From the Departamento de Fisiología, Facultad
de Medicina, University of Santiago de Compostela, 1 Calle San
Francisco, Santiago de Compostela, 15705 A Coruña, Spain and
§ Unidad de Inmunología and ¶ Unidad de
Biología Molecular, Complejo Hospitalario Universitario de
Santiago,
Santiago de Compostela, 15705 A Coruña, Spain
Received for publication, July 26, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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We show in this work that the inhibition of Cdk4
(6) in Rb Progression through the mammalian cell cycle is controlled by the
sequential activation of a series of cell cycle-dependent kinases (Cdks)1 (1). The Cdks
active in G1 include the cyclin E-Cdk2 complex and cyclin D
complexes with Cdk4 and Cdk6 (2). The enzymatic activity of a Cdk can
be regulated at three levels (3): cyclin binding and activation,
subunit phosphorylation, and inhibition by one of a group of small
regulatory proteins, called Cdk inhibitors (4).
The cyclin D1-Cdk4 complex plays an important role in oncogenesis.
Cyclin D1 and Cdk4 genes are often amplified or
overexpressed in many types of cancer (5). Besides, experimental
overexpression of cyclin D1 can induce oncogenic transformation both in
cultured cells and in transgenic mice (6, 7). The importance of cyclin D1-Cdk4 (and of cyclin D-Cdk4 (6) complexes in general) in tumorigenesis is further demonstrated by the frequent deregulation of
the downstream effectors and upstream regulators of the complex (8).
One of the targets of cyclin D-Cdk4 (6) is pRB, the product of a known
tumor suppressor gene. p16ink4a (referred to hereafter as p16),
a regulator of cyclin D-Cdk4 (6), is also an important tumor suppressor
gene product. It is noteworthy that very few human tumors have
mutations in more than one of these elements. For example, mutations in
p16 and Rb seem to be mutually exclusive, with very few
tumors carrying mutations in both genes (9). This has been taken as
evidence that p16-cyclin D-Cdk4-pRB form part of a biological pathway. Alteration of this path contributes to the oncogenic phenotype of the
cancer cell, but once the path is altered, the mutation of another
element confers no further selective advantage to the cell bearing it.
Despite the relevance of the cyclin D-Cdk4 (6) complex in
tumorigenesis, its role in cell cycle progression is still not clear.
This complex can in principle play two different roles in this respect.
On one side, it has a kinase activity per se that is known
to phosphorylate some substrates, at least pRB and its two related
proteins, p107 and p130 (10-13). On the other hand, it can bind to the
kinase inhibitors p27 and p21 and sequester them from Cdk2-containing
complexes (13, 14). The relative importance of these two activities in
cell cycle progression is still not completely settled.
Several experiments support the idea that an important role of cyclin
D-Cdk4 (6) in cell cycle progression is to be a titrator of Cdk2
inhibitors. The Cdk4 (6) inhibitor p16 not only can inhibit the Cdk4
and Cdk6 kinase activity; it also disrupts cyclin D-Cdk4 (6) complexes
and displaces the p27 bound by these complexes, leaving it free to
inhibit Cdk2 (14). This indirect inhibition of Cdk2 is essential for
p16-mediated growth arrest (13). A dominant negative mutant of Cdk4,
Cdk4N158 (dnCdk4), can inhibit the kinase activity of
endogenous Cdk4, but it does not induce displacement of p27 to Cdk2
complexes, and it does not induce growth arrest when overexpressed (13, 15). Moreover, overexpression of cyclin E can override the growth arrest imposed by p16 (16). These results suggest that growth arrest
mediated by p16 is due to its inhibition of cyclin E-Cdk2 complexes.
Fibroblasts derived from Cdk4 knockout mice show a delay in
G1 progression when stimulated to proliferate from
quiescence that has also been suggested to be related to the
displacement of p27 to Cdk2-containing complexes (17). Furthermore,
substitution of the cyclin D1 gene with cyclin E restores the defects
caused by cyclin D1 deficiency in mice, showing that if there is cyclin E activity, cyclin D1 is dispensable (18).
