Oncogene (2005) 24, 277–286
& 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00
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Regulation of cell cycle checkpoints by polo-like kinases
Suqing Xie1, Bin Xie2, Marietta Y Lee2 and Wei Dai*,1
1
Molecular Carcinogenesis Division, Department of Medicine & Brander Cancer Research Institute, New York Medical College,
Valhalla, NY 10595, USA; 2Department of Biochemistry, Molecular Carcinogenesis Division, New York Medical College,
Valhalla, NY 10595, USA
Protein kinases play a pivotal role in execution of cell
division. Polo and Polo-like kinases have emerged as
major regulators for various cell cycle checkpoints. Early
genetic studies have demonstrated that CDC5, a budding
yeast counterpart of vertebrate Plks, is essential for
successful mitotic progression. Mammalian Plks localize
primarily to the centrosome during interphase and the
mitotic apparatus during mitosis. Many key cell cycle
regulators such as p53, Cdc25C, cyclin B, components of
the anaphase-promoting complex, and mitotic motor
proteins are directly targeted by Plks. Although the exact
mechanism of action of these protein kinases in vivo
remains to be elucidated, Plks are important mediators for
various cell cycle checkpoints that monitor centrosome
duplication, DNA replication, formation of bipolar
mitotic spindle, segregation of chromosomes, and mitotic
exit, thus protecting cells against genetic instability during
cell division.
Oncogene (2005) 24, 277–286. doi:10.1038/sj.onc.1208218
Keywords: polo; polo-like kinases; cell cycle; checkpoint; cell division
Introduction
The progression of the cell cycle is tightly regulated in
order to maintain genetic integrity and to ensure that
genetic information is correctly passed on to daughter
cells. Surveillance mechanisms, known as checkpoints,
have thus been discovered, that monitor the integrity of
the cell cycle progression (Elledge, 1996). By definition,
cell cycle checkpoints inhibit cell cycle progression until
the specific cellular processes under surveillance are
completed with high fidelity. In eukaryotes, both DNA
replication and chromosome segregation are highly
regulated and are monitored at several steps in the cell
cycle. A loss of checkpoint function can result in
infidelity of DNA replication or of chromosome
segregation, and thereby predispose cells to genetic
instability.
Reversible phosphorylation is a fundamental molecular mechanism that is critical for regulating cell
division. Deregulated phosphorylation of the components important to cell cycle control frequently leads to
*Correspondence: W Dai; E-mail: [email protected]
abnormal cell division and/or transformation. To date,
several families of protein kinases such as cyclindependent kinases (Cdks), polo-like kinases (Plks), and
Aurora family of kinases have been characterized, which
are important to cell cycle regulation. Extensive research
in the past decade or so has demonstrated that Polo and
Plks are involved in regulation of various cell cycle
checkpoints that ensure the timing and order of cell
cycle events such as DNA repair, bipolar spindle
formation, chromosome segregation, and mitotic exit
(Nigg, 1998; Dai et al., 2002; Barr et al., 2004).
In this review, we summarize the properties of Polo
and Plks in various cell cycle checkpoint controls. We
also present new lines of evidence supporting the role of
mammalian Plks in regulating both an intra-S-phase
checkpoint and a spindle checkpoint.
Plks’ role in intra-S-phase checkpoint
Checkpoint activation pathway
During the S phase, the genome is most susceptible to
damage caused by environmental stresses and/or internal perturbations. Thus, it is natural that cells have
evolved sophisticated mechanisms to monitor DNA
damage and initiate repair processes if the damage is not
extensive. Extensive research in the past decade has
shown that the signaling pathways that underlie the
cellular response to DNA damage (or genotoxic stress)
consist of sensors, signal transducers, and effectors
(Zhou and Elledge, 2000). Although the identities of the
damage sensors remain unclear, the molecular entities
responsible for transducing the damage signals to
specific effectors are relatively well characterized.
ATM (ataxia telangiectasia mutated) and its homolog
ATR (ATM-related) function early in the signaling
pathways and are central to the DNA damage response
(Sancar et al., 2004). Downstream mediators/transducers of ATM and ATR include the protein kinases
Chk1, Chk2, and likely Plk3 (Xie et al., 2001b; Sancar
et al., 2004). The effectors of the checkpoint include p53
and Cdc25 (Sancar et al., 2004).
Both CDC5 in the budding yeast and mammalian
Plks are known to participate in the DNA damage
response. An early study shows that the electrophoretic
mobility of Cdc5 in denaturing gels is affected when the
cells are exposed to DNA damage agents, and this
Plks and cell cycle checkpoints
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278
modification of Cdc5 is dependent on Mec1, Rad53 (a
yeast Chk1 homolog), and Rad9 (Cheng et al., 1998). In
addition, a functionally defective Cdc5 mutant protein
suppresses a Rad53 checkpoint defect, whereas overexpression of Cdc5 overrides checkpoint-induced cell
cycle arrest (Sanchez et al., 1999), suggesting that Cdc5
acts downstream of Rad53. Several recent studies show
that Plk1 activity is inhibited upon DNA damage (Smits
et al., 2000; van Vugt et al., 2001; Ando et al., 2004). The
DNA damage-induced inhibition of Plk1 appears to be
mediated by blocking its activation because expression
of activation mutants of Plk1 can override the G2/M
arrest induced by DNA damage (Smits et al., 2000).
Subsequent studies by this group reveal that DNA
damage-induced inhibition of Plk1 is at least in part
dependent on ATM or ATR because caffeine treatment
blocks the inhibition of Plk1 after IR- or UV-induced
DNA damage (van Vugt et al., 2001). The activity of
p53 is greatly enhanced upon DNA damage checkpoint
activation.
