 |
INTRODUCTION |
The basic cell cycle machinery is composed of regulatory
cyclin subunits complexed to cyclin-dependent kinase
(cdk)1 subunits (1). When
active, the cyclin·cdk complex phosphorylates specific substrates in
a cell cycle-dependent manner. Cyclin E·cdk2 is active in
mid G1 close to the restriction point, cyclin
A·cdk2 from the beginning of S to M, whereas cyclin B·cdk1 is
active at the G2-M transition (2). After binding of the
cyclin to the cdk, the resulting heterodimer remains inactive because
of the presence of inhibitory phosphorylation and/or lack of activating phosphorylation. cdk2 contains two sites of inhibitory phosphorylations on threonine 14 and on tyrosine 15. It also contains a site for activating phosphorylation on threonine 160 (3). The activation of
cyclin E or A·cdk2 requires the dephosphorylation of Thr-14 and
Tyr-15 and the phosphorylation of Thr-160. The former is accomplished through the action of the dual specificity phosphatase CDC25 (4) and
the latter through the action of the Cdk-activating enzyme; CAK (5).
Inhibitory proteins, which associate with and inactivate the kinase,
further regulate cyclin·cdks (6). p21 and its close relatives p27 and
p57 belong to this class of cdk inhibitors (CKIs).
Regulation of cdk in response to different stimuli has until now been
described at the level of CKI induction or changes in the inhibitory
phosphorylation on Thr-14 and Tyr-15. Following gamma radiation or
exposure to other DNA-damaging agents, p21 is induced in a
p53-dependent manner to inhibit cdk2 and block the cell
cycle at the G1-S transition (7). Intra-S phase checkpoints activated by DNA damage and mitotic checkpoints induced by DNA damage
or incomplete DNA replication lead to inhibitory phosphorylation of
cdks (cdk2 or cdk1, respectively) on Thr-14 and Tyr-15. In contrast,
regulation of a cyclin·cdk complex at the level of Thr-160 phosphorylation appears to be rare.
Because of the pivotal role of cyclin E or A·cdk2 in cell cycle
progression, molecules that inhibit their activities are likely to have
antineoplastic properties. Inhibitors of the rate-limiting enzyme in
cholesterol synthesis, hydroxymethyl-glutaryl-CoA reductase, have
growth deterrent effects in a number of normal and cancer cell lines
(8, 9). Collectively known as "statins," these drugs are used for
treatment of hypercholesterolemia (10). One explanation for their
growth-altering effects is that the lack of isoprenylation of key
regulatory proteins like Ras, Rap, and others leads to defective
subcellular localization of these key proteins. Lovastatin, however,
inhibits cdk2 through a ras-independent pathway (11). Growth
inhibition was accompanied by an induction of CKIs, p21, and/or p27
(9), due to the inhibition of proteasomes by statins (12). The
increased association of p21 with cyclin·cdk2 that accompanies the
cdk2 kinase inhibition is believed to account for the cell cycle block.
However, definitive evidence that p21 and/or p27 are necessary for
growth inhibition by statins has been lacking, and alternate pathways
by which statins could inhibit cdks have not been explored.
The studies reported here evaluate the molecular mechanism of
inhibition of cell proliferation by mevastatin. Mevastatin, an analogue
of lovastatin, leads to a profound G1 block in a prostate (PC3) cancer cell line. Although p21 levels are elevated and its association with cdk2 increased, it is unlikely that the inhibition of
kinase activity and cellular proliferation are caused by p21. Instead a
decrease in the activating phosphorylation of cdk2 likely accounts for
the antiproliferative effects of this drug.
 |
MATERIALS AND METHODS |
Materials, Cell Culture, and Flow Cytometry--
PC3 cells from
the ATCC (Manassas, VA) were grown in RPMI medium supplemented with
10% fetal bovine serum and antibiotics.
Mevastatin (compactin, Sigma) was dissolved in Me2SO,
and 10 µM was added to subconfluent cells.
TGF-
1 (Invitrogen) was dissolved in deionized water and used at 80 pM. Cells for flow cytometry were processed as previously
described (13). Antibodies to cdk2, cyclin E, cyclin A, p57, p21, p27,
and phosphotyrosine were from Santa Cruz Biotechnology. Anti-Rb
antibody was the kind gift of Dr. E. Harlow (Harvard University).
