Regulation of Cell Cycle Checkpoints by Polo-Like Kinases

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Regulation of cell cycle checkpoints by polo-like kinases

Suqing Xie1, Bin Xie2, Marietta Y Lee2 and Wei Dai*,1

1Molecular 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 abnormal cell division and/or transformation. To date, division. Polo and Polo-like kinases have emerged as several families of protein kinases such as cyclin- major regulators for various cell cycle checkpoints. Early dependent kinases (Cdks), polo-like kinases (Plks), and genetic studies have demonstrated that CDC5,a budding Aurora family of kinases have been characterized, which yeast counterpart of vertebrate Plks,is essential for are important to cell cycle regulation. Extensive research successful mitotic progression. Mammalian Plks localize in the past decade or so has demonstrated that Polo and primarily to the centrosome during interphase and the Plks are involved in regulation of various cell cycle mitotic apparatus during mitosis. Many key cell cycle checkpoints that ensure the timing and order of cell regulators such as p53,Cdc25C,cyclin B,components of cycle events such as DNA repair, bipolar spindle the anaphase-promoting complex,and mitotic motor formation, chromosome segregation, and mitotic exit proteins are directly targeted by Plks. Although the exact (Nigg, 1998; Dai et al., 2002; Barr et al., 2004). mechanism of action of these protein kinases in vivo In this review, we summarize the properties of Polo remains to be elucidated,Plks are important mediators for and Plks in various cell cycle checkpoint controls. We various cell cycle checkpoints that monitor centrosome also present new lines of evidence supporting the role of duplication,DNA replication,formation of bipolar mammalian Plks in regulating both an intra-S-phase mitotic spindle,segregation of chromosomes,and mitotic checkpoint and a spindle checkpoint. exit,thus protecting cells against genetic instability during cell division. Oncogene (2005) 24, 277–286. doi:10.1038/sj.onc.1208218 Plks’ role in intra-S-phase checkpoint Keywords: polo; polo-like kinases; cell cycle; checkpoint; cell division Checkpoint activation pathway During the S phase, the genome is most susceptible to damage caused by environmental stresses and/or inter- nal perturbations. Thus, it is natural that cells have Introduction evolved sophisticated mechanisms to monitor DNA damage and initiate repair processes if the damage is not The progression of the cell cycle is tightly regulated in extensive. Extensive research in the past decade has order to maintain genetic integrity and to ensure that shown that the signaling pathways that underlie the genetic information is correctly passed on to daughter cellular response to DNA damage (or genotoxic stress) cells. Surveillance mechanisms, known as checkpoints, consist of sensors, signal transducers, and effectors have thus been discovered, that monitor the integrity of (Zhou and Elledge, 2000). Although the identities of the the cell cycle progression (Elledge, 1996). By definition, damage sensors remain unclear, the molecular entities cell cycle checkpoints inhibit cell cycle progression until responsible for transducing the damage signals to the specific cellular processes under surveillance are specific effectors are relatively well characterized. completed with high fidelity. In eukaryotes, both DNA ATM (ataxia telangiectasia mutated) and its homolog replication and chromosome segregation are highly ATR (ATM-related) function early in the signaling regulated and are monitored at several steps in the cell pathways and are central tothe DNA damage response cycle. A loss of checkpoint function can result in (Sancar et al., 2004). Downstream mediators/transdu- infidelity of DNA replication or of chromosome cers of ATM and ATR include the protein kinases segregation, and thereby predispose cells to genetic Chk1, Chk2, and likely Plk3 (Xie et al., 2001b; Sancar instability. et al., 2004). The effectors of the checkpoint include p53 Reversible phosphorylation is a fundamental mole- and Cdc25 (Sancar et al., 2004). cular mechanism that is critical for regulating cell Both CDC5 in the budding yeast and mammalian division. Deregulated phosphorylation of the compo- Plks are known to participate in the DNA damage nents important to cell cycle control frequently leads to response. An early study shows that the electrophoretic mobility of Cdc5 in denaturing gels is affected when the *Correspondence: W Dai; E-mail: [email protected] cells are exposed to DNA damage agents, and this Plks and cell cycle checkpoints SXieet al 278 modification of Cdc5 is dependent on Mec1, Rad53 (a 2001a, b). Plk3 interacts with and phosphorylates p53, yeast Chk1 homolog), and Rad9 (Cheng et al., 1998). In targeting serine-20 of p53 in vitro (Xie et al., 2001b). In addition, a functionally defective Cdc5 mutant protein response to DNA damage, the kinase activity of Plk3 is suppresses a Rad53 checkpoint defect, whereas over- rapidly increased in an ATM-dependent manner (Xie expression of Cdc5 overrides checkpoint-induced cell et al., 2001a, b). Peptide mapping, as well as in vitro cycle arrest (Sanchez et al., 1999), suggesting that Cdc5 phosphorylation followed by immunoblot analysis with acts downstream of