A recent study shows that Plk1 binds p53 and inhibits
the transactivating activity of this tumor suppressor
protein; the kinase activity of Plk1 is required for
inhibition of p53 activity; besides, Plk1 also negatively
affects the proapoptotic function of p53 in human lung
tumor cell lines (Ando et al., 2004). Again, ATM is
capable of attenuating Plk1-mediated suppression of
p53 activity (Ando et al., 2004). Given the potential role
of Plk1 in promoting cell proliferation and tumorigenesis (Dai and Cogswell, 2003), these observations are
consistent with the negative role of Plk1 in DNA
damage-induced cell cycle arrest. On the other hand,
there are also reports suggesting that Plk1 may play a
positive role in DNA damage checkpoint activation.
Chk2 co-immunoprecipitates with Plk1, overexpression
of which enhances phosphorylation of Chk2 at T68
(Tsvetkov et al., 2003), a site primarily targeted by
ATM, and its phosphorylation is correlated with its
activation (Ahn et al., 2000); in addition, Plk1 phosphorylates recombinant Chk2 in vitro at the same site
and colocalizes with Chk2 to a certain mitotic apparatus
(Tsvetkov et al., 2003). In a separate study, Drosophila
Polo has also been implicated to be a substrate of Chk2
as well (Xu and Du, 2003).
Other mammalian Plks are also involved in the DNA
damage checkpoint activation pathway. Expression of
Plk2 mRNA is rapidly induced in human thyroid cells
upon X-ray irradiation; a radiation-responsive element
has been identified as p53RE, a p53-binding homology
element, in the basal promoter region of this gene
(Shimizu-Yoshida et al., 2001). Consistent with that
observation, a separate study shows that Plk2 expression is partly mediated by p53 (Burns et al., 2003). A
recent study by Swallow et al shows that Plk4 interacts
with p53 and that this interaction is mediated by the
polo box (this review issue). Several lines of evidence
indicate that Plk3 is an important mediator in the DNA
damage checkpoint response pathway. Plk3 kinase
activity is activated upon oxidative stress and DNA
damage induced by ionizing-radiation mimetic drugs,
and its activation is ATM-dependent (Xie et al.,
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2001a, b). Plk3 interacts with and phosphorylates p53,
targeting serine-20 of p53 in vitro (Xie et al., 2001b). In
response to DNA damage, the kinase activity of Plk3 is
rapidly increased in an ATM-dependent manner (Xie
et al., 2001a, b). Peptide mapping, as well as in vitro
phosphorylation followed by immunoblot analysis with
antibodies specific for phosphorylated forms of p53, also
indicates that Plk3 phosphorylates p53 on physiologically relevant sites. Immunoprecipitation and ‘pulldown’ assays reveal that Plk3 physically interacts with
p53 and that the extent of this interaction is increased in
response to DNA damage (Xie et al., 2001b). The role of
Plk3 in regulation of serine-20 phosphorylation of p53 in
vivo is further supported by our recent RNAi study.
Downregulation of endogenous Plk3 through transfection of small-interfering RNA (siRNA) to Plk3, but not
to luciferease, significantly compromises phosphorylation of p53 on serine-20 before and after oxidative stress
(Figure 1). Plk3 also physically interacts with Chk2
(Bahassi et al., 2002; Xie et al., 2002), and there exists a
functional connection as well between these two
enzymes during DNA damage checkpoint activation.
Plk3 phosphorylates Chk2 on a residue different from
threonine-68, and it may contribute to the full activation
of Chk2 although ATM is necessary for phosphorylation and activation of Chk2 in vivo (Bahassi et al., 2002).
Together, these combined studies suggest that Plk3
functionally links DNA damage to the induction of cell
cycle arrest or apoptosis.
DNA damage repair
A possible role for Plks in DNA synthesis or repair is
implied from several early studies. Expression of both
mammalian Plk2 and Plk3 is rapidly induced by mitogen
treatment (Simmons et al., 1992; Li et al., 1996).
Induction of Plk3 mRNA by mitogens is protein
synthesis-independent, suggesting that it is an immediate-early gene product (Li et al., 1996). Furthermore,
Figure 1 Silencing of Plk3 results in significantly reduced p53
phosphorylation on serine-20. GM00637 cells, which constitutively
express high levels of p53, were transfected with siRNA duplexes to
Plk3 (Plk3-siRNA) or to luciferase GL3 (Luc-siRNA) (SMARTpool, Dharmacon) for 72 h. The transfected cells were treated with
H2O2 (100 mM) for 15 min and then collected for preparation of
lysates. Equal amounts of cell lysates were blotted for Plk3, serine20-phosphorylated p53, and actin
Plks and cell cycle checkpoints
S Xie et al
279
microinjection of sense Plk1 mRNA, but not antisense
RNA, into NIH3T3 cells that have been serum-starved
results in incorporation of 3H-thymidine (Hamanaka
et al., 1994). The latter observation suggests that Plk1 is
required for DNA synthesis and overexpression of Plk1
appears to be sufficient for induction of DNA synthesis.
Our recent study suggests that Plk3 may directly
participate in DNA repair by regulating the activity of
DNA polymerase d (pol d). DNA pol d is thought to be
a major enzyme involved in the DNA damage repair
process (Hubscher et al., 2002; Haracska et al., 2003).
Pol d has been identified as four subunits in Schizosaccharomyces pombe and four subunits in mammalian
cells (p125, p68, p50 and p12) (Hubscher et al., 2002).
Post-translational modifications by phosphorylation are
well documented for DNA polymerases; Pol d is a
phosphorylatable protein whose phosphorylation peaks
during the S phase (Zeng et al., 1994). In order to
identify proteins that either are targets of Plk3 or
regulatory components of Plk3, we have employed the
antibody array approach to screen a potential interaction between Plk3 and cell cycle or signaling proteins.