Anti-phospho-Rb antibody was from Cell Signaling Technologies (Beverly,
MA). Duplex oligoribonucleotides were from Dharmacon (Lafayette,
CO). Radioactive isotopes, [
-32P]ATP,
[32P]orthophosphate, and
[35S]methionine were from PerkinElmer Life
Sciences. Recombinant cyclin H·cdk7 was the kind gift of Dr. Robert
Fisher (Sloan-Kettering). The expression plasmid PGEX-cak1p was the
kind gift of Dr Mark Solomon (Yale University).
Immunoblots--
Cells were treated with lysis buffer (50 mM, Tris-HCl pH 7.4, 0.2% Nonidet P40, 150 mM
NaCl, 1 mM EDTA, and protease inhibitors). Lysates were
clarified by centrifugation, and protein concentration was determined
by the Bradford assay method. 30 µg of total cellular protein was
loaded per lane and separated by SDS-PAGE. Proteins were transferred to
nitrocellulose membranes, and immunoblots were performed as
previously described (14). 10- × 8-cm 12% SDS-polyacrylamide gels
were used to resolve samples except in experiments for the resolution
of the two forms, where a 13- × 15-cm 11% SDS-polyacrylamide gel was used.
Immunoprecipitations and Kinase Assays--
Cells were treated
with lysis buffer supplemented with 50 mM NaF, 1 mM Na3V04. Equal amounts of lysates
(200-1500 µg of protein) were immunoprecipitated with anti-cdk2,
cyclin E, or cyclin A antibodies. The pellet was washed three times
with lysis buffer and resuspended in 1× sample buffer. For kinase
assays, the pellet was washed twice in lysis buffer and twice in kinase
wash buffer (50 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, and 5 mM MnCl2). The pellet was resuspended in 20 µl of kinase reaction buffer (50 mM
Tris-HCl, pH 7.4, 10 mM MgCl2, 5 mM
MnCl2, 5 mM dithiothreitol, 10 µM
ATP, 0.5 µCi of [
-32P]ATP, 2 µg of histone H1).
The mixture was incubated at 30 °C for 30 min, and the reaction was
stopped by the addition of equal volume of 2× sample buffer. For cdk2
activation, kinase assays were carried out as previously described (15,
16). For phosphatase assays, cells were solubilized in lysis buffer
devoid of EDTA and phosphatase inhibitors (unless otherwise indicated).
Substrates were incubated with cell lysates in the presence of either
10 mM MgCl2 or 10 mM
CaCl2 for 30 min at 30 °C, and the reaction was stop by
addition of sample buffer.
[35S]Methionine and 32P
Labeling--
Subconfluent cells were grown with or without mevastatin
for 42 h. For [35S]methionine labeling, the medium
was removed, and the cells were washed 2× in phosphate-buffered saline
and incubated in methionine free medium with or without mevastatin for
1 h. Cells were labeled for 4 h in methionine-free medium,
supplemented with 5% dialyzed fetal calf serum, 25 µCi/ml
[35S]methionine (specific activity, 1175 Ci/mmol) with or
without mevastatin. Proteins were extracted with lysis buffer as above, and protein concentration was determined by the Bradford assay. For
32P labeling of cellular proteins, cells were incubated in
phosphate free medium for 2 h and then in medium containing 350 µCi/ml [32P]orthophosphate for 3 h prior to harvesting.
RNAi Transfections--
A p21 oligonucleotide corresponding to
nucleotides 1938-1960 (p21-2) was synthesized. A control RNAi to
cyclin H-(194-206) was also synthesized. This RNAi had no
effect on cellular levels of cyclin H and was used as a control. Cells
were transfected twice with 60 nM oligonucleotide at 0 and
24 h. 4 h after the second transfection, cells were
washed and fresh media were supplemented with either mevastatin or
Me2SO. Cells were harvested 48 h later.
 |
RESULTS |
Mevastatin Treatment Leads to a G1 Block and Decrease
in Kinase Activity of cdk2--
As has been reported with lovastatin
treatment of PC3-M cells (17), mevastatin treatment leads to a
significant decrease in the S phase population with a concurrent
increase in G1 noted by 24 h, and a more pronounced
G1 block by 36 h (Fig.
1A). This inhibition of cell
growth is paralleled by a dose-dependent inhibition by
mevastatin on the activity of cdk2 (not shown). Immunoblot analysis of
cell lysates after 36 h of mevastatin treatment shows that,
although the levels of the cell cycle-regulated proteins cdk2, p27, and
p57 remained unchanged, the amount of p21 was increased (Fig.
1B). Immunoprecipitation of cell lysates with antibodies to
cdk2 confirm that, although the amount of cdk2 remained relatively unchanged, there was a small but perceptible increase in p21 associated with cdk2 (Fig. 1C). This rather modest increase in p21 was
accompanied by a dramatic inhibition of kinase activity of cdk2
following mevastatin treatment. These experiments confirm previous
observations that the increase in cellular p21 and the subsequent
inhibition of cdk2 parallel growth inhibition by statins.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Cell cycle block by mevastatin.
