Small GTPases and Cell
Cycle Regulation
C.J. Marshall
CRC Centre for Cell and Molecular Biology, Institute of Cancer Research,
237 Fulham Road, London SW3 6JB, U.K.
Introduction
Novartis Medal Lecture
T h e Ras family of small GTPases was originally
discovered as viral oncogenes ; subsequent studies
demonstrated that the cellular proto-oncogenes
are mutated in a high proportion of human
tumours such as colorectal and pancreatic cancer
[l]. Oncogenic mutations create forms of the Ras
proteins that are constitutively active because they
are locked in the active GTP-bound state [2].
Since the discovery of Ras oncogenes, a central
issue in cancer research has been to understand the
function of these proteins. T h e expression of
oncogenic Ras proteins in established cell lines
liberates the cells from the requirement for growth
factors for proliferation [3] ; microinjection of
recombinant Ras proteins into quiescent cells
drives the cells into DNA synthesis [4,5]. These
experiments indicate that oncogenic Ras proteins
alter cell cycle regulation. Furthermore, classic
experiments performed by Stacey and co-workers,
with the use of microinjection of antibody to
neutralize the function of normal Ras proteins in
cells, showed that Ras is required for growth
factors to stimulate cell proliferation [ 6 ] . T h e
localization of Ras proteins to the inner surface of
the plasma membrane suggests that they are
involved in early steps of growth factor signalling
mechanisms. T h e argument that Ras protein
function was required for growth factors to stimulate cell proliferation was strengthened further by
the demonstration that the treatment of quiescent
cells with growth factors leads to the normal Ras
proteins being converted into the active GTPbound state [7,8]. T h u s Ras proteins act as a
molecular switch to transduce signals from growth
Delivered at the University of Glasgow,
on 7 April 1999
CHRIS J. MARSHALL
factor receptors to intracellular signalling pathways.
Ras and cell cycle regulation
T h e observations by Stacey and co-workers that
ablating Ras function blocks growth factors from
stimulating quiescent cells to enter the cell cycle
shows that activation of Ras is a key regulatory
event in the control of cell proliferation by growth
factors. However, these experiments did not demonstrate which events in the cell cycle Ras regulates. Control of the proliferation of mammalian
cells is a complex process. As far as we know,
the key regulatory events in growth factor stimulation of cell cycle entry take place in G , and involve the activation of the G , cyclin-dependent
Abbreviations used: CDK, cyclin-dependent kinase; CDKI, CDK
inhibitor; ER oestrogen receptor; ERK extracellular signalregulated protein kinase: GDS, guanine nucleotide dissociation
stimulator; MAP, mitogen-activated protein; MAPKK MAP kinase
kinase: P13-k, phosphoinositol 3-kinase.
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Biochemical Society Transactions ( 1999) Volume 27, part 4
kinase (CDK) complexes. These consist of the Dtype cyclins ( D l , D2 and D3) complexed to CDK4
or CDK6 and CDK2 complexed to either cyclin E
or cyclin A. In many systems the expression of the
D- and E-type cyclins is dependent on growth
factor and their expression might be important
control points on which signalling pathways activated by growth factor receptors impinge [9]. In
addition to the levels of cyclins regulating CDK
activity, the C D K inhibitors (CDKIs) exert additional control. There are two classes of these :the
p16 family (p15, p16 and p19), which act only on
CDK4 and CDK6, and the p21 family
(p21 Waf'iCtp1, ~ 2 7 ~and
' ~ p57),
'
which act on all the
G, CDKs [lo]. In quiescent growth-factor-arrested cells the levels of ~ 2 7 " ' ~are
' high and the
addition of growth factor leads to a decrease in
levels. A key biochemical activity of the G, CDKs
is to regulate through phosphorylation the function of pRb'", the product of the retinoblastoma
susceptibility locus [ l 11. In the underphosphorylated form, pRb"' binds to the E2F family of
transcription factors and prevents E2F-mediated
gene expression, either by sequestering E2F from
the promoters that it activates or by the binding of
pRb'OS-E2F complexes to DNA and the repression of gene expression [9]. Phosphorylation of
pRblo4 by G, CDKs liberates E2F from pRb'"
and thus permits gene expression that drives cells
into S-phase.
