From the Departments of Pharmacological and
Physiological Sciences and § Molecular Microbiology and
Immunology, St. Louis University School of Medicine, St. Louis,
Missouri 63104
Received for publication, January 17, 2001, and in revised form, April 12, 2001
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ABSTRACT |
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Activation of
cyclin-dependent kinase 2 (CDK2)-cyclin E in the late
G1 phase of the cell cycle is important for transit
into S phase. In Chinese hamster embryonic fibroblasts (IIC9)
phosphatidylinositol 3-kinase and ERK regulate
Cyclin-dependent kinase 2 (CDK2)1 is a member of a
family of serine/threonine kinases essential for ordered progression
through the cell cycle (2). Activation of CDK2 requires assembly of CDK2 with its regulatory subunit cyclin E. The synthesis of cyclin E
occurs late in G1 and is controlled by CDK4,6-cyclin D
activity, which increases in early G1 (3-5). CDK4,6-cyclin
D phosphorylates retinoblastoma (pRb) protein partially inactivating
its negative regulatory functions, allowing for cyclin E expression and
the activation of CDK2-cyclin E. The ordered activation of these CDKs results in the sequential phosphorylation of pRb (6-8), the activation of the E2F family of transcription factors (9, 10), and controlled progression through the G1 phase of the cell cycle.
Mitogenic stimulation of quiescent cells is essential for cyclin D
expression and activation of CDK4,6-cyclin D (11-13). In late
G1 cells become refractory to the removal of extracellular growth factor (11-13). Advancement through the restriction point requires sequential phosphorylation of pRb by CDK4-cyclin D and then by
CDK2-cyclin E (11-13). Phosphorylation of pRb by CDK2-cyclin E results
in full inactivation of pRb (11-13). Although the only known substrate
for CDK4-cyclin D is pRb, CDK2-cyclin E affects G1
progression by phosphorylating substrates other than pRb. For example,
in cells in which pRb is inactivated by expression of SV40 large T
antigen, inactivation of CDK2-cyclin E still results in G1
arrest (14). However, inactivation of CDK4-cyclin D is without effect.
In addition, although mice in which the coding sequences of the cyclin
D1 gene are deleted show several developmental abnormalities, including
hypoplastic retinas, replacement of the missing cyclin D1 coding
sequences with cyclin E rescues the wild type phenotypes (15). In these
mice expression of cyclin E is under control of the cyclin D1 promoter
and, therefore, independent of regulation by Rb phosphorylation.
Furthermore, protein lysates from the developing retinas of the cyclin
E "knockin" mice show only 20% of the levels of Rb phosphorylation
when compared with wild type (15). These data indicate that ectopic
expression of cyclin E can rescue the wild type phenotype without the
need for full inactivation of pRb (15). Furthermore, passage of
pRb Activation of CDK2 is regulated by dephosphorylation of
Thr14 and Tyr15 by cdc25A (16) and
phosphorylation of Thr160 by CDK-activating enzyme
(CAK) (17, 18). Ectopic expression of cdc25A shortens the time
of G1 by dephosphorylating CDK2-cyclin E (19). Because both
cdc25A and CAK are localized to the nucleus, translocation of
CDK2-cyclin E to the nucleus is important for interaction of the
CDK2-cyclin E complex with these regulatory factors. The mechanism
responsible for CDK2 translocation from the cytoplasm to the nucleus is
still poorly understood. In this study we show ERK activity regulates CDK2-cyclin E activity
independent of its effect on cyclin D1 expression and CDK4-cyclin D1
activity. Both ERK and PI 3-kinase are essential for cyclin E
expression by their effects on the expression of cyclin D1 and
CDK4-cyclin D1 activation (1). Although transient expression of cyclin
E rescues Cell Culture and Reagents--
IIC9 cells were maintained in
Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and
2 mM L-glutamine (BioWhittaker, Walkersville,
MD) supplemented with 5% (v/v) fetal calf serum, 100 units/ml
penicillin, and 100 mg/ml of streptomycin (all from Sigma).