A, mevastatin leads to a G1 blocks in PC3 cells.
Percentage of cells in G1 (black bars), S
(hatched bars), and G2/M (white bars)
at indicated times following addition of Me2SO
(Control) or mevastatin (MEV). Experiments are
the average of three separate experiments. Arrow bars
represent mean ± S.D. B, immunoblot of lysates
of PC3 cells in the absence ( ) and presence (+) of mevastatin.
36 h after addition of mevastatin, cells were lysed and 30 µg of
total protein was loaded per lane. Although levels of cdk2, p27, and
p57 remained relatively unchanged, a significant increase in p21 level
was noted. C, Cdk2 complex from PC3 cells. 200 µg of total
cellular protein from PC3 cells was immunoprecipitated with an antibody
to cdk2 and immunoblotted with antibodies to cdk2 and p21. cdk2 levels
remained unchanged (top), whereas a small increment in
associated p21 was noted (middle). The bottom
panel shows the dramatic reduction in the activity of cdk2 from
mevastatin-treated cells.
|
|
p21 Is Induced in PC3 Cells following TGF-
1 Treatment but
without Concurrent Inhibition of cdk2 in Vitro--
Although
mevastatin treatment clearly led to induction of p21, the modest
(~2-fold) increase in the amount of p21 associated with the inhibited
cdk2 made us question whether this association was the only mechanism
of kinase inhibition. In support of the existence of other mechanisms,
treatment of PC3 cells with TGF-
1 induces p21 (Fig.
2A) and increased association
of p21 with cdk2 (Fig. 2B),
but no inhibition of kinase activity (Fig. 2B) or induction of a G1-S block (data not shown). The increase in p21
association with cdk2 is more profound in TGF-
1-treated than with
mevastatin-treated cells (compare with Fig. 1C). This
observation suggests that a simple association of p21 with cdk2 does
not explain the entire antiproliferative effect of mevastatin. Because
it is possible that a factor in TGF-
-1-treated cells abrogates the
inhibition of cdk2 by p21, we pretreated cells with TGF-
1 for
12 h prior to the addition of mevastatin. The addition of
mevastatin to TGF-
1-treated cells inhibited cdk2 activity (Fig.
2C) and stopped cell proliferation (data not shown).
Therefore, the kinase activity of p21 associated cdk2 in
TGF-
1-treated cells was not caused by the induction of a novel p21
resistance factor by this cytokine.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
TGF- 1 enhances p21 levels and its
association with cdk2. A, 30 µg of cell lysates
derived from cells grown in the absence ( ) or presence (+) of 80 pM TGF- 1 was subjected to immunoblot analysis. Cdk2
level remained unchanged with a significant induction of p21.
B, immunoprecipitation with an anti-cdk2 antibody shows an
increased amount of p21 associated with cdk2 (middle panel)
but no significant reduction in histone kinase activity
(bottom). C, kinase activities of cdk2 from PC3
cells treated with TGF- 1 for 12 h prior to mevastatin addition.
Cells were harvested 36 h after addition of mevastatin. Cdk2
kinase activity is inhibited despite pretreatment and continued
presence of TGF- 1.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 3.
p21-depleted cdk2 complexes are inhibited in
mevastatin-treated cells. A, anti-p21 Western blot.
Mevastatin-treated and untreated lysates were depleted using an
anti-p21 antibody. Five sequential immunoprecipitates were performed on
each sample. Lanes 1 and 2, lysates (5% of
immunoprecipitations); lanes 3 and 4, first and
fifth immunoprecipitates of the untreated sample; lanes 5 and 6, first and fifth immunoprecipitates of the
mevastatin-treated sample. Note that there is no detectable p21 by the
5th immunoprecipitate. Lanes 1 and 2 represent
loading controls; lanes 7 and 8, p21 content of
cdk2 immunoprecipitated from depleted supernatants. B,
anti-cdk2 Western blot and cdk2 kinase activity. Top:
lanes 1 and 2, cdk2 immunoblot of total lysates
(5% of immunoprecipitations); lanes 3-6, the cdk2
immunoblot of p21 immunoprecipitates. Cdk2 is barely detectable but is
most distinct in lane 5, upper band (first p21
immunodepletion of mevastatin-treated cells). Lanes 7 and
8, cdk2 Western blot of immunoprecipitates of cdk2 on
p21-depleted supernatant. Similar levels of cdk2 remained in the
supernatant after the depletion of p21. Bottom, the histone
H1 kinase activity of the cdk2 from mevastatin-treated cells was still
inhibited.
