition to the control of cyclinD1
kinase complexes, cell anchorage may also indirectly modulate
cyclinE-CDK2 via the ERK pathway. Although anchorage has no significant
effect on either the levels or formation of these complexes in G1, it
does affect the levels of CKIs, which act to inhibit their activity. In
suspended cells, expression of both p21CIP1/WAF1
and p27KIP1 are increased dramatically, the
latter being a consequence of decreased degradation (66
, 67
, 72)
. Together with the decrease in cyclinD1 expression, this
contributes to cell cycle arrest at G1/S.
Finally, a novel role for the ERK MAP kinase pathway in G2/M cell cycle
control has recently been suggested. Although growth factors are the
primary ERK-activating stimuli, ionizing radiation has also been shown
to be a potent ERK pathway agonist in contrast to other stress stimuli
(73
, 74)
. This promotes ERK-dependent cell cycle recovery
after DNA repair since cells expressing dominant-negative MEK2, but not
MEK1, are unable to recover from a radiation-induced G2/M checkpoint
arrest (75)
. In addition, treatment of cells with PD98059,
a pharmacological inhibitor of endogenous MEK, provokes a G2/M arrest
and concomitant reduction of cyclinB-CDK1 kinase activity, supporting a
role for the ERK pathway in mitotic control (76)
.
ERK5/BMK1, a novel member of the MAP kinase family, was recently
identified. Although ERK5 contains unique carboxyl-terminal and loop 12
sequences, it possesses the characteristic T-E-Y tripartite
phosphorylation motif unique to this subfamily of enzymes
(77)
. The catalytic activity of ERK5 is increased after
phosphorylation by its upstream activator MEK5 and in certain cell
types by agonists such as serum, hydrogen peroxide, and high osmolarity
(see Fig. 1
) (78
, 79)
. Although there appears to be no
direct catalytic cross talk, at the level of cell proliferation there
is significant synergy between the ERK1/2 and ERK5 pathways. For
example, Raf-1-dependent transformation of fibroblasts is either
enhanced or blocked by expression of constitutively active or kinase
dead mutants of MEK5, respectively (80)
. Furthermore, ERK5
can play a direct role in cell cycle progression mediated by EGF, an
ERK5 agonist. Expression of a dominant negative mutant of ERK5
abolishes EGF- and serum-dependent proliferation of epithelial cells
and prevents entry into S-phase. These effects appear to be independent
of Ras. Conversely, activated forms of Ras can enhance ERK5 catalytic
activity significantly, and in certain cell types Ras is required for
ligand-dependent activation (81)
. This suggests that both
Ras-dependent and -independent mechanisms of ERK5 activation may exist,
although the exact mechanisms are currently unclear (82)
.
One clue, however, comes from observations with Raf-1. This kinase
plays a role in ERK5 regulation since expression of wild-type and
activated forms are able to rescue loss of ERK5 activity caused by
expression of dominant-negative Ras effector mutants (80)
.
The catalytic activity of Raf-1, however, appears not to be required
(80)
. One mechanism by which Raf-1 might therefore
regulate ERK5 is by modulation of protein complexes. This is suggested
by the observation that Raf-1 and ERK5 interact physically and that
this interaction is enhanced by increasing ERK5 activity
(80)
. Exactly how the ERK5 pathway directly controls
proliferation is unclear. The observation that the transcription factor
MEF2C is a direct phosphorylation target of ERK5 and its
trans-activation is increased dramatically by serum in an
ERK5-dependent manner suggests a possible mechanism (79)
.
Numerous immediate-early genes harbor MEF2 binding sites in their
promoters, including c-jun, strongly suggesting that genes
involved directly in cell proliferation may be transcriptional targets
of the ERK5 pathway (79)
. This hypothesis is supported by
the observation that the c-fos promoter is also modulated by
ERK5 after direct phosphorylation of the ternary complex factor Sap1a,
a member of the ETS domain-containing transcription factor family
(81)
. Its should be stressed, however, that the extent of
ERK5 activation and consequently its physiological role in both cell
cycle regulation and the stress response may be cell type specific
(78)
.