Our initial screen results reveal that p125, the major Pol
d subunit, is one of the proteins that interacted with Plk3
(data not shown). The physical interaction between
these two proteins was further confirmed by coimmunoprecipitation (data not shown) and pull-down
experiments. Ni-NTA resin, having a high affinity to
proteins with a stretch of histidine residues, is capable of
pulling down p125 antigen from lysates prepared from
cells co-infected with His6-Plk3 and p125 baculoviruses,
but not from parental cells with no viral infection or
cells infected with p125 baculovirus alone (Figure 2a).
Plk3’s kinase activity is high during the S phase
(Ouyang et al., 1997). Therefore, in vitro kinase assays
were performed to examine whether recombinant Plk3
directly phosphorylates p125. We first made a series of
truncated forms of p125 expressed as glutathione s
transferase (GST) fusion proteins. Purified proteins were
subjected to kinase assays in vitro. We observed that a
deletional form of p1251365 encompassing amino acids
1–365 (of the total 1107 amino acids) is phosphorylated
by His6-Plk3, whereas other truncated forms were not
phosphorylated (Figure 2b). Additional deletions revealed that the phosphorylation site(s) resides in amino
acids 1–115 (Figure 2c). Two-dimensional peptide
mapping of this fragment shows that there is only one
major phosphorylation site in the p1251115 fragment
(Figure 2d), although five serine residues could be
potential targets of Plk3. To identify p125’s Plk3
phosphorylation site, we constructed GST expression
constructs coding for mutant peptide fragments with
each of five serine/threonine residues replaced with
alanines, respectively. Various phosphorylation site
mutant proteins of p1251115 were then expressed and
purified (Figure 2e). Approximately equal amounts of
the purified mutant proteins were used for in vitro kinase
assays in the presence of His6-Plk3. The mutant
p1251115 with serine-60 substituted with alanine is not
phosphorylated by His6-Plk3, whereas all other mutants
are phosphorylated (Figure 2f), confirming that serine-
Figure 2 Plk3 interacts and phosphorylates the p125 subunit of
Pol d. (a) Sf9 cells either infected with p125 baculovirus or both
His6-Plk3 and p125 baculoviruses for 48 h. Cell lysates were
prepared from the infected cells, as well as from uninfected control
cells. Ni-NTA resin, which was capable of binding His6-Plk3, was
added to each lysate. After thorough washing, proteins bound to
Ni-NTA resin, along with cell lysates, were blotted for p125. (b, c)
Various fragments of p125 were expressed as GST fusion proteins.
Approximately equal amounts of purified proteins, as well as GST
alone and casein, were used for in vitro kinase assays in the presence
of purified His6-Plk3. Kinase buffer alone was also used as a
negative control. Reaction mixtures were fractionated on denaturing SDS–polyacrylamide gels, followed by autoradiography. Each
number denotes a peptide fragment encompassing the particular
region of p125 protein. (d) The phosphorylated band (arrow GST1-110) as shown in (c) was excised from the gel and eluted. The
eluted protein was subjected to two-dimensional peptide analysis.
The letters C and E correspond to chromatography and electrophoresis, respectively, and the letter o denotes the origin. (e) Each
of five serine/threonine residues in the GST-1-110 fragment was
replaced individually with alanine in five mutants through sitedirected mutagenesis. Mutant proteins were expressed and purified
and the purified proteins were analysed on an SDS–polyacrylamide
gel, which was stained with Coomasie Brilliant blue (f). Roughly
equal amounts of the purified mutant proteins as shown in e, along
with casein, were used for in vitro kinase assays in the presence of
His6-Plk3
60 is the site targeted by Plk3. The neighboring sequence
of the identified phosphorylation site, L–Q–S*–V–L–E,
partially conforms to the consensus motif, D/E–X–S/
T*–U–X–D/E (X, any amino acid; U, a hydrophobic
amino acid), for phosphorylation by vertebrate Plk1.
The significance of physical interaction and phosphorylation of Pol d by Plk3 remains to be elucidated. We
speculate that the phosphorylation may regulate the
subcellular localization of pol d because the region of
p1251115 contains a nuclear localization signal.
Together with various observations reported in the
literature, a simple model is proposed to illustrate the
role of vertebrate Plks in an intra-S-phase checkpoint
(Figure 3). Although downstream of ATM, Plk1 and
Plk3 appear to be differentially regulated during the
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Plks and cell cycle checkpoints
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Replication
Error
DNA Damage
lex
mp
1
-1-
9
P
P
Co
F
Rad7-RC
ATM/ATR
DNA Repair
PlK1
Chkl/2
PlK3
Claspin
p53
Cdc25
Polδ
Plks’ role in the G2/M checkpoint
PlK2
G2
M
Figure 3 Major pathways where Plks may play a role in intra-Sphase checkpoint in mammalian systems. Arrows and bars denote
the positive and negative regulations, respectively. Dotted lines
denote where the regulatory role in vivo between these proteins
remains to be elucidated
DNA damage response. Whereas Plk3 positively regulates p53 activity, Plk1 functions to inactivate p53
during DNA damage response. Plk2 is a transcriptional
target of p53 and its function in the checkpoint response
remains unclear. Both Plk1 and Plk3 interact and
phosphorylate Chk2 in vitro, although their regulatory
hierarchy in vivo remains to be elucidated. Plk3 may also
participate in DNA damage repair by regulating Pol d
activity through phosphorylation.
