From the Departments of Pharmacological and
Physiological Science and § Molecular Microbiology and
Immunology, Saint Louis University School of Medicine,
St. Louis, Missouri 63110
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ABSTRACT |
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RhoA has been identified as an important
regulator of cell proliferation. We recently showed that the Ras/RhoA
pathway regulates the degradation of p27Kip and the
progression of Chinese hamster embryo fibroblasts (IIC9 cells) through
G1 into S phase (Weber, J. D., Hu, W., Jefcoat, S. C.,
Raben, D. M., and Baldassare, J. J. (1997) J. Biol. Chem. 272, 32966-32971). In this report, we have demonstrated that, in
IIC9 cells, RhoA regulates cyclin E/CDK2 activity, which is required
for p27Kip degradation. As previously shown in several
fibroblasts cell lines, expression of dominant-negative CDK2 in IIC9
cells blocked serum-induced cyclin E/CDK2 activity and
p27Kip degradation. In the absence of serum, expression of
constitutively active RhoA(63) resulted in significant stimulation of
cyclin E/CDK2 activity and degradation of p27Kip.
Cotransfection of dominant-negative CDK2 and RhoA(63) inhibited RhoA(63)-induced cyclin E/CDK2 activity and p27Kip
degradation. In addition, expression of dominant-negative RhoA blocked
serum-induced cyclin E/CDK2 activity and p27Kip
degradation. Finally, expression of catalytically active cyclin E/CDK2
rescued the effect of expression of dominant-negative RhoA. Taken
together, these data show that RhoA regulates p27Kip
degradation through its regulation of cyclin E/CDK2 activity.
Cyclin-dependent kinases and their inhibitors are
important for ordered progression through the cell cycle (1).
Progression through G1 into S phase requires activation of
both cyclin D/CDK4 and cyclin E/CDK2 activities (1). In addition to the
cyclins, specific inhibitory proteins (CKI proteins) associate with
distinct cyclin kinase complexes and regulate their activities. These
CKI proteins are divided into two families on the basis of sequence homology: the Cip/Kip and Ink4 families. The Cip/Kip family, which includes p21Cip (2), p27Kip1 (3, 4), and
p57Kip2 (5), inhibits CDK4, CDK6, and CDK2 activities. The
Ink4 family, which includes p16, p15, p18, and p19, specifically
inhibits CDK4 and CDK6 activities (6-8).
p27Kip is expressed in most cells and is thought to be an
important negative regulator of mitogen-induced progression through G1 (9-11). Expression of p27Kip results in
G1 arrest in a variety of cell types (12). The level of
p27Kip increases when cells are growth-arrested by contact
inhibition, mitogen withdrawal, or other anti-proliferative signals (9, 10, 14-18). Consistent with the notion that p27Kip is
important in G1 progression, mitogens stimulate elimination of p27Kip during late G1 or as cells enter
G1/S. Interestingly, decreases in p27Kip
protein levels are not a result of any changes in mRNA levels (18,
19), but mostly a result of decreased translation concomitant with
increased ubiquitin-directed degradation (18-20). Recently, considerable effort has been directed at understanding the pathway important for ubiquitin-directed degradation and the upstream signaling
pathways that regulate targeting to ubiquitin-dependent proteolysis (21-23).
Mitogens stimulate p27Kip degradation by inducing cyclin
E/CDK2 phosphorylation of p27Kip at Thr-187 (21-23).
Expression of dominant-negative CDK2 in NIH-3T3 cells blocks
serum-induced phosphorylation of p27Kip at Thr-187 and
p27Kip degradation (21). Mutation of Thr-187 to alanine
results in a stable form of p27Kip, which is resistant to
phosphorylation by cyclin E/CDK2 and degradation (21, 22) and, when
overexpressed, induces G1 arrest. Taken together, these
data strongly indicate that inhibition of p27Kip
degradation alone results in elevated levels of p27Kip and
inhibition of G1 progression.