On the other hand, some experiments suggest that the kinase activity of
cyclin D-Cdk4 (6) plays a role in cell cycle progression, even if it is
not essential for the completion of the cell cycle. Specifically,
overexpression of dnCdk4 induces a delay in G1 progression in quiescent cells stimulated to proliferate (13), a result that we
have reproduced in our system. However, the mechanism that mediates
this effect has not been studied in detail.
p27kip1 (p27) is a Cdk inhibitor that plays a role in the
establishment of quiescence. The levels of p27 are elevated in
quiescence (19), although the exact mechanisms that mediate this
induction are still not completely understood. The levels of this
protein can be regulated in several different ways (19-24). The best
studied of them is the modulation of its half-life. Nevertheless,
regulation of p27 translation has also been suggested as an important
means of p27 induction during quiescence (19).
In this work, we report that inhibition of Cdk4 (6) kinase activity
induces accumulation of p27 in quiescent cells and a delay in cell
cycle progression through G1 in serum-stimulated cells. p27
accumulation is produced by enhanced translational activity of its
mRNA, an effect mediated by the 3'-untranslated region of the p27
mRNA. These results show that the kinase activity of Cdk4 (6) is
important for cell cycle progression at least in part because it
modulates the translation efficiency of an important cell cycle
regulator, p27.
Cell Culture and
Synchronization--
Rb Cell Cycle Analysis--
For flow cytometry analysis of DNA
content, cells were stained with propidium iodide, and DNA fluorescence
was measured with a Becton Dickinson device. To determine incorporation
of 5-bromo-2'-deoxyuridine (BrdUrd), cells plated on coverslips were
incubated with BrdUrd for 1 h, fixed, and processed for
immunofluorescence according to the manufacturer's instructions
(in situ cell proliferation kit; Roche Molecular Biochemicals).
Transfections and Retroviral Infections--
For retroviral
infections, human p16 or Cdk4N158
were cloned into the retroviral vector pBABEpuro by standard
techniques. The resulting construct was transfected into Phoenix cells.
Retroviral supernatants were obtained and used to infect
Rb Antibodies--
The following antibodies from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA) were used: anti-Cdk4 (sc-260);
cyclin D1 (sc-450); cyclin E (sc-481); p107 (sc-318); human p16 (sc-
468); Cdk2 (sc-163); p27kip1 (sc-528); Sp1 (sc-59).
Immunoprecipitations and Western Blot Analysis--
Cells were
lysed in EBC buffer (50 mM Tris, pH 8, 120 mM
NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol, 1 mM
orthovanadate, aprotinin, 50 mM NaF) and cleared by
centrifugation. For immunoprecipitation, equal protein quantities were
incubated with appropriate antibodies. Immune complexes were collected
with protein G-Sepharose beads (AP Biotech) and washed three times with
EBC buffer. Resuspended samples were resolved by SDS-PAGE and
transferred to nitrocellulose membranes. Detection of the immune
complexes was performed with a chemiluminescence assay (Tropix)
according to the manufacturer's instructions. For detection of
proteins in cell lysates, a 100-µg sample of the total cell lysate
was separated on SDS-polyacrylamide gels and processed for Western blotting.
Protein Labeling--
Cells were growth-arrested and then
preincubated for 1 h in methionine- and cysteine-deficient
Dulbecco's modified Eagle's medium, pulse-labeled for 90 min with
fresh medium containing 1.5 mCi/ml Redivue Promix L-35S (AP
Biotech), and then incubated in Dulbecco's modified Eagle's medium.
At the time points shown, samples of cells were collected and lysed in
radioimmune precipitation buffer (in phosphate-buffered saline; 1%
Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease
inhibitors). Protein concentrations were determined by Bradford assay
(Bio-Rad), and equal amounts of protein extracts were used for protein
immunoprecipitation. Samples were resolved by SDS-PAGE (12%), and the
gels were transferred to nitrocellulose membranes and exposed.
Kinase Assays--
Cyclin E kinase assays were performed as
described (28). Cdk4 kinase assays and preparation of substrate
glutathione S-transferase-RB were carried out essentially as
described by Matsushime et al. (29). In both
assays, the phosphorylated substrate was detected initially by
autoradiography and quantitated by cutting bands off the gel and
measuring their radioactivity.
