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How do small GTPase signal transduction pathways regulate cell
cycle entry?
Chris Marshall
A variety of studies have shown that activation of the cell cycle
machinery requires the participation of multiple signalling
pathways. These pathways include Ras-dependent effectors
such as the extracellular-signal related kinases, otherwise
known as mitogen-activated protein kinases (ERKs, MAPKs),
phosphatidylinositol 3 (PI3)-kinase and p21Ral pathways, as
well as other signalling pathways regulated by the small
GTPases p21Rho, p21Rac and p21Cdc42.
Addresses
Cancer Research Campaign Centre for Cell and Molecular Biology,
Chester Beatty Laboratories, Institute of Cancer Research, London
SW3 6JB, UK; e-mail: [email protected]
pathways interface with the cell cycle machinery it is
important to identify which cell cycle events are directly
regulated by intracellular signals. Considerable evidence
suggests that signal transduction pathways from the small
GTPases of the Ras and Rho family play an important role
in cell cycle control. This review provides an outline of
how growth-factor-activated small GTPase signalling pathways control entry into the cell cycle in mammalian
somatic cells. The reader is referred to other reviews for a
comprehensive description of signalling pathways regulated by small GTPases [2,3].
Figure 1
Current Opinion in Cell Biology 1999, 11:732–736
0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
CAK
Cdk-activating kinase
Cdk
cyclin-dependent kinase
CKI
Cdk inhibitor
ERK
extracellular-signal-regulated kinase
GSK
glycogen synthase kinase
MAPK mitogen-activated protein kinase
PI3
phosphatidylinositol 3
pRb
retinoblastoma protein
PO 4
PO 4
PO 4
pRb
Cdk4/6
The regulation of the activity of these kinases requires an
interplay between the synthesis of the cyclins, formation
of cyclin/Cdk complexes, association with the cyclindependent-kinase inhibitors (CKIs), phosphorylation of
Cdks by activating kinases (CAKs) and transport of cyclin
Cdk complexes to the nucleus (Figure 1; for a review, see
[1••]). Some of these events may be regulated directly by
signal transduction pathways, whereas others are a consequence of the cell cycle machinery becoming activated.
Thus, in trying to understand how signal transduction
cyclin D
cyclin E
Cdk2
SKP2
Introduction
Numerous studies with cultured cells have demonstrated
that entry of quiescent Go cells into S-phase is dependent
on the activities of the G1 cyclin-dependent kinases
(Cdks). These kinases consist of Cdk4 and Cdk6 complexed with the D-type cyclins D1, D2, D3, and Cdk2
complexed with cyclin E. A key regulatory event mediated by the G1 Cdks is the phosphorylation of the ‘pocket’
proteins and in particular the retinoblastoma protein
pRb105, which is the product of the Rb tumour suppressor
locus. In its hypophosphorylated form pRb105 associates
with the E2F family of transcription factors and actively
promotes transcriptional repression or sequesters E2F
from genes required for entry into S-phase. Following
phosphorylation of pRb105 by the G1 Cdks, E2F is released
from pRb105 and transcription results.
S -PHASE
E2F
p16 CKIs
p21Waf1 p27Kip1
Current Opinion in Cell Biology
Relationship between cyclins and CKIs in the regulation of Cdk
activity, pRb105 phosphorylation and S-phase entry. The
cyclinD/Cdk4/6 and the cyclinE/Cdk2 complexes phosphorylate pRb,
which releases the transcription factor E2F, resulting in entry into Sphase. The activity of Cdk4/6 is inhibited by the p16 family of CKIs,
whereas the p21 family (p21Waf1, p27Kip1) inhibits Cdk2 and Cdk4/6.
The cyclinE/Cdk2 complex phosphorylates p27Kip1, which is then
degraded by p45Skp2 (SKP2). Degradation of p27Kip1 is an important
control point for entry into the cell cycle. The double-headed arrow
between Cdk4/6 and the p21 family of CKIs reflects the potential dual
role of these CKIs in assembly of active cyclinD/Cdk4/6 complexes
and in their inhibition.
Signal transduction and cell cycle progression
A characteristic of mitogenic signalling is that it requires
prolonged exposure to growth factors until the ‘restriction
point’ is reached and growth factors are no longer necessary. This requirement for prolonged exposure to growth
factors implies that signal transduction also must be sustained. The seminal experiments of Stacey and
collaborators [4], using microinjection of a Ras neutralising
How do small GTPase signal transduction pathways regulate cell cycle entry? Marshall
antibody, showed that cellular Ras function is required
throughout most of G1 in order for growth factors to stimulate quiescent cells to enter DNA synthesis. These
observations are nicely paralleled by the demonstration
that the activation of Ras occurs in two waves following
growth factor stimulation: the first immediately following
addition of growth factors and the second in mid G1 [5].