Although significantly less well characterized, an involvement in cell
cycle control of the SAPKs has recently emerged. JNK1 has been shown to
bind to and phosphorylate the E2F1 transcription factor in
vitro and consequently reduce its DNA binding affinity
(83)
. Thus, JNK activation in vivo may have the
consequence of disrupting E2F-containing transcriptional complexes and
imparting a stress-induced G1 arrest subsequent to CDK-mediated
phosphorylation of pRB. This JNK-mediated arrest, however, can be
separated from the role in cell cycle control of its primary
transcription factor target, c-Jun. Phosphorylation of c-Jun on serines
63 and 73 by JNK enhances its trans-activation in response
to stress stimuli. In contrast, the proliferative functions of
c-Jun—for example, in elevating the expression of cyclinD1—appear to
be independent of these phosphorylations (84)
. In
addition, c-Jun has also recently been shown to promote proliferation
by acting as a transcriptional repressor of p53 and consequently its
target gene, the CDK inhibitor p21CIP1/WAF1
(85)
. Similarly, an involvement of p38 and its upstream
kinases MKK3, MKK6, and MEKK3 in the establishment of a G1/S cell cycle
arrest has been well documented (86
, 87)
and has been
attributed to antagonism of ERK-mediated expression of cyclinD1
(41)
. Intriguingly, another mechanism by which p38 MAP
kinase may negatively regulate the cell cycle is by activation of the
mitotic spindle assembly checkpoint pathway that monitors the correct
formation of the spindle and attachment of kinetochores. Treatment of
cells with nocodazole, which disrupts the spindle, causes activation of
p38 during M-phase but not other phases of the cell cycle. Moreover,
checkpoint activation can be suppressed by treatment with the p38
inhibitor SB203580 (88)
. The significance and molecular
mechanisms of these observations have yet to be established although
they do strongly suggest that stress-activated MAP kinase pathways play
a role in arresting the cell cycle, possibly to prevent inappropriate
execution of key transitions in the presence of DNA damaging agents or
other stressful stimuli.
|
INTEGRATION OF THE ERK PATHWAY WITH OTHER RAS EFFECTOR PATHWAYS IN
CELL CYCLE CONTROL
|
|---|
Although in this review we have focused specifically on MAP kinase
pathways, it is unclear exactly how other Ras effector pathways are
integrated in vivo to control the cell cycle and how these
contribute to tumorigenesis when deregulated. The duration of ERK
activation after G0 release is significantly less than that of Ras,
which appears to be biphasic, peaking again in late G1. At this time
effector pathways such as the PI 3-kinase/PKB pathway, which in
addition to the ERK pathway also regulates cyclinD1 and S-phase entry,
may be the primary target of Ras signaling (89
, 90)
.
Indeed, the PI 3-Kinase/PKB pathway provides a good example of how
multiple effector pathways contribute to the complexity of Ras
signaling. Although the transcriptional regulation of cyclinD by the
ERK pathway has been well characterized, it has recently been shown
that cyclinD phosphorylation at threonine 286 by GSK3 negatively
regulates its stability. GSK3 is itself negatively regulated by PKB.
Consequently, after activation of PI 3-Kinase by Ras in response to
growth factor stimulation, cyclinD protein would be stabilized due to
the absence of GSK3 phosphorylation (91)
. CyclinD protein
is therefore up-regulated by Ras after stimulation via two independent
effector pathways: the ERK pathway, contributing to increased mRNA
synthesis, and the PI 3-kinase pathway, contributing to increased
protein stability. As with the ERK pathway, components of the PI
3-kinase/PKB pathway are able to stimulate transcription from a cyclinD
reporter construct; however, peak activation requires synergistic ERK
activity indicating the degree of cooperation necessary for maximal Ras
signaling (90)
.