The importance of pRb"' phosphorylation to
cell cycle regulation suggests that this might be
one of the events controlled by Ras-mediated
signal transduction. To test this hypothesis we
examined whether the deletion of pRblo5would
alter the requirements for Ras-dependent signalling. Asynchronously growing mouse embryo
fibroblasts, derived from embryos in which the
pRb'05 locus had been inactivated by homologous
recombination, were injected with the Rasneutralizing antibody Y13-259 and the effects
on cell proliferation were examined. Whereas the
injection of Y13-259 into control pRb'" fmouse
embryo fibroblasts almost completely inhibited
proliferation, in the pRb'" - / - cells the inhibition was much less marked [12]. These results
suggested that a major function of Ras-dependent
signalling in cell cycle control is to regulate the
activity of the G, cyclins that phosphorylate and
inactivate pRblo5. The importance of Ras signalling to the activation of the D-type CDK
complexes is further illustrated by a similar experiment in which, instead of deleting pRb'",
mouse embryo fibroblasts were used from which
0 I999 Biochemical Society
CDKIs had been deleted. Whereas the deletion of
p21
or ~ 2 7 ~ "(G.
' D'Abaco, H. Paterson
and C.J. Marshall, unpublished work) had little
effect on the inhibition of cell proliferation after
microinjection of the Ras neutralizing antibody,
cells from which p16 had been deleted had a
decreased requirement for Ras-mediated signalling [12].
Although the deletion of pRb'" decreases the
requirements for Ras-dependent signalling it does
not completely abrogate them. That pRb'05- / cells still have a requirement for Ras-dependent
signalling can be most easily demonstrated if,
instead of injecting asynchronously growing cells
with the Ras-neutralizing antibody, quiescent
growth arrested cells are injected and then stimulated with serum growth factors. Under such
conditions the serum stimulation of DNA synthesis is completely blocked (G. D'Abaco, H.
Paterson and C.J. Marshall, unpublished work).
We interpret these experiments through a model
in which Ras-dependent signalling is required for
several events in cell cycle control. As demonstrated by Stacey and co-workers with the use of
timed injections of Y13-259, Ras function is
required for most of G, but a point is reached at
which injection no longer inhibits entry into Sphase [13]. In pRblo5--/ - cells the point at which
Ras function is no longer required might occur
earlier so that there is a smaller 'window' in which
Y 13-259 injection into asynchronous growing
cultures inhibits entry into S-phase. In the synchronous cultures produced by serum stimulation
of quiescent cells, all the cells in the culture have to
pass through the period in which Ras signalling is
required, so the injection of Y13-259 into the cells
before they are stimulated with serum completely
blocks entry into S-phase. It is possible that the
multiple events, which Ras regulates in the stimulation of cell cycle entry with growth factors, are
the activation of the cyclin D-dependent kinases
and the activation of CDK2. Results from
Nevins's laboratory show that oncogenic Ras
together with overexpressed c-Myc leads to
CDK2 expression [14]. Thus in growth-factorstimulated cells the activation of Ras might be
required for the activation of CDK2.
Ras, Raf and the extracellular signalregulated protein kinase
(ERK)/mitogen-activated protein
(MAP) kinase pathway
The first clue to the nature of the intracellular
signalling pathways that were controlled by p21 ras
3 64
Novartis Medal Lecture
insensitive to expression of the N17Ras interfering
mutant [21]. Given the hypothesis that Ras might
lie between tyrosine kinases and Raf in a signal
transduction cascade we examined whether Raf
could phosphorylate and activate MAPKK. This
experiment showed that Raf-1 was likely to be a
direct activator of a MAPKK mutant [21], a result
that was confirmed and extended when recombinant versions of Raf and MAPKK became available [22]. These experiments therefore suggested
that there was a signal transduction cascade initiated by membrane-bound tyrosine kinases that
resulted in Ras activation, which then caused Raf
to become activated, which phosphorylated and
activated MAPKK, which in turn phosphorylated
and activated the ERKs.
