MOLECULAR AND CELLULAR BIOLOGY, Feb. 2010, p. 694–710
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 30, No. 3
Cdk2 and Cdk4 Regulate the Centrosome Cycle and Are Critical
Mediators of Centrosome Amplification in p53-Null Cells?
Arsene M. Adon,1Xiangbin Zeng,1Mary K. Harrison,1Stacy Sannem,1
Hiroaki Kiyokawa,2Philipp Kaldis,3and Harold I. Saavedra1*
Department of Radiation Oncology, Emory University School of Medicine, 1365C Clifton Rd., Room C3084, Atlanta,
Georgia 303221; Northwestern University, Department of Molecular Pharmacology and Biological Chemistry,
Feinberg School of Medicine, Mail Code S215, 303 East Chicago Avenue, Chicago, Illinois 60611-30082;
and Institute of Molecular and Cell Biology (IMCB) Cell Division and Cancer Laboratory (PRK),
61 Biopolis Drive, Proteos 3-10B, Singapore 138673, Singapore3
Received 25 February 2009/Returned for modification 24 March 2009/Accepted 11 November 2009
The two mitotic centrosomes direct spindle bipolarity to maintain euploidy. Centrosome amplification—the
acquisition of >3 centrosomes—generates multipolar mitoses, aneuploidy, and chromosome instability to
promote cancer biogenesis. While much evidence suggests that Cdk2 is the major conductor of the centrosome
cycle and that it mediates centrosome amplification induced by various altered tumor suppressors, the role
played by Cdk4 in a normal or deregulated centrosome cycle is unknown. Using a gene knockout approach, we
report that Cdk2 and Cdk4 are critical to the centrosome cycle, since centrosome separation and duplication
are premature in Cdk2?/?mouse embryonic fibroblasts (MEFs) and are compromised in Cdk4?/?MEFs.
Additionally, ablation of Cdk4 or Cdk2 abrogates centrosome amplification and chromosome instability in
p53-null MEFs. Absence of Cdk2 or Cdk4 prevents centrosome amplification by abrogating excessive centriole
duplication. Furthermore, hyperactive Cdk2 and Cdk4 deregulate the licensing of the centrosome duplication
cycle in p53-null cells by hyperphosphorylating nucleophosmin (NPM) at Thr199, as evidenced by observations
that ablation of Cdk2, Cdk4, or both Cdk2 and Cdk4 abrogates that excessive phosphorylation. Since a mutant
form of NPM lacking the G1Cdk phosphorylation site (NPMT199A) prevents centrosome amplification to the
same extent as ablation of Cdk2 or Cdk4, we conclude that the Cdk2/Cdk4/NPM pathway is a major guardian
of centrosome dysfunction and genomic integrity.
The centrosome maintains genomic integrity by enforcing
euploidy (20). A centrosome consists of two centrioles, con-
taining proteins such as ?-tubulin; structural proteins including
pericentrin, ?-tubulin, and centrin-2; and cell cycle-regulatory
proteins, which include p53 and cyclin E/Cdk2. Normal cells
have one mature centrosome during early G1(20). At late G1,
each of the centrioles composing the mature centrosome sep-
arates and duplicates to form a new (or daughter) centriole
between late G1and late S phase, culminating in two fully
mature centrosomes at G2. The two mitotic centrosomes asso-
ciate with spindle fibers and migrate toward opposite sides of
the spindle pole to establish bipolarity. This ensures that sister
chromatids segregate toward each spindle pole. Following cy-
tokinesis, each daughter cell receives one centrosome and an
equal complement of chromatids. Normal centrosome dupli-
cation must be strictly controlled and strictly coordinated with
S-phase initiation and progression (68). When this control
fails, centrosome amplification occurs, leading to aberrant and
multipolar mitotic spindles, increased frequency of chromo-
some segregation errors, aneuploidy, and chromosome insta-
bility (12, 20). Centrosome amplification, aneuploidy, and
chromosome instability contribute to cancer biogenesis and
progression by triggering reduced expression of tumor sup-
pressors and overexpression of proto-oncogenes.
One of the pathways contributing to centrosome amplifica-
tion is deregulated centrosome duplication triggered by the G1
cyclin-dependent kinases (Cdks) (27, 51, 59). The Cdks, a
family of serine/threonine protein kinases, control the onset of
the major cell cycle events, such as DNA synthesis and mitosis
(65). Cdk activities are positively regulated by association with
different cyclins, which are temporally expressed at specific
phases of the cell cycle; they are negatively regulated by a
variety of Cdk inhibitors (CKIs) (65). Individual and combina-
torial gene knockouts of the cyclins and Cdks have uncovered
redundancy in the regulation of DNA synthesis and specificity
in their abilities to control development and tumorigenesis (2,
5, 6, 24, 25, 38, 52, 55, 61, 72, 77). However, how the cyclins and
Cdks individually or cooperatively impinge on centrosome du-
plication is poorly understood. Biochemical and pharmacolog-
ical evidence pointed to Cdk2 as the only Cdk coordinating the
centrosome duplication and cell cycles (32, 33, 36, 40, 44).
Cdk2 was proposed to coordinate the cell and centrosome
duplication cycles by phosphorylating Rb to promote S phase
(28) and by phosphorylating various centrosomal proteins to
regulate the centrosome duplication cycle (10, 19, 51). Cyclin
E/Cdk2 phosphorylates nucleophosmin (NPM)/B23 at T199 to
regulate centrosome licensing; this phosphorylation allows
centrioles to separate and centriole duplication to commence
(70). Cdk2 directly promotes centrosome duplication by phos-
phorylating Mps-1 and CP110 and by modulating the activity of
Plk4 (10, 19, 26). However, gene knockout approaches de-
* Corresponding author. Mailing address: Department of Radiation
Oncology, Emory University School of Medicine, 1365C Clifton Rd.,
Room C3084, Atlanta, GA 30322. Phone: (404) 778-5509. Fax: (404)
778-5520. E-mail: firstname.lastname@example.org.
?Published ahead of print on 23 November 2009.
throned Cdk2 as the sole Cdk coordinating the cell and cen-
trosome duplication cycles, since mouse embryonic fibroblasts
(MEFs) from which Cdk2 (14) or cyclins E1 and E2 (25) had
been deleted showed only a minor deviation from normal cen-
trosome ratios and proliferated. These results implied that, as
with the cell cycle, there is redundancy among the Cdks regu-
lating the centrosome duplication cycle. These results were
unexpected, given the involvement of Cdk2 in the regulation of
two central steps in the centrosome duplication cycle: licensing
and duplication. To date, the identity of the Cdks supporting
Cdk2 in regulating normal centrosome duplication is unknown.
As the cyclins, Cdks, and CKIs control centrosome duplica-
tion, altered tumor suppressors and oncogenes deregulate
those cell cycle-regulatory molecules, leading to centrosome
amplification (12, 21). Ablated genes that result in elevated
Cdk2 activity and elevated frequencies of centrosome amplifi-
cation include E2F3, p53, Skp2, and p21Waf1; similarly, ectopi-
cally expressed cyclins E and A result in elevated Cdk2 activity
and centrosome amplification in p53?/?MEFs (13, 27, 44, 49,
59, 68). Likewise, oncogenes and altered tumor suppressors
that hyperactivate Cdk4 and result in high frequencies of cen-
trosome amplification include ectopically expressed Her2 (47),
H-RasV12, v-Mos (57), MEK1Glu217/Glu221(58), cyclin D1 (50),
and silenced MEK2 (73). Conversely, p16 restricts excessive
centriole reduplication (42, 44). However, the relationships
between altered genes, ectopic activities of specific Cdks, and
centrosome amplification are correlational, as they deregulate
cyclin/Cdk activities as well as complex signal transduction
cascades that control a plethora of transcripts.
The abilities of the cell cycle and centrosomal checkpoints—
signaling pathways that monitor the integrity and replication
status of the genome and the centrosome—to inhibit entry into
S phase and centrosome duplication are closely associated with
the function of the p53 tumor suppressor (45, 74). The p53
transcription factor is inactivated in approximately 50% of
human cancers (71). p53 regulates the transcription of a large
number of genes to prevent entry into S phase in the presence
of DNA or centrosome damage (45, 74). Indeed, ablation of
p53 allows centrosome amplification, aneuploidy, and chromo-
some instability (22). A gene product central to centrosome
duplication control is p21Waf1, expressed at low levels in a
p53-dependent manner (48) to inhibit the cyclin E/Cdk2 com-
plex (65). In addition, p21Waf1has been implicated in the
assembly of the cyclin D1/Cdk4 complex, and its overexpres-
sion inhibits the activity of Cdk4 at higher concentrations (30,
35, 76). The continual presence of p21Waf1guards against
premature activation of cyclin E/Cdk2 and perhaps against that
of cyclin D/Cdk4, ensuring the coordinated initiation of cen-
trosome and DNA duplication. In p21Waf1?/?MEFs, initiation
of centrosome and DNA duplication is uncoupled, much like
that in cells with constitutively active cyclin E/Cdk2 (13, 48,
68). Importantly, observations that the reintroduction of wild-
type p53 into p53?/?cells resulted in nearly complete restora-
tion of the centrosome duplication cycle while ectopic expres-
sion of p21Waf1in p53?/?cells only partially restored that cycle
(68) suggest that p53 controls centrosome duplication through
multiple pathways, one of which is mediated by the negative
regulation of Cdk2 by p21Waf1.
Since direct evidence linking Cdk2 or Cdk4 to centrosome
amplification in p53?/?MEFs was lacking, we used a genetic
approach to test whether Cdk4 and Cdk2 mediate that abnor-
mal process. Our results revealed that p53 knockout does not
signal centrosome amplification and chromosome instability
exclusively through Cdk2, as suggested previously (21). We
propose a new paradigm: ablation of p53 requires the presence
of both Cdk2 and Cdk4 activities in order to induce high
frequencies of centrosome amplification and chromosome in-
MATERIALS AND METHODS
Generation of mouse embryonic fibroblasts. Mice were crossed as Cdk2?/??
Cdk2?/?, Cdk4?/?? Cdk4?/?, Cdk2?/?Cdk4?/?? Cdk2?/?Cdk4?/?(meiot-
ically recombined ), p53?/?Cdk2?/?? p53?/?Cdk2?/?, p53?/?Cdk4?/??
p53?/?Cdk4?/?, and p53?/?Cdk2?/?Cdk4?/?? p53?/?Cdk2?/?Cdk4?/?.