Ras has long been known to exert both positive and negative effects on
cell growth and proliferation. The molecular basis of these opposing
actions are now beginning to be understood based on the particular
effector pathway targeted by Ras in response to a given stimulus or
with respect to the particular cellular, biological, or temporal
context. For example, in contrast to the growth-promoting effects of
transient Ras activation associated with growth factor stimulation, in
many cell types prolonged Ras activation will lead to cell cycle arrest
or apoptosis. Activation of the JNK pathway is commonly associated with
apoptosis. Despite being viable, mice lacking both alleles of the
brain-specific JNK3 kinase display a marked decrease in both seizure
activity and hippocampal neuron apoptosis associated with the
excitotoxic glutamate-receptor agonist kainic acid (92)
.
In addition, JNK1/JNK2 double mutant mice, but not other JNK compound
mutants, are embryonic lethal, displaying hindbrain exencephaly and a
reduction in cellular apoptosis along the neural folds
(93)
. Exactly whether Ras promotes apoptosis via
activation of the JNK pathway is currently unclear. Ras has been shown
to signal to the JNK pathway, possibly by a mechanism involving MEKK1
(94
95
96)
. In addition, both oncogenic Ras and an activated
form of Raf-1 promote the expression and secretion of heparin binding
epidermal growth factor in fibroblasts that can activate JNK in an
autocrine manner (97)
. Conversely, Ras may mediate
pro-survival signals via activation of the PI 3-kinase/PKB pathway.
Several substrates have been identified downstream of PKB, such as Bad
and members of the forkhead transcription factor family, which
contribute to cell survival after PKB activation (98)
.
Opposing effects of different Ras effector pathways have also been
observed with regard to cell cycle progression. For example, the
expression of the CKI p21CIP1/WAF1 is regulated
by Ras via the Raf kinase in response to serum or growth factors.
However, the small GTPase Rho, which is essential for Ras-induced
transformation, was shown to inhibit Raf-mediated expression of
p21CIP1/WAF1 at the promoter level, despite being
a downstream Ras effector molecule. Conversely, inhibition of Rho leads
to cell cycle arrest at S-phase due to Raf-mediated up-regulation of
p21CIP1/WAF1 (99)
. Consequently, the
cellular outcome with regard to survival vs. apoptosis, or
proliferation vs. cell cycle arrest, may depend entirely on the
particular effector pathway(s) engaged with Ras at a given time or in a
particular cell type and may also be a consequence of the degree to
which Ras is activated at that time.
|
OTHER MECHANISMS OF MAPK-DEPENDENT MODULATION OF GROWTH AND
PROLIFERATION
|
|---|
One additional intriguing feature of the MAP kinase pathway that
has emerged recently is its ability to influence growth and
proliferation by modulating protein synthesis, independently of
transcriptional regulation by the phosphorylation of transcription
factors. The translation of mRNA into protein requires several
cofactors, including initiation and elongation factors. Eukaryotic
translation initiation factor-4E (eIF-4E) binds to the mRNA 5'-terminal
7-methyl-GTP cap and facilitates its complex formation with ribosomes
and other components of the multisubunit initiation factor eIF-4F, thus
influencing rates of global translation. Consistent with this function,
eIF-4E is required for cell cycle progression and is oncogenic when
overexpressed. In quiescent cells, the binding of eIF-4E to other
components of eIF-4F is inhibited due to sequestration by interaction
with eIF-4E binding proteins (4EBPs) that act as translational
repressors. After mitogenic stimulation, however, phosphorylation of
4EBPs by p70S6K, independently of ERKs, promotes
release of eIF-4E. Subsequently, direct phosphorylation of eIF-4E and
concomitant stimulation of translation initiation occurs by an
ERK-dependent but p70S6K-independent mechanism.