RhoA is a member of the Ras-like small G protein superfamily (24). In
addition to its role in the formation of stress fibers and focal
adhesion (25), RhoA also regulates Ras-dependent cell cycle
progression and cell transformation (26, 27). Activation of RhoA is
required for G1 cell cycle progression in Swiss 3T3 cells
(26, 27), and expression of geranylgeranylated RhoA facilitates the S
phase entry in rat thyroid FRTL-5 cells (28). Moreover, Ras-mediated
transformation in NIH-3T3 cells requires activation of both RhoA and
Raf (26, 29-31). We previously demonstrated that overexpression of
dominant-negative Ras or dominant-negative RhoA inhibits
PDGF1-induced DNA synthesis
of IIC9 cells (32).
Recent data suggest that RhoA regulates IIC9 cell cycle progression by
stimulating p27Kip degradation. Expression of
geranylgeranylated RhoA promotes the degradation of p27Kip
in rat thyroid FRTL-5 cells (28). In IIC9 cells, we found that expression of dominant-negative RhoA inhibits p27Kip
degradation (32). In the present study, we show that RhoA-induced activation of cyclin E/CDK2 precedes decreases in p27Kip
protein amounts. This is the first study to demonstrate a requirement for RhoA in the activation of cyclin E/CDK2 activity and clearly identifies a role for RhoA in growth and transformation.
Cells and Cell Culture--
IIC9 cells, a subculture of Chinese
hamster embryo fibroblasts, were grown and maintained in Dulbecco's
modified Eagle's medium (DMEM) containing 10% (v/v) fetal calf serum.
Subconfluent cultures were growth-arrested by incubation for 2 days in
serum-free DMEM. Serum-arrested cells were washed twice and
equilibrated in basal medium for 30 min prior to the addition of serum.
Dominant-negative (dnCDK2) and wild-type CDK2 were the generous gifts
Dr. Ed Harlow. Cyclin E was a generous gift from the laboratory of Dr.
James Roberts. Dominant-negative RhoA (dnRhoA) and constitutively
active RhoA (RhoA(63)) were constructed as described previously by
site-directed mutagenesis of Thr to Asn at codon 19 or Gln to Leu at
codon 63, respectively (32).
Transient Transfection--
The cDNAs encoding wild-type
CDK2, dnCDK2, wild-type cyclin E, dnRhoA, and RhoA(63) were transfected
into subconfluent IIC9 cells using LipofectAMINETM
according to the manufacturer's protocol (Life Technologies, Inc.).
Following expression of the cDNAs by incubation for 24 h in
DMEM plus 10% serum, the cells were serum-arrested by incubation in
DMEM for 48 h before agonist stimulation. Transient transfection using LipofectAMINETM resulted in 80-90% expression
efficiency as visualized by Cyclin E/CDK2 Kinase Assay--
Subconfluent (80%)
growth-arrested IIC9 cells were stimulated with 10% fetal calf serum.
Cells were harvested at the indicated times after serum addition by
scraping into 150 µl of ice-cold lysis buffer (50 mM
Hepes, 150 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mM NaF, 0.1 mM sodium vanadate, 50 mM Western Blot Analysis--
Serum-deprived cell cultures were
incubated in the absence or presence of serum or PDGF. At the indicated
times, the samples were scraped into 150 µl of cold
phosphate-buffered saline, centrifuged at 4 °C for 5 min at
14,000 × rpm, and then lysed on ice in 30 µl of solubilization
buffer (25 mM Hepes, 300 mM NaCl, 2 mM EDTA, 1.5 mM MgCl2, 1% Triton
X-100, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml pepstatin). The samples were
subjected to brief sonication and centrifuged for 5 min at 4 °C.
Protein concentrations of the supernatants were determined by the
Bio-Rad protein assay as recommended by the manufacturer. Typically, 50 µg of lysate protein were resolved by 12% SDS-polyacrylamide gel
electrophoresis, and the separated proteins were transferred to
polyvinylidene difluoride membranes (Immobilon, Millipore Corp.,
Bedford, MA). The membranes were probed with the appropriate antibody.
Immunoreactive bands were visualized by chemiluminescence using the ECL
Western blotting system (Amersham Pharmacia) as recommended by the manufacturer.