After mating, embryos were isolated from females 13.5 days after detection of
seminal plugs. Embryos were collected under sterile conditions and their livers
extirpated for extraction of DNA and PCR genotyping. MEFs were generated
using established methods (59). The individual genotypes were assessed by PCR
genotyping with primers specific for the wild-type and knockout p53, Cdk2, and
Cdk4 alleles (6). All experiments were performed on passage 2 (p2) MEFs.
Cell culture. MEFs were maintained under proliferating conditions with 10%
fetal bovine serum (FBS)–Dulbecco’s modified Eagle medium (DMEM). For
serum arrest experiments, cells were cultured in 0.2% FBS–DMEM for 60 h.
Centriole reduplication assay. Three independent, proliferating MEFs of the
genotypes indicated in Fig. 4E and F, plated in two-well chamber slides, were
either left untreated or treated with 2 mM hydroxyurea (HU) for 48 h. For
coimmunostaining of ?- and ?-tubulins in order to examine centrioles, cells were
first incubated on ice for 30 min, to destabilize microtubules nucleated at the
centrosomes, and then briefly extracted (?1 min) with cold extraction buffer
[0.75% Triton X-100, 5 mM piperazine-N,N?-bis(2-ethanesulfonic acid) (PIPES),
2 mM EGTA (pH 6.7)]. Cells were then briefly washed in cold phosphate-
buffered saline (PBS) and were fixed as previously described (59). Cells were
immunostained with anti-?-tubulin monoclonal (DMA1; Sigma) and anti-?-tu-
bulin polyclonal (ab11317; Abcam) antibodies. The antibody-antigen complexes
were detected with the appropriate Alexa Fluor-conjugated antibodies (Molec-
ular Probes), and the frequencies of centrosome amplification were calculated by
counting 200 cells per group.
Serum starvation and BrdU incorporation assay. MEFs of the indicated
genotypes plated in 60-mm-diameter dishes were first grown to confluence in
10% FBS–DMEM and then split into three groups. The cells in groups 1 and 2
were plated onto two-well tissue culture chamber slides, and those in the third
group were plated onto 60-mm-diameter petri dishes. MEFs of different groups
were first plated at high densities, then starved for 60 h by culturing in medium
supplemented with 0.1% FBS, and finally released by the addition of 10% FBS
for various times. The cells in group 1 were immediately fixed for centrosome
staining at the time points indicated in Fig. 2. To measure the S-phase entry of
the indicated genotypes, MEFs in group 2 were pulse-labeled with 20 ?M
bromodeoxyuridine (BrdU) (51-7581KZ; BD Pharmingen) and incubated for 30
min as described previously (64, 75). BrdU-positive cells were detected using
primary antibodies against BrdU (NA61; Calbiochem) and an Alexa Fluor 555-
conjugated secondary antibody (Molecular Probes). We counted 200 cells per
group for the BrdU and centrosome assays. Lysates from cells in group 3 were
obtained for Western blotting at the time points indicated in Fig. 2.
Immunofluorescence. Immunofluorescence was performed by following our
published protocols (59). MEFs were plated at 4 ? 104per well into two-well
tissue culture chamber slides and were grown for 2 to 3 days. Cells were fixed in
cold 4% paraformaldehyde, washed in PBS, permeabilized in a 1% NP-40–PBS
solution, and blocked in 5% bovine serum albumin (BSA) in PBS. Centrosomes
were stained overnight at 4°C with monoclonal antibodies against pericentrin
(611814; BD Biosciences) and/or ?-tubulin (ab11317; ABCAM). Chromosome
breaks were detected using phosphorylated histone 2A variant X (?-H2AX)
(07-164; Upstate) with the appropriate Alexa Fluor-conjugated antibodies (Mo-
lecular Probes). Cells were also counterstained with 4?,6-diamidino-2-phenylin-
dole (DAPI). For each experiment involving calculations of the frequencies of
centrosomes, at least 200 cells from each chamber were counted per group.
Western blotting. Western blotting was performed according to published
protocols (59). Protein lysates were obtained by incubating cells in lysis buffer (50
mM HEPES [pH 7.9], 250 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1%
NP-40, 10% glycerol, 0.5 mM NaF, 0.1 mM NaVO4, 0.1 mM phenylmethylsul-
fonyl fluoride, 10 mM ?-glycerophosphate, 0.1 mM dithiothreitol, 0.1 mg/ml
VOL. 30, 2010Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION 695
aprotinin, 0.1 mg/ml leupeptin) for 30 min at 4°C. Samples were denatured at
95°C for 5 min in sodium dodecyl sulfate (SDS) sample buffer, resolved by
SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene diflu-
oride membranes (Bio-Rad, Hercules, CA). The blots were incubated in block-
ing buffer (5% [wt/vol] nonfat dry milk or BSA in Tris-buffered saline plus 0.1%
Tween 20 [TBS-T]) for 1 h and were then probed overnight at 4°C with the
primary antibodies. The blots were then rinsed in TBS-T and incubated with the
appropriate horseradish peroxidase-conjugated secondary antibodies at room
temperature. The blots were then rinsed in TBS-T, and the antibody-antigen
complex was visualized with the chemiluminescent HRP Antibody Detection
reagent (Denville Scientific Inc., Metuchen, NJ). Western blotting for the de-
tection of phosphorylated nucleophosmin was performed similarly, except that
the serine/threonine phosphatase inhibitor calyculin A (Upstate, CA) was in-
cluded in the culture medium 10 min prior to harvest at a concentration of 100
nM. The antibodies used in the various Western blotting experiments were as
follows: anti-Cdk2 (sc-163; Santa Cruz), anti-Cdk4 (2906; Cell Signaling), anti-
p53 (sc-6243; Santa Cruz), anti-p57Kip2(sc-8298; Santa Cruz), anti-p16INK4A
(sc-1207; Santa Cruz), anti-p21Waf1(sc-397; Santa Cruz), anti-p27Kip1(sc-528;
Santa Cruz), anti-?-actin (4970; Cell Signaling), anti-cyclin A (ab38; Abcam),
anti-cyclin D1 (2922; Cell Signaling), and anti-cyclin E (sc-481; Santa Cruz).
The micronucleus assay. The micronucleus assay was performed as described
previously (59). Briefly, 4 ? 104cells were plated into each well of a two-well
chamber slide (177380; Nalge Nunc International). After 2 to 3 days in culture,
cells were fixed in 4% paraformaldehyde and were stained with DAPI. Micro-
nuclei appear as spherical structures with a morphology similar to that of the
nucleus, except that their sizes range from 1/10 to 1/100 the size of a nucleus;
1,000 cells were counted for each genotype analyzed.
Transfections. For transient transfection of wild-type and mutant NPM/B23,
three independent p53?/?MEFs were cotransfected with a plasmid carrying
either FLAG-tagged wild-type NPM/B23 or a FLAG-tagged substitution mutant
(Thr1993 Ala) with a neomycin resistance gene (pcDNA3.1) by using Lipo-
fectamine (Invitrogen, Carlsbad, CA). As a negative control, the empty vector
was transfected. After transfection at 37°C overnight in a 5% CO2incubator,
cells were fed with fresh complete medium for 24 h. The cells were then treated
with complete medium containing 2.5 mg/ml neomycin (Sigma, St. Louis, MO)
for 7 days. G418-resistant cells were maintained in complete medium containing
neomycin (1 mg/ml) for an additional 2 days and were replated for further
culture in fresh complete medium for an additional 24 h.
Three independent Cdk4?/?MEFs were also transfected with a plasmid car-
rying an empty vector (pBABE-hygro) or encoding Cdk2 (pBABE-Hygro-Cdk2)
by using Lipofectamine (Invitrogen) according to the manufacturer’s protocol.
After transfection (37°C overnight), cells were fed with fresh complete medium
for 24 h and were then switched to selective medium (150 ?g/ml hygromycin) for
4 days. Selected cells were directly plated onto two-chamber slides, fixed as
previously described (59), and stained for centrosome analysis.
RNA interference. Cdk2 and Cdk4 small interfering RNAs (siRNAs) (cata-
logue no. SC-29260 and SC-29262) were purchased from Santa Cruz Biotech-
nology, Inc. Three independent MEFs of each genotype were grown in a six-well
plate until they became 60 to 80% confluent. The cells were then transfected with
each siRNA according to the manufacturer’s protocol, and 72 h after the trans-
fection, the cells were used to prepare cell lysates and were plated onto a
two-chamber slide for centrosome analysis.
We also knocked down p53 with two synthesized siRNA duplex sequences by
transfecting three independent wild-type MEFs using Lipofectamine 2000 (In-
vitrogen) according to the manufacturer’s protocol. The two siRNA duplex
sequences targeting the p53 mRNA are ACCAF161020_1 (sense, 5?-rCrArCrA
ACCAF161020_2 (sense, 5?-rGrGrUrGrArArArUrArCrUrCrUrCrCrArUrCrA
rArGrUrGrGrUrUrU-3?; antisense, 5?-rArCrCrArCrUrUrGrArUrGr GrArGr
All the siRNAs were prepared using a transcription-based method with the
Silencer siRNA construction kit (Integrated DNA Technologies) according to
the manufacturer’s instructions. Three days after the addition of complete me-
dium, cell lysates were prepared for appropriate assays.
Cyclin-dependent kinase 4 activity assay. The cyclin-dependent kinase 4 assay
was performed according to published methods (67). Briefly, the NPM peptide is
phosphorylated because of Cdk4 activity, which is measured using luminometric
estimation of ATP depletion. Immunoprecipitated (Ip) Cdk4, obtained from 150
?g of total-protein extract from wild-type and Cdk2?/?MEFs at different time
points, was washed three times in cell lysis buffer and twice in kinase buffer and
was then resuspended in 30 ?l of kinase buffer, optimized to preserve the Cdk4
activity. A kinase reaction was performed by adding 20 ?l of a mixture containing
0.5 ?M ATP and 2 ?g of NPM peptide (catalog no. ab39518; Abcam) as a
substrate in kinase buffer. The reaction mixture was incubated for 30 min at 30°C,
and then an equal volume of Kinase-Glo reagent was added. As reaction con-
trols, the kinase reaction was performed with samples in the absence of the
peptide substrate (no NPM), with no Ip-Cdk4, and with Ip-Cdk4 obtained from
total-protein extracts from Cdk4?/?MEFs. Samples were then incubated for 20
min at room temperature, and the developed luminescence was recorded using
the SpectraMax Gemini XS luminometer and expressed as relative light units.