Growth-arrested (Go) cells were established by
washing subconfluent (80%) cells once with phosphate-buffered saline
(PBS) followed by a 48-h incubation with Transient Transfections--
IIC9 cells were grown to
subconfluency (80%) in Dulbecco's modified Eagle's medium containing
4.5 g/liter glucose and 2 mM L-glutamine
(BioWhittaker) supplemented with 5% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 mg/ml of streptomycin (Sigma). The cells
were transfected with a solution containing 5 µl/ml
PlusTM, 4 µl/ml LipofectAMINETM (Life
Technologies, Inc.), and 2 µg/ml DNA in Opti-MEM medium (Life
Technologies, Inc.) following the manufacturer's protocol. 5 h
post-transfection, Dulbecco's modified Eagle's medium supplemented with calf serum (final serum concentration of 0.1% v/v), 100 units/ml penicillin, and 100 mg/ml of streptomycin were added. After 12 h
the cells were growth-arrested for 24 h prior to stimulation. Transfection efficiency was determined by co-transfecting the cells
with BiogreenTM pRK5 (Pharmingen, San Diego, CA), and
quantitating the number of transfected cells using a fluorescence microscope.
Western Blot Analysis--
Growth-arrested IIC9 cells were
incubated in the presence or absence of 1 units/ml CDK2-Cyclin E Assay--
Growth-arrested IIC9 cells were
incubated in the absence or presence of 1 units/ml CDK4-Cyclin D1 Assay--
Growth-arrested IIC9 cells were
incubated in the absence or presence of 1 units/ml Thymidine Incorporation--
Growth-arrested IIC9 cells were
incubated in the absence or presence of 1 units/ml Immunocytochemistry--
Growth-arrested IIC9 cells grown on
chamber slides (Nalgeen® Labware, Rochester, NY) were
incubated in the absence or presence of 1 units/ml Co-immunoprecipitations--
Growth-arrested IIC9 cells were
incubated in the presence or absence of 1 units/ml Both ERK and PI 3-kinase Activities Are Required for CDK2-Cyclin E
Activity--
In IIC9 cells ERK and PI 3-kinase activities are
essential for Ectopic Expression of Cyclin E Rescues the Inhibition of
CDK2-Cyclin E Activity by Inhibitors of PI 3-kinase but Not
ERK--
Because the synthesis of cyclin E is essential for
CDK2-cyclin E activity, we reasoned that ectopic expression of cyclin E would rescue the inhibition of CDK2-cyclin E activity that is seen with
treatment with either LY294002 or PD98059 (Fig. 1B). Consistent with this hypothesis, replacing the cyclin D1 gene with
cyclin E gene restores the wild type phenotype in animals deficient in
cyclin D1 expression (15). We next examined whether the lack of
CDK2-cyclin E activity is rescued by ectopic cyclin E expression and,
therefore, is dependent on the decrease in cyclin E protein levels that
we observed in the presence of either LY294002 or PD98059. In IIC9
cells transient expression of cyclin E results in marked increases in
the amounts of cyclin E protein in the presence or absence of either
PD98059 or LY294002 (Fig. 2). Ectopic expression of cyclin E rescues the LY294002-induced decrease in CDK2-cyclin E activity (Fig. 2) as determined by in vitro
histone phosphorylation. Surprisingly, expression of cyclin E does not rescue the inhibition of CDK2-cyclin E activity by PD98059 (Fig. 2),
although the levels of cyclin E are similar to those in cells treated
with LY294002 (Fig. 2). These data suggest that ERK activity has an
additional role in regulating CDK2-cyclin E activity, in addition to
controlling cyclin E expression via its effect on cyclin D1 levels
(22-25).
Expression of Cyclin E Rescues Growth in IIC9 Cells in Which
CDK2 Nucleocytoplasmic Translocation Is Dependent upon ERK
Activity--
Subcellular compartmentalization of protein complexes is
an underlying regulatory process that affects many cellular events. The
subcellular distribution of CDKs and their regulatory cyclins is an
important factor for regulating their activities and their ability to
control cell cycle progression. The nucleocytoplasmic redistribution of
CDK2 is essential for activation, and indeed both the regulatory CAK
required for Thr160 phosphorylation and cdc25A, the
phosphatase responsible for dephosphorylation of Thr14 and
Tyr15, are constitutively located in the nucleus (26, 27).