Work from Cooper and colleagues has shown that Mnk1, a kinase acting
downstream of ERK, is a member of the eIF-4F complex and directly
phosphorylates eIF-4E in vivo by an ERK-dependent mechanism
after mitogenic stimulation (100
, 101)
. Mnk1 is also a
substrate for the p38 MAP kinase; however, many stimuli that activate
p38, such as heat shock, hydrogen peroxide, and sorbitol, fail to
induce phosphorylation of eIF-4E. Under these conditions, eIF-4E
appears to be protected from Mnk1-mediated phosphorylation by its
increased interaction with 4EBP1. This suggests a mechanism by which
protein synthesis, and therefore cell growth, may be attenuated by p38
in response to stress (102)
.
In addition to protein synthesis, the ERK MAP kinase pathway has
recently been shown to stimulate another key mechanism involved in cell
growth, the synthesis of pyrimidine nucleotides, critical in the
de novo production of DNA and RNA. The rate-limiting step in
this process is catalyzed by an enzyme known as carbomoyl phosphate
synthetase (CPS II), which is part of a larger tripartite protein
complex called CAD. CPS II activity and phosphorylation have been shown
to be stimulated by treatment of cells with the ERK agonists PDGF and
EGF, respectively, and ERK is able to phosphorylate CPS II in
vitro. Furthermore, stimulation by EGF of uridine 5'-triphosphate
synthesis in vivo is abolished after treatment with the MEK
inhibitor PD98059 (103)
. These results indicate that MAP
kinase pathways may play a much broader role in the regulation of cell
growth and proliferation than was hitherto apparent. Regulation of the
cell cycle control apparatus may therefore emerge as only one of many
mechanisms by which these ubiquitous signaling pathways couple
extracellular signals to growth and proliferation.
|
PERSPECTIVE
|
|---|
The mechanism of ligand-dependent activation of signal
transduction pathways has been a major focus of biomedical research
over the past few years and has provided significant insights into the
mechanisms by which cells sense and respond to their outside
environment. In particular, the MAP kinase pathway has been extensively
characterized and its role in a vast array of cellular processes is now
beginning to be understood. Its involvement in cell growth and
proliferation has long been recognized and since its upstream
activator, Ras, is mutated in around one-third of all human tumors, an
exquisite understanding of its mechanism of action and relationships
with other mitogenic signaling pathways is of major importance. In
addition, our understanding of the molecular mechanisms that couple the
cell cycle machinery to MAP kinase pathways in general has
increased dramatically over the past several years. The emerging theme
suggests that the ERK pathway acts primarily to positively regulate the
cell cycle during G1 at the level of cyclinD synthesis, assembly of
cyclinD-dependent kinase complexes and subsequent phosphorylation of
pRb, while the SAPK pathways may oppose these functions and elicit a
stress-induced cell cycle arrest. The wealth of data accumulated thus
far have centered largely on pathway analyses in immortalized cell
lines using dominant negative or interfering mutants. Transformation of
normal primary cells, however, requires additional cooperating
oncogenes or loss of tumor suppressor genes. Cell type- and biological
context-dependent differences in activation of Ras effector pathways
also appear to play key roles in the determination of the cellular
response elicited by a given stimulus in addition to the strength and
duration of that stimulus. To gain a more complete understanding of
exactly how MAP kinase pathways influence the cell cycle in
vivo, it will be necessary to determine exactly how they are
integrated with other signaling pathways in specific cell types.
Further developments in the use of specific chemical inhibitors, animal
models, and genetics should complement more traditional approaches. In
particular, the molecular dissection of more genetically tractable
model organisms in which signaling components are exquisitely conserved
such as the fruit fly, nematode, and yeast will continue to provide not
only fascinating new insights but, more important, should provide
additional scope for pharmacological intervention in disease
therapy.
|
ACKNOWLEDGMENTS
|
|---|
We apologize to those whose publications could not be cited or
discussed due to space limitations. For critical reading of the
manuscript, we would like to acknowledge Drs. Karen Philpott, Oliver
Rausch, Alastair Reith, Alison Rowles, and Andreas Sewing (SmithKline
Beecham), and Vicky Buck and Leland Johnston (NIMR). J.M. is supported
by the Medical Research Council.
|
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ABSTRACT
INTRODUCTION