RNA Analysis--
Northern blot was performed by standard
techniques using the appropriate cDNA probes. 18 S RNA analysis was
performed with an oligonucleotide probe as described (30).
Luciferase, We began this work trying to determine whether p16 overexpression
had an effect in cell cycle progression in Rb-negative
cells, even if this effect was not a complete arrest of the cycle. To this end, we have used Rb Rb/
3T3 cells enhances the
accumulation of the p27kip1 cyclin-dependent kinase
inhibitor when these cells are induced into quiescence. Two different
forms of inhibition of Cdk4 (6), namely overexpression of the Cdk4 (6)
inhibitor p16 and overexpression of a dominant negative mutant of Cdk4
(Cdk4N158), result in this effect. This suggests that the
relevant activity of Cdk4 (6) that has to be inactivated in this
setting is its kinase activity. The accumulation of p27kip1
is due to enhanced translation of the protein, mediated by the 3'-untranslated region of the p27kip1 mRNA. Moreover, the
cells that overexpress p16ink4a or Cdk4N158 show a
delay in G1 when made quiescent and restimulated to
proliferate. This delay is overcome by transfection of a plasmid
expressing antisense p27kip1, which shows that the accumulation
of p27kip1 in these cells is related to their G1
delay. In summary, we report a new functional link between two
important cell cycle regulators, Cdk4 and p27kip1, and provide
a mechanistic explanation to the previously reported epistatic
relations between these two proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
3T3 cells (25) as
well as the retroviral packaging cell line Phoenix (26) were
cultured as described. To obtain a G0 arrest, cells were
allowed to reach confluence and then starved in medium with
0.1% serum for 48 h. Serum-deprived cells were stimulated to
reenter the cycle by the addition of medium with 20% serum. When
specified, aphidicolin was added at a concentration of 5 µg/ml. For
synchronization of continuously proliferating cells, cells were
arrested in mitosis by treatment with 0.4 µg/ml nocodazole (Sigma)
for a maximum of 12 h. Mitotic cells were collected by shake off,
washed, and replated to enter the cell cycle in a synchronized form.
/
3T3 (26). Rb
/
3T3 cells were selected in 4.5 µg/ml puromycin, and clones were isolated from the resulting populations by limiting dilution. Transient
transfections were performed with FuGene 6 Transfection Reagent (Roche
Molecular Biosciences) according to the manufacturer's instructions,
using the plasmids pRcCMV-p16, pCMV dnCdk4 (15), and pCMV
(Clontech), together with the SvL series of vectors (27). Cells were harvested in 200 µl of reporter lysis buffer (Promega). Luciferase and
-galactosidase activities were assayed with standard methods. The p27 depletion experiments were performed using the pECE-ASp27 plasmid (34).
-galactosidase, and hypoxanthine
phosphoribosyltransferase mRNAs were quantitated by reverse
transcriptase-PCR analysis, using oligonucleotides derived from the
coding sequence of the three genes for the PCR.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
3T3 cells as a
model system (25). This is a cell line derived from mice in which the
Rb gene has been deleted by genetic targeting (31). Contrary
to Rb-negative tumor cells, Rb
/
3T3 cells show cell cycle regulation similar to normal
Rb-positive cells. Specifically, they can be arrested in
G0 when serum-deprived and can also be contact-inhibited
(25). Therefore, they seem to be a good model to study cell cycle
regulation of Rb-negative cells.
/
3T3 cells have endogenous p16 (data not
shown). Nevertheless, they also have cyclin D1-Cdk4 complexes formed in
a cell cycle-dependent fashion and active as a kinase (see
Fig. 1, B and C, first
two lanes of each). However, both the amount of
cyclin D1-Cdk4 complexes and the activity of Cdk4 as a kinase are
clearly lower in Rb
/
3T3 cells than in
NIH3T3 cells, which are known to be functionally p16-negative (data not
shown). We infected Rb
/
3T3 cells with a
retrovirus encoding human p16 and isolated individual colonies from the
transduced populations. These clones expressed the exogenous p16 at
high levels (Fig. 1A). The two clones shown in Fig.