Image acquisition and manipulation. Slides were analyzed using a Zeiss
Axioplan II microscope with a Plan-Apochromat 100? (numerical aperture, 1.4)
oil immersion objective. Images were taken using a color digital camera (Axio-
cam HRC) and Zeiss Axiovision software. Confocal images were acquired with
a Zeiss LSM 510 META point scanning laser confocal microscope mounted on
a Zeiss Axioplan II upright microscope equipped with a Plan-Apochromat 63?
(numerical aperture, 1.4) oil immersion objective. Images were captured by Zeiss
Image Browser. All the samples were mounted in Fluoromount-G mounting
medium (Southern Biotech) and were analyzed at room temperature.
Mouse embryonic fibroblasts proliferate despite the absence
of Cdk2 and Cdk4. To investigate how Cdk2 and Cdk4 indi-
vidually or synergistically contribute to the regulation of nor-
mal centrosome duplication and how they mediate centrosome
amplification, wild-type, p53?/?, Cdk2?/?, Cdk4?/?, Cdk2?/?
Cdk4?/?, p53?/?Cdk2?/?, p53?/?Cdk4?/?, and p53?/?
Cdk2?/?Cdk4?/?embryonic day 13.5 (E13.5) mouse embry-
onic fibroblasts (MEFs) were generated. Genotypes were de-
termined by allele-specific PCR (Fig. 1A). Western blotting
confirmed the presence or absence of p53, Cdk2, and Cdk4
(Fig. 1B). To establish whether Cdk2 and Cdk4 were upregu-
lated as compensatory mechanisms for their loss, expression
levels of Cdk2 in Cdk4?/?cells and of Cdk4 in Cdk2?/?cells
were examined using wild-type cells as controls (Fig. 1C).
There is no compensatory upregulation of Cdk2 or Cdk4 in
Cdk4?/?or Cdk2?/?MEFs; their levels are the same as those
in wild-type controls.
Published observations indicate that early-passage Cdk2?/?
and Cdk4?/?MEFs proliferate and that the kinetics of entry
into S phase in Cdk2?/?or Cdk4?/?MEFs are moderately
delayed relative to those of wild-type MEFs (5, 72). Addition-
ally, Cdk2?/?Cdk4?/?MEFs senesce at earlier passages than
the individual knockouts (5, 6, 52, 72). Ablation of p53 abro-
gates senescence associated with the single or combined loss of
Cdk2 and Cdk4 at late passages (53). To rule out the possibility
that any reductions in the frequency of centrosome amplifica-
tion in p53?/?MEFs lacking Cdks are associated with major
changes in the frequencies of proliferation, the G1/S transition
in each genotype was investigated. The MEFs indicated in Fig.
2 were plated, serum starved, stimulated with serum, and har-
vested at the indicated time points for measurement of S-phase
entry by BrdU incorporation (data available on request). Most
MEFs peaked with 30 to 50% of cells in S phase at 12 h (Fig.
2A). In contrast, Cdk2?/?Cdk4?/?MEFs entered S phase
with delayed kinetics, and fewer than 10% of the cells were
actively proliferating at 16 h. Likewise, all MEFs lacking p53
and the Cdks reached their peak frequency of cells in S phase
at 12 h (Fig. 2B).
Our goal was to identify deregulated cell cycle-regulatory
molecules that may trigger the centrosome amplification ob-
served in p53?/?MEFs. Various defects in the cell cycle-
regulatory machinery result in deregulated Cdk activities and
centrosome amplification. Those include overexpression of cy-
696 ADON ET AL.MOL. CELL. BIOL.
clins A, E, and D (27, 48–50, 59). To further investigate the
molecular consequences of Cdk loss for regulatory molecules
governing the G1/S transition, and whether deregulation of
various cyclins accounted for centrosome amplification in p53-
null cells, Western blotting was performed to analyze the ex-
pression of cyclin A, cyclin D1, and cyclin E at the indicated
time points (Fig. 2C). Levels of cyclin E were robust through-
out the cell cycle, but its accumulation was decreased in
Cdk2?/?and Cdk2?/?Cdk4?/?MEFs from 4 to 12 h post-
serum addition. Cyclin D1 levels reached maximal accumula-
tion at 12 and 16 h. No major changes in cyclin D1 levels were
observed, except for moderately diminished levels in Cdk4?/?,
Cdk2?/?Cdk4?/?, p53?/?Cdk4?/?, and p53?/?Cdk2?/?
Cdk4?/?MEFs relative to those in MEFs of other genetic
groups at 12 and 16 h. Cyclin A expression was low in all the
MEFs before 8 h post-serum addition; its expression levels
peaked at 16 h post-serum addition, with similar expression in
all MEFs. We conclude that except for minor changes in the
expression of cyclins E and D1 when their respective catalytic
partners, Cdk2 and Cdk4, are ablated, the temporal cyclin
expression patterns are what we would expect of a normal cell
cycle, consistent with the similar kinetics of entry into S phase
for all genetic groups.
Reduced expression of certain CKIs can also result in ele-
vated frequencies of centrosome amplification, and their over-
expression can suppress centrosome amplification (42, 66, 68).
For example, ectopic expression of p16INK4A, a Cdk4-specific
inhibitor, prevents centriole duplication and centrosome am-
plification (42, 44). On the other hand, p27Kip1, recently re-
ported to be a dual Cdk2 and Cdk4 inhibitor (54), prevents
centrosome amplification triggered by gamma-irradiation (36,
66). In addition, ablation of p21Waf1, a Cdk2-specific inhibitor
at physiological levels and a Cdk4 inhibitor at higher levels (29,
30, 35), results in centrosome amplification; its ectopic expres-
sion partly suppresses centrosome amplification in p53?/?
MEFs (13, 39, 68). To determine whether CKIs may deregu-
late Cdk2 and Cdk4 in p53?/?MEFs, leading to centrosome
amplification, we probed Western blots with antibodies against
p21Waf1, p27Kip1, p57Kip2, and p16INK4A(Fig. 2D). To detect
constitutive signaling triggered by the absence of p53, Western
blotting was performed on serum-starved cells. Those analyses
showed that in p53?/?MEFs, endogenous p21Waf1was unde-
tectable. In contrast, p27Kip1levels in p53?/?MEFs were un-
changed from those in wild-type and other MEFs. p57Kip2and
p16INK4Awere overexpressed in p53?/?MEFs.
We also examined the expression level of p57Kip2and
p16INK4Aby siRNA-mediated silencing of p53 in wild-type
MEFs (Fig. 2E). p53-specific siRNA duplex sequences were
synthesized and used to knock down the p53 gene in three
independent wild-type MEFs. Western blot analysis revealed
that depletion of p53 in wild-type cells did not lead to a major
elevation of p57Kip2levels but that steady-state p16INK4Alevels
were moderately increased. We conclude that the only major
alteration in a cell cycle-regulatory molecule associated with
centrosome amplification in p53?/?MEFs is the absence of
p21Waf1, consistent with published results (68). We also con-
clude that the reported high frequencies of centrosome ampli-
fication in p53?/?MEFs (22) occurred despite robust levels of
FIG. 1. Genetic ablation of p53, Cdk2, or Cdk4 leads to absence of the respective protein expression. (A) PCR-based genotyping. Results of
PCR analysis of genomic liver DNA from E.13.5 embryos generated by crossing p53?/?Cdk2?/?or p53?/?Cdk4?/?mice are shown. These gels
included five double mutants (p53?/?Cdk2?/?[left panel] or p53?/?Cdk4?/?[right panel]), one Cdk2?/?mutant, one Cdk4?/?mutant, wild-type
(Wt) embryos, and a control lacking DNA (H2O). M, molecular size marker; KO, knockout. (B) To confirm the genotyping data generated in panel
A, Western blotting was performed using antibodies specific to p53, Cdk2, and Cdk4. ?-Actin was used as a loading control (bottom). (C) Western
blotting was conducted to determine the expression levels of Cdk4 in Cdk2?/?MEFs and of Cdk2 in Cdk4?/?MEFs. To ensure that equal amounts
of proteins were loaded, ?-actin was used to probe the same membrane.
VOL. 30, 2010 Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION697
CKIs controlling Cdk2 and Cdk4 activities, including p16INK4A
and p27Kip1, potent inhibitors of centrosome amplification and
centriole duplication (36, 42, 44, 66).
Cells lacking Cdk2 and Cdk4 display abnormal centrosome
cycles. MEFs devoid of Cdk2 undergo minor defects in cen-
triole duplication (14). This suggested to us that either the
Cdks regulating the centrosome duplication cycle are redun-
dant, as is the case with the Cdks regulating S-phase entry (2,
5, 6, 38, 52, 61, 72), or other Cdks or centrosomal kinases are
solely responsible for orchestrating normal centrosome dupli-
FIG. 2. Ablation of Cdk2 or Cdk4 does not significantly alter the cell cycle. (A and B) Cells of the indicated genotypes were arrested in G0and
subsequently stimulated by addition of serum. Cells were pulse-labeled with BrdU 30 min prior to harvest and were harvested at the indicated time points
after serum stimulation. Cells were stained with anti-BrdU antibodies and the appropriate secondary antibodies and were visualized using confocal
microscopy and a 63? objective. Nuclei were counterstained with DAPI. This experiment was repeated twice; results of a representative experiment are
presented. Frequencies represent BrdU-positive cells in a population of at least 200 cells per group. Wt, wild type. (C) Whole-cell extracts were prepared
D1, cyclin E, and ?-actin as a control. Cyc, cyclin. (D) Western blotting was performed with MEFs cultured in DMEM containing 0.2% FBS for 48 h.
The numbers 1 and 2 above the lanes represent the loading of the protein lysates of two independent MEFs of the indicated genotypes. Western blots
were probed with antibodies against p21Waf1, p27Kip1, p57Kip2, and p16INK4A. ?-Actin served as a loading control. (E) Western blots of proteins extracted
from controls (wild-type MEFs) or of wild-type MEFs transfected with siRNAs specific to p53 were probed with p53, p57, p16, and ?-actin (control).