To determine whether the inability of ectopically expressed cyclin E to
rescue CDK2-Cyclin E Complex Formation Is Independent of PI 3-kinase and
ERK--
The association of CDK2 with the regulatory subunit, cyclin
E, is a rate-limiting step in the formation of active CDK2-cyclin E
complexes (28, 29), indicating that formation of these complexes with
cyclin E in late G1 are essential for activation. At
present little is known concerning the formation of CDK2-cyclin E
complexes in vivo. A possible explanation for the ability of
expression of cyclin E to rescue CDK2-cyclin E activity in cells in
which PI 3-kinase activity is blocked, but is ineffectual when ERK
activation is prevented, is that ERK plays an important role in complex
formation in vivo. We investigated this possibility by
determining whether endogenous CDK2 associates with ectopically
expressed cyclin E (Fig. 5). Cyclin E
immunoprecipitates from ERK Is Essential for CDK2-Cyclin E Activity Independent of Its
Effect on CDK4-Cyclin D1--
Activation of CDK2-cyclin E in late
G1 enables cells to progress into S phase and replicate
cellular DNA (14, 30, 31). Because cyclin E expression depends on
CDK4-cyclin D1 activity, CDK2-cyclin E activity is downstream of
CDK4-cyclin D1 (1). Accumulation of cyclin D1 is controlled by both ERK
(23-25) and PI 3-kinase (1, 32, 33); therefore both signaling pathways are important for mitogen activation of cyclin D1 expression and CDK4-cyclin D1 activity. Furthermore, these pathways have similar effects on pRb inactivation, cyclin E expression, and CDK2-cyclin E
activation. Because of their similar effects on cyclin E expression, we
expected that ectopic expression of cyclin E would rescue the inhibition of The Role of ERK in CDK2-Cyclin E Nucleocytoplasmic
Translocation--
It is likely that the effect of ERK on CDK2-cyclin
E translocation to the nucleus is not solely a direct effect as has
recently been proposed (35). These investigators put forward that
nuclear translocation of CDK2 is associated with complexes containing active ERK. In this scheme CDK2-cyclin E associates with active ERK and
is carried into the nucleus along with ERK, which acts as a nuclear
transport factor. In IIC9 cells CDK2 translocation occurs several hours
after the addition of -thrombin-induced G1 transit by their effects on cyclin
D1 protein accumulation (Phillips-Mason, P. J., Raben,
D. M., and Baldassare, J. J. (2000) J. Biol.
Chem. 275, 18046-18053). Here, we show that ERK also affects
CDK2-cyclin E activation by regulating the subcellular localization of
CDK2. Ectopic expression of cyclin E rescues the inhibition of
-thrombin-induced activation of CDK2-cyclin E and transit into S
phase brought about by treatment of IIC9 cells with LY29004, a
selective inhibitor of mitogen stimulation of phosphatidylinositol
3-kinase activity. However, cyclin E expression is ineffectual in
rescuing these effects when ERK activation is blocked by treatment with
PD98059, a selective inhibitor of MEK activation of ERK.
Investigation into the mechanistic reasons for this difference found
the following. 1) Although treatment with LY29004 inhibits
-thrombin-stimulated nuclear localization, ectopic expression of
cyclin E rescues CDK2 translocation. 2) In contrast to treatment with
LY29004, ectopic expression of cyclin E fails to restore
-thrombin-stimulated nuclear CDK2 translocation in IIC9 cells
treated with PD98059. 3) CDK2-cyclin E complexes are not affected by
treatment with either inhibitor. These data indicate that, in addition
to its effects on cyclin D1 expression, ERK activity is an important controller of the translocation of CDK2 into the nucleus where it is activated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/
cells through G1 still requires
CDK2-cyclinE activation suggesting CDK2-cyclin E phosphorylates targets
other than pRb, and these are essential for progression through
G1 into S (14).