|
|
p21-depleted cdk2 from Mevastatin-treated Cells Is
Inhibited--
Because the p21-containing complexes of cyclin·cdk2
from TGF-
1-treated cells is active, we wondered if p21 solely
accounted for the inhibition of cdk2 in mevastatin-treated cells. We
prepared lysates from mevastatin-treated and untreated cells and
subjected equal amounts of the lysate to sequential
immunoprecipitations with anti-p21 antibody until the supernatant was
depleted of p21. Fig. 3A shows no detectable p21 in the
supernatant of the fifth immunoprecipitate of mevastatin-treated and
untreated lysates (lanes 4 and 6). The
p21-depleted supernatants were then immunoprecipitated with antibody to
cdk2. The cdk2 immunoprecipitated from the depleted supernatants was
devoid of p21 (Fig. 3A, lanes 7 and
8). In Fig. 3B, anti-cdk2 immunoblot of total
lysates (top, lanes 1 and 2) show that
cdk2 content is similar. Cdk2 immunoblots of p21 immunoprecipitates (lanes 3-6) show that only a small amount of cdk2 is
associated with p21 (upper band best seen in lane
5). Parallel cdk2 immunodepletion experiments show that most of
the total p21 is non-cdk2 bound (not shown). Thus, it is not surprising
that cdk2 immunoprecipitates of p21-depleted lysates (top,
lanes 7 and 8) show that similar amounts of the
protein are present in mevastatin-treated and untreated cells. However,
kinase assays against histone H1 show that the cdk2 from
mevastatin-treated cells is still inhibited (bottom). This
suggests that enhancement of p21 levels and its increased association
with cdk2 may not be the only mechanism of cdk2 inhibition by mevastatin.
Mevastatin Blocks Cell Proliferation in the Absence of
p21--
The above observations led us to question whether mevastatin
can block p21-deficient PC3 cells. We used RNA interference (RNAi) to
deplete PC3 cells of p21. Fig.
4A shows that p21
siRNA-transfected cells treated with mevastatin have lower levels of
p21 when compared with untreated cells (lanes 1 and
4). The decrease of p21 was not accompanied by a
compensatory increase of p27 or p57 levels. In addition a control
oligonucleotide duplex did not prevent the mevastatin-induced elevation
of p21 (Fig. 4A, lanes 5 and 6)
confirming the specificity of the synthetic oligonucleotide for p21.
Fig. 4B shows an example of a quantitative Western blot of
mevastatin-treated cells with or without transfection of p21 siRNA.
Although p21 is detectable in 5 µg of lysate from untransfected
cells, it is barely detectable in 50 µg of
oligonucleotide-transfected cell lysate. An average of multiple
experiments suggests that RNAi decreases p21 level by 10- to 16-fold in
mevastatin-treated cells. Immunoprecipitations with antibodies to cdk2
show no associated p21 in oligonucleotide-transfected cells (Fig.
4C), confirming that the cdk2 complex is devoid of p21.
Despite the lack of p21, the Rb protein is hypophosphorylated (Fig.
4D, top two panels, lanes 3 and
4) and cdk2 kinase activity blocked (bottom two
panels, lanes 3 and 4) upon mevastatin
treatment. The kinase inhibition and hypophosphorylation of Rb is
equivalent to that in mevastatin-treated but untransfected (lane
2) or control oligonucleotide-transfected cells (lane
6). FACS analysis of p21 siRNA-transfected cells shows a
decrease in the S phase population upon mevastatin treatment that is
similar to that of untransfected and control RNAi-transfected cells
(Fig. 5E). These results show
that inhibition of cdk2 is not dependent on the ability of mevastatin
to elevate p21 protein levels in PC3 cells.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Elimination of p21 protein does not prevent
mevastatin-induced inhibition of cell growth. PC3 cells were
transfected with p21 RNA interfering oligonucleotide (p21
RNAi) or a control interfering RNAi (control RNAi).
Parallel mock transfections (no RNAi) were also performed.
Transfected cells were then treated with mevastatin (+) or
Me2SO ( ). A, immunoblot of cell lysates with
antibodies to p21 (top) and cdk2 (bottom). Note
the specific inhibition of p21 levels in mevastatin-treated cells (lane 4). The p21 level in
lane 4 is less than that in untreated cells (lane
1). The control RNAi does not affect basal or induced p21 levels.
cdk2 levels are unchanged. B, quantitation of the decrease
in p21 upon RNAi. Immunoblot of lysates from mevastatin-treated PC3
with and without transfection of p21 RNAi. Although p21 protein was
detectable in 5 µg of lysate from untransfected cells, it was not
detectable in 50 µg of lysate from p21 RNAi-transfected cells.