1A were chosen for in-depth study of their cell cycle regulation. These cells had disrupted cyclin D1-Cdk4 complexes compared
with parent cells (Fig. 1B), and their Cdk4 activity was not
detectable above background levels, defined as the kinase activity of
immunoprecipitates in which the anti-Cdk4 antibody had been
preincubated in the presence of a Cdk4-blocking peptide (Fig.
1C). We repeated this experiment three times. The average kinase activity of Cdk4 in p16.1 cells was 5.5% that of control Rb
/
3T3 cells, with an upper 95% confidence
interval of 22.2%, whereas the kinase activity of p16.2 cells was
0.67% that of control Rb
/
3T3 cells, with
an upper 95% confidence interval of 4.2%.
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Fig. 1.
Overexpression of p16 disrupts cyclin D-Cdk4
and down-regulates cyclin E-Cdk2 complexes in
Rb /
3T3 cells. A, overexpression of p16. Extracts were
prepared from parental Rb
/
3T3 cells or
cells transduced with human p16 (p16.1 and p16.2), and a Western blot
was performed with an anti-human p16 antibody. B, disruption
of cyclin D1-Cdk4 complexes. Parental Rb
/
3T3 cells or p16 colonies were serum-deprived (0 h) and restimulated for 14 h after serum deprivation
(14 h). Extracts were prepared and immunoprecipitated with
an anti-Cdk4 antibody. The immunoprecipitate (IP) was
processed for Western blots (WB) against cyclin D1
(upper panel) or Cdk4 as a loading control
(lower panel). C, inhibition of Cdk4
kinase activity. Anti-Cdk4 immunoprecipitates of p16-overexpressing or
parental Rb
/
3T3 cells were incubated with
glutathione S-transferase-RB (residues 792-928) in the
presence of [
-32P]ATP, and the reaction was run on
SDS-PAGE and exposed to autoradiography. The background activity of the
assay was determined by preincubating the anti-Cdk4 antibody with a
Cdk4-blocking peptide (lanes marked with a plus
sign). D, lowering of cyclin E-associated kinase
activity. Anti-cyclin E immunoprecipitates of parent
Rb
/
3T3 or p16 cells, serum-deprived
(0 h) or stimulated for 14 h (14 h), were
incubated with histone H1 in the presence of
[
-32P]ATP. The reactions were run in an SDS-PAGE and
exposed by autoradiography. E, redistribution of p27 from
cyclin D1 to cyclin E complexes. Cyclin D1 (left
panels), or cyclin E (right panels)
were immunoprecipitated from Rb
/
3T3 or p16
cells, which had been serum-deprived and restimulated for 14 h.
Immunoprecipitates were run in an SDS-PAGE and Western blots performed
for p27 and either cyclin D1 (right panel) or
Cdk2 (left panel) as loading controls.
The kinase activity of cyclin E-containing complexes in serum-stimulated cells overexpressing p16 was clearly lower than in parent cells, but it was not completely inhibited (Fig. 1D). In p16 overexpressors, p27 was displaced from cyclin D-containing to cyclin E-containing complexes in p16-overexpressing cells (Fig. 1E). We concluded that p16 was acting as described (13, 14), displacing p27 from cyclin D to cyclin E, and this was contributing to the inhibition of cyclin E-dependent kinase activity. Overexpression of p16 also affected the phosphorylation of a cyclin D-Cdk4 target, the Rb-related protein p130 (data not shown), which shows that the inhibition of this complex was functionally significant in vivo as well as in vitro.
We then studied the effect of p16 overexpression on the cell cycle of
proliferating Rb-negative cells. Specifically, we determined the length of G1 in control and p16 cells. For this, we
synchronized cells by nocodazole treatment followed by mitotic shake
off and measured DNA synthesis at different times after mitotic release by incorporation of BrdUrd. Fig.