-Thrombin, a potent mitogen of IIC9 cells,
is a serine protease that activates a G-protein-coupled receptor known
as PAR-1 (protease-activated receptor) (20). Receptor activation and subsequent
dissociation of the
-GTP subunit from the
subunits results in
coordinate stimulation of several mitogenic signaling pathways, cell
cycle re-entry, and progression through the cell cycle (21). Recent data from our laboratory (1) demonstrate a role for ERK and phosphatidylinositol (PI) 3-kinase activities in cell growth. Interestingly, both ERK and PI 3-kinase regulate the expression of
cyclin D1 and activation of CDK4-cyclin D1 activity in Chinese hamster
embryonic fibroblasts (IIC9).
-thrombin-induced CDK2-cyclin E activity and
G1 progression in IIC9 cells treated with LY294002, an
inhibitor of PI 3-kinase activation, transient expression of cyclin E
is without effect in IIC9 cells treated with PD98059, an inhibitor of
MEK activation of ERK. We also provide evidence that the ectopically
expressed cyclin E associates with CDK2 in cells treated with PD98059,
but the complex does not translocate into the nucleus. These data
clearly demonstrate that in addition to its ability to control cyclin
D1 expression, ERK also regulates CDK2-cyclin E nucleocytoplasmic translocation.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-MEM medium
containing 2 mM L-glutamine (BioWhittaker)
supplemented with 100 units/ml penicillin and 100 mg/ml of streptomycin
(basal medium). Growth-arrested IIC9 cells were stimulated with
1 unit/ml of human
-thrombin and incubated for the indicated times
at 37 °C in 5% CO2. PD98059 (New England Biolabs,
Beverly, MA) was used at 15 µM. LY294002 (Calbiochem) was
used at 10 µM.
-thrombin after
pretreatment in the presence or absence of 10 µM LY294002
or 15 µM PD98059 for 30 min. At the indicated times,
cells were washed twice with cold PBS and lysed in cold lysis buffer
(50 mM HEPES, pH 7. 5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 2 µM sodium vanadate, 20 mM sodium
fluoride, 50 µM
-glycerolphosphate, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). The lysates
were sonicated briefly, and insoluble material was pelleted by
microfugation at 14,000 × g at 4 °C for 4 min.
Protein concentrations were determined using Coomassie®
Plus (Pierce) as recommended by the manufacturer. Protein lysates (15-20 µg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Boston, MA) as recommended by the manufacturer. Membranes were
probed with polyclonal antibodies to cyclin D1, CDK2, CDK4 (Santa Cruz
Biotechnology, Santa Cruz, CA), and cyclin E (Upstate Biotechnology,
Lake Placid, NY). Immunoreactive bands were visualized by ECL detection
(Amersham Pharmacia Biotech) as recommended by the manufacturer.
-thrombin after
preincubation in the absence or presence of 10 µM
LY294002 or 15 µM PD98059 for 30 min. After 17 h,
cells were washed twice with cold PBS and lysed in cold lysis buffer
(50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 2 µM sodium
vanadate, 20 mM sodium fluoride, 50 µM
-glycerolphosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and
10 µg/ml pepstatin). The lysates were sonicated briefly, and
insoluble material was pelleted by microfugation at 14,000 × g at 4 °C for 4 min. Protein concentrations were
determined using Coomassie® Plus (Pierce) as recommended
by the manufacturer. Cell lysates (50 µg protein) were incubated with
2 mg of polyclonal CDK2 antibody (Santa Cruz Biotechnology) at 4 °C
with gentle rocking for 2 h. The CDK2 immune complexes were then
immunoprecipitated by overnight incubation with protein G-agarose
(Sigma) at 4 °C with gentle rocking. The CDK2 immune complexes were
pelleted by microfugation at 14,000 × g and washed
four times with cold PBS supplemented with 1 mM PMSF, 2 µM sodium vanadate, 20 mM sodium fluoride, 50 µM
-glycerolphosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin and further washed twice
with cold wash buffer (50 mM HEPES, pH 7.5, 1 mM dithiothreitol, and 10 mM
MgCl2). The CDK2 immune complexes were resuspended in 30 ml
of reaction buffer (50 mM HEPES, pH 7.5, 1 mM
dithiothreitol, 10 mM MgCl2, 2.5 mM
EGTA, 10 µM
-glycerolphosphate, and 20 µM ATP) and incubated with 4 µg of histone H1
(Calbiochem) and 0.5 µCi of [32P-
]ATP at 30 °C
for 45 min. The reaction was stopped by the addition of 10 µl of 4×
Laemmli sample buffer. Samples were subjected to SDS-polyacrylamide gel
electrophoresis. The gels were dried, and CDK2-cyclin E activity was
quantified using a PhosphorImagerTM (Molecular Dynamics).