Cyclin E/CDK2 Activation Precedes Degradation of
p27Kip--
Recent studies have demonstrated that
phosphorylation of p27Kip at Thr-187 by cyclin E/CDK2 is
required for ubiquitin-dependent degradation of
p27Kip (21-23). To determine whether cyclin E/CDK2
activity precedes serum-induced p27Kip degradation, we
examined the time course of p27Kip degradation and cyclin
E/CDK2 activity. Serum stimulated a rapid increase in cyclin E/CDK2
activity (Fig. 1A). Increased
activity was detectable within 30 min, was maximal at 17 h, and
remained elevated for 24 h (Fig. 1A). In contrast to
cyclin E/CDK2 activity, a change in p27Kip protein was not
detectable at 30 min (Fig. 1B). A significant decrease in
p27Kip could be detected at 4 h, and
p27Kip was barely detectable at 24 h (Fig.
1B). These data demonstrate that cyclin E/CDK2 activity
precedes any detectable decrease in p27KIP.
The rapid increase in cyclin E/CDK2 activity was unexpected since, in
most cell types, this activity does not increase until late in
G1 subsequent to activation of cyclin D/CDK4/CDK6.
Previously, we showed that cyclin D/CDK4 activity is not detectable
until 1-2 h after the addition of PDGF or serum (33), yet cyclin
E/CDK4 activity increased within 30 min (Fig. 1A),
indicating that, in IIC9 cells, cyclin E/CDK2 increases prior to a
detectable increase in cyclin D/CDK4 activity. The timing of an
increase in cyclin E/CDK2 in most cell types in late G1 is
likely a result of the dependence of cyclin E expression on cyclin
D/CDK4 activity (34). To understand the early activation of cyclin
E/CDK2 observed with IIC9 cells, we examined whether expression of
cyclin E in IIC9 cells was independent of cyclin D/CDK4 (Fig.
1C). Significant amounts of cyclin E were detectable in
quiescent IIC9 cells (Fig. 1C). A modest increase in cyclin
E protein was detectable within 4 h, was maximal (1.5-2-fold) at
17 h, and remained elevated for 24 h (Fig. 1C).
Cyclin E is therefore detectable in quiescent IIC9 cells and remains
elevated throughout G1 into S phase. The high level of
cyclin E, which is expressed in quiescent IIC9 cells, is markedly
different from the level of cyclin D1 protein (32, 33). Cyclin D1
protein is very low but detectable in quiescent IIC9 cells and
increases at least 5-fold with PDGF (32, 33). Significant levels of
cyclin E are present in quiescent cells, and serum stimulates only
small increases. These data suggest that there are sufficient amounts
of cyclin E in quiescent cells and indicate that the increases in this
cyclin E/CDK2 activity occur independent of increased expression of
cyclin E.
RhoA Regulates the Activity of Cyclin E/CDK2 and the Degradation of
p27Kip--
Data obtained from overexpression of
dominant-negative mutants of both RhoA and CDK2 indicate that
mitogen-induced degradation of p27Kip requires activation
of RhoA (32) and cyclin E/CDK2 (21-23). To determine whether RhoA
regulates cyclin E/CDK2 activity, we next examined the effect of
blocking RhoA activation on cyclin E/CDK2 activity and
p27Kip protein (Fig. 2).
Expression of dnRhoA inhibited serum-stimulated cyclin E/CDK2 activity
(Fig. 2A) as well as p27Kip degradation (Fig.
2B). To further demonstrate that RhoA regulates cyclin
E/CDK2 activity, IIC9 cells were treated with botulinum C3 exoenzyme,
which specifically ADP-ribosylates RhoA and blocks its function (35).
This treatment significantly inhibited serum activation of cyclin
E/CDK2 activity and the serum-induced decrease in p27Kip
(data not shown).
Our data show that cyclin E levels were significantly elevated in
quiescent IIC9 cells (Fig. 1C). Furthermore, expression of
dnRhoA inhibited serum-induced cyclin E/CDK2 activity (Fig. 2A) and p27Kip degradation (Fig. 2B).