An important feature of signalling by the
ERK/MAP kinases is that the ERKs have both
cytosolic and nuclear substrates. Once the ERKs
have been activated by phosphorylation they
translocate to the nucleus, where they phosphorylate and activate transcription factors such as Elk-1
and Ets [23,24]. I n addition to directly regulating
the activity of pre-existing transcription factors by
phosphorylation, the activation of ERKs has a role
in the induction of expression of transcription
factors such as C-FOS,Fra-1, Fra-2 and Jun [25].
Thus the ERK/MAP kinase pathway is an important mediator of changes in gene expression. A
key regulatory event that impinges on the effects of
ERK activation in changing patterns of gene
expression is the length of time that ERK activity
persists. Although many stimuli elicit E R K activation, some give very transient activation that
persists only for a few minutes, whereas others
give activation that can last for several hours
[26,27]. For example, in PC12 phaeochromocytoma cells the transient activation of ERKs by
stimulation with epidermal growth factor does not
elicit the pattern of gene expression that causes
neurite differentiation, which results only from
the sustained activation of the pathway that follows
from stimulation with nerve growth factor [28].
Not only does the duration of ERK activation
have a role in the type of signalling events that are
activated; it is also apparent that the magnitude of
ERK activation can have profound effects. In
experiments performed with J. Smith we were
able to show that the pattern of gene expression
was altered by a 2-fold change in the amount of
activated MAP kinase/ERK kinase (Mek) injected into Xenopus eggs [29]. This result suggests
that apparently small changes in the level of
activation of the ERKs have profound effects on
came from antibody neutralization experiments in
which it was shown that tyrosine kinase oncogenes
such as v-Src required the function of normal Ras
proteins to transform cells, whereas serine kinase
oncogenes such as v-Raf or v-Mos did not seem to
require Ras function to transform cells [15]. These
results, although open to other interpretations,
suggested that Ras might act as an intermediate
between membrane-bound tyrosine kinases and
cytoplasmic serine/threonine kinases in signal
transduction pathways. T o identify kinases that
are activated in response to Ras signalling, we used
scrape-loading to introduce recombinant Ras
proteins into cells followed by in-gel renaturation
kinase assays. These studies showed that two
kinases of molecular masses 42 and 44 kDg were
rapidly activated in response to activated g a s [16].
T h e similarity in molecular mass of these kinases
to the MAP kinases, also known as ERKs, which
Ray and Sturgill [17] and others had shown were
activated in response to growth factor signalling,
led us to investigate whether Ras was an activator
of the MAP kinases. By raising antibodies against
the ERKs we were able to show that oncogenic
Ras activates ERKs [16]. Because oncogenic Ras
activated ERKs in the absence of stimulation with
growth factors, we examined whether growthfactor-mediated activation of ERKs was mediated
by the activation of endogenous Ras. Expression
of the interfering mutant of Ras, N17Ras, which
blocks the activation of Ras to the GTP-bound
state by growth factors, prevented ERK activation
mediated by epidermal growth factor, plateletderived growth factor or insulin [18]. Subsequent
studies by a number of laboratories have shown
that growth-factor-mediated activation of ERKs
is usually mediated by Ras activation. There seem
to be very few examples of ERK activation that do
not involve Ras activation; indeed, we showed
recently that the activation of ERKs by phorbol
ester, which had previously been thought to be
independent of Ras activation, is actually mediated
by activated Ras [19].
Although the introduction of oncogenic Ras
rapidly activates the ERKs, it was apparent when
these experiments were performed that Ras was
unlikely to activate the ERKs directly. Work from
P. Cohen’s and E. Krebs’s laboratories had suggested that the ERKs are themselves phosphorylated by an upstream kinase or MAP kinase kinase
(MAPKK) [20,20a]. We were able to show
that the expression of an oncogenic version of Raf
led to the activation of ERKs but, unlike the
activation of ERKs by tyrosine kinases, was
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Biochemical Society Transactions ( 1999) Volume 27, part 4
different transcription factors. Another illustration of the importance of the magnitude of ERK
activation in cell regulation is provided by experiments with regulatable versions of Raf. Such
experiments show that, although D N A synthesis
in quiescent cells can be stimulated by the activation of ERKs, if the ERKs are activated to
a greater magnitude, cell cycle arrest results
through the induction of the C D K I p2lWaf1
[30,31]. Whereas some cell types respond to
graded stimuli with a graded response of ERK
activation, in some systems a graded response
does not occur; rather, E R K activation is an
‘all-or-none’ phenomenon [32]. What determines
whether E R K activation is a graded response or an
all-or-none phenomenon will be an interesting
area of future enquiry.