2A shows that the
behavior of control and p16 cells was identical. However, the situation
was different in cells that had been made quiescent and then stimulated to proliferate. As shown in Fig. 2B, when cells were
synchronized using this protocol and analyzed by flow cytometry, those
that overexpressed p16 progressed through the cell cycle clearly slower than control cells. In fact, at 23 h after stimulation, between 25 and 30% of control cells had already attained the 4n DNA
content characteristic of G2/M cells, but there was no
enrichment of cells with 4n DNA content in cells overexpressing
p16, even 26 h after serum stimulation. The same difference was
seen when we analyzed another p16-overexpressing colony, compared with
a different control (data not shown). We also determined the moment of
the beginning of DNA synthesis by measuring BrdUrd incorporation in
control and p16 cells, at different times after serum stimulation. We used the two colonies that had been analyzed previously, together with
a third colony, and another control (a puromycin-resistant colony from
an infection of Rb/
3T3 cells with empty
pBABEpuro). This experiment showed that the delay occurred before S
phase entry in p16 cells (Fig. 2C). Furthermore, the effect
was evident in the three colonies analyzed, which showed a delay of
several h with respect to the two control cell lines
(Rb
/
3T3 and puro), despite the slight
variability in S phase entry between these two. We concluded that p16
induced a marked delay in cell cycle progression in serum-stimulated,
Rb-negative cells.
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The delay in cell cycle progression in serum-stimulated cells we
observed was very similar to that reported in cells overexpressing a
dominant negative Cdk4 mutant or derived from Cdk4/
mouse embryos (13, 17). We wanted to know whether the effect could be reproduced by affecting only the kinase activity of Cdk4. We infected Rb
/
cells with
Cdk4N158, a dominant negative Cdk4 (dnCdk4) that has been
shown to inhibit the kinase activity of Cdk4 but not to affect the
distribution of p27 between Cdk4 and Cdk2 complexes (13) and studied
the cell cycle of the resulting colonies. Inhibition of Cdk4 kinase activity had the same effect on the cell cycle as overexpression of
p16. Cells overexpressing a dominant negative version of the kinase
showed a delay in cell cycle progression when serum-stimulated and
entered S phase several h later than control cells (Fig.
3).
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We then studied the regulation of cell cycle-regulated genes and
proteins in cells overexpressing p16 or dnCdk4. The most striking
difference we found between these cells and controls related to p27.
The levels of this protein were higher in quiescent than in
proliferating cells, as has been described (see Fig.
4C). Surprisingly, its levels
in quiescence were higher in p16 and dnCdk4 cells than in parental
cells (Fig. 4A) or cells that had been transduced with an
empty vector (Fig. 4C). Given that the levels of this
protein are induced upon entry into quiescence, we determined whether
the inhibition of Cdk4 activity influenced the withdrawal of cells from
the cell cycle when serum was removed. We measured BrdUrd incorporation
by immunofluorescence in cells at different times after serum
withdrawal. A slightly higher percentage of parental cells
(Rb/
cells) incorporated BrdUrd in the first
12 h of serum deprivation, compared with cells without Cdk4
activity. Nevertheless, there was no significant difference between
cells transduced with p16 or dnCdk4 and cells transduced with an empty
vector (puro cells). At later times, the differences were no longer
evident, and from 48 h onward, there were no cells positive for
BrdUrd in any of the populations tested (Fig. 4B). Despite
the fact that the differences in parental and transduced cells in serum
deprivation were very small, we were concerned about the effects that
difference might have on the regulation of p27. For this reason, we
used a puromycin-resistant cell line (puro) as a control in the
following experiments. We performed a time course of the induction of
the p27 in puro cells and two cell lines overexpressing either dnCdk4
or p16 (Fig. 4C). The induction of p27 with respect to
proliferating cells was evident after only 3 h of serum withdrawal
in the three cell lines, but there was no appreciable difference
between the control and transduced cells until 48 h of serum
deprivation had passed. We concluded that the higher levels of p27 in
cells overexpressing p16 or dnCdk4 were unlikely to be a consequence of
different rates of cell cycle withdrawal upon serum deprivation.
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We then began to study the mechanisms that mediate the induction of p27
in p16 and dnCdk4 cell lines. We first determined the levels of the p27
mRNA in quiescent parental and transduced cells. To our surprise,
the levels of p27 mRNA were clearly lower in quiescent p16 and
dnCdk4 cells than in parental cells (Fig. 5A). We are at present
studying the reason for this difference. Nevertheless, it is clear that
the high levels of p27 in transduced cells are not a consequence of
higher levels of p27 mRNA.