Checkpoint adaptation
Adaptation to an intra-S-phase checkpoint is the
molecular process that alleviates the cell cycle arrest
even in the presence of a damaged genome or replication
errors. It is best described in the budding yeast as the
response to DNA strand breaks. CDC5, a Polo
homologue in the budding yeast, is required for the
adaptation (Toczyski et al., 1997). A recent study shows
that a similar molecular mechanism also exists in
vertebrates and Polo-like kinase is involved in this
process (Yoo et al., 2004). Plx1, the Xenopus counterpart
of mammalian Plk1, physically interacts with the
checkpoint protein claspin that is phosphorylated on
threonine-906; this interaction leads to additional
phosphorylation of claspin on serine-934 by Plx1 (Yoo
et al., 2004). Xenopus egg extracts are capable of
adaptation after a long interphase delay and the
adaptation is characterized by having incompletely
replicated DNA, dissociation of claspin from chromatin,
and inactivation of Chk1. Interestingly, egg extracts
treated with the DNA replication poison aphidicolin
Oncogene
supplemented with claspin mutants with threonine-906
and serine-934 residues replaced with alanines are
unable to undergo adaptation (Yoo et al., 2004),
strongly suggesting a role for Plx1 in alleviating DNA
replication checkpoint response by inactivation of
claspin through phosphorylation. For unicellular organisms, continued cell proliferation through adaptation is
a better alternative than being permanently arrested in
the presence of extensive DNA damage. From the
evolutionary point of view, this mechanism may even
offer certain selective advantages. However, the biological significance of checkpoint adaptation in multicellular organisms such as Xenopus remains a mystery
because it is difficult to imagine why an organism would
allow the survival of a cell with a damaged or
incompletely replicated genome, a condition suitable
for malignant transformation.
The G2/M checkpoint prevents inappropriate initiation
of mitosis when there exist abnormalities in the genome.
One major mechanism of this checkpoint in vertebrates
involves regulation of the activity of Cdc2/cyclin B,
whose activation requires the combined action of the
upstream phosphatase Cdc25 and a Cdk-activating
kinase. Plks have been reported to phosphorylate and
regulate two key molecular players involved in controlling mitotic onset, although their biological significance
remains to be elucidated. Early work has shown that
Plx1 indirectly regulates Cdc2/cyclin B, the activity of
which is required for initiation of mitosis (Abrieu et al.,
1998). Recombinant Plx1 is capable of phosphorylation
of Cdc25, an activator of Cdc2, and stimulation of its
activity in vitro; Cdc25 phosphorylated by Plx1 exhibits
strong MPM-2 epitopes (Qian et al., 2001). The
activation of Cdc25 by Plk1 also occurs in a mammalian
system. Both Plk1 immunoprecipitates obtained from
G2/M-arrested human cells and recombinant human
Plk1 phosphorylate recombinant Cdc25C in vitro and
this phosphorylation leads to the activation of the
phosphatase (Roshak et al., 2000). Plk3 also interacts
and phosphorylates Cdc25C in vitro (Ouyang et al.,
1999; Bahassi et al., 2004) although the sites phosphorylated by Plk3 appear to be different from those of Plk1
(Toyoshima-Morimoto et al., 2002). The biological
consequence of phosphorylation of Cdc25C by Plk1 or
Plk3 in vivo remains largely unknown. However, a
couple of studies suggest that Plk-mediated phosphorylation of Cdc25C may promote its nuclear translocation (Toyoshima-Morimoto et al., 2002; Bahassi et al.,
2004). For example, Plk1 phosphorylates Cdc25C on
serine-198, which is located in a nuclear export signal
sequence (Toyoshima-Morimoto et al., 2002). Constitutively active Plk1 promotes the nuclear localization of
this dual-specific protein phosphatase in vivo, whereas a
mutant Cdc25C in which serine-198 is replaced by
alanine, but not the wild-type one, remains in the
cytoplasm during prophase (Toyoshima-Morimoto
et al., 2002). Interestingly, a recent study shows that
Plks and cell cycle checkpoints
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Plk3 phosphorylates Cdc25C mainly on serine-191,
which is also located within the nuclear exclusion motif
(Bahassi et al., 2004). Further analysis of ectopically
expressed mutant forms of Cdc25 as well as Plk3 leads
authors to conclude that Plk3 mediates Cdc25C nuclear
translocation by phosphorylating it primarily on serine191 (Bahassi et al., 2004).
Plk1 and its orthologue are reported to regulate the
nuclear translocation of cyclin B, resulting in activation
of Cdc2 as well. Plx1 appears to be the primary enzyme
that phosphorylates serine-147 in vitro within the
nuclear export signal sequence of cyclin B1, because
antibody depletion abolishes the activity of Plx1
towards cyclin B1 (Toyoshima-Morimoto et al., 2001).
Serine-147 is involved in both retention of cyclin B in the
nucleus and maintenance of the activity of Cdc2,
because a phospho-specific antibody to this residue
detects cyclin B1 only during the G2/M phase and
because a mutant cyclin B1 with serines 133 and 147
replaced with alanines remains in the cytoplasm
(Toyoshima-Morimoto et al., 2001). Consistently, Yuan
et al. (2002) demonstrate that human cyclin B1 contains
four major serine residues (serines 126, 128, 133, and
147), phosphorylation of which is important for its
nuclear translocation. Moreover, Plk1 is primarily
responsible for phosphorylation of serine-133 (Yuan
et al., 2002). On the other hand, Walsh et al. (2003)
report that cyclin B1 is phosphorylated by Plx1 on
serine-101 (equivalent to serine-133 of human cyclin
B1), but not on serine-113 (equivalent to serine-147 of
human cyclin B1), within the cytoplasmic retention
sequence. Furthermore, Jackman et al. (2003) demonstrate that human Plk1 neither phosphorylates cyclin B1
in the nuclear export sequence nor causes translocation
into the nucleus; using phospho-specific antibodies to
cyclin B1, these authors show that centrosome-localized
cyclin B is first phosphorylated during prophase,
suggesting that Cdc2/cyclin B1 may be first activated
in the cytoplasm and that the centrosomes may be the
primary sites for activation of proteins that are essential
for mitotic entry.