Activation of Raf
T h e activation of Raf is a key component in the
activation of the ERK cascade by Ras. Work in the
laboratories of Avruch, Cooper, Downward and
Wigler showed that there was a direct interaction
between Raf-1 and Ras in the GTP-bound state
[33-361. Together with the observation that in
Ras-transformed cells Raf-1 is recruited to the
plasma membrane [37], this led us to propose that
Ras-GTP recruits Raf-1 to the plasma membrane, where it becomes activated. It was possible
Figure I
Ras-GTP recruits Raf- I to the plasma membrane for
two phosphorylation events required for activation
When Ras is in the inactive GDP-bound form, Raf- I is cytosolic
and inactive. In the presence of Ras-GTP, either owing t o the
presence of oncogenic Ras or after the stimulation of normal Ras
by growth factor, Raf- I is recruited t o the plasma membrane for
phosphorylation on serine 338 and tyrosine 34 I . Abbreviations:
RBD, Ras-GTP-binding domain of Raf- I ; CRD, cysteine-rich
domain of Raf- I ; cat, catalytic domain of Raf- I . Pak3 is likely to be
one of the serine 338 kinases [42].
Tk
ERK
0 1999 Biochemical Society
366
to test this hypothesis by targeting Raf-1 directly
to the plasma membrane; such experiments show
that membrane-targeted Raf-1 is activated independently of Ras [38,39]. Although the mechanism by which membrane recruitment activates
Raf-1 is not totally understood, it can be shown to
depend at least in part on phosphorylation at
tyrosine 341 and serine 338 [39,40]. Phosphorylation of these residues is also required for the
activation of Raf-1 by Ras-GTP, and phosphorylation at these two sites can be shown to be
dependent on the interaction between Ras and
Raf-1 [41] (Figure 1). T h u s one role of Ras-GTP
in the activation of Raf-1 is to recruit Raf-1 to the
plasma membrane to enable it to be phosphorylated at serine 338 and tyrosine 341. Work in M .
Marshall’s laboratory suggests that Pak3 is a
candidate serine 338 kinase [42]. Because Pakfamily kinases are regulated by members of the
Rac/Cdc42 family of small GTPases, these results
suggest a requirement for both Ras and Rac/
Cdc42 in the activation of Raf-1 . T h e identity of
the tyrosine kinase that phosphorylates tyrosine
341 is not known, although it is clear that Src
family kinases are strong candidates. However,
phosphorylation at serine 338 and tyrosine 341
cannot be the only role of Ras-GTP in the
activation of Raf-1 because work in M . Marshall’s
laboratory has shown that the replacement of
serine 338 and tyrosine 341 with aspartic residues,
to mimic phosphorylation, generates a Raf-1 protein with elevated basal kinase activity that still
responds to Ras-GTP by activation [40]. T h u s
there must be at least one other Ras-dependent
step in the activation of Raf-1; because Raf-1
directly targeted to the plasma membrane cannot
be further activated by Ras, it is likely that this
step depends on recruitment to the plasma membrane and takes place at the membrane.
In mammalian cells there are three Raf genes :
Raf-1, A-Raf and B-Raf; however, much of the
work attempting to study the activation of the
ERK/MAP kinase pathway has concentrated on
Raf-1 . Interestingly, there are clear differences
between the regulation of B-Raf and that of Raf-1
and A-Raf. Although Raf-1 and A-Raf require a
Ras signal and a tyrosine kinase signal for maximal
activation, B-Raf requires only a Ras signal [43].
T h e lack of a requirement for tyrosine phosphorylation for the activation of B-Raf is explained by BRaf having an aspartic residue at the position of
the critical tyrosine residues in Raf-1 and B-Raf.