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It is known that an important mechanism of regulation of the levels of p27 depends on the modulation of its half-life. We suspected that the effect we were seeing was due to differences in protein stability and determined the half-life of p27 in our cells with a pulse-chase experiment using medium labeled with [35S]methionine and [35S]cysteine (Fig. 5B). p27 was very stable in the three cell lines analyzed, consistent with previous reports on its half-life in serum-deprived cells (23, 32). The experiment clearly showed that the differences in p27 protein levels were not the consequence of its differential stability. In fact, the half-life of p27 was longer in puro cells (>12 h), than in either p16.1 cells (half-life of 8 h) or Cdk4dn.1 cells (half-life of 7.5 h). Surprisingly, however, the intensity of the band at time 0 was clearly higher in transduced cells than in control cells, which suggested an effect in protein translation. The intensity of p27 labeling after a 90-min incubation in [35S]methionine and [35S]cysteine is also shown in Fig. 5C. The lack of enhancement in the labeling of an unrelated protein, the transcription factor Sp1, shows that the effect is specific to p27.
Translation of p27 has been proposed as an important mechanism of
regulation of this protein (22). Using translation reporter plasmids
with the untranslated regions of p27 flanking a luciferase reporter
gene, Vidal et al. (33) have identified a region in the
3'-untranslated region (UTR) of p27 that responds to extracellular signals and may be at least partially responsible for the accumulation of p27 upon serum deprivation. We have used the assay system they have
developed to confirm the effect of the inhibition of Cdk4 on
translation of p27. The reporter plasmids we have used express the
luciferase gene under the control of the SV40 early promoter, SV40
polyadenylation signal, and the 5'-UTRs (nucleotides 303-466) of the p27 mRNA (5' plasmid); the 3'-UTR (nucleotides 1063-2404) of p27 mRNA (3' plasmid); or both UTRs (5', 3' plasmid). We have transfected these plasmids in control cells and cells overexpressing dnCdk4 or p16, together with a -galactosidase expression vector, and
compared the corrected luciferase activity of this plasmid in the
different cells to that of a plasmid expressing luciferase under the
same promoter but without the p27 mRNA UTRs. Fig.
6A shows that both in dnCdk4
and p16 cells, the activity of the reporter was enhanced with respect
to control cells. This enhancement appears to depend mainly on the
3'-UTR of p27, since a plasmid containing only this sequence from the
p27 mRNA showed the same effect. The higher luciferase activity in
the cells without Cdk4 activity was not the result of a higher
transcription or mRNA stability, since the mRNA of luciferase
did not vary significantly when analyzed by reverse transcription-PCR
in any given cell line, when transfected with the different reporter
plasmids (Fig. 6A, lower panel).
Significantly, these effects were observed only in quiescent cells. The
same assays in actively proliferating cells did not give any
statistically significant differences between control and transduced
cells.
|
We wanted to know whether the inhibition of Cdk4 could affect the
translation of p27 mRNA in a transient assay. To this end, we used
the same reporter plasmids in parental cells
(Rb/
3T3), cotransfecting them with p16 or
dnCdk4 (Fig. 6B). In these experiments, both p16 and dnCdk4
enhanced the activity of the reporter plasmids. Again, the effect was
seen only in quiescent cells, and the enhancement was dependent on the
3'-UTR of the p27 mRNA. We concluded that the inhibition of Cdk4
activity enhances the translation of p27 mRNA through its 3'-UTR in
quiescent cells.