698 ADON ET AL.MOL. CELL. BIOL.
cation. Since centrosome duplication licensing must be coor-
dinated with entry into S phase (the latter initiated by cyclin
D/Cdk4 and continued by cyclin E/Cdk2), and based on the
brief expression overlap between cyclin D/Cdk4 and cyclin
E/Cdk2 activities at the G1/S transition (37), we hypothesized
that cyclin D1/Cdk4 is involved in that coordination. To assess
the involvement of Cdk4 in normal centrosome duplication,
and to explore whether Cdk2 and Cdk4 cooperate to affect
centrosome duplication, we measured the frequencies of cells
with 1, 2, or ?3 centrosomes in early-passage (passage 2)
MEFs devoid of Cdk2 and/or Cdk4 by using immunohisto-
chemistry with antibodies against pericentrin and ?-tubulin,
core components of the centrosome (Fig. 3A). Previous studies
showed that normal ratios of centrosomes in wild-type MEFs
are 60% cells with one centrosome to 40% cells with two
centrosomes (68). Any deviation in the ratios of centrosomes
within a population is indicative of defects in the various steps
driving the centrosome duplication cycle: licensing, separation
of centrioles, and duplication of centrioles (59, 68). A centro-
some ratio favoring cells with one centrosome is indicative of
defective licensing of the centrosome cycle, or centriole sepa-
ration, while a centrosome ratio favoring cells with two cen-
trosomes is indicative of premature centriole separation and
duplication. Consistent with published results, Cdk2?/?MEFs
did not display a statistically significant deviation in centro-
some ratios from wild-type MEFs (48:46% versus 58:38%,
respectively). In contrast, Cdk4?/?MEFs showed a significant
divergence from wild-type MEFs in the ratio of cells with one
centrosome to cells with two centrosomes (77:20% versus 58:
38%). This accumulation of cells with one centrosome is not
due to a longer G1phase, since Cdk4?/?MEFs entered S
phase with kinetics similar to those of wild-type MEFs (as
presented in Fig. 2A). In addition, Cdk2?/?Cdk4?/?MEFs
also displayed a severe deviation from wild-type MEFs in cen-
trosome ratios (35:55% versus 58:38%).
To assess the phase in the cell cycle at which the various
centrosome defects occurred, we dissected the centrosome du-
plication kinetics in cells by comparative analysis of synchro-
nized wild-type, Cdk2?/?, Cdk4?/?, and Cdk2?/?Cdk4?/?
cells (Fig. 3B). MEFs of the indicated genotypes were grown in
duplicate, followed by serum starvation for 60 h. Quiescent
cells were stimulated with serum, and BrdU was included in
the medium to monitor S-phase entry. Every 4 h for a period
of 16 h, BrdU incorporation and the number of cells within the
population with one or two centrosomes were scored. In ac-
cordance with the kinetics of entry into S phase, the percentage
of wild-type MEFs with two centrosomes peaked at 12 h post-
serum stimulation. Cdk2?/?MEFs reached maximal ratios of
cells with two centrosomes earlier than wild-type MEFs, at 8 h
poststimulation. In contrast to wild-type or Cdk2?/?MEFs,
the percentages of Cdk4?/?MEFs with two centrosomes de-
creased throughout the cell cycle, as the cells steadily accumu-
lated a centrosome content of one. Another intriguing defect
was that in Cdk2?/?Cdk4?/?MEFs, in which ablation of Cdk2
overrode the defect in centrosome ratios in Cdk4?/?MEFs.
Even though Cdk2?/?Cdk4?/?MEFs were unable to replicate
their DNA efficiently, their centrosome duplication peaked at
8 h post-serum stimulation; thus, the centrosome and cell cy-
cles were uncoupled in those cells.
We then explored whether transient downregulation of
Cdk2 and Cdk4 with siRNAs in wild-type MEFs recapitulated
the centrosome cycle defects in Cdk2?/?or Cdk4?/?MEFs
(Fig. 3C and D). Wild-type MEFs were transfected with
siRNA duplexes against Cdk2 or Cdk4. Seventy-two hours
after transfection, cell lysates were obtained, and centrosome
analyses were performed. Western blot analysis of the extracts
from the transfected cells showed significant depletion of Cdk2
and Cdk4 (Fig. 3C). In contrast to the Cdk2?/?centrosome
profile, which showed normal ratios of centrosomes, siRNA-
mediated downregulation of Cdk2 led to the accumulation of
cells with two centrosomes; however, as with Cdk4?/?MEFs,
depletion of Cdk4 promoted more cells with one centrosome
(Fig. 3D). Our results have identified one of the major triggers
for normal centrosome duplication, which involves Cdk4, as
well as the cooperation of Cdk2 and Cdk4 activities. Experi-
ments that knocked out and knocked down Cdk2 or Cdk4
suggested that the functions of those Cdks are unique. To
address whether ectopic expression of Cdk2 rescued the accu-
mulation of cells with one centrosome observed for Cdk4?/?
MEFs, we overexpressed Cdk2 in Cdk4?/?cells. As shown in
Fig. 3E, Cdk2 significantly rescued the centrosome defects
imposed by ablation of Cdk4, demonstrating that the centro-
some defect imparted by ablated Cdk4 is reversed by overex-
pression of Cdk2; in this scenario, Cdk2 and Cdk4 are redun-
Individual ablation of Cdk2 or Cdk4 abolishes centrosome
amplification in p53?/?MEFs by preventing excessive centri-
ole duplication. The current models attempting to explain how
the absence of p53 allows centrosome amplification propose
that elevated Cdk2 activity is primarily responsible for centro-
some amplification in those MEFs (21, 68). This was suggested
by observations that p21Waf1?/?cells have elevated frequen-
cies of centrosome amplification or that ectopic expression of
p21Waf1partly restored normal centrosome frequencies in
p53?/?MEFs (13, 68). Since p21Waf1influences cyclinD/Cdk4
positively by promoting its assembly but inhibits it at higher
concentrations (3, 29, 30, 35), we speculated that Cdk4 may
also mediate centrosome amplification in p53?/?MEFs.
Therefore, we set out to explore the relative contributions of
Cdk2 and/or Cdk4 to centrosome amplification in p53?/?
MEFs. While wild-type cells did not display elevated frequen-
cies of centrosome amplification, loss of p53 resulted in 40% of
the cells displaying centrosome amplification (Fig. 4A and B).
As predicted, ablation of Cdk2 in p53?/?MEFs prevented
centrosome amplification (by approximately 70%). To our sur-
prise, since no one has reported elevated Cdk4 activity in
p53?/?MEFs, and since our Western blots presented in Fig. 2
did not reveal any changes in any cyclins or CKIs that might
promote the deregulation of Cdk4, ablation of Cdk4 sup-
pressed centrosome amplification in p53?/?MEFs to the same
extent as ablation of Cdk2. To establish whether Cdk2 and
Cdk4 cooperated to further decrease centrosome amplifica-
tion, we calculated the frequencies of centrosome amplifica-
tion in p53?/?Cdk2?/?Cdk4?/?MEFs. Indeed, p53?/?
Cdk2?/?Cdk4?/?MEFs displayed frequencies of centrosome
amplification similar to those of p53?/?Cdk2?/?or p53?/?
Cdk4?/?MEFs. We next used siRNAs to silence the expres-
sion of Cdk2 or Cdk4 in p53?/?MEFs (Fig. 4C and D).
Western blotting indicated that most of the Cdk2 or Cdk4 was
depleted relative to the levels in the controls (Fig. 4C). Indeed,
VOL. 30, 2010Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION 699
FIG. 3. Ablation of Cdk2 and Cdk4 or siRNA-mediated silencing of Cdk2 and Cdk4 leads to distinct centrosome cycle defects. (A) Proliferating
E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, processed, and coimmunostained with anti-pericentrin, anti-?-tubulin,
and the appropriate secondary antibodies. Averages ? standard deviations of percentages of cells with one, two, and three centrosomes are shown.
Exactly 8 wild-type (Wt), 3 Cdk2?/?, 4 Cdk4?/?, and 3 Cdk2?/?Cdk4?/?embryos were analyzed. The statistical significance of the averages (P ?
0.05) was established by an unequal-variance t test. t test values for the percentages of cells in each population containing one centrosome relative
to that for the wild type were 0.159885 for Cdk2?/?MEFs, 0.000518 for Cdk4?/?MEFs, and 0.000544 for Cdk2?/?Cdk4?/?MEFs. The P values
for the percentages of cells in each population containing two centrosomes relative to that of wild-type MEFs were 0.122182 for Cdk2?/?MEFs,
0.000172 for Cdk4?/?MEFs, and 0.000528 for Cdk2?/?Cdk4?/?MEFs. The P values for the percentages of cells with ?3 centrosomes relative
to that of wild-type MEFs were 0.091487 for Cdk2?/?MEFs, 0.06122 for Cdk4?/?MEFs, and 0.000808 for Cdk2?/?Cdk4?/?MEFs. (B) MEFs
of the indicated genotypes were grown in duplicate to confluence, followed by serum starvation for 60 h. Quiescent cells were stimulated with
serum, and every 4 h for a period of 16 h, the numbers of cells with one and two centrosomes were scored. This experiment was repeated twice;
results of a representative experiment are presented. The BrdU data are the same as those presented in Fig. 2A and B; they are shown here for
purposes of clarity. (C) Western blots of proteins extracted from nontransfected wild-type cells, or from cells transfected with siRNAs against Cdk2
or Cdk4, and probed with antibodies against Cdk2 or Cdk4. The same membrane was probed with ?-actin as a control. (D) Wild-type MEFs
700ADON ET AL.MOL. CELL. BIOL.
as with the combinatorial knockouts, siRNA-mediated inhibi-
tion of Cdk2 or Cdk4 suppressed centrosome amplification in
p53?/?MEFs (Fig. 4D). We conclude that Cdk2 and Cdk4 are
individually required to mediate centrosome amplification.