C, immunoblot of cdk2 immunoprecipitates with antibodies to
p21 and cdk2. Note that p21 is not detectable in mevastatin-treated,
p21 RNAi-transfected cells (lane 4). D, Rb is
hypophosphorylated in mevastatin-treated cells even in the absence of
significant levels of p21. Top two panels, immunoblot with
antibodies to Rb and phospho-Rb. Only the faster migrating Rb
(hypophosphorylated) was present in mevastatin-treated cells devoid of
p21 (lane 4). Bottom two panels, histone H1
phosphorylation by immunoprecipitates shows inhibition of total cdk2
and cyclin E-associated cdk2 in mevastatin-treated, p21-deficient cells
(lane 4). E, fluorescence-activated cell sorting
analysis shows a decrease in the S phase population that is similar
between untransfected, p21 RNAi-transfected, and control
RNAi-transfected cells that have been treated with mevastatin. The
bars show the mean ± S.D. of three experiments.
|
|

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
The cyclin E·cdk2 complex is inhibited by
mevastatin. A, immunoblot of mevastatin-treated lysates
with cyclin E or A antibodies. Cells were treated with mevastatin and
collected at the indicated times. Note that the cyclin A level declined
following mevastatin treatment (upper panel), whereas the
cyclin E level remained relatively unchanged. B,
immunoprecipitations with either anti-cyclin A or anti-cyclin E
antibody. Top two panels, phosphorylation of histone H1.
Note the marked inhibition of both cyclin E·cdk2 and cyclin A·cdk2
in mevastatin-treated cells. Bottom, immunoblot with
anti-cdk2 antibody indicated that inhibition of cyclin E-associated
kinase activity are not due to failure to associate with cdk2.
C, immunoprecipitation with cyclin E antibody and subsequent
immunoblot with cdk2 (top) and p21 (middle) shows
that the discrepancy in the kinase activity (bottom) of
cyclin E·cdk2 from TGF- 1 and mevastatin-treated cells was not
caused by differences in the cdk2:p21 ratio.
|
|
The Cyclin E·cdk2 Complex Is Intact but Inhibited in
Mevastatin-treated PC3 Cells--
We next evaluated changes in cyclins
A and E, the regulatory cyclins of cdk2, upon mevastatin treatment.
Following mevastatin treatment, the level of cyclin E protein remained
relatively constant, but the amount of cyclin A declined within 48 h (Fig. 5A) in parallel with a decline in cyclin A mRNA
(data not shown). Immunoprecipitation with antibodies to cyclin A and E
and subsequent histone H1 kinase assays show that cyclin A·cdk2 was
inhibited (Fig. 5B, top panel). Interestingly,
cyclin E·cdk2 was also inhibited (middle panel), despite
the presence of an intact cyclin E·cdk2 complex (bottom panel). Because the cyclin D1·cdk4 kinase activity was unchanged (data not shown), the mevastatin-induced cell cycle block appears to be
a result of the inhibition of cyclin E·cdk2. Previous studies have
shown that cyclin A transcription is dependent on E2F (18), so that
inhibition of cyclin E·cdk2 and underphosphorylation of Rb could
explain the decrease in cyclin A mRNA, protein levels, and lack of
cyclin A-associated kinase activity.
Immunoprecipitation of mevastatin and TGF-
1-treated cell lysates
with cyclin E antibody and immunoblots of the precipitates with
anti-cdk2 and p21 antibodies were performed to examine the amount of
p21 associated with cyclin E·cdk2 in the two lysates (Fig.
5C). In support of the hypothesis that p21 is not
responsible for the inhibition of cdk2, the ratio of cdk2 to p21 in
each complex was not different despite the dramatic difference in
kinase activity (Fig. 5C, bottom panel).
Decrease in Activating Phosphorylation May Account for Inhibition
of cdk2 in PC3 Cells--
The inhibition of cyclin E·cdk2 kinase
activity was seen even when p21 induction was prevented by RNAi (Fig.
4D, bottom panel). Because cyclin E·cdk2 was
inhibited despite an intact complex and independent of p21, we examined
whether there were changes in the regulatory phosphorylation of cdk2.
Immunoprecipitates of cdk2 probed with antiphosphotyrosine antibody
showed no increase in the content of phosphotyrosine, a marker of
inhibition through Thr-14 and Tyr-15 phosphorylation (Fig.