Together, these studies all indicate the importance of
Plk1 or its orthologue in promoting mitotic onset by
controlling the activity of Cdc2/cyclin B. Plk1 activity in
turn is partly regulated through ubiquitination by Chfr
(Kang et al., 2002), an E3 ubiquitin ligase commonly
referred to as a checkpoint protein. Chfr contains a ringfinger domain which auto-ubiquitinates and is required
for the ligase activity towards substrates such as Plk1;
Chfr-mediated ubiquitination of Plk1 impairs both
activation of Cdc25C and inactivation of Wee1, resulting in a delayed activation of Cdc2 (Kang et al., 2002).
Chfr activity is negatively regulated through phosphorylation by PKB both in vivo and in vitro; a phosphorylation mutant of Chfr that cannot be phosphorylated
by PKB causes significant reduction of Plk1 as well as
inhibition of mitotic entry in the presence or absence of
DNA damage (Shtivelman, 2003). In addition to DNA
damage and incompletion of DNA replication, insufficient chromatid decatenation also triggers the activation
of a G2/M checkpoint (Deming et al., 2002). Treatment
of cells with ICRF-193, an inhibitor of topoisomerase II
that does not cause DNA damage, suppresses Plk1
kinase activity, which is correlated with a reduction in
cyclin B phosphorylation; the ICRF-193-mediated
suppression of Plk1 appears to be ATR-dependent
(Deming et al., 2002).
Plks’ role in spindle checkpoint
The requirement of Plks in metaphase/anaphase transition is evident from the study of RNAi. Silencing Plk1
via RNAi alone induces a significant increase in the 4N
cell population, as shown by the analysis of flow
cytometry (Figure 4a and b). A further examination
by fluorescence microscopy reveals that a majority of
cells are arrested at metaphase when they are transfected
with siRNA targeting Plk1, but not luciferease (Figure
4c and d). Similar observations are also reported in
several independent studies (Liu and Erikson, 2002; van
Vugt et al., 2004). Silencing of Plk1 by RNAi causes the
accumulation of almost half of the cells with incompletely separated chromatids (Liu and Erikson, 2002).
What is intriguing is that silencing of Plk1 apparently
does not cause G2/M arrest because these cells exhibit a
high level of Cdc2/cyclin B activity (Liu and Erikson,
2002). In addition, a separate study shows that Plk1
depletion via RNAi leads to prometaphase arrest (van
Vugt et al., 2004). A detailed analysis of the timing of
mitotic entry indicates that the majority of cells with
Plk1 deletion initiate mitotic entry with kinetics similar
to that of control cells, indicating that Plk1 is not
required for mitotic entry (van Vugt et al., 2004).
Furthermore, overexpression of kinase-dead Plk1 or
Plk1 with an amino-terminal deletion also causes preanaphase arrest, which is characterized by an elevated
Cdc2/cyclin B activity with DNA content X4N (Seong
et al., 2002). Together, these observations indicate that,
whereas Plk1 is essential for progression of mitosis, its
activity may not be essential for the onset of mitosis in
mammalian cells.
Bipolar mitotic spindle
The spindle checkpoint (also known as the spindle
assembly/integrity checkpoint or mitotic checkpoint)
ensures that cells with a defective spindle or with
defective spindle–kinetochore interactions do not initiate anaphase entry. The accurate segregation of
chromosomes during mitosis is a crucial cellular process
that depends on the formation of intact bipolar spindles.
Faithful transmission of chromosomes is at least partly
regulated by the spindle checkpoint, a mechanism
preventing the cell from prematurely entering anaphase
until all replicated and condensed chromosomes have
attached to functional bipolar spindles. Early studies
have demonstrated the importance of Plks in the
formation of bipolar spindle (Sunkel and Glover,
1988; Ohkura et al., 1995). In fission yeast, either
disruption of the Plo1 or its overexpression results in the
formation of monopolar spindles due to the failure of
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Plks and cell cycle checkpoints
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282
Figure 4 Silencing Plk1 results in metaphase arrest. (a) A549 or
HeLa cells were transfected with Plk1 siRNA duplex (Plk1-siRNA,
50 -AAGGGCGGCUUUGCCAAGUGC-30 ) or with a nonspecific
siRNA duplex (CNTL-siRNA) for 3 days. Equal amounts of
lysates prepared from the transfected and untransfected parental
cells were blotted for Plk1 and tubulin. Recombinant flag-tagged
Plk1 was used as a positive control for blotting. (b) HeLa cells were
transfected with siRNA duplex to Plk1 (Plk1-siRNA) or to
luciferase (Luc-siRNA, 50 -CUUACGCUGAGUACUUCGA-30 )
for 3 days. Transfected cells, as well as the untransfected cells,
were analysed for DNA content by flow cytometry. (c) HeLa cells
seeded on chamber slides were transfected with or without siRNA
duplex to Plk1 or to luciferase for 3 days. Cells were then fixed,
stained with DAPI, and examined for their cell cycle status using
fluorescence microscopy. The brightly stained cells are in various
stages of mitosis. (d) The number of mitotic cells as shown in (c)
were scored and the percentage of cells in various stages of mitosis
was summarized. P, M, and A denote prophase, metaphase, and
anaphase, respectively. (e) HeLa cells were fixed and stained with
DAPI (blue), and the antibody with BubR1 (green). Representative
cells of various cell cycle stages (interphase, prophase and
metaphase) are shown. Arrows indicate the location of spindle
poles. Bar, 5 mM
the spindle pole body to complete either its duplication
or separation (Ohkura et al., 1995). Vertebrate Plks are
also involved in regulating bipolar spindle formation. In
HeLa cells, neutralizing Plk1 activity by antibody
injection leads to a mitotic arrest, with a monopolar
spindle formed around a centrosome that is reduced in
size (Lane and Nigg, 1996). Moreover, human cells with
Plk1 deficiency are incapable of formation of a bipolar
spindle, resulting in mitotic arrest (van Vugt et al.,
2004).