In normal centrosome duplication, cells enter G1with a
single centrosome composed of two centrioles: the mother
centriole (older) and the daughter centriole (newer). To di-
rectly test whether Cdk2 and Cdk4 regulate centriole duplica-
tion, we performed a centriole reduplication assay. This assay
involves challenging cells with hydroxyurea (HU) for 48 h,
which inhibits DNA synthesis at late G1/early S phase. Since
centrosomal checkpoints are functional in wild-type cells, cen-
trosome reduplication is predicted to be absent in those cells
(44). On the other hand, cells lacking certain checkpoint con-
trols, such as those cells in which p53 is ablated, continue
duplicating their centrioles within the centrosomes, resulting in
multiple centrioles. For the centriole reduplication assay, pro-
liferating MEFs of the indicated genotypes were either left
untreated or treated with HU for 48 h. To determine the
presence of centrioles, the cells were subjected to cold treat-
ment and brief extraction prior to fixation. This treatment
destabilizes microtubules nucleated at centrosomes; hence,
centrioles can be microscopically visualized by immunostaining
for ?-tubulin, a major component of centrioles, at a high mag-
nification. Coimmunostaining of cells subjected to cold treat-
ment and brief extraction with anti-?-tubulin (which detects
the pericentriolar material [PCM]) and anti-?-tubulin revealed
that wild-type cells stopped centrosome duplication after 48 h
in culture in the presence of HU, while p53?/?cells continued
centrosome duplication (Fig. 4E and F). On the other hand,
ablation of either Cdk2 or Cdk4 in p53?/?cells completely
halted centriole reduplication. The same experiment was re-
peated with 48-h cultures in the presence of mimosine or HU,
with immunostaining done with ?-tubulin, and the results were
the same: we detected the accumulation of centrosomes in
p53?/?MEFs, and ablation of Cdk2 or Cdk4 prevented that
accumulation (data not shown). These experiments demon-
strated that ablation of Cdk2 and Cdk4 suppresses centrosome
amplification by normalizing the centrosome reduplication de-
fect triggered by ablation of p53. Importantly, this result iden-
tified one of the specific steps in the centrosome cycle affected
by the absence of Cdk2 or Cdk4: centriole duplication.
Genetic ablation of Cdk2 or Cdk4 suppresses chromosome
instability in p53?/?MEFs. Ablation of p53 generates chro-
mosome instability through centrosome amplification (22, 23,
43) and by allowing the generation of reactive oxygen species,
which are predicted to result in double-strand DNA breaks
(60). To establish how the absence of Cdk2 and Cdk4 modu-
lates active chromosome instability in p53?/?MEFs, we used
two assays: the micronucleus assay and ?-H2AX immunostain-
ing. One of the initial cellular responses to the introduction of
double-strand breaks is the phosphorylation of serine 139 of
the carboxy-terminal tail of H2AX (56). The number of phos-
phorylated H2AX (?-H2AX) molecules increases linearly with
the severity of damage. Therefore, this assay represents a way
to mark double-strand DNA breaks, a precursor to the chro-
mosome breaks and recombinations leading to structural chro-
mosomal abnormalities, the second major form of chromosome
instability. Control experiments revealed that Adriamycin, a
chemical that promotes DNA breaks, resulted in most cells in
the population containing ?-H2AX foci (not shown). As shown
in Fig. 5A and B, loss of p53 resulted in a significant elevation
of the percentage of cells with ?-H2AX foci over that for
wild-type controls. In contrast, ablation of Cdk2 or Cdk4 in
p53?/?cells decreased the number of cells containing ?-H2AX
relative to that observed for wild-type cells, consistent with
published observations that loss of Cdk2 reduces DNA repair
Our second assay to detect active genomic instability was the
micronucleus assay. A micronucleus is a chromosome or chro-
mosome fragment missegregated during mitosis as a conse-
quence of spindle damage or as a consequence of lost centro-
meric sequences (acentric chromosomes are unable to bind
mitotic fibers and are excluded from the segregating chroma-
tids) (31, 46, 69). Following cytokinesis, micronuclei appear in
the cytoplasm as DNA-containing spheres surrounded by a
nuclear membrane (Fig. 5C, arrows). The extent of micronu-
cleus formation reflects the frequency of cells in a population
actively losing whole chromosomes (a type of chromosome
instability dependent on centrosome amplification), as well as
the frequency of fragmented chromosomes, which arise as a
result of DNA breaks (57, 58). Our results indicated that
ablation of Cdk2 or Cdk4 reduced micronucleus formation in
p53?/?MEFs to wild-type levels (Fig. 5D). We conclude that
the absence of Cdk2 or Cdk4 prevents chromosome instability,
an abnormal phenotype strongly associated with tumor biogen-
esis and progression (1, 11, 16, 17, 20).
Cdk2 and Cdk4 signal centrosome amplification and chro-
mosome instability through a common phosphorylation site in
nucleophosmin, Thr199. The nucleophosmin (NPM) protein,
located at the centrosome, prevents premature centriole sep-
aration and duplication during early G1similarly to the way in
which nuclear Rb prevents premature entry from early G1into
S phase. Both proteins are phosphorylated in late G1by the
Cdks to relieve negative regulation (37, 51). Unphosphorylated
NPM binds to unduplicated centrosomes (centrosomes with
that either were left untransfected or were transfected with siRNAs against Cdk2 or Cdk4 were immunostained with anti-?-tubulin antibodies, and
frequencies were established by counting cells with one and two centrosomes in a population of at least 200 cells per group. Three independent
MEF groups were used. The statistical significance of the averages (P ? 0.05) was established by an unequal-variance t test. t test values for the
percentage of cells in each population containing one centrosome relative to the percentage with two centrosomes were 0.215535 for wild-type
MEFs, 0.003271 for MEFs transfected with a siRNA against Cdk2, and 0.008772 for those transfected with a siRNA against Cdk4. (E) Three
independent Cdk4?/?MEFs transfected with plasmids carrying either a control vector (pBABE-hygro) or pBABE-hygro-Cdk2 and plated after
selection with hygromycin were immunostained using anti-?-tubulin antibodies and the appropriate secondary antibodies. Frequencies were
established by counting cells with one and two centrosomes in a population of at least 200 cells per group. t test values of the percentage of cells
in each population containing one centrosome relative to the percentage with two centrosomes were 0.345271 for pBABE-hygro-transfected MEFs
and 0.136406 for pBABE-hygro-Cdk2-transfected MEFs.
VOL. 30, 2010Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION 701
FIG. 4. Ablation or siRNA-mediated silencing of Cdk2 and Cdk4 prevents centriole reduplication and centrosome amplification in p53?/?
MEFs. (A) MEFs of the indicated genotypes were coimmunostained with antibodies recognizing pericentrin (red) (b, f, j, n, and r) and ?-tubulin
(green) (c, g, k, o, and s). Nuclei were stained with DAPI (blue) (a, e, I, m, and q). (d, h, l, p, and t) Overlay images of the pericentrin and ?-tubulin
immunostaining. Wt, wild type. (B) Proliferating E13.5 mouse embryonic fibroblasts of the indicated genotypes were fixed, processed, and
coimmunostained with anti-pericentrin, anti-?-tubulin, and the appropriate secondary antibodies. The graph presents averages ? standard
deviations of the percentages of cells with one, two, and three or more centrosomes. Exactly 8 wild-type, 8 p53?/?, 5 p53?/?Cdk2?/?, 5 p53?/?
Cdk4?/?, and 4 p53?/?Cdk2?/?Cdk4?/?embryos were analyzed. t test values for the percentage of cells in each population containing one
centrosome (relative to that for the wild type) were 0.017174 for p53?/?MEFs, 0.137854 for p53?/?Cdk2?/?MEFs, 0.358121 for p53?/?Cdk4?/?
MEFs, and 3.95E-05 for p53?/?Cdk2?/?Cdk4?/?MEFs. P values for the percentage of cells in each population containing two centrosomes
relative to that for wild-type MEFs were 0.860687 for p53?/?MEFs, 0.9713 for p53?/?Cdk2?/?MEFs, 0.024679 for p53?/?Cdk4?/?MEFs, and
2.69E-05 for p53?/?Cdk2?/?Cdk4?/?MEFs. P values for the percentage of cells with ?3 centrosomes relative to that for wild-type MEFs were
0.006967 for p53?/?MEFs, 0.232114 for p53?/?Cdk2?/?MEFs, 0.706722 for p53?/?Cdk4?/?MEFs, and 0.051209 for p53?/?Cdk2?/?Cdk4?/?
MEFs. (C) Western blots of extracts from untransfected p53?/?MEFs or p53?/?MEFs transfected with Cdk2- or Cdk4-specific siRNAs were
probed with the indicated primary antibodies. (D) Frequencies of centrosome amplification in control p53?/?MEFs and in p53?/?MEFs in which
Cdk2 or Cdk4 was knocked down. Three independent MEFs were used. Centrosomes were detected as for panels A and B. The statistical
significance of the averages (P ? 0.05) was established by an unequal-variance t test. P values for the percentage of cells with ?3 centrosomes
relative to that of control p53?/?MEFs were 0.002445 for MEFs transfected with a Cdk2-specific SiRNA and 0.006696 for MEFs transfected with
a Cdk4-specific siRNA. (E) Proliferating MEFs of the indicated genotypes (3 per group) were either left untreated (NT) or treated with 2 mM
HU for 48 h. To determine the presence of centrioles, the cells were subjected to cold treatment and brief extraction prior to fixation. This
treatment destabilizes microtubules nucleated at centrosomes; hence, centrioles can be microscopically visualized by immunostaining for ?-tubulin
(a major component of centrioles) at a high magnification. Cells were coimmunostained with anti-?-tubulin polyclonal (green) (b, f, j, and n) and
anti-?-tubulin monoclonal (red) (c, g, k, and o) antibodies and were counterstained with DAPI (blue) (a, e, i, and m). (d, h, l, and p) Overlaid
images of ?-tubulin and ?-tubulin immunostaining. (Insets) Magnified images of the areas indicated. (F) Frequencies of centriole reduplication
were established by counting cells with ?3 separated centrioles in a population of at least 200 cells per group. P values for HU-treated compared
to NT cells were 0.791492 for wild-type MEFs, 1 for p53?/?Cdk2?/?MEFs, 0.507158 for p53?/?Cdk4?/?MEFs, and 0.012161 for p53?/?MEFs.
closely associated centrioles) to prevent premature centriole
separation and duplication (51, 70). Upon phosphorylation by
Cdk2 at the G1/S transition on T199, NPM disassociates from
the centrosome, allowing centriole separation and duplication
(51, 70). Our laboratory previously correlated unregulated cy-
clin E/Cdk2 activity with phosphorylation of NPM at T199
(59). We demonstrated that cyclin E/Cdk2 phosphorylation of
NPMT199in G0rather than in late G1rendered NPM func-
tionally inactive, since it no longer had the ability to bind
centrosomes at G0. This inability to bind centrosomes allowed
a constitutive centrosome duplication cycle, where centrioles
separated and duplicated uncontrollably, resulting in centro-
some amplification. Since the loss of Cdk4 prevented centro-
some amplification in p53?/?MEFs as efficiently as the abla-
tion of Cdk2, we set out to establish whether they shared the
same phosphorylation site in NPM to signal centrosome am-
plification and chromosome instability. Specifically, we tested
whether suppression of centrosome amplification by ablation
of Cdk2 and Cdk4 correlated with restoration of normal phos-
phorylation of NPMT199. MEFs were serum starved to mimic
G0,and the phosphorylation status of NPMT199was assessed
by Western blotting (Fig. 6A). The following results provided
important clues as to the mechanism by which ablation of Cdk2
or Cdk4 prevented centrosome amplification in p53?/?MEFs.