6A). However upon metabolic labeling of cells with [32P]orthophosphate,
phosphorylation of cdk2 was suppressed following mevastatin treatment
but not in untreated or TGF-
1-treated cells (Fig. 6B).
Because this finding could be explained on the basis of a decreased
pool of cyclin A·cdk2 complexes, we repeated these experiments by
immunoprecipitation with an antibody to cyclin E. Fig. 6C
shows that there is a decrease in the phosphorylation of cyclin
E-associated cdk2 from mevastatin-treated cells. It has been previously
reported that cdk2 can be resolved on SDS-PAGE into a fast migrating
band containing the activating phosphate on Thr-160 and a slower
migrating band devoid of this phosphate (19). Western blot of cell
lysates with anti-cdk2 antibody showed a decrease in the faster
migrating phosphorylated band in mevastatin-treated PC3 cells (Fig.
6C, upper panel). To selectively examine the
cyclin E·cdk2 population, the mobility of cdk2 associated with cyclin E was examined (Fig. 6D, lower panel). Consistent
with our previous results, the de-phosphorylated form of cdk2 (slower
migrating, labeled with an asterisk) was increased upon
mevastatin treatment. Because of the similar content of p21 in the
cyclin E·cdk2 from TGF-
1 and mevastatin-treated cells (Fig.
6C), the difference in phosphorylation could not be ascribed
to an unequal amount of p21 in the kinase complex.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Reduced phosphorylation of cdk2 upon
mevastatin treatment of PC3 cells. A, Western blot of
cdk2 immunoprecipitates with antityrosine phosphate antibody
(lower panel) shows no change in phosphotyrosine content.
The upper panel is a cdk2 immunoblot as a loading control.
B, immunoprecipitation of lysates from
[32P]orthophosphate-labeled PC3 cells with anti-cdk2
antibody. Proteins were transferred to nitrocellulose and processed for
autoradiography (top) and immunoblotted with anti-cdk2
antibody (bottom). Phosphate incorporation into cdk2 was
decreased with mevastatin treatment. The immunoblot confirms equal
loading of the three lanes. C, immunoprecipitation of cyclin
E·cdk2 from [32P]orthophosphate or
[35S]methionine-labeled cells. The band shown is the
32-kDa cdk2 protein. Note the decreased incorporation of radiolabeled
phosphate into cdk2 derived from mevastatin. D,
immunoblot of cell lysates and cyclin E immunoprecipitates from
untreated, TGF- 1, and mevastatin-treated PC3 cells. Samples were analyzed on a SDS-polyacrylamide gel under conditions that
resolve the two forms of cdk2. Samples were electrophoresed an
additional 30 min after the dye front had exited the gel (for better
resolution of the two forms of cdk2). Western blot was performed with
anti-cdk2 antibody. The relative amount of the slower migrating band
(asterisk) corresponding to cdk2 not phosphorylated on
Thr-160 was increased in mevastatin-treated cells. The
arrowheads indicate the phospho-Thr-160 content of cdk2.
E, H1 kinase assay of cyclin E·cdk2 following in
vitro activation by GST-Cak1. Immunoprecipitates of cyclin
E·cdk2 from untreated (UNT), TGF- 1, and
mevastatin-treated PC3 cells were treated with either GST or GST-Cak1
in the presence of 1 mM cold ATP for 30 min at 30 °C.
The pellet was washed, and histone H1 and [ -32P]ATP
were then added. cdk2 kinase activity inhibited with mevastatin
treatment (lane 5) was restored by GST-Cak1 (lane
6). Lanes 1 and 2 contained no lysate from
PC3 cells and confirm that Cak1 has no intrinsic kinase activity
against histone H1. F, H1 kinase assay of cyclin E·cdk2
following in vitro activation by recombinant cyclin
H·cdk7. Immunoprecipitates of cyclin E·cdk2 from PC3 cells were
incubated as in D with or without recombinant cyclin
H·cdk7. The pellet was washed, and histone H1 and
[ -32P]ATP were then added. Cyclin H·cdk7 restores
the cdk2 kinase activity (compare lanes 5 and 6).
Lanes 1 and 2 are mock immunoprecipitations
devoid of lysate and confirm that the enhanced kinase activity against
histone H1 is not secondary to the presence of cdk7.
|
|
The activating phosphorylation of cdks is under the control of the
cdk-activating kinase, Cak. In yeast, a monomeric protein Cak1p
(15) is required for activation of cdks. In mammalian cells a trimeric
enzyme consisting of Cdk7, cyclin H, and MAT1 is felt to be the
putative Cak (20, 21). We evaluated the ability of recombinant yeast
Cak to activate the inhibited cyclin E·cdk2 from mevastatin-treated
cells (Fig. 6E). As previously reported (15), GST-Cak1 by
itself has no intrinsic histone H1 kinase activity (lane 2).