-thrombin after
preincubation in the absence or presence of 10 µM
LY294002 or 15 µM PD98059 for 30 min, and CDK4-cyclin D1
was assayed as previously reported (22, 23).
-thrombin for
17 h after preincubation in the absence or presence of 10 µM LY294002 or 15 µM PD98059 for 30 min.
Following the 17 h incubation, 1 µCi/ml
[3H]thymidine (PerkinElmer Life Sciences) was
added to the cells for an additional 3-h incubation.
3H-Labeled cells were washed twice with cold PBS, and the
DNA was precipitated by incubating the cells in 5% (v/v)
trichloroacetic acid for 30 min at 4 °C. The trichloroacetic
acid-precipitated DNA was washed twice with cold 5% trichloroacetic
acid and solubilized with 500 ml of 2% (w/v) sodium bicarbonate
in 0.1 N NaOH. The solution was neutralized with 100 µl of 5%
trichloroacetic acid, and the trichloroacetic acid-precipitated was
[3H]DNA quantified by scintillation counting.
-thrombin for
17 h after preincubation in the absence or presence of 10 µM LY294002 or 15 µM PD98059 for 30 min.
Subsequent to activation, the cells were fixed in a 3.7% Formalin
(Sigma) solution for 10 min at room temperature followed by a 6-min
incubation in ice-cold methanol at
20 °C. The cells were washed in
PBS and then blocked in 1 ml of blocking buffer (0.8 g of fatty
acid-free bovine serum albumin (Sigma) in 100 µl of PBS) for 2 h
at room temperature. Polyclonal CDK2 antibody (Santa Cruz
Biotechnology) was added at a 1:35 dilution (antibody:blocking buffer)
and incubated at room temperature for 2 h. The cells were washed
three times with PBS. The secondary antibody, Texas Red-linked
anti-monoclonal Ig (Amersham Pharmacia Biotech), was added at 1:50
dilution in blocking buffer for 45 min at room temperature. Again the
cells were washed three times with PBS and then mounted using gel mount (Biomeda Corp., Foster City, CA) and microscope coverslips (Fisher). Images were visualized using a fluorescent microscope.