Previously, we found that PDGF stimulates p27Kip
degradation in the absence of cyclin D expression and cyclin D/CDK4
activity (32), suggesting that, in IIC9 cells, RhoA itself regulates
the levels of p27Kip. We therefore decided to determine
whether expression of a constitutively active mutant of RhoA (RhoA(63))
stimulates cyclin E/CDK2 activity in the absence of agonist (Fig.
3A). Expression of RhoA(63)
induced a significant increase in cyclin E/CDK2 activity in the absence of serum (Fig. 3A). This activity was ~80% of the maximal
activity seen with serum-stimulated IIC9 cells. Although expression of RhoA(63) could induce cyclin E/CDK2 activity in quiescent IIC9 cells,
serum stimulated a further increase (~1.4-fold) in the activity of
cyclin E/CDK2 in IIC9 cells expressing RhoA(63) (Fig. 3A).
This increase in activity is consistent with the ability of serum to
induce a 2-fold increase in cyclin E (Fig. 1C). In addition
to increasing cyclin E/CDK2 activity and consistent with the notion
that cyclin E/CDK2 activity is necessary for p27Kip
degradation, expression of RhoA(63) reduced p27Kip protein
to levels comparable to those seen with serum stimulation (Fig.
3B). These data clearly demonstrate that expression of
constitutively active RhoA alone can markedly increase cyclin E/CDK2
activity and induce p27Kip degradation. To further examine
the ability of RhoA(63) to mediate the loss of p27Kip, we
next determined whether coexpression of dnCDK2 with RhoA(63) blocks
p27Kip breakdown. In contrast to expression of RhoA(63)
alone, coexpression of dnCDK2 and RhoA(63) did not result in increased
activity (data not shown) or a decrease in p27Kip protein
in quiescent IIC9 cells (Fig. 4). These
data are in agreement with the results demonstrating that serum-induced
degradation of p27Kip is dependent on cyclin E/CDK2
activity and suggest that cyclin E/CDK2 activity is downstream of
RhoA.
Expression of Cyclin E/CDK2 Rescues dnRhoA-dependent
Inhibition of p27Kip Degradation--
We next examined
whether expression of cyclin E/CDK2 induces the degradation of
p27Kip protein in cells expressing dnRhoA. Expression of
cyclin E/CDK2 results in significant cyclin E/CDK2 activity in
quiescent IIC9 cells (Fig. 5A)
and loss of p27Kip (Fig. 5B). These changes
occurred in the absence of cyclin D/CDK4 activity (data not shown).
Therefore, up-regulation of cyclin E/CDK2 activity itself induced
p27Kip degradation. To determine whether up-regulation of
cyclin E/CDK2 activity rescues the effects of dnRhoA, we examined
cyclin E/CDK2 activity and p27Kip levels in cells
cotransfected with dnRhoA and cyclin E/CDK2 (Fig. 6). Cyclin E/CDK2 activity was markedly
increased in cells overexpressing dnRhoA and cyclin E/CDK2 (Fig.
6A). Furthermore, p27Kip levels were reduced in
these cells (Fig. 6B). Taken together, these data strongly
suggest that RhoA is upstream of cyclin E/CDK2.
Expression of Cyclin E/CDK2 Rescues dnRhoA-induced Growth
Arrest--
Inhibition of RhoA by expression of dnRhoA or treatment
with botulinum C3 exoenzyme resulted in significant inhibition of growth (Fig. 7A). This is
consistent with the observation that RhoA is important in mammalian
cell growth and transformation (26, 27). Our data indicate that RhoA
regulates cell cycle progression at least partially through its
regulation of cyclin E/CDK2 activity and p27Kip levels. If
cyclin E/CDK2 activity is the major cell cycle downstream effector,
cotransfection of cyclin E/CDK2 should rescue the arrest observed with
expression of dnRhoA. Indeed, serum stimulated significant growth in
IIC9 cells expressing dnRhoA and cyclin E/CDK2 (Fig. 7A). In
IIC9 cells coexpressing dnRhoA and cyclin E/CDK2, serum was less
mitogenic than in wild-type IIC9 cells (Fig. 7A). However, growth under these conditions was virtually identical to growth in the
presence of a potent mitogen, PDGF (Fig. 7B), indicating that cyclin E/CDK2 activation is a target for
RhoA-dependent regulation of cell cycle progression through
G1 into S phase. Whereas expression of dnRhoA could block
PDGF-stimulated growth, overexpression of cyclin E/CDK2 rescued growth
to near identical values seen in cells treated with PDGF alone (Fig.