Another interesting feature of the activation of BRaf is that the phosphorylation of serine 445,
Novartis Medal Lecture
turn out to be true effectors of Ras, considerable
evidence suggests that P13-ks and the Ral-GDS
family of guanine nucleotide exchange factors are
effectors of Ras. T h u s the expression of oncogenic
Ras elevates the levels of 3-phosphorylated inositol lipids [46] and Ral-GTP [47] in cells and the
expression of dominant-negative N17Ras blocks
the ability of growth factors to elevate levels of 3phosphorylated inositol lipids [46] and Ral-GTP
[48]. Dominant-negative versions of Ral have been
shown to block Ras transformation, and activated
versions of Ral and P13-k will synergize with
activated Raf to transform cells [49].
Given that Ras signalling seems to activate
multiple signal transduction pathways, a key issue
is to delineate which pathways are involved in
mediating different Ras-dependent cellular responses. A number of different approaches can be
taken to address this problem; for example, the
effects of inhibiting one arm of Ras-dependent
signalling can be investigated through the expression of dominant-negative versions of Rasdependent signalling components, or through the
use of chemical inhibitors. Extensive use of dominant-negative versions of Mek-1 [SO] and the
chemical inhibitors of Meks, PD098059 [51] and
U0126 [52], has been made to study the requirements for ERK/MAP kinase activation in signalling. For example, such studies show that ERK
activation is an important effector pathway in Rasmediated oncogenic transformation and is required for growth factors to stimulate cell proliferation [Sl]. A second approach is to express
constitutively activated versions of signalling components and study which cellular responses are
elicited [SO].
equivalent to serine 338 in Raf-1, is constitutive
and not inducible [41]. T h u s the regulation of BRaf seems to be rather simpler than that of Raf-1
and A-Raf, in which there are multiple Rasdependent events. Such results suggest that B-Raf
could be a primary target of oncogenic Ras in
tumour cells. Little work has been done to characterize activation in tumour cells, although work of
M. Rosario in this laboratory clearly shows that BRaf and Raf-1 are activated in Ras-transformed
N I H 3T3 cells.
Phosphoinositol 3-kinases (PI3-ks),
Ral-GEFs and other effectors of Ras
Although Raf-1 was the first effector of Ras to be
identified, investigations of other proteins that
bound only to ~ 2 1 ' "in
~ the active GTP-bound
state have led to the discovery of a number of
potential partners. These candidate effectors include protein kinases such as protein kinase C - ( ,
MEKK-1, the P13-k family of lipid kinases, the
Ral-guanine nucleotide dissociation stimulator
(GDS) family of guanine nucleotide exchange
factors for the Ral family of GTPases, and other
proteins of unknown function [44] (Figure 2).
Strong support for Ras signalling being mediated by multiple effectors is provided by the
genetic experiments of White and Wigler. They
modified the two-hybrid system to select for
mutants of Ras that could interact with some proteins but not others. Using this system they were
able to show that the interaction between Ras and
Raf was essential for transformation but that other
effector interactions were also required [45]. Although some of the candidate effectors might not
Figure 2
Signalling pathways to and from Ras
The activation of p2 I to the GTP-bound form leads to the activation of multiple signalling pathways. Abbreviations: API , transcription
factor complex comprising memberj ofthe Fos and Jun protein families; GEF, guanine nucleotide exchange factor; PDKI, PIP,-dependent
kinase I ; PKB. protein kinase B; PKC, protein kinase C: PL42. phospholipase A,: Ra-BPI, Ral binding protein I.
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0 1999 Biochemical Society
Biochemical Society Transactions ( I 999) Volume 27, part 4
Cell cycle entry mediated by the
activation of Mek-I
additional growth factors. However, this induction of DNA synthesis occurs with a considerable
delay compared with the induction of DNA
synthesis after the addition of serum growth
factors to tamoxifen-untreated cells. This observation suggested that one or more additional
events was taking place that was not a direct
consequence of ERK activation. Experiments with
effector mutants of Ras had suggested that the
ERK/MAP kinase signal must combine with
other signals to induce DNA synthesis [25,55].
Therefore we investigated whether the activation
of the P13-k pathway, which is known to be a key
requirement for the stimulation of DNA synthesis
by growth factors, was also required for the
induction of DNA synthesis by the activated
Mek.