Ras function is also required for cell cycle progression in
asynchronously growing cycling cells, and recent observations indicate that Ras function is actually required in the
previous cell cycle presumably to provide cell cycle components, such as cyclin D1, for the following cell cycle [6•].
One approach to understanding how signal transduction
pathways are coupled to the cell cycle is to investigate
whether genetic ablation of key regulatory components
alters the requirements for cell signalling. Two research
groups have investigated whether pRb105 null cells have a
reduced requirement for Ras-dependent signalling.
Although blocking Ras almost completely inhibited proliferation of asynchronous cultures of wild-type cells, it had a
very much reduced effect on the null cells [7,8]. These
experiments highlight the importance of Ras signalling in
activating the G1 Cdks that phosphorylate and inactivate
pRb105. Loss of pRb105 does not completely overcome the
requirement for Ras-dependent signalling because
Mittnacht et al. [8] found that pRb105 null cells still showed
some inhibition of proliferation when injected with a Ras
neutralising antibody, and Peeper et al. [7] found that
pRb105 null cells still require Ras function for exit from Go
into G1. Possibly this requirement for Ras signalling may
reflect the need for cyclinE/Cdk2 activity which is still
required in pRb105 null cells. Experiments with oncogenic
Ras show that in combination with overexpressed c-Myc it
can lead to the activation of cyclinE/Cdk2 in the absence
of growth factor stimulation [9].
Regulation of cyclin D1 expression
A key event in stimulating cell cycle entry in quiescent
cells is the activation of the kinases Cdk4 and Cdk6. In quiescent cells, expression of the D-type cyclins is low and is
stimulated by mitogenic growth factor treatment. Studies
from Pouyssegur and collaborators [10] established that
growth factor stimulation of cyclin D1 levels requires activation of the ERK MAP kinase pathway. This observation
has been extended to a variety of systems and demonstrates
that one component of Ras-dependent mitogenic signalling
is activation of the ERK MAP kinases. Sustained activation
of ERK MAP kinases is required for entry into DNA synthesis and has been shown to be required for cyclin D1
expression [11,12]. The ERK MAP kinase pathway appears
to regulate transcription of the cyclin D1 gene as growth
factor stimulation of cyclin D1 mRNA levels is blocked by
inhibiting activation of ERK with the MAP kinase
kinase (Mek1) inhibitor PD98059 [11]. Furthermore, activated Raf, a component of the Ras pathway, can stimulate
expression from cyclin D1 reporter constructs [10]. The
precise mechanism by which ERK MAP kinase signalling
733
leads to activation of cyclin D1 transcription is not clear. A
clear transcriptional effector of ERK MAP kinase signalling
is the AP1 complex of transcription factors, which consists
of Fos related proteins c-Fos, Fos B, Fra-1, Fra-2, and Jun
related proteins c-Jun, JunB and JunD. Thus the link
between ERK MAP kinase signalling and cyclin D1 transcription might be AP1. Evidence to support such a
mechanism comes from the observation that mouse embryo
fibroblasts null for c-Jun grow poorly in culture and fail to
induce cyclin D1 mRNA expression following growth factor stimulation [13•]. However, others have found that
cyclin D1 expression is not reduced in c-Jun null cells and
have argued that the proliferative defect of the null cells
results from elevated levels of p53 in these cells inducing
expression of the CKI p21Waf1 [14•]. A second transcriptional effector of ERK MAP kinase signalling is c-Ets-2 and
this may provide the route to cyclin D1 transcription [15].