Although the molecular mechanism by which Plk1, or
its orthologue, regulates mitotic spindle formation
remains nebulous, several proteins appear to play a
central role in this process (Budde et al., 2001; Yarm,
2002; Casenghi et al., 2003). (i) Plk1 phosphorylates
Oncogene
NIp, ninein-like protein, and the phosphorylation
disrupts the ability of NIp to be associated with ãtubulin, as well as with the centrosome (Casenghi et al.,
2003). As NIp is an important component of the
centrosome and essential for stimulating microtubule
nucleation, it is proposed that at the onset of mitosis
Plk1 displaces NIp through phosphorylation, thus
establishing a condition for assembly of the mitotic
spindle (Casenghi et al., 2003). (ii) Plk1 interacts with
and phosphorylates the microtubule-stabilizing protein
TCTP; two serine residues of TCTP are apparently
targets of Plk1 both in vivo and in vitro; ectopic
expression of these TCTP phosphorylation site mutants
impairs mitotic progression and chromosomal segregation, resulting in an increase in multinucleation (Yarm,
2002). Therefore, Plk1-mediated phosphorylation of
TCTP may reduce its ability to stabilize microtubules,
leading to increased microtubule dynamics needed
during mitosis. (iii) Plx1 is capable of phosphorylating
stathmin (also termed Op18), a microtubule-destabilizing protein, and thereby negatively regulates its activity
(Budde et al., 2001). Phosphorylation of stathmin by
Plx1 peaks at the onset of mitosis, promoting microtubule stabilization and mitotic spindle assembly. Thus,
stathmin may be another substrate, whose phosphorylation by Plks plays a key role in mitotic progression.
Spindle checkpoint machinery
Extensive research in the past several years has
delineated the molecular pathway by which spindle
checkpoint activation leads to metaphase arrest
(Figure 5). Spindle checkpoint is fully activated when
there exists a defective spindle or a defective spindle–
kinetochore interaction (Musacchio and Hardwick,
2002; Bharadwaj and Yu, 2004). Activated checkpoint
components, including Bub1, BubR1, Bub3, Mad1,
Mad2, and CENP-E, inhibit anaphase-promoting complex/cyclone (APC/C) via direct interaction with Cdc20,
an APC/C activator (Bharadwaj and Yu, 2004). Besides,
it has been shown that early mitotic inhibitor 1 (Emi1)
also functions to block the activity of APC/C (Moshe
et al., 2004). When the checkpoint is inactivated, APC/
C, a ubiquitin E3 ligase, poly-ubiquitinates securin (a
separase inhibitor) for degradation by the proteasomal
pathway. After dissociation from securin, separase (a
caspase-like protease) becomes activated, which cleaves
cohesin subunit Scc1/hRad21 that connects sister
chromatids, thus allowing mitotic progression from
metaphase into anaphase. In other words, the cohesin
complex must be removed by the proteolytic action of
separase to allow the onset of anaphase. Both occupancy at the kinetochore and tension generated between
the kinetochores and the spindle poles are enough to
satisfy (inactivate) the spindle checkpoint (Musacchio
and Hardwick, 2002). Plks appear to regulate the
spindle checkpoint at least at four different levels
(Figure 5). (i) Plk1 facilitates the degradation of Emi1
by SCFbTrCP ubiquitin ligase. This may be mediated
through phosphorylation of a sequence in Emi1 that is
required for polyubiquitin conjugation (Moshe et al.,
Plks and cell cycle checkpoints
S Xie et al
283
BubR1
Plks
Bub3
these two mitotic kinases may cooperate to promote
mitotic progression.
Mad2
Cdc20
Emi 1
Ub
Ub
Ub
APC/C
Plk1
Plks’ role in the cytokinesis checkpoint
Ub
Separase Securin
Securin
Degradation
Cohesin
Separase
Plks
p
Cohesin
Metaphase
Cohesin
Anaphase
Figure 5 A model illustrating Plks’ role in the spindle checkpoint
response pathway. Four potential levels of regulation of the
pathway by Plks are indicated
2004). Thus, Plk1 promotes mitotic progression through
removal of an APC/C inhibitor; (ii) Plks may directly
regulate the activity of spindle checkpoint components.
For example, Plk3 and Plk1 interact with BubR1 and
Plk1 is capable of phosphorylating BubR1 in vitro
(unpublished results). We have also observed that
BubR1 exhibits spindle pole subcellular localization
during metaphase but not in prophase or prometaphase
(Figure 4e). Plks are known to reside at centrosomes and
spindle poles (Arnaud et al., 1998; Wang et al., 2002). It
is possible that the transit of BubR1 through spindle
poles is a necessary step for its inactivation at the onset
of anaphase. (iii) Plk1 is associated with APC components at the metaphase–anaphase transition, thus,
indirectly regulating its ubiquitination activity (Kotani
et al., 1998; Golan et al., 2002). Mouse Plk1 specifically
phosphorylates at least three components of APC
(subunits Cdc16, Cdc27, and Tsg24) and activates
APC/C in vitro (Kotani et al., 1998). In fission yeast,
Plo1 physically interacts with Cut23, a subunit of APC/
C, through its noncatalytic domain; the physical
interaction is compromised by a Cut23 mutation that
is accompanied by a metaphase arrest, while overexpression of Plo1 can rescue this phenotype (Golan
et al., 2002). (iv) A Plk1 homologue in yeast plays an
important role in degradation of a cohesin subunit. In
Saccharomyces cerevisiae, Cdc5 phosphorylates serine
residues adjacent to the cleavage site of Scc1, a subunit
of cohesin, thereby enhancing its cleavage by separase at
the onset of anaphase (Alexandru et al., 2001).