7B). These data demonstrate that cyclin E/CDK2 activity is a
major target of RhoA.
Observations from several independent studies suggest that
oncogenic Ras may mediate its action through both
Raf-dependent and RhoA-dependent pathways (29,
31). Expression of constitutively active Ras results in transformation
in NIH-3T3 cells; expression of constitutively active Raf or RhoA alone
only weakly transforms NIH-3T3 cells (29). Furthermore, cotransfection
with either dominant-negative Rac or RhoA blocks Ras-induced
transformation (29). Our previous studies have shown that the
Ras/Raf/mitogen-dependent protein kinase pathway is
important for the induction of cyclin D and that RhoA regulates
p27Kip degradation (32, 33). In this study, we have
demonstrated for the first time that RhoA induces p27Kip
degradation through regulation of cyclin E/CDK2 activity. Our data
therefore indicate that the Ras/Erk pathway regulates cyclin D/CDK4/CDK6 activity and that the Ras/RhoA pathway regulates cyclin E/CDK2 activity. Both these activities are crucial for passage through
G1 into S phase (Fig. 8).
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
-galactosidase staining.
-glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml
pepstatin, and 10 µg/ml leupeptin). The lysates were briefly
sonicated, and insoluble material was pelleted by centrifugation at
14,000 × g for 5 min at 4 °C. Protein concentrations were determined using the Bio-Rad protein assay according to the manufacturer's specifications. Cyclin E-CDK2 immune
complexes were isolated by incubation with anti-cyclin E antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) plus protein G. After
several washes, the cyclin E-CDK2 complexes were incubated for 45 min
at 30 °C with histone and 0.5 µCi of [
-32P]ATP.
The reactions were terminated by the addition of 5 µl of 4× Laemmli
sample buffer. The samples were boiled for 5 min and subjected to
SDS-polyacrylamide gel electrophoresis. The gels were dried, and cyclin
E/CDK2 activities were quantified by PhosphorImager analysis (Molecular
Dynamics, Inc.).
RESULTS
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Fig. 1.
Cyclin E/CDK2 activation precedes
p27Kip degradation. Growth-arrested IIC9 cells were
stimulated with 10% fetal calf serum, and cell lysates were prepared
as described under "Experimental Procedures." Cyclin E immune
complexes were immunoprecipitated from lysates containing 50 µg of
protein and assayed for their ability to phosphorylate histone protein
(Histone-P) as described under "Experimental Procedures"
(A). Similar results were obtained in four separate
experiments. Lysates containing 40 µg of protein were electrophoresed
on 12% SDS-polyacrylamide gels and immunoblotted with
anti-p27Kip1 antibody (Santa Cruz Biotechnology)
(B) or anti-cyclin E antibody (C) . Similar
results were obtained in four separate experiments.
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Fig. 2.
RhoA regulates cyclin E/CDK2
activity and p27Kip degradation. Subconfluent
IIC9 cells were transfected with 1 µg of dnRhoA as described under
"Experimental Procedures." The transfected cells were grown for
36 h in DMEM plus 10% fetal calf serum and then growth-arrested
for 48 h in DMEM. Cell lysates were isolated from growth-arrested
cells and serum-stimulated cells. A, cyclin E immune
complexes were isolated from 50 µg of cell lysate and assayed for
their ability to phosphorylate histone protein (Histone-P)
as described under "Experimental Procedures." Similar results were
obtained in four separate experiments. B, lysates containing
40 µg of protein were electrophoresed on 12% SDS-polyacrylamide gels
and immunoblotted with anti-p27Kip1 antibody. Similar
results were obtained in four separate experiments. WT, wild
type.