Although the ERK MAP kinase pathway appears to be
required for growth factor signalling to activate cyclin D1
expression in a variety of systems, it is clear that it is not the
only signalling pathway that is required. Activation of the
PI3-kinase pathway is also required since activation of ERK
MAP kinases resulting from an inducible Mek construct
only results in cyclin D1 expression when a PI3-kinase signal is present [16]. Furthermore, inhibition of the
PI3-kinase pathway blocks growth factor stimulated cyclin
D1 expression through a rapamycin-insensitive pathway
[17]. Transcription from cyclin D1 reporter constructs can
be stimulated by signals from PI3-kinase and the Ral and
Rac GTPases [18,19•]. In addition, signalling from Cdc42
elevates cyclin D1 expression, although it is not clear
whether this is due to an effect on cyclin D1 transcription
[20]. These observations demonstrate that multiple small
GTPase signalling pathways are required for cyclin D1
expression. The signalling pathways activated by Ral, Rac
and Cdc42 that contribute to cyclin D1 expression have not
been elucidated, however. As the activation of PI3-kinase
can lead to the activation of Rac, at least part of the effects
of PI3-kinase on the activation of cyclin D1 reporters may
be mediated by Rac. However, other PI3-kinase signalling
pathways may be involved such as protein kinase B (PKB)
[19]. Although these observations suggest that a variety of
GTPase signalling pathways impinge on cyclin D1 transcription some caution must be exercised. Many of these
studies have been carried out with reporter constructs in
transient assays and using overexpression of constitutively
activated signalling components, so there is still much to
learn about the mechanism by which the endogenous
D-type cyclin genes are regulated by signalling pathways.
As well as the effects of the PI3-kinase pathway on cyclin
D1 transcription, PI3-kinase post-transcriptionally regulates the cyclin D1 protein. Phosphorylation of cyclin D1 at
Thr286 leads to its degradation with the phosphorylated
protein having a half life of around 10 minutes. Sherr and
collaborators [21••] have identified glycogen synthase
kinase-3β (GSK-3β) as the Thr286 kinase and argued that
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Cell multiplication
Figure 2
+Myc
Ras
PI3-K
PKB
Cdk2
cyclin E
Raf
RalGEFs
MEK
Ral
p21Waf1
ERK
GSK3β
Rho
p27Kip1
Stability
Cyclin D1 Transcription
Small GTPase signalling and the regulation of
G1 Cdk activity. Inhibitory pathways are
indicated with a hammerhead, and stimulatory
pathways are indicated with an arrow. Ras
activates the PI3-kinase (PI3-K) signalling
pathway, resulting in cyclin D1 transcription
and stability, and degradation of p27Kip1.
Cyclin D1 transcription is also activated by
the Ras-activated Ral and MEK/ERK
signalling pathways. It is not clear whether
Cdc42 acts transcriptionally or posttranscriptionally to regulate cyclin D1
expression. Activation of the ERK pathway
leads to expression of p21Waf1, which inhibits
cyclinE/Cdk2 complex activity. Rho signalling
is thought to control the cell’s response to
activation of the ERK pathway.
Rac/
Cdc42
Current Opinion in Cell Biology
inactivation of GSK-3β through phosphorylation by PKB
provides a PI3-kinase- and Ras-dependent route to the stabilisation of cyclin D1. PI3-kinase, together with mTor, the
mammalian target of rapamycin, may also regulate the
translation of the cyclin D1 mRNA [22]. In cells where
cyclin D1 mRNA levels are high, even in the absence of
growth factors, the PI3-kinase-dependent regulation of
cyclin D1 protein stability and translation may be particularly important for cell cycle progression and such cells may
show no requirement for the ERK MAP kinase pathway.
Such situations may arise where cyclin D1 transcription is
elevated due to gene amplification or translocation.
Signal transduction and CKIs
As well as the regulation of Cdk4 and Cdk6 activity through
the synthesis of D-type cyclins, the cell cycle machinery is regulated by the CKIs. In quiescent-serum-starved fibroblasts the
levels of the CKI p27Kip1 are high and drop following growth
factor stimulation. The degradation of p27Kip1 appears to be an
important control point for entry into the cell cycle and may be
a key regulator of cyclinE/Cdk2 activity [1••]. Studies with
inducible dominant negative Ras constructs demonstrate that
the downregulation of p27Kip1 that occurs late in G1 phase
requires Ras [23]. At this time point ERK MAP kinase signalling is low and the degradation of p27Kip1 appears to be
mediated by the PI3-kinase pathway. Phosphorylation of
p27Kip1 at Thr187 by cyclinE/Cdk2 leads to its degradation.
Since PI3-kinase activity stabilises cyclin D1 expression, this
may permit cyclinD1/Cdk4 complexes to titrate out p27Kip1
resulting in cyclinE/Cdk2 activity and phosphorylation of
p27Kip1. Recent results show that p27Kip1 phosphorylated at
Thr187 is recognised by the F-box protein p45Skp2 and this
leads to ubiquitin-dependent proteolysis [24•,25•]. The level
of p45Skp2 is low in quiescent cells and is induced following
growth factor stimulation, perhaps this is through a PI3-kinasedependent step. Another point at which PI3-kinase activity
may be required for p27Kip1 degradation is in the induction of
cyclin E. Cyclin E is an E2F-responsive gene, and PI3-kinase
may elevate cyclin E expression through PKB mediated activation of E2F-dependent transcription [26]. In interleukin-2
stimulated T cells, PI3-kinase-mediated activation of E2F
transcription also involves a rapamycin-sensitive event [27],
which may explain why p27Kip1 degradation in T cells is
blocked by rapamycin treatment.