FIG. 5. Ablation of Cdk2 and Cdk4 inhibits chromosome instability in cells lacking p53. (A and B) The frequencies of ?-H2AX foci (arrows)
in cells with the indicated genotypes were calculated. Bars, 10 ?m. The graph (B) shows the average percentage of ?-H2AX foci in each population
of at least 200 cells. Error bars, standard deviations. Each group included 4 different MEFs. P values (relative to the wild-type control) were
0.015218 for p53?/?MEFs, 0.126173 for p53?/?Cdk2?/?MEFs, and 0.346771 for p53?/?Cdk4?/?MEFs. (C and D) Proliferating E13.5 mouse
embryonic fibroblasts of the indicated genotypes were fixed, and nuclei were visualized with DAPI. Frequencies of micronucleus (insets and
arrows) formation were calculated for at least 500 cells of each of the indicated genotypes. Each group included 4 different MEFs. P values (relative
to the wild-type control) were 0.016122 for p53?/?MEFs, 0.137054 for p53?/?Cdk2?/?MEFs, and 0.370282 for p53?/?Cdk4?/?MEFs.
VOL. 30, 2010Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION 703
FIG. 6. Cdk2 and Cdk4 affect the centrosome cycle and centrosome amplification through NPM. (A) E13.5 MEFs of the indicated genotypes
were serum starved for 60 h. Cells were preincubated with calyculin A, a serine/threonine phosphatase inhibitor. (Top) Western blot analysis of
protein fractions of G0-arrested MEFs probed with antibodies against phospho-NPMT199(p-NPMT199). (Center and bottom) Blots were probed
with antibodies against total nucleophosmin and ?-actin to show equal loading. (B) MEFs of the indicated genotypes were treated with 2 mM HU
for 48 h and were then preincubated with calyculin A, a serine/threonine phosphatase inhibitor, before protein extraction. Western blots of the
protein extracts were probed with antibodies against NPMT199or against total NPM (control). The MEFs for the left panel are independent of
those for the right. (C) Western blot analyses of MEFs of the indicated genotypes that were serum arrested and released into the cell cycle for
various times. Western blots of the protein extracts were probed with antibodies against NPMT199or against total NPM (control). (D) Western
blots of Cdk4 immunoprecipitated (Ip-Cdk4) from extracts of wild-type, Cdk2?/?, or Cdk4?/?MEFs were probed with antibodies against Cdk4
and cyclin D1. (E) Cdk4 kinase assays of protein lysates from wild-type, Cdk2?/?, and Cdk4?/?MEFs were carried out at various time points
following serum addition. Results for the 0-, 4-, and 8-h time points are shown. The results are from three independent MEFs. The experiment
was repeated at least twice, and results of a representative experiment are presented. The reaction mixtures either contained NPM peptide
(? NPM) or contained no NPM peptide (no NPM). Luminescence was recorded by the SpectraMax Gemini XS luminometer using the SoftMax
program. P values of kinase assays comparing NPM to no NPM at each indicated time point were 0.107203829 for lysates from wild-type MEFs
at 0 h, 0.037437678 for those from Cdk2?/?MEFs at 0 h, 0.000111335 for those from wild-type MEFs at 4 h, 0.002861355 for those from Cdk2?/?
MEFs at 4 h, 0.000761355 for those from wild-type MEFs at 8 h, and 0.000449084 for those from Cdk2?/?MEFs at 8 h. The P values from the
kinase assays performed with Cdk4?/?MEFs were greater than 0.05 at any given time point.
704 ADON ET AL.MOL. CELL. BIOL.
First, while wild-type cells displayed a baseline level of NPM
phosphorylation, ablation of Cdk2 resulted in a level of phos-
phorylation lower than that in wild-type cells. The relative level
of phosphorylation of NPM in Cdk4?/?MEFs was identical to
that in wild-type MEFs. Second, ablation of p53 resulted in
constitutive phosphorylation of NPMT199; importantly, the ab-
sence of Cdk2 or Cdk4 reduced the hyperphosphorylation of
NPMT199in p53?/?MEFs to wild-type levels. These results
suggested that ablation of Cdk2 or Cdk4 abrogated centrosome
amplification in p53?/?MEFs by restoring the normal phos-
phorylation of NPMT199and reestablishing normal centrosome
duplication licensing. To test whether the Thr-199 phosphory-
lation of NPM in p53?/MEFs was reduced by ablation of Cdk2
or Cdk4 under the conditions used for the centriole redupli-
cation assay, MEFs were treated with HU for 48 h, followed by
protein extraction. As shown in Fig. 6B, Western blotting in-
dicated that in HU-arrested cells, ablation of Cdk2 and/or
Cdk4 reduced the level of NPMT199phosphorylation from that
NPM/B23 is a direct substrate of Cdk2 (51). To establish the
status of NPMT199phosphorylation in wild-type, Cdk2?/?, and
Cdk4?/?MEFs throughout the cell cycle, we performed West-
ern blotting at various time points following release from se-
rum starvation (Fig. 6C). Phosphorylation of NPMT199was
moderately lower in Cdk4?/?MEFs than in wild-type MEFs at
4 and 8 h. In contrast, phosphorylation of NPMT199in Cdk2?/?
MEFs between 0 and 12 h was severely diminished. Because
Western blotting suggested that NPM was indeed a target of
Cdk4, we set out to demonstrate that Cdk4 can directly phos-
phorylate NPM. To that end, wild-type, Cdk2?/?, and Cdk4?/?
MEFs were serum starved, stimulated by the addition of
serum, and harvested at different time points, followed by
protein extraction. The presence of Cdk4 (34 kDa) and its
cofactor cyclin D1 (36 kDa) in Ip-Cdk4 was demonstrated by
analysis of Western blots probed with anti-cyclin D1 and anti-
Cdk4 antibodies (Fig. 6D). As expected, immunoprecipitations
using antibodies against Cdk4 and cyclin D1 with Cdk4?/?
MEFs did not pull down any Cdk4 or cyclin D1, demonstrating
the specificity of the antibodies. After showing that the anti-
bodies specifically pulled down Cdk4, we performed lumines-
cent kinase assays with the Kinase Glo kit (Promega), as pre-
viously described (67), immunoprecipitating cyclin D/Cdk4
complexes with antibodies against Cdk4 and incubating immu-
noprecipitates with purified NPM (Fig. 6E). In this assay, re-
duction of luminescence by the addition of kinase buffer and
purified NPM peptide to the Cdk4 immunoprecipitates indi-
cates kinase activity. To rule out the possibility that any con-
taminating Cdk2 activity would result in phosphorylation of
NPM, we performed immunoprecipitation with synchronized
Cdk2?/?MEFs. As an additional control, we performed kinase
assays with Cdk4?/?MEFs. For extracts of wild-type or
Cdk2?/?MEFs from 4 and 8 h post-serum stimulation, con-
siderable kinase activity was detected in samples containing
NPM peptide compared to samples without the substrate (no
NPM) (Fig. 6E). This phosphorylation was slightly reduced,
but still statistically significant, in wild-type MEFs at 12 and
16 h after serum addition (data available on request). We then
validated our experiment by including Ip-Cdk4 from extracts of
Cdk4?/?MEFs at the indicated time points; the extract from
the Cdk4?/?MEFs where no Cdk4 was precipitated showed no
kinase activity compared to samples without the substrate (no
NPM). These results demonstrated that NPM is a Cdk4 target
and that the maximal phosphorylation corresponded to the
point of the cell cycle where centrosome cycle licensing occurs:
mid/late G1. However, unlike the phosphorylation of NPM by
Cdk2, which occurs both in the nucleus and in the cytoplasm
(59), due to a centrosome localization signal in cyclin E (41),
phosphorylation of NPM by Cdk4 is predicted to be nuclear,
because localization of Cdk4 and of cyclin D1 was strictly
nuclear, and we did not find any Cdk4 or cyclin D1 within
centrosomes (data available on request).
To gather more-direct evidence that the phosphorylation of
NPMT199by Cdk2 and Cdk4 is critical to centrosome amplifi-
cation, plasmids encoding FLAG epitope-tagged wild-type
NPM and the mutant NPM/B23 lacking the T199 phosphory-
lation site (NPMT199A) were transfected into E13.5 MEFs de-
rived from p53 mutant embryos (Fig. 7). As control, the vector
alone was transfected. Following neomycin selection, cells
were examined for the level of expression of exogenous NPM/
B23 by Western blot analysis using an anti-FLAG antibody.
This analysis revealed that NPM- and NPMT199A-transfected
cells expressed similar protein levels (Fig. 7A). In addition, we
set out to establish whether wild-type NPM or NPMT199A
modulated the frequencies of centrosome amplification in
p53?/?MEFs (Fig. 7B). This analysis revealed that while
p53?/?MEFs carrying the vector control and those expressing
wild-type NPM had the same frequencies of centrosome am-
plification, the mutant lacking the G1Cdk phosphorylation site
displayed greatly reduced frequencies of centrosome amplifi-
cation. The extent of inhibition was almost identical to that in
p53?/?Cdk2?/?or p53?/?Cdk4?/?MEFs (as presented in
Fig. 4B). In summary, we have demonstrated that Cdk2 and
Cdk4 hyperphosphorylate NPM and that this hyperphosphor-
ylation is critical to centrosome amplification.