Preincubation of cyclin E·cdk2 from mevastatin-treated cells with
GST-Cak1, however, induced the histone H1 kinase activity of the former
(compare lanes 5 and 6). Fig. 6F shows
a similar experiment performed with recombinant cyclin H·cdk7, the
putative mammalian Cak. In accordance with the observation with yeast
Cak, cyclin H·cdk7 activates the inhibited cdk2 from mevastatin-treated cells(compare lanes 5 and 6).
Cdk7 does not phosphorylate histone H1 (lane 2). These
observations support the notion that the inhibition of cyclin E·cdk2
upon mevastatin treatment of PC3 cells is due to a decrease in
activating phosphorylation on Thr-160.
The decrease in activating phosphorylation on Thr-160 could be a result
of 1) decrease in level or activity of the trimeric cyclin
H·cdk7·mat1 complex (Cak), 2) decrease in level or activity of a
novel Cak, or 3) increase of a phosphatase targeted to phospho-Thr-160. Immunoblots to evaluate the levels of cdk7 and cyclin H showed no
changes in their respective cellular levels following mevastatin treatment (data not shown). Immunoprecipitation of the complex with
cdk7 antibodies followed by analysis of its ability to phosphorylate bacterially produced GST-cdk2 showed no decrease in the Cak activity following mevastatin treatment (Fig.
7A). We have been unable to
see any change in cdk2 phosphatase activity in mevastatin-treated lysates. For example, in Fig. 7B, cyclin·cdk2 was
immunoprecipitated from untreated cells and incubated with increasing
amounts of mevastatin-treated lysate supplemented with 10 mM MgCl2. After incubation, the pellets were
washed and the ability of the kinase to phosphorylate histone H1 was
determined. If cdk2 was dephosphorylated on Thr-160, there would be a
decrease in kinase activity. No decrease in the activity of cdk2 was
seen. In Fig. 7C, GST-cdk2 was phosphorylated in
vitro with yeast Cak1p. The phosphorylated product was then used
as the substrate for phosphatase assays. Cell lysate was incubated with
phosphorylated GST-cdk2. Lane 1 shows the control with no
added lysate, lanes 2 and 3 show reactions
containing untreated lysates, and lanes 4 and 5 are reactions containing lysates from mevastatin-treated cells.
Lanes 3 and 5 represent cell lysates with or
without phosphatase inhibitors (Ptase
). There is no
perceptible difference in the phosphatase activity of lysates from
mevastatin-treated and untreated cells. We have performed similar
reactions, with supplemental calcium, with and without ATP and have
been unable to see enhanced cdk2 phosphatase activity in
mevastatin-treated cells. We are therefore left with the possibility
that a novel Cak is responsible for the phosphorylation of cyclin
E·cdk2 in vivo, and this is suppressed upon mevastatin treatment. Alternatively, the cyclin E·cdk2 is sequestered from cyclin H·cdk7 Cak in mevastatin-treated cells.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Cdk7 and phosphatase activity in
mevastatin-treated cells. A, immunoprecipitation of
mevastatin-treated and untreated lysates with cdk7 antibody and
subsequent evaluation for kinase activity against GST-cdk2 in
vitro. The cyclin H·cdk7 Cak activity was retained in
mevastatin-treated cells (lane 4). Immunoprecipitation with
non-immune serum (NIS) serves as a negative control
(lanes 1 and 3). B, lysates from
untreated cells (UNT) were immunoprecipitated with anti-cdk2
antibody. Pellets were incubated in phosphatase buffer supplemented
with 0, 5, 20, and 50 µg of mevastatin-treated lysate. Samples were
resolved on SDS-PAGE and processed for radiography. Note the lack of
reduction in kinase activity of cdk2. C, GST-cdk2 was
incubated with cell lysates in the presence or absence of phosphatase
inhibitors (Ptase and +). Lane 1,
phosphorylated GST-cdk2 content without added lysate; lanes
2 and 3, addition of lysate from untreated cells;
lanes 4 and 5, addition of lysate from
mevastatin-treated cells. There is no enhanced phosphatase activity
against phosphorylated cdk2 in mevastatin-treated cells.
|
|
 |
DISCUSSION |
Like its analog, lovastatin, mevastatin inhibits cell growth. In
PC3 cells, this antiproliferative effect parallels an increase in
cellular levels of p21, leading to the assumption that the increased
association of p21 with cdk2 is sufficient to account for the kinase
inhibition and cell-cycle block. The p21 and p27 levels are enhanced in
lovastatin-treated cells via the inhibition of the proteasomes (12).