Induction of DNA synthesis by the treatment
of the N I H 3T3 ER-Mek cells with tamoxifen
was completely blocked by treatment with the
P13-k inhibitor LY294008. Furthermore, as demonstrated by the activation of protein kinase B,
activation of ERKs in the N I H 3T3 ER-Mek
cells led to the activation of P13-k but with a delay
of approx. 16 h. This delay corresponded to the
induction of cyclin D1 expression, which itself
was LY 294008-sensitive. These experiments
therefore suggest that activation of ERK/MAP
kinase and P13-ks is required for the induction of
cyclin D1. To test this idea we have examined the
consequences of inhibiting ERK activation, P13-k
activation, or both, on the elevation of cyclin D1
Inhibition of the activation of the ERK/MAP
kinases by the Mek inhibitor PD098059 blocks
the ability of growth factors to stimulate DNA
synthesis in quiescent cells [51,53]. Growth factor
activation of ERKs is required for the expression
of cyclin D1, a key regulatory event in the stimulation of DNA synthesis in quiescent cells by
growth factors. Because the activation of ERKs is
necessary for the stimulation of DNA synthesis,
it is reasonable to ask whether the activation
of ERKs is sufficient to induce DNA synthesis
in the absence of other growth factor signals.
T o do this we constructed an inducible form
of a constitutively activated version of Mek-1,
which cannot be further activated by growth factor
signals. This was fused to a mutant of the oestrogen receptor (ER) that responds to tamoxifen
rather than oestrogen [25]. As originally shown
by McMahon and co-workers for Raf-1 [54],
such constructs are inactive in the absence of
steroid but become active on the addition of
steroid. When expressed in N I H 3T3 cells the
ER-Mek construct is inactive but, on the addition
of tamoxifen, ERK activity is elevated 2-%fold
within a few hours and remains elevated for at
least 48 h. If tamoxifen is added to serum-starved
growth-arrested N I H 3T3 cells expressing ERMek, the activation of Mek leads to the induction of DNA synthesis without the need for
Figure 3
Interactions between signalling pathways and GTPases in cell cycle control
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0 1999 Biochemical Society
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S
I
G2/M
@=
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expression after stimulation with growth factors.
T o ablate the induction of cyclin D1 completely,
both inhibitors are required (G. D'Abaco and C.J.
Marshall, unpublished work). This argues that the
expression of cyclin D1 integrates the signals from
two different signal transduction pathways (Figure
3). An attractive basis for this signal integration is
that the ERK/MAP kinase pathway is required to
induce the transcription of the cyclin D1 gene,
whereas the P13-k pathway regulates the stability
of the cyclin D1 protein [56].
Integration of signalling events in cell
cycle control
In addition to the induction of cyclin D1 expression requiring input from two different
signalling pathways, it is likely that other key
events in the regulation of cell proliferation require
inputs from more than one pathway. We have
shown that optimal proliferation of cytokinedependent BAF3 cells requires both the ERK/
MAP kinase pathway and the p38SAPK pathway
[57]. Although signalling pathways can co-operate
to activate key cell cycle components, other interactions between signalling pathways are possible.
For example one signalling pathway can operate to
oppose the potential growth-inhibitory properties
of another. Induction of DNA synthesis in quiescent cells by serum is dependent on the activation of
both the Ras and the Rho signalling pathways. We
have shown that a requirement for signalling
through Rho is to regulate the induction of the
C D K I p21Waf1by Ras [58]. When Rho signalling
is low, Ras induces high levels of p21
and Ras
is unable to induce DNA synthesis ; however,
when Rho signalling is high, Ras fails to induce
p2lWaf1and DNA synthesis ensues (Figure 3). In
this interaction the requirement for Rho signalling
for the induction of cell proliferation might act as
a ' checkpoint ' to ensure that activation of the Ras
pathway occurs only in conditions under which
Rho signalling is high, for example when cells are
attached.
It is an honour t o receive the Novartis Medal, which has had so
many distinguished recipients.The work of my laboratory has been
generously supported by the Cancer Research Campaign for
nearly 20 years. Without the contributions of past and present
members of my laboratory none of this work would have been
possible. I thank all of them, but especially Hugh Paterson for his
insight and countless microinjection experiments.
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