To further understand the relationship between the levels of p27 and
the length of the G1 phase after serum stimulation, we cotransfected a plasmid that expresses a mouse p27 antisense mRNA (p27 AS) and a -galactosidase expression vector into
p16-overexpressing cells. This plasmid has been previously shown to
lower the levels of p27 (34). After transfection, cells were
serum-deprived and stimulated to reenter the cell cycle in the presence
of BrdUrd. At different times after stimulation, cells were stained for
-galactosidase and BrdUrd, and the percentage of
-galactosidase-positive cells that had incorporated BrdUrd was
determined (Fig. 7A). About
10% of cells transfected with p27 AS incorporated BrdUrd after 48 h of serum deprivation, compared with less than 1% for those cells that did not receive p27 AS. When cells were stimulated to proliferate, the serum-deprived p27 AS cells entered S phase clearly earlier than
untransfected cells or cells transfected only with
-galactosidase. We concluded that p27 is important for cell cycle withdrawal on serum
deprivation and that the levels of p27 influence the length of
G1 after serum stimulation in p16-overexpressing cells.
When we repeated the experiments in a cell line overexpressing dnCdk4, the results were virtually identical (Fig. 7B), showing that
p27 is important also in the lengthening of G1 in these
cells.
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DISCUSSION |
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In this work, we describe a new link between two important cell cycle regulators. Inhibition of Cdk4 enhances the accumulation of p27 in quiescence, and this is important in the lengthening of G1 after quiescent cells are stimulated to proliferate.
Both p16 and a dominant negative mutant of Cdk4 have the same effect, suggesting that the important activity of Cdk4 (or Cdk6) that has to be inhibited to produce the effect on p27 is its kinase activity and not the ability of cyclin D-Cdk4 (6) complexes to sequester p27. The kinase activity of Cdk4 had been shown to be important for the phosphorylation of several cell cycle-related substrates, the most relevant of which are pRB and the Rb-related proteins. Despite this, the only effect on cell cycle regulation of the inhibition of Cdk4 kinase activity that had been described is the lengthening of G1 in the reentry in the cell cycle after quiescence (13). We propose in this work a mechanism for this effect, namely the enhancement of p27 accumulation in quiescent cells.
Lowering of the levels of p27 by antisense technology in cells without Cdk4 activity shortens the length of G1 after quiescent cells are stimulated to proliferate, which shows that the enhancement of the accumulation of p27 in quiescence is related to the lengthening of G1 in these cells. The fact that the levels of p27 in quiescence determine the length of G1 after stimulation with serum is not surprising, since one of the principal events that has to take place in the progression of the G1 phase of the cell cycle is the activation of cyclin E-Cdk2 complexes, and a limiting step for this is the inactivation of p27 (35-37). Another effect of the transfection of p27 antisense is the high percentage of p27 AS-transfected cells that remain BrdUrd-positive when serum-deprived. This may be due to a high DNA repair activity in these cells or to a deficient withdrawal from the cell cycle. We favor the latter possibility, given the intensity of the BrdUrd signal and previous reports that showed the role of p27 in serum deprivation (34, 36, 37). Despite this difference in behavior upon serum deprivation, the degree of synchronization attained in the experiments with antisense technology is enough to make the shortening of G1 after restimulation in transfected cells evident.
The higher levels of p27 in cells without Cdk4 activity are due to a translational effect mediated by the 3'-UTR of p27 mRNA. In contrast, the levels of p27 mRNA are lower in cells without Cdk4 activity when induced into quiescence, and the half-life of the protein is not prolonged by the inhibition of Cdk4. Translation of p27 mRNA has been proposed to be an important mechanism for the accumulation of the protein in quiescent cells, and the 3'-UTR of the messenger of p27 has also been shown to be regulated by extracellular signals. Specifically, Vidal et al. have identified a region of 300 bp in the 3'-UTR that is responsible of the accumulation p27 after inhibition of Rho activity (33). It will be interesting to further delineate the region of p27 mRNA modulated by Cdk4 and study the relation between the Rho and the Cdk4 pathways.
The inhibition of Cdk4 kinase activity cannot stimulate the translation of p27 alone. Cells need to be made quiescent for the effect to be evident. At first sight, this might be due to the instability of p27 protein in proliferating cells, which could preclude the accumulation of the protein even if translation was enhanced. Nevertheless, transfection experiments with the translational reporter plasmids show that in proliferating cells, the inhibition of Cdk4 cannot enhance the translation of p27. This suggests that the effect of Cdk4 on the translation of p27 needs a second signal, probably the lack of growth factor signaling characteristic of quiescence. Furthermore, the effect on p27 is only evident after 48 h of serum deprivation, a time when cells do not have any detectable cyclin D-Cdk4 complexes or Cdk4 activity (Fig. 1). In our view, this suggests that the effect of Cdk4 on translation of p27 is indirect and mediated by an as yet unidentified factor or factors with a long half-life. We expect that further analysis of the relation between Cdk4 and p27 will shield some light on this issue.