-thrombin after
pretreatment in the presence or absence of 10 µM LY294002
or 15 µM PD98059 for 30 min. At the indicated times,
cells were washed twice with cold PBS and lysed in cold lysis buffer
(50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 2 mM sodium
vanadate, 20 mM sodium fluoride, 50 µM
-glycerolphosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and
10 µg/ml pepstatin). The lysates were sonicated briefly, and the
insoluble material was pelleted by microfugation at 14,000 × g at 4 °C for 4 min. Protein concentrations were
determined using Coomassie® Plus (Pierce) as recommended
by the manufacturer. Protein lysates (100 µg) were incubated with 5 µg of polyclonal cyclin E antibody (Upstate Biotechnology) or
monoclonal cyclin D1 antibody (Santa Cruz Biotechnology) at 4 °C
with gentle rocking for 2 h. The immune complexes were then
immunoprecipitated by overnight incubation with protein G-agarose
(Sigma) at 4 °C with gentle rocking. The immune complexes were
pelleted by microfugation at 14,000 × g and washed
four times with cold PBS supplemented with 1 mM PMSF, 2 mM sodium vanadate, 20 mM sodium fluoride, 50 µM
-glycerolphosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin and further washed twice
with cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA,
0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM PMSF, 2 mM sodium vanadate, 20 mM sodium fluoride, 50 µM
-glycerolphosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). Immune complexes were
resuspended in cold lysis buffer, resolved by SDS-polyacrylamide gel
electrophoresis, and transferred to a polyvinylidene difluoride membrane (Millipore Corp.) as recommended by the manufacturer. Membranes were probed with polyclonal antibodies to CDK2 or CDK4 (Santa
Cruz Biotechnology). Immunoreactive bands were visualized by ECL
detection (Amersham Pharmacia Biotech) as recommended by the manufacturer.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-thrombin-induced cyclin D1 expression, CDK4-cyclin D
kinase activity, and cell growth (1). Because pRb, which is the only known substrate of CDK4-cyclin D, binds to and represses the E2F family
of transcription factors (9), and the cyclin E gene promoter is under
E2F regulation, we expect that cyclin E expression and CDK2-cyclin E
activity will be blocked in cells treated with either PD98059, an
inhibitor of MEK activation of ERK, or LY294002, a selective inhibitor
of PI 3-kinase (Fig. 1). We initially
examined by Western blot analysis the time course of cyclin E protein
expression (Fig. 1A). Cyclin E protein increases ~8 h
after
-thrombin addition reaching maximal protein levels after
24 h (Fig. 1A). Consistent with the observed increases
in cyclin E protein, CDK2-cyclin E activity does not increase until 10 to 12 h (Fig. 1A). We next examined the effects of the
inhibition of ERK and PI 3-kinase activities on cyclin E expression and
CDK2-cyclin E activity. As found previously (1), pretreatment with
either PD98059 or LY294002 dramatically prevents
-thrombin-mediated
cyclin D1 expression and CDK4-cyclin D1 activity (data not shown).
Consistent with the prevailing notion that the phosphorylation of pRb
by CDK4-cyclin D is essential for cyclin E expression, pretreatment
with PD98059 or LY294002 blocks
-thrombin-stimulated cyclin E
expression (Fig. 1B). As expected, treatment with either
inhibitor prevents
-thrombin-induced CDK2-cyclin E stimulation (Fig.
1B).
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Fig. 1.
Treatment with PD98059 and LY294002 inhibits
cyclin E protein accumulation and CDK2-cyclin E activity.
Growth-arrested IIC9 cells were stimulated with 1 units/ml -thrombin
for 0, 4, 8, 12, 17, or 24 h. At the indicated times cells lysates
were prepared by scraping into cold lysis buffer (see "Materials and
Methods"). A, an aliquot from each time point was analyzed
for cyclin E protein accumulation or CDK2-cyclin E activity. Lysate
proteins (25-35 µg) were separated by SDS-polyacrylamide gel
electrophoresis (9.75%) and immunoblotted with a polyclonal cyclin E
antibody, or CDK2-cyclin E complexes were immunoprecipitated from
lysates containing equal amounts of protein with a polyclonal CDK2
antibody, and CDK2-cyclin E activity was assayed for their ability to
phosphorylate histone H1 protein in vitro as described under
"Materials and Methods." B, growth-arrested IIC9 cells
were stimulated with 1 units/ml
-thrombin for 17 h in the
absence or presence of 10 µM LY294002 or 15 µM PD98059. Cyclin E levels and CDK2-cyclin E activities
were assayed as described above. The data are representative of three
independent experiments
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Fig. 2.
Ectopic expression of cyclin E rescues the
inhibition of CDK2-cyclin E activity by inhibitors of PI 3-kinase but
not ERK. Growth-arrested IIC9 cells were stimulated with 1 units/ml -thrombin for 17 h in the absence or presence of 10 µM LY294002 or 15 µM PD98059. Where
indicated, cells were transiently transfected with cyclin E as
described under "Materials and Methods." Cells were harvested by
scraping into cold lysis buffer (see "Materials and Methods").