Although there is no doubt that p21 levels are elevated in
mevastatin-treated cells, our studies suggest that p21 may not always
be the active inhibitor of the cdk2 kinase. In fact, a model where p21
interacts with the cyclin·cdk to inhibit its activity cannot explain
the observations we report here, for the following reasons. 1) The p21
associated with cdk2 appears out of proportion with the dramatic
reduction of kinase activity noted with mevastatin treatment. TGF-
1
enhances the levels of p21 and increases its association with cdk2 but
does not inhibit the kinase activity. Thus a mere 2-fold increase in
the association of p21 with the kinase complex upon treatment with
mevastatin is unlikely to be enough to inhibit the cdk2. 2) Elimination
of p21 from PC3 cells by RNA interference does not prevent the
mevastatin-induced cell cycle block and cdk2 inhibition, suggesting
that p21 is dispensable for these effects of mevastatin. 3) Lastly, in
experiments to be reported elsewhere we find that, consistent with the
above observations, overexpression of p21 in PC3 cells does not result in cell cycle arrest, despite elevated levels and association of p21
with cyclin E·cdk2. This implies that the p21 protein in PC3 cells is
inactive in the inhibition of the cdk2. The inactivity of p21 in these
cells is thus the primary reason for unmasking a second pathway by
which mevastatin inhibits cdk2.
In PC3 cells a decrease in activating phosphorylation of cdk2 is the
critical factor that suppresses cyclin E·cdk2 kinase. This might be
secondary to decreased levels or activity of the cdk2-activating kinase
(Cak). There is some debate regarding the identity of the physiologic
Cak in mammalian cells. Although the trimeric complex of cyclin H,
cdk7, and mat1 (20, 21) was long felt to assume this role, a number of
lines of experimental evidence suggest that there might well be a
second Cak in mammalian cells. First, in yeast the physiologic cak1p is
a monomer that has no significant homology to any of the members of the
trimeric mammalian Cak. Second, the trimeric Cak complex is a
constituent of the basal transcriptional unit TFII-H, whereas the yeast
cak1p plays no role in transcription (22). Lastly, recent experimental evidence suggests that a second Cak activity can be identified in
chromatographic fractions of HeLa cell lysates (23) and that an
immunologically similar protein can be identified in HepG2 cells with
antibodies to the yeast Cak1p (24).
We have not yet detected enhanced cdk2 phosphatase activity in cell
extracts following mevastatin treatment. Furthermore, the
constitutively active phosphatase (protein phosphatase type 2C)
that has been identified in mammalian cells targets Thr-160 of
monomeric cdk2 for dephosphorylation. Because the decreased phosphorylation of cdk2 from mevastatin-treated cells is seen on cyclin
E-complexed cdk2, it is unlikely that enhanced protein phosphatase type
2C activity is responsible. As a result, we favor the
possibility that a novel mammalian Cak is suppressed or that cdk2 is
sequestered from cyclin H·cdk7·mat1 Cak in mevastatin-treated PC3 cells.
The failure to phosphorylate Rb upon mevastatin treatment leads to
decreased cyclin A transcription, decreased cyclin A complexed with
cdk2, and loss of cyclin A·cdk2 kinase activity, further contributing
to the cell cycle block. Therefore, the decrease in cdk2 kinase
activity occurs through two different mechanisms: 1) a primary decrease
in Thr-160 phosphorylation of cyclin E·cdk2 and 2) a secondary
decrease of cyclin A mRNA and protein.
In summary, we provide evidence for a novel pathway by which mevastatin
inhibits cyclin-dependent kinases and cell cycle
progression in PC3 cells. Treatment of hepatoma-derived HepG2 cells
with TGF-
was reported to decrease the activating phosphorylation on
cyclin E·cdk2 without any increase in p21 or other cdk inhibitors
(24). Our results provide a second example where the activating
phosphorylation on cdk2 is regulated independently of changes in
associated cyclin or cdk inhibitors. Because p21 is a potent inhibitor
of cdk2 in vitro, there are many examples where the
induction of p21 occurring concurrently with inhibition of cdk2 has led
to the assumption that the p21 inhibits the kinase by simple and direct
association in the affected cells. Our studies suggest that, in some of
these cells, p21 may be dispensable for the inhibition of the kinase and that additional inhibitory pathways may be in use.