Similarly, Cdc5 is also required for phosphorylation
and removal of cohesin from chromosomes during
meiosis (Lee and Amon, 2003). In Xenopus, Plx1
destabilizes the linkage of sister chromatids because,
when Plx1 is depleted from Xenopus egg extracts, the
release of cohesin during prophase is blocked (Losada
et al., 2002). Interestingly, this blockage is more
pronounced when both Plx1 and aurora B are simultaneously depleted (Losada et al., 2002), suggesting that
In low eukaryotes
The essential role of Plks in cytokinesis is first
established from the study in yeast. Several studies
report that CDC5 in the budding yeast and Plo1 in the
fission yeast are involved in controlling mitotic exit
(Heitz et al., 2001; Hu et al., 2001). (i) CDC5 is one of
the components that form the machinery called the
mitotic exit network, which consists of additional genes
the GTPase TEM1, the phosphatase CDC14, CDC15,
LTE1, MOB1, and DBF2 (Hu et al., 2001). (ii) Cdc5 is
also part of the FEAR (Cdc fourteen early anaphase
release) network, which also includes the separase Esp1
and the kinetochore-associated proteins Slk19 and
Spo12 (Stegmeier et al., 2002). The FEAR network
initiates Cdc14 release from Cfi1/Net1 during early
anaphase, whereas the MEN maintains Cdc14 in the
released state during late anaphase and telophase
(Stegmeier et al., 2002). In fact, Cdc5 is capable of
affecting the phosphorylation state of Net1, a nucleolar
inhibitor of Cdc14, thus reducing its affinity with Cdc14
(Yoshida and Toh-e, 2002). Given that Cdc14 is
required for the timely activation of MEN, Cdc14
releasing through the FEAR network is an important
step for initiating mitotic exit. This notion is supported
by several lines of additional evidence obtained from the
study in low eukaryotes. By using temperature-sensitive
mutants of CDC5 with carboxyl-terminal defects, Park
et al. (2004) show that, at semi-permissive temperatures,
certain mutants exhibit chained cell morphology and
shared cytoplasms between connected cell bodies; a
further examination reveals that septin assembly at the
incipient bud site is delayed with a loosely organized
septin ring at the neck of the mother bud, indicating a
defect in cytokinesis. A similar requirement for Plk in
the fission yeast has also been reported. Plo1 is a key
regulator of cytokinetic actomyosin ring (CAR) formation and recruitment of Plo1 to the spindle pole body, as
well as a functional spindle checkpoint, is required for
CAR formation and septation (Mulvihill and Hyams,
2002).
Overexpression of wild-type Cdc5, or a catalytically
inactive form, results in the formation of multinucleated
cells in the budding yeast, which is apparently due to
negative regulation of Swe1, a Wee1 kinase, by Cdc5
(Bartholomew et al., 2001). Moreover, ectopic expression of wild-type mammalian Plk1 complements the cell
division defect associated with the CDC5-1 mutation in
S. cerevisiae and the degree of complementation
correlates closely with the Plk1 activity measured in
vitro; expression of an activated Plk1 (Plk1T210D) also
induces a class of cells with unusually elongated buds
which develop multiple septal structures (Lee and
Erikson, 1997). In addition, a loss of S. pombe Plo1
function leads to the failure of septation both in the
Oncogene
Plks and cell cycle checkpoints
S Xie et al
284
formation of an F-actin ring and in the deposition of
septal material, suggesting that Plo1 function is required
in the regulatory cascade that controls septation
(Schwab et al., 2001). The overexpression of Plo1 also
induces the formation of multiple septa without nuclear
division (Schwab et al., 2001). A more detailed analysis
on Plo1 indicated that it plays a role in the positioning
of division sites by regulating Mid1p, a known gene
product regulating cytokinesis in the fission yeast
(Bahler et al., 1998). A recent study on CAR has
revealed that recruitment of Plo1 to the spindle pole
body is substantially reduced in the presence of the
microtubule-depolymerizing agent thiabendazole, causing an extensive delay in CAR formation (Mulvihill and
Hyams, 2002).
In high eukaryotes
Cytokinesis in animal cells is achieved by the formation
of an actin ring that contracts to divide the cytoplasm.
Polo and vertebrate Plks are essential for this process. A
Drosophila genetic study reveals the requirement of Polo
in the regulation of cytokinesis (Carmena et al., 1998).
In fruit flies harboring hypomorphic alleles of the Polo
gene, a number of defects in cytokinesis are observed,
which include a failure to form the correct mid-zone and
mid-body structures at telophase and the frequent
formation of binuclear or tetranuclear spermatids
during meiosis. There are also defects in correct
localization of the Pavarotti kinesin-like protein to
function in cytokinesis, and incorporation of the septin
Peanut and actin molecules into a contractile ring
(Carmena et al., 1998). In Xenopus, it has been
demonstrated that the addition of a catalytically inactive
Plx1 mutant to M-phase-arrested Xenopus egg extracts
inhibits the proteolytic destruction of several APC/C
targets, the inactivation of Cdc2 protein kinase activity,
and the entry into interphase induced by Ca2 þ (Brassac
et al., 2000).