In addition, we investigated whether the suppression of cen-
trosome amplification by NPMT199Arestored genomic stabil-
ity. Cells expressing the vector and wild-type NPM had the
same frequencies of micronucleus formation, while those ex-
pressing NPMT199Ahad reduced frequencies (Fig. 7C). Like-
wise, NPMT199Ainhibited the formation of ?-H2AX double-
stranded foci (Fig. 7D). We conclude from these experiments
that ablation of either Cdk2 or Cdk4 prevents the generation of
chromosome breaks and chromosome losses in p53?/?MEFs.
We propose a new paradigm for how centrosome amplification
and chromosome instability arise in p53?/?MEFs: the pres-
ence of both Cdk2 and Cdk4 is absolutely required in order to
hyperphosphorylate NPMT199to generate centrosome ampli-
fication and chromosome instability (Fig. 8).
In this study, we investigated the relative contributions of the
G1Cdks—Cdk2 and Cdk4—to centrosome amplification in
p53?/?MEFs and to the regulation of the centrosome cycle.
How do Cdk2 and Cdk4 affect the centrosome cycle? Ablation
of Cdk2 leads to moderate defects in centrosome duplication,
suggesting redundancy in the control of this process (14); our
results with asynchronously growing Cdk2?/?MEFs confirmed
those results. However, experiments measuring the centro-
some cycle at various time points throughout the cell cycle in
VOL. 30, 2010Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION 705
Cdk2?/?and Cdk4?/?MEFs, as well as transient downregu-
lation of Cdk2 and Cdk4 using siRNAs, uncovered distinct
centrosome cycle defects, suggesting that the functions of these
Cdks are nonredundant. For example, while Cdk2 deficiency
promoted the early separation and duplication of centrosomes,
the absence of Cdk4 promoted the accumulation of cells with
one centrosome that failed to separate and duplicate. The
accumulation of cells with one centrosome in Cdk4?/?MEFs
was not compensated for by passage, since the accumulation of
cells with one centrosome was also observed in MEFs silenced
with a siRNA directed against Cdk4. The accumulation of cells
with one centrosome in Cdk4?/?MEFs was not due to a block
in G1; these MEFs entered S phase with kinetics similar to
FIG. 7. NPMT199Asuppresses centrosome amplification and chro-
mosome instability. (A) Passage 2 p53?/?MEFs were transiently trans-
fected with plasmids encoding FLAG epitope-tagged NPM and the
NPM/B23 mutant (NPMT99A). As a control, an empty vector was
transfected. After neomycin selection, cell lysates were obtained and
then probed with anti-FLAG antibodies. (B) The transfectants de-
scribed in the legend to panel A were fixed, immunostained with
anti-?-tubulin polyclonal antibodies, and detected with Alexa Fluor
488-conjugated secondary antibodies. Cells were counterstained with
DAPI. The number of cells with ?3 centrosomes in a population of at
least 200 cells was statistically analyzed by fluorescence microscopy.
Each group included three transfected MEFs. P values (relative to the
result for the transfected vector control) were 0.006963 for NPMT199A
and 0.560677 for wild-type NPM. (C) Proliferating E13.5 mouse em-
bryonic fibroblasts of the indicated genotypes were fixed, and nuclei
were visualized with DAPI. The frequencies of micronucleus forma-
tion in a population of 500 cells were calculated for the indicated
genotypes. P values (relative to the result for the transfected vector
control) were 0.011338 for NPMT199Aand 0.353737 for wild-type
NPM. (D) Frequencies of ?-H2AX foci in cells with the indicated
genotypes were calculated. P values (relative to the result for the
transfected vector control) were 0.002591 for NPMT199Aand 0.476327
for wild-type NPM.
FIG. 8. Model explaining how the ablation of Cdks prevents cen-
trosome amplification. (A) Ablation of p53 results in undetectable
levels of p21Waf1, leading to hyperactive Cdk2 and Cdk4. Hyperactive
Cdks cross talk to the centrosome in two modes. First, hyperactive
Cdks hyperphosphorylate Rb in the nucleus, leading to uncontrolled
E2F-dependent transcription of molecules that influence various steps
in the centrosome duplication cycle: those involved in centriole split-
ting, as well as centriole duplication kinases (CtDKs). Second, hyper-
active Cdks constitutively phosphorylate NPMT199, resulting in exces-
sive licensing of centrosome duplication. Uncontrolled expression of
CtDKs and the inability of NPM to suppress normal centrosome du-
plication lead to faster centrosome duplication cycles within a single
cell cycle, resulting in the formation of multiple centrosomes.
(B) When Cdk2 or Cdk4 is deleted from p53?/?MEFs, Rb is under-
phosphorylated, and the E2F-dependent transcription of CtDKs is
restored. In addition, underphosphorylated NPM restores normal cen-
trosome licensing and prevents excessive centriole duplication. This
restricts the centrosome duplication cycle to one per cell cycle, thus
resulting in normal centrosome numbers.
706ADON ET AL.MOL. CELL. BIOL.
those of wild-type and Cdk2?/?MEFs. On the other hand,
inhibition of Cdk2 with siRNAs led to many cells in the pop-
ulation harboring two centrosomes, suggesting that that defect
was compensated for in Cdk2?/?MEFs by Cdk4, other Cdks,
or centrosomal kinases. Our observations showed that con-
comitant ablation of Cdk2 and Cdk4 caused a severe accumu-
lation of cells with two centrosomes; experiments following the
cell and centrosome cycles in Cdk2?/?Cdk4?/?MEFs con-
firmed that centriole separation and duplication were prema-
ture. The fact that the centrosome cycle defect in Cdk2?/?
Cdk4?/?MEFs was much more severe than that in other
genetic groups suggests that Cdk2 and Cdk4 cooperate to
regulate the centrosome cycle. The accumulation of cells with
two centrosomes in asynchronously growing Cdk2?/?Cdk4?/?
MEFs was not a complete block, since 40% of cells still con-
tained one centrosome. Upon serum arrest, the ratio of the
number of Cdk2?/?Cdk4?/?MEFs with one centrosome to
that with two centrosomes was similar to that for wild-type
MEFs, and upon serum addition, centrosomes separated and
duplicated prematurely, indicating an active centrosome cycle
in Cdk2?/?Cdk4?/?MEFs. In those MEFs, the cell and cen-
trosome cycles were uncoupled: centriole separation and du-
plication preceded entry into S phase. Thus, as in the cell cycle,
in the absence of Cdk2 and Cdk4, other Cdks or centrosome
duplication kinases also play a role in driving the centrosome
duplication cycle. That redundancy is also supported by the
baseline phosphorylation of NPMT199in p53?/?Cdk2?/?
MEFs cultured with HU, demonstrating that
NPMT199is phosphorylated by another kinase in the absence
of Cdk2 and Cdk4. Those results indicated that the same re-
dundancy operating within the kinases controlling S phase also
exists in the regulation of the centrosome cycle.
Because NPM phosphorylation at Thr199 by Cdk2 is essen-
tial to signal entry into the centrosome cycle (51, 70), we
assessed whether the centrosome defects observed in Cdk2?/?
and Cdk4?/?MEFs were due in part to changes in NPMT199
phosphorylation. As reported previously, NPMT199is a canon-
ical target of Cdk2; our Western blot analyses revealed that at
early time points (0 to 8 h) following the addition of serum to
Cdk2?/?MEFs, the phosphorylation of NPMT199was greatly
diminished from that for wild-type MEFs. The phosphoryla-
tion of NPMT199increased in Cdk2?/?MEFs at 12 h following
serum addition. The fact that cyclin D1 levels peaked at 12 h of
serum addition in Cdk2?/?MEFs indicated that perhaps cyclin
D1/Cdk4 was responsible for that phosphorylation. On the
other hand, levels of NPMT199were moderately reduced in
Cdk4?/?MEFs at early time points after serum addition (0 to
8 h). Our Cdk4 kinase assays using NPM as a substrate con-
firmed that NPM is indeed a Cdk4 target and that its phos-
phorylation is cell cycle regulated. In serum-arrested cells we
did not detect phosphorylation of NPM; that phosphorylation
commenced at 4 and 8 h following serum addition and was
detected up to 16 h after serum addition. However, the fact
that the reduction in NPMT199phosphorylation was not nearly
as great in Cdk4?/?MEFs as in Cdk2?/?MEFs suggests that
the Cdk2 present in Cdk4?/?MEFs actively phosphorylates
NPMT199and that the phosphorylation of NPMT199is more
efficiently carried out by Cdk2 than by Cdk4.
We speculate that cells have evolved compensatory mecha-
nisms such that when they are devoid of Cdk2, Cdk4 can
indeed license the centrosome cycle. However, at this point we
cannot rule out the possibility that Cdk2 phosphorylates
NPMT199to prime Cdk4 phosphorylation of NPM in other
sites that would control centrosome licensing at late G1. In-
deed, there is evidence in the literature that phosphorylation of
NPM at Thr234 and Thr237 by Cdk1 stabilizes NPM in the
centrosome during M phase (9). A second possible reason why
Cdk4?/?MEFs display cells with one centrosome is that Cdk4
may directly phosphorylate and regulate the activity of a cen-
triole separase, or it may regulate the Rb/E2F-dependent tran-
scription of a centriole separase. Centriole separases are
poorly understood; one centrosome separase normally active
at M phase that displays centriole separase activity when
ectopically expressed during interphase is Nek2A (18). A third
possibility is that Cdk4 modulates other licenser factors besides
NPM. The only other known centrosome-regulatory protein
that may display licensing activity is CP110. Like NPM, CP110
is a phosphorylation target of Cdk2, and inhibition of its ac-
tivity causes centrosome amplification (10). However, unlike
NPM, which disassociates from centrosomes upon Cdk phos-
phorylation, CP110 is present in the centrosome to cap the
synthesis of a new centriole (34). Thus, we think it unlikely that
Cdk4 utilizes CP110 to license the centrosome cycle. Never-
theless, the activity inhibited by the ablation of Cdk4 that leads
to the accumulation of cells with one centrosome is reversible,
since ablation of Cdk2 (in Cdk2?/?Cdk4?/?MEFs), ectopic
expression of Cdk2, or ablation of p53 does not allow the
accumulation of cells with one centrosome.
What causes the accumulation of two centrosomes in cells in
which Cdk2 is knocked down, and in cells in which Cdk2 and
Cdk4 are ablated? Our experiments following the cell and
centrosome cycles in Cdk2?/?MEFs and in Cdk2?/?Cdk4?/?