The activity of cyclin D-Cdk4 complexes had been previously reported to be more important in cells growing at a suboptimal rate than in actively growing cells. For example, embryonic stem cells do not have a detectable Cdk4 activity, and are refractory to p16 expression, when they are actively proliferating. When they are induced to differentiate, the kinase activity of Cdk4 is activated, and they become susceptible to p16 overexpression (38). Furthermore, when all Rb family members are inactivated in embryonic stem cells, there is no consequence while they retain a high proliferative state, but their differentiation is inhibited (39). Likewise, and as stated above, fibroblasts derived from mice in which the Cdk4 gene is inactivated by gene targeting grow normally when continuously proliferating but show a delay in S phase entry when serum-deprived and restimulated to proliferate (17). Our results are consistent with this view of Cdk4 activity being important in nonproliferating cells and provide a possible explanation for the effect. Translation of p27 is modulated by Cdk4 activity in conjunction with growth factor-generated signals, and this influences the regulation of the cell cycle.
Overexpression of p16 has been previously shown to result in a stimulation of translation of another important cell cycle regulator, the Cdk inhibitor p21 (40). There are several differences between that effect and the one reported here. Only p16 has been shown to enhance translation of p21, which suggests that the relevant function of Cdk4 that has to be inactivated in this case is its ability to sequester p27. Besides, the induction of p21 occurred even in proliferating cells and contributed to the p16-mediated growth arrest. Despite these differences, it will be interesting to delineate the regions of p21 mRNA responsible for its translational induction and compare them with those of p27 with an equivalent function.
Previous reports have underscored the relation between cyclin D-Cdk4
complexes and p27. Relevantly, Tsutsui et al. have shown that proliferative defects in in vitro cultured
Cdk4/
mouse embryo fibroblasts can be
corrected by deletion of p27 (17), and both Geng et al. (41)
and Tong et al. (42) have reported that deletion of the p27
gene can overcome the developmental abnormalities characteristic of
cyclin D1
/
mice. These results have been explained in
terms of the importance of redistribution of p27 from Cdk4- to
Cdk2-containing complexes. Our results provide an alternative
explanation. The rescue of the cell cycle effects of a dominant
negative Cdk4, a mutant that does not induce redistribution of p27 to
Cdk2 complexes, by antisense p27, suggests that the relevance of this
inhibitor as a downstream target of Cdk4 may be related to its
accumulation in cells without Cdk4 activity when quiescent, rather than
to the redistribution of the existing p27 to Cdk2 complexes.
In summary, we have described a new function of the kinase activity of
Cdk4, important for the effects that its inhibition has on the cell
cycle regulation of cells and related to the accumulation of the Cdk
inhibitor p27 during quiescence. In depth study of the pathway leading
from Cdk4 to the translation of p27 will yield important insight into
how cells withdraw from the cell cycle when deprived of growth factors.
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ACKNOWLEDGEMENTS |
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We are grateful to G. Peters, R. Watson, T. Jacks, and A. Koff for materials and reagents, to A. Vidal for helpful discussions, and to C. Pombo and V. Arce for critically reading the paper.
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FOOTNOTES |
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* This work was supported by Xunta de Galicia Grants XUGA20003A98 and PGIDT00PX12801PR and Ministerio de Ciencia y Tecnología of Spain Grant SAF2001-3021.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.:
34-981-582658; Fax: 34-981-574145; E-mail: fszalvid@usc.es.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M207530200
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ABBREVIATIONS |
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The abbreviations used are: Cdk, cell cycle-dependent kinase; dnCdk4, dominant negative Cdk4; BrdUrd, 5-bromo-2'-deoxyuridine; UTR, untranslated region; CMV, cytomegalovirus.
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