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Fig. 3.
Expression of RhoA(63) induces cyclin E/CDK2
activity and p27Kip degradation in quiescent IIC9
cells. Subconfluent IIC9 cells were transfected with 1 µg of
RhoA(63) and growth-arrested as described for Fig. 2. A,
cyclin E immune complexes were immunoprecipitated from lysates (50 µg
of protein) of untransfected cells. The immune complexes were assayed
for their ability to phosphorylate histone protein
(Histone-P) as described under "Experimental
Procedures." Similar results were obtained in four separate
experiments. B, lysates from samples described in
A containing 40 µg of protein were electrophoresed on 12%
SDS-polyacrylamide gels and immunoblotted with anti-p27Kip1
antibody. Similar results were obtained in three separate experiments.
WT, wild type.
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Fig. 4.
Expression of dnCDK2 blocks RhoA(63)-induced
p27Kip degradation. Subconfluent IIC9 cells were
transfected with 1 µg of RhoA(63) plus 1 µg of dnCDK2 and
growth-arrested as described for Fig. 2. Lysates containing 40 µg of
protein were electrophoresed on 12% SDS-polyacrylamide gels and
immunoblotted with anti-p27Kip1 polyclonal antibody.
Similar results were obtained in four separate experiments.
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Fig. 5.
Expression of cyclin E/CDK2 induces cyclin
E/CDK2 activity and p27Kip degradation in quiescent IIC9
cells. Subconfluent IIC9 cells were transfected with 1 µg of
cyclin E plus 1 µg of CDK2 and growth-arrested as described for Fig.
2. Lysates containing 50 µg of protein were prepared from
growth-arrested IIC9 cells or from cells stimulated for 17 h with
10% fetal calf serum as described under "Experimental Procedures."
A, cyclin E immune complexes were prepared from lysates
containing 50 µg of protein and assayed for their ability to
phosphorylate histone protein (Histone-P) as described under
"Experimental Procedures." Similar results were obtained in four
separate experiments. B, lysates (40 µg of protein) were
electrophoresed on 12% SDS-polyacrylamide gels and immunoblotted with
anti-p27Kip1 antibody. Similar results were obtained in
three separate experiments. WT, wild type.
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Fig. 6.
Expression of cyclin E/CDK2 rescues
dnRhoA-dependent inhibition of p27Kip
degradation. Subconfluent IIC9 cells were transfected with dnRhoA
alone or dnRhoA plus cyclin E/CDK2 and then growth-arrested as
described for Fig. 2. Lysates were prepared from quiescent cells or
from cells stimulated with 10% fetal calf serum for 17 h.
A, cyclin E immune complexes were isolated from lysates
containing 50 µg of protein and assayed for their ability to
phosphorylate histone protein (Histone-P) as described under
"Experimental Procedures." Similar results were obtained in four
separate experiments. B, lysates (40 µg of protein) were
electrophoresed on 12% SDS-polyacrylamide gels and immunoblotted with
anti-p27Kip1 antibody. Similar results were obtained in
four separate experiments.
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Fig. 7.
Expression of cyclin E/CDK2 rescues
dnRhoA-induced growth arrest. Subconfluent IIC9 cells were
transfected with empty vector, 1 µg of dnRhoA alone, or 1 µg of
dnRhoA plus 1 µg of cyclin and 1 µg of CDK2 and then
growth-arrested as described for Fig. 2. A, growth-arrested
cells were incubated for 17 h with DMEM alone or with DMEM plus
10% fetal calf serum. In those samples treated with botulinum C3
transferase, growth-arrested IIC9 cells were preincubated for 2 h
with 40 µg/ml botulinum C3 transferase and then treated for 17 h
with 10% fetal calf serum. Following the 17-h incubation, the cells
were assayed for [3H]thymidine as described under
"Experimental Procedures." The data indicate means ± S.D.