In addition to the PI3-kinase pathway, other signalling pathways may contribute to p27Kip1 degradation. In vitro p27Kip1
can be phosphorylated by ERK MAP kinases, although the
site(s) of phosphorylation has not been identified. It has
been argued that the ERK MAP kinases are involved in the
degradation of p27Kip1 [28]. This requirement for ERK MAP
kinase signalling in p27Kip1 degradation could be ascribed
merely to a requirement of the cyclinD/Cdk4/6 pathway for
subsequent cyclinE/Cdk2 activity. However, support for a
direct role for ERK MAP kinase activity is provided by
experiments in which activation of ERK in a MEK1inducible cell line leads to p27Kip1 degradation many hours
before any cyclinE/Cdk2 activity is measurable [16]. Thus
the ERK MAP kinase pathway may play a direct role in
p27Kip1 degradation. Such a role could be especially important in cells where the ERK MAP kinase pathway is
constitutively active as a consequence of oncogenic Ras
expression. Interestingly the small GTPase p21RhoA has
been shown to be involved in growth factor stimulated
degradation of p27Kip1 as blocking Rho inhibits growth factor
mediated degradation of p27Kip1 [29]. Rho signalling may be
involved in the activation of cyclinE/Cdk2 as expression of
constitutively active RhoA results in stimulation of
cyclinE/Cdk2 activity in the absence of growth factors [30•].
These results indicate a RhoA signalling pathway leading to
p27Kip1 degradation, but an alternative interpretation (see
below) is that the blockade of Rho signalling results in
elevated levels of p21Waf1, which inhibits cyclinE/Cdk2
activity and blocks p27Kip1 degradation.
How do small GTPase signal transduction pathways regulate cell cycle entry? Marshall
Although the levels of p27Kip1 are high in quiescent cells,
p21Waf1 levels are low, but show modest increases following
growth factor stimulation. Why the expression of a CKI
should increase following growth factor stimulation might be
explained by an important role for p21Waf1 and p27Kip1 as
assembly factors for Cdk4 and Cdk6 with D-type cyclins and
as providers of signals for nuclear import. As these Cdk complexes assemble they titrate out p21Waf1 and p27Kip1, and
permit activation of cyclinE/Cdk2. In some systems growth
factor stimulation of p21Waf1 levels is mediated by the ERK
MAP kinase pathway [31]. Thus activation of the ERK MAP
kinase pathway may be required to stimulate p21Waf1 expression to mediate assembly of the cyclinD1/Cdk4 complex.
However, there may be another ERK MAP kinase dependent step in assembly of cyclinD1/Cdk4 complexes [32].
Signalling events that lead to high levels of p21Waf1 cause cell
cycle arrest. Elevated levels of p21Waf1 can result from sustained high level activation of the ERK MAP kinase pathway
[33] or as a consequence of ERK MAP kinase activity under
conditions of low p21Rho signalling [34•]. The requirement
for Rho signalling may ensure that anchorage-dependent cells
only respond to activation of the ERK MAP kinase pathway
when p21Rho is in the active GTP-bound state, for example,
when cells are attached to the extracellular matrix [35].
Conclusions
It is now clear that multiple signal transduction pathways
from small GTPases impinge on the activation of the cell
cycle machinery (see Figure 2). Thus, cell cycle progression
can be viewed as an integrator of signalling pathways. This
integration arises because activation of the cell cycle
requires multiple events (e.g. D-type cyclin expression and
p27Kip1 degradation) each of which may themselves require
multiple signalling inputs. For example, cyclin D1 expression can be affected by the ERK MAP kinase, PI3-kinase,
Rac and Ral signalling pathways. The regulation of cyclin
D1 expression provides an important model, see Figure 2,
for how signal pathway integration can occur, since some
signals may regulate transcription, whereas others regulate
protein stability or translation [21••]. How much redundancy there is between different signalling pathways remains
to be determined. But it remains a reasonable hypothesis
that a requirement for the activation of multiple signalling
pathways may act as a checkpoint to ensure that cell proliferation only occurs when it is appropriate.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
••
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•
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•
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•
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Cell multiplication
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•
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•
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•
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