MEFs revealed premature separation and duplication of cen-
trosomes. The premature accumulation of cells with two cen-
trosomes was present in early cell cycle stages of Cdk2?/?
MEFs, but the premature accumulation was restored at later
passages cycle. Perhaps Cdk2 alone and Cdk2 and Cdk4 to-
gether impose negative regulation of centriole separases or of
other centrosome kinases. For example, Cdk2 targets other
than NPM may prevent the binding of centriole separases to
the centriole pair. When Cdk2 alone or both Cdk2 and Cdk4
are ablated, those centriole separases and/or centriole dupli-
cation kinases may associate with the centrosomes prematurely
to signal early centriole separation and duplication. Other tar-
gets of Cdk2 that regulate various steps within the centrosome
duplication cycle include CP110 and mMps-1 (10, 19). Plk4 has
not been reported to be directly regulated by Cdk2 but re-
quires Cdk2 activity for its maximum activity (26). Our future
experiments will identify the centriole separases, centrosome
cycle licensers, or centriole duplication kinases deregulated in
Cdk2?/?, Cdk4?/?, and Cdk2?/?Cdk4?/?MEFs at the tran-
scriptional or posttranslational level.
Our first major finding was that Cdk2 and Cdk4 regulate
distinct steps in the centrosome cycle. The second major find-
ing is that either Cdk2 or Cdk4 signals centrosome amplifica-
tion in p53-null MEFs. Ablation of p53 was proposed to lead to
centrosome amplification, in part by preventing p21Waf1from
controlling Cdk2 (13, 39, 68). Using a genetic approach, we
demonstrated that, indeed, Cdk2 is a major mediator of cen-
trosome amplification in p53?/?MEFs. A unique finding was
VOL. 30, 2010 Cdk2 AND Cdk4 IN CENTROSOME AMPLIFICATION707
that ablation of Cdk4 was equally effective at suppressing cen-
trosome amplification. This was unexpected, since no one has
reported elevated Cdk4 activity in p53?/?MEFs. Western blot
analysis assessing the relative levels of cyclins or CKIs that
promote or inhibit Cdk2 or Cdk4 activities failed to identify
any deregulated cell cycle-regulatory molecule that would pro-
mote Cdk4 activity. For example, levels of cyclin D1 were
similar in all groups. On the other hand, p16 was upregulated
in p53?/?MEFs, which would be consistent with decreased
How does Cdk4 mediate centrosome amplification in p53?/?
MEFs? At low concentrations, p21Waf1is a specific inhibitor of
Cdk2 and promotes the assembly of cyclin D/Cdk4 (35), while
at higher concentrations it equally inhibits Cdk2, Cdk3, Cdk4,
and Cdk6 (30, 35). In fact, there is precedent for the involve-
ment of p21Waf1in inhibiting Cdk2 and Cdk4 activities trig-
gered by the Ras oncogene, since mice lacking p21Waf1and
overexpressing MMTV-H-RasG12Vdisplay higher Cdk2 and
Cdk4 activities than those expressing H-RasG12Valone (4).
Perhaps a similar scenario exists within the centrosome, as
p21Waf1may inhibit both Cdk2 and Cdk4 activities to regulate
centrosome duplication; thus, ablation of p53 and the concom-
itant absence of endogenous p21Waf1may hyperactivate Cdk2
and Cdk4. Another possibility is that ablation of Cdk2 or Cdk4
changes the CKI/Cdk ratios within the cell, allowing more
p21Waf1to bind to and inhibit the Cdk that is still present in the
cell; this compensatory mechanism has been observed in
Cdk4?/?MEFs, in which more p27Kip1is available to bind and
inhibit Cdk2 activity (72). However, observations that p53?/?
cells are devoid of detectable endogenous p21Waf1render un-
likely the possibility that more p21Waf1is available to inhibit
Cdk2 activity in p53?/?Cdk4?/?MEFs to prevent centrosome
amplification. Other Cdks that may impose that kind of com-
pensatory regulation are p16 and p57, which are overexpressed
in p53?/?MEFs. Nevertheless, since p16 and p57 did not
accumulate upon transient knockdown of p53, and because the
silencing of Cdk2 or Cdk4 was able to suppress centrosome
amplification in the same cells, that level of indirect compen-
satory inhibition of Cdk2 or Cdk4 by the CKIs is unlikely. Still,
because our experiments have not ruled out that type of com-
pensatory mechanism, we will develop composite p53, Cdk,
and CKI knockdowns and knockouts to directly address the
individual roles of those CKIs in preventing the hyperactivity
of the remaining Cdk.
The following evidence supports a direct role for Cdk2 and
Cdk4 in mediating centrosome amplification in p53-null cells.
First, concomitant loss of Cdk2 and Cdk4 in p53?/?MEFs did
not reduce the frequencies of centrosome amplification more
than the ablation of each individual kinase, rendering unlikely
the possibility that the compensatory mechanism imposed by
CKIs on Cdk2 or Cdk4 kinase activities is responsible for the
suppression of centrosome amplification when either kinase is
ablated. Second, concomitant ablation of Cdk2 and Cdk4 in
p53-deficient MEFs cultured in HU reduced NPMT199hyper-
phosphorylation to the same extent as ablation of Cdk2 or
Cdk4 alone. In addition, rendering the NPMT199site nonphos-
phorylatable by mutating the Cdk2/Cdk4 phosphorylation site
(NPMT199A) suppressed centrosome amplification to the same
extent as ablation of Cdk2 or Cdk4, demonstrating that abla-
tion of Cdks prevents maximum centrosome amplification.
Therefore, we conclude that both Cdk2 and Cdk4 are individ-
ually required to signal centrosome amplification in cells in
which p53 has been deleted.
How do Cdk2 and Cdk4 deregulate centrosome duplication,
resulting in centrosome amplification? The model depicted in
Fig. 8, based on published observations (44, 51, 59, 70) and the
observations reported here, shows two ways in which the G1
Cdks could affect centrosome amplification: first, by triggering
unregulated phosphorylation of Rb and the release of E2Fs,
and second, by the direct phosphorylation of centrosomal tar-
gets. Although we did not perform a wide screen for all the
E2F targets that may result in centrosome amplification in
p53?/?MEFs, those E2F targets that we analyzed by Western
blotting were unchanged (cyclins A, E, and D). Thus, in the
present study, we tested the second scenario.
In this study, we resolved how Cdk2 and Cdk4 directly
impinge on centrosome amplification through NPM. Our ex-
periments indicated that ablation of Cdk2 or Cdk4 decreased
NPMT199hyperphosphorylation in p53?/?MEFs arrested in
G0(serum arrested) or in late G1(under an HU block). Those
ylation of NPMT199normalized the licensing of the centrosome
cycle, as well as the excessive centriole reduplication, in p53?/?
MEFs to prevent centrosome amplification. Our experiments
showing that the introduction of a mutant NPM lacking Cdk2
and Cdk4 phosphorylation sites (NPMT199A) abrogated cen-
trosome amplification in p53?/?MEFs to the same extent as
the ablation of Cdk2 or Cdk4 demonstrated that, indeed, the
Cdk2, Cdk43NPMT199pathway is central to the prevention of
centrosome amplification. Based on our observations that
Cdk2 and Cdk4 cross talk to the centrosome through the same
phosphorylation site in NPM, Thr199, we propose a novel
paradigm: that full-fledged phosphorylation of NPM by both
Cdk2 and Cdk4 in p53?/?MEFs is critical to the induction of
centrosome amplification. How do Cdk2 and Cdk4 prevent
centriole reduplication? Our experiments showing that abla-
tion of Cdk2 or Cdk4 prevented centriole reduplication clearly
identified an important step within the centrosome cycle that
impinges on centrosome amplification. Western blot analyses
showing that the hyperphosphorylation of NPMT199is abro-
gated in p53?/?Cdk2?/?, p53?/?Cdk4?/?, and p53?/?
Cdk2?/?Cdk4?/?MEFs indicated that the restoration of nor-
mal centrosome cycle licensing is key to the prevention of
centriole reduplication. Another putative mechanism is that
the Cdks control the expression or activity of centriole dupli-
cation kinases, including Plk4, Plk2, and mMps-1. So far, West-
ern blotting and real-time PCR have not detected increased
levels of Plk4 in p53?/?MEFs; we have yet to explore whether
loss of p53 influences Plk4’s kinase activity. Thus, future ex-
periments will address how the activities of centriole duplica-
tion kinases are modulated by Cdk2 and Cdk4.
A third major finding was that ablation of Cdk2 or Cdk4 in
p53?/?MEFs inhibits two precursors to the two major types of
chromosome instability: the formation of micronuclei (precur-
sors to aneuploidy) and double-strand DNA breaks (precur-
sors to chromosome rearrangements). We conclude that both
G1Cdks are critical to centrosome amplification and chromo-
some instability in p53?/?MEFs. Our data have important
implications for cancer therapy, since the 50% of human can-
cers that harbor p53 mutations can be treated with small-
708 ADON ET AL.MOL. CELL. BIOL.
molecule inhibitors of Cdk2 or Cdk4, currently under devel-
opment or undergoing clinical trials (63). By using inhibitors
specific to either Cdk, we can remove the ability of p53 ablation
to generate chromosome instability, an abnormal phenotype
strongly associated with a poor prognosis and resistance to
chemotherapy (8, 15), thus stopping cancer progression in its
tracks. One could accomplish this inhibition without the pro-
liferative toxicity predicted to occur when Cdk2 and Cdk4 are
concomitantly inhibited and without the mitotic and develop-
mental defects triggered by Cdk1 inhibition (6, 61, 63).
We thank Carla G. Saavedra for scientific editing and Joi Car-
michael for assistance with mouse husbandry. Thanks to Peter J. Stam-
brook and Paul W. Doetsch for critical discussions of the manuscript.
Special thanks to Kenji Fukasawa for NPM constructs, and to Gustavo
W. Leone for Cdk2 expression vectors. We acknowledge Adam Mar-
cus from the Winship Cancer Institute’s imaging core facility for as-
H. I. Saavedra was supported by NCI award K01CA104079, a Geor-
gia Cancer Coalition Distinguished Scholar Award, and the Depart-
ment of Radiation Oncology, Emory University School of Medicine.
H. Kiyokawa was supported by NIH-R01-CA100204 and NIH-
R01CA112282. M. K. Harrison was supported by the Genetics and
Molecular Biology Program’s NIH Predoctoral Training Grant
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