Lysates were assayed for cyclin E and CDK2 protein expression. Lysates
were also assayed for CDK2-cyclin E activity. Lysate proteins (15-35
µg) were separated by SDS-polyacrylamide gel electrophoresis (9.75%
or 12.75%) and immunoblotted with polyclonal cyclin E or CDK2
antibodies. CDK2-cyclin E complexes were immunoprecipitated from
lysates containing equal protein with a polyclonal CDK2 antibody and
assayed for CDK2-cyclin E activity as described under Fig. 1. The data
are representative of three independent experiments.
-Thrombin-induced PI 3-kinase but Not ERK Activation Is
Blocked--
Ectopic expression of cyclin E rescues CDK2-cyclin E
activity in cells in which cyclin D expression and CDK4-cyclin D1
activity are repressed. To address the effects of the activation of
CDK2-cyclin E in the absence of CDK4-cyclin D1 activity on cell cycle
progression, we examined the ability of expression of cyclin E to
rescue cell cycle re-entry and G1 progression in cells
treated with the PI 3-kinase or ERK inhibitors (Fig.
3). As previously shown (1), treatment of
IIC9 cells with either PD98059 or LY294002 blocks growth as determined
by [3H]thymidine incorporation (Fig. 3). Although ectopic
expression of cyclin E rescues
-thrombin-stimulated growth in IIC9
cells treated with LY294002, it is ineffective in cells in which ERK activity is blocked by pretreatment with PD98059 (Fig. 3). These data
are consistent with the observation that in mice in which the coding
sequences of the cyclin E gene replace those of cyclin D1 the mice
appear to be normal (15). Furthermore, these results suggest that ERK
activation controls CDK2-cyclin E activity independent of its effects
in regulating cyclin D1 expression and CDK4-cyclin D1 activation.
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Fig. 3.
Expression of cyclin E rescues growth in IIC9
cells in which -thrombin-induced PI 3-kinase
but not ERK activation is blocked. Growth-arrested IIC9 cells were
stimulated with 1 units/ml
-thrombin for 17 h in the absence or
presence of 10 mM LY294002 or 15 mM PD98059.
Where indicated, cells were transiently transfected with cyclin E. Cells were then incubated for an additional 3 h with 1 µCi/ml of
[3H]thymidine. Cells were washed, and DNA was
precipitated as described under "Materials and Methods."
[3H]DNA was quantified by scintillation counting. The
data are representative of three independent experiments.
-thrombin-induced CDK2-cyclin E activation in the presence of PD98059 is a result of the failure of CDK2 to translocate to the nucleus, we examined the effects of both PD98059 and LY294002 on CDK2
redistribution in
-thrombin-stimulated IIC cells transiently transfected with cyclin E (Fig. 4). In
serum-arrested IIC9 cells CDK2 is detected in the cytoplasm (Fig.
4A). We next asked whether translocation of significant
amounts of CDK2 is seen in cells stimulated with
-thrombin for
17 h. We selected 17 h, because significant CDK2-cyclin E
activity is found at this time (Fig. 1A). After 17 h
significant amounts of CDK2 are found in the nucleus (Fig.
4B). In IIC9 cells stimulated with
-thrombin but
pretreated with either PD98059 or LY294002, CDK2 is localized
exclusively to the cytoplasm (Fig. 4, C and E).
In agreement with the ability of ectopic expression of cyclin E to
rescue CDK2-cyclin E activity in IIC9 cells treated with LY294002,
ectopic expression of cyclin E induces significant nuclear localization
of CDK2 in
-thrombin-stimulated cells treated with LY294002 (Fig.
4D). However, ectopic cyclin E expression is ineffective in
rescuing CDK2 translocation in cells preincubated with PD98059 (Fig.
4F). Thus, ERK promotes CDK2-cyclin E activation by two
mechanisms, the regulation of CDK4-cyclin D1 activity, which in turn
controls the synthesis of cyclin E, and the translocation of the
CDK2-cyclin E complex into the nucleus.
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Fig. 4.