(n = 3). B, growth-arrested cells were
incubated for 17 h with DMEM alone or with DMEM plus 10 ng/ml
PDGF. After 17 h, the cells were assayed for
[3H]thymidine incorporation as described under
"Experimental Procedures." The data indicate means ± S.D.
(n = 3).
DISCUSSION
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Fig. 8.
Proposed summary of Ras/Erk and
Ras/RhoA pathways and their importance in G1
progression.
Several recent reports (21-23) and the data in this paper clearly demonstrate that mitogen-induced p27Kip degradation is dependent on cyclin E/CDK2 activity. Previously, we found that, in IIC9 cells, PDGF stimulates p27Kip degradation in the absence of cyclin D/CDK4/CDK6 activity (32). This is likely a result of the high level of cyclin E expressed in quiescent IIC9 cells (Fig. 1C). Interestingly, progression through G1 in these cells still requires cyclin D expression, suggesting that, in cells containing normal levels of retinoblastoma protein,2 progression through G1 requires both cyclin D- and cyclin E-dependent kinases. These results are in agreement with data that indicate that retinoblastoma protein must be phosphorylated by cyclin D-dependent kinases prior to cyclin E/CDK2 (37).
How RhoA regulates cyclin E/CDK2 activity is still an open question. Olson et al. (36) found that RhoA blocks the induction of p21Cip1, which is an inhibitor of cyclin/CDK activity and an adapter protein for assembly of D cyclins with their respective CDK proteins (37). Microinjection of constitutively active Ras into NIH-3T3 cells in the absence of active RhoA induces p21Cip1 expression and does not stimulate DNA synthesis (36). However, in the presence of active RhoA, p21Cip1 expression is repressed, and cells enter S phase and synthesize DNA. More important, in cells from transgenic mice nullizygous for the gene encoding p21Cip1, active RhoA is not required for Ras-induced DNA synthesis, suggesting that the suppression of p21Cip1 is the only role of RhoA in Ras-induced DNA synthesis. Although this is an intriguing suggestion for the role of RhoA, it does not likely account for our results and therefore is not the only function for RhoA in progression through G1. Ras-induced p21Cip1 induction is likely a result of activation of the Ras/Erk pathway (39). This suggests that Ras stimulates cyclin D and p21Cip1 expression. Previously, we showed that the Ras/Rho (but not the Ras/Erk) pathway is required for PDGF-induced p27Kip degradation (32). The data in this paper clearly show that RhoA stimulates p27Kip degradation by activation of cyclin E/CDK2. This occurs in the absence of Erk activation2 and likely in the absence of p21Cip1 expression. These distinct roles of RhoA in G1 progression may possibly be determined by careful analysis of RhoA protein with mutations of the effector-binding sites and therefore await further investigation.
Oddly, p27Kip is an inhibitor and substrate of cyclin
E/CDK2. Phosphorylation of p27Kip by cyclin E/CDK2 is
required for its degradation in G1 (21). In
vitro kinetic analysis of the association of p27Kip
with cyclin E/CDK2 suggests that p27Kip binds to cyclin
E/CDK2 with high and low affinities (21). Initially, it binds with low
affinity, and then slowly, the binding shifts to high affinity (21).
Furthermore, when it binds with low affinity, it is a substrate, and at
high affinity, an inhibitor (21). At equilibrium, p27Kip
inhibits the activity. It is possible that, in vivo, certain conditions favor high or low affinity binding and thus determine whether it is a substrate or inhibitor. RhoA may be an important regulator of this association. The present data clearly demonstrate that RhoA regulates cyclin E/CDK2 activation and identify a role for
RhoA in progression through G1 of the cell cycle.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ed Harlow for wild-type CDK2 and dnCDK2 cDNAs and Drs. James Roberts and Steve Coats for cyclin E cDNA.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant R01 DK46814 (to J. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 314-577-8543; Fax: 314-577-8233; E-mail: Baldasjj{at}slu.edu.
The abbreviations used are: PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle's medium; dnCDK2 dominant-negative CDK2, dnRhoA, dominant-negative RhoA.
2 W. Hu, C. J. Bellone, and J. J. Baldassare, unpublished data.
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REFERENCES |
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