Degradation of mammalian Plks such as Plk1
contributes to inactivation of this kinase, a necessary
step for normal mitotic exit (Lindon and Pines, 2004),
which may explain why overexpression of kinase-active
Plk3 causes the accumulation of cells with cytokinetic
failure (Wang et al., 2002). Putative destruction boxes
have been identified in Plk1 (Lindon and Pines, 2004) as
well as in the N-terminus of Cdc5 (Golsteyn et al., 1994),
indicating that at least some Plks may function as the
activator as well as the target of APC/C. Interestingly,
Plk1 also interacts with 20S and 26S proteasome
subunits and phosphorylates the 20S proteasome,
leading to its enhanced proteolytic activity (Feng et al.,
2001). Thus, Plk1 functions as a mitotic regulator of
proteolytic activities through its effect on both APC/C
and proteasomes.
Several mammalian Plks are found at the midbody
region during late mitosis (Conn et al., 2000; Tsvetkov
et al., 2003; Ruan et al., 2004). It has been shown
that the functional Polo box domain of Plk4 localizes
the enzyme to the cleavage furrow during cytokinesis
(Hudson et al., 2001). It has also been demonstrated
Oncogene
that EGFP–Plk3 fusion protein concentrates at the
midbody and is associated with the cellular cortex
(Conn et al., 2000), suggesting a potential role for
Plk3 during mitotic exit. Our independent study
confirms that ectopic expression of kinase-active Plk3,
but not the kinase-defective one, causes a defect in
cytokinesis, eventually resulting in apoptosis (Wang
et al., 2002).
The exact molecular mechanism by which mammalian
Plks regulate cytokinesis remains to be elucidated.
However, a series of recent studies begin to shed light
on the possible role of Plk1 in the regulation of
cytokinesis. (i) Plk1 colocalizes with Pavarotti, a
kinesin-related motor protein that is required for the
organization of the central spindle, the formation of a
contractile ring, and cytokinesis (Adams et al., 1998).
Consistent with a role in regulating microtubule motor
proteins, Plk1 also phosphorylates mitotic kinesin-like
protein 2 (MKlp), the phosphorylation of which is
essential for cytokinesis in mammalian cells (Neef et al.,
2003). It is proposed that Plk1-mediated phosphorylation of MKlp2 may facilitate its spatial restriction to the
spindle region during late mitosis, a necessary step for
cytokinesis (Neef et al., 2003). Together, these studies
suggest that motor proteins are targets of Plk1 during
exit from mitosis. (ii) Plk1 physically interacts with
nuclear distribution gene C (NudC) (Zhou et al., 2003).
In fact, Plk1 phosphorylates NudC at conserved serine274 and serine-326 both in vivo and in vitro; silencing
NudC via RNAi causes multiple defects in mitosis,
including arresting cells at the midbody stage, which can
be rescued by wild-type NudC, but not NudC with
serines 274 and 326 mutated into alanines (Zhou et al.,
2003). As NudC, as well as Plk1, concentrates at the
midbody during cytokinesis (Tsvetkov et al., 2003; Zhou
et al., 2003), it is possible that NudC phosphorylated by
Plk1 may generate signals that are essential for
completion of cell division. (iii) Some recent studies on
the role of Plks in Golgi dynamics may provide new
insights into the mechanism of action of these kinases in
cytokinesis (Lin et al., 2000; Litvak et al., 2004; Ruan
et al., 2004; Xie et al., 2004). Plk1 is shown to interact
with Golgi-specific protein GRASP65 and participate in
regulation of fragmentation of the Golgi apparatus
during mitosis (Lin et al., 2000). Similarly, human Plk3
is Golgi-localized during interphase and distributes in a
manner similar to that of Golgi components during
mitosis; moreover, during telophase, Plk3 and Golgi
components exhibit two clusters, with one localized at
the spindle pole and the other close to the midbody
(Ruan et al., 2004). Furthermore, Nir2, a peripheral
Golgi protein essential for cytokinesis (Litvak et al.,
2002), is phosphorylated by Cdk1 during mitosis and
this phosphorylation provides a binding site for Plk1
(Litvak et al., 2004). Given that phosphorylation of Nir2
during mitosis is an essential modification mediating
normal cytokinesis (Litvak et al., 2004), it is tempting to
speculate that Plk1 and perhaps other Plks may play a
major role in coordinating Golgi fragmentation and
assembly, mitotic spindle assembly and disassembly,
and chromosome segregation during mitosis, deregula-
Plks and cell cycle checkpoints
S Xie et al
285
tion of which would compromise cytokinesis, resulting
in formation of multinucleated cells.
Conclusions
Cell cycle checkpoints monitor specific cell cycle-related
processes and block cycle progression until these
processes are completed with high fidelity. Plks participate in almost all known cell cycle checkpoints that
protect cells against genetic instability during cell
division. Although it remains unclear as to their exact
mechanism of action in controlling the intra-S-phase
checkpoint, spindle checkpoint, and cytokinesis checkpoint, an increasing amount of evidence suggests that
protein kinases of the polo family are central players in
the temporal and spatial coordination of cell division.
As tumor cells invariably harbor defects in various cell
cycle checkpoints, further characterization of the role of
Plks in cell cycle checkpoint control may lead to the
identification of new molecular targets for the development of drugs that are more effective and specific for
killing tumor cells.
Acknowledgements
We apologize to many authors whose articles could not be
cited in this review due to space limitations. We thank Dr
Frank Traganos for critical reading of the manuscript and the
members in Dr Dai’s laboratory for helpful discussions. We
also thank Ms Lisa Buerle for editorial and administrative
assistance. The work is supported in part by grants from the
National Institutes of Health to WD (CA90658 and CA74229)
and to ML (GM31973).
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