CDK2 nucleocytoplasmic translocation is
dependent on ERK. Growth-arrested IIC9 cells were stimulated with
1 units/ml -thrombin (B-F) for 17 h in the absence
of (B) or presence of 10 µM LY294002
(C and D) or 15 µM PD98059
(E and F). Where indicated, cells were
transiently transfected with cyclin E (D and F).
Cells were fixed in a 3.7% Formalin solution (see "Materials and
Methods"), and CDK2 was visualized with a fluorescent microscope. The
data are representative of three independent experiments.
-thrombin-stimulated IIC9 cells
overexpressing cyclin E contain CDK2 as detected by Western blot
analysis (Fig. 5, lane 2). To ensure the selectivity of the
association of ectopically expressed cyclin E with CDK2, we examined
the ability of overexpressed cyclin E to bring down endogenous CDK4.
Although IIC9 cells contain levels of endogenous CDK4 comparable with
endogenous CDK2 (data not shown), CDK4 is undetectable in cyclin E
immunoprecipitates (Fig. 5, lanes 2-4). Furthermore, in
cells ectopically expressing cyclin D1, cyclin D1 immunoprecipitates
contain CDK4 but not CDK2 (Fig. 5, lane 1). We next asked
whether in cells stimulated with
-thrombin treatment with either
LY294002 or PD98059 affects the association of ectopically expressed
cyclin E with CDK2. In the presence of LY294002 and PD98059 cyclin E
immunoprecipitates contain levels of CDK2 comparable with that found in
the absence of the inhibitors (Fig. 5, compare lane 2 with
lanes 3 and 4). Taken together these data
indicate that in the presence of
-thrombin these inhibitors do not
affect the association of CDK2 with cyclin E.
View larger version (9K):
[in a new window]
Fig. 5.
CDK2-cyclin E complex formation is
independent of PI 3-kinase and ERK. Growth-arrested IIC9 cells
were stimulated with 1 units/ml -thrombin for 17 h following
preincubation in the absence or presence of 10 µM
LY294002 or 15 µM PD98059. Where indicated, cells were
transiently transfected with cyclin E or cyclin D1. Cells were
harvested by scraping into cold lysis buffer (see "Materials and
Methods"). Cyclin E or cyclin D were immunoprecipitated from lysates
containing equal protein with a polyclonal antibody for cyclin E or
monoclonal cyclin D antibody, respectively. Proteins from each
immunoprecipitate were separated by SDS-polyacrylamide gel
electrophoresis (12.75%) and immunoblotted with a polyclonal antibody
to either CDK2 or CDK4. The data are representative of three
independent experiments.
-thrombin-induced CDK2-cyclin E activity observed with
pretreatment with either inhibitor. However, whereas the ectopic
expression of cyclin E rescues the effects of the inhibition of PI
3-kinase on growth and CDK2-cyclin E activity, it is ineffectual in
rescuing the inhibition of CDK2-cyclin E seen when ERK activation is blocked. These data suggest an additional role for ERK in the regulation of translocation of CDK2-cyclin E into the nucleus where it
is activated by dephosphorylation by cdc25A and phosphorylation by CAK
(34).
-thrombin. However, mitogens including
-thrombin (36, 37) activate ERK and induce ERK translocation to the
nucleus within 10 to 15 min. Importantly, in cells in which cyclin E is
ectopically expressed prior to
-thrombin addition, the earliest we
are able to detect CDK2 translocation is ~12 h after
-thrombin
stimulation (data not shown). This suggests that CDK2 translocation is
dependent on some other function of ERK, possibly its effect on
Elk-1-dependent transcriptional activation. Therefore, we
propose it likely that ERK activation results indirectly in the
translocation of CDK2-cyclin E. Although the mechanism of CDK2 nuclear
translocation awaits further investigation, the data in this paper
clearly show that there is a role for ERK activity, in addition to
regulation of cyclin E expression.
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FOOTNOTES |
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* 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; E-mail: baldasjj@slu.edu.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M100409200
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; pRb, phosphorylated retinoblastoma; CAK, CDK-activating enzyme; ERK, extracellular signal-regulated kinase; PI, phosphatidylinositol; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.
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