Rad53. Several recent studies show antibodies specific for phosphorylated forms of p53, also that Plk1 activity is inhibited upon DNA damage (Smits indicates that Plk3 phosphorylates p53 on physiologi- et al., 2000; van Vugt et al., 2001; Ando et al., 2004). The cally relevant sites. Immunoprecipitation and ‘pull- DNA damage-induced inhibition of Plk1 appears to be down’ assays reveal that Plk3 physically interacts with mediated by blocking its activation because expression p53 and that the extent of this interaction is increased in of activation mutants of Plk1 can override the G2/M response to DNA damage (Xie et al., 2001b). The role of arrest induced by DNA damage (Smits et al., 2000). Plk3 in regulation of serine-20 phosphorylation of p53 in Subsequent studies by this group reveal that DNA vivo is further supported by our recent RNAi study. damage-induced inhibition of Plk1 is at least in part Downregulation of endogenous Plk3 through transfec- dependent on ATM or ATR because caffeine treatment tion of small-interfering RNA (siRNA) to Plk3, but not blocks the inhibition of Plk1 after IR- or UV-induced to luciferease, significantly compromises phosphoryla- DNA damage (van Vugt et al., 2001). The activity of tion of p53 on serine-20 before and after oxidative stress p53 is greatly enhanced upon DNA damage checkpoint (Figure 1). Plk3 alsophysically interacts with Chk2 activation. (Bahassi et al., 2002; Xie et al., 2002), and there exists a A recent study shows that Plk1 binds p53 and inhibits functional connection as well between these two the transactivating activity of this tumor suppressor enzymes during DNA damage checkpoint activation. protein; the kinase activity of Plk1 is required for Plk3 phosphorylates Chk2 on a residue different from inhibition of p53 activity; besides, Plk1 also negatively threonine-68, and it may contribute to the full activation affects the proapoptotic function of p53 in human lung of Chk2 although ATM is necessary for phosphoryla- tumor cell lines (Ando et al., 2004). Again, ATM is tion and activation of Chk2 in vivo (Bahassi et al., 2002). capable of attenuating Plk1-mediated suppression of Together, these combined studies suggest that Plk3 p53 activity (Ando et al., 2004). Given the potential role functionally links DNA damage to the induction of cell of Plk1 in promoting cell proliferation and tumorigen- cycle arrest or apoptosis. esis (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, DNA damage repair there are alsoreportssuggesting that Plk1 may play a A possible role for Plks in DNA synthesis or repair is positive role in DNA damage checkpoint activation. implied from several early studies. Expression of both Chk2 co-immunoprecipitates with Plk1, overexpression mammalian Plk2 and Plk3 is rapidly induced by mitogen of which enhances phosphorylation of Chk2 at T68 treatment (Simmons et al., 1992; Li et al., 1996). (Tsvetkov et al., 2003), a site primarily targeted by Induction of Plk3 mRNA by mitogens is protein ATM, and its phosphorylation is correlated with its synthesis-independent, suggesting that it is an immedi- activation (Ahn et al., 2000); in addition, Plk1 phos- ate-early gene product (Li et al., 1996). Furthermore, phorylates 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 alsoinvolved 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 Figure 1 Silencing of Plk3 results in significantly reduced p53 polo box (this review issue). Several lines of evidence phosphorylation on serine-20. GM00637 cells, which constitutively indicate that Plk3 is an important mediator in the DNA express high levels of p53, were transfected with siRNA duplexes to damage checkpoint response pathway. Plk3 kinase Plk3 (Plk3-siRNA) or to luciferase GL3 (Luc-siRNA) (SMART- activity is activated upon oxidative stress and DNA pool, Dharmacon) for 72 h. The transfected cells were treated with H2O2 (100 mM) for 15 min and then collected for preparation of damage induced by ionizing-radiation mimetic drugs, lysates. Equal amounts of cell lysates were blotted for Plk3, serine- and its activation is ATM-dependent (Xie et al., 20-phosphorylated p53, and actin

Oncogene Plks and cell cycle checkpoints SXieet al 279 microinjection of sense Plk1 mRNA, but not antisense RNA, intoNIH3T3 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 Schizo- saccharomyces 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. Figure 2 Plk3 interacts and phosphorylates the p125 subunit of Our initial screen results reveal that p125, the major Pol Pol d.(a) Sf9 cells either infected with p125 baculovirus or both d subunit, is one of the proteins that interacted with Plk3 His6-Plk3 and p125 baculoviruses for 48 h. Cell lysates were (data not shown). The physical interaction between prepared from the infected cells, as well as from uninfected control these two proteins was further confirmed by co- cells. Ni-NTA resin, which was capable of binding His6-Plk3, was added to each lysate. After thorough washing, proteins bound to immunoprecipitation (data not shown) and pull-down Ni-NTA resin, along with cell lysates, were blotted for p125. (b, c) experiments. Ni-NTA resin, having a high affinity to Various fragments of p125 were expressed as GST fusion proteins. proteins with a stretch of histidine residues, is capable of Approximately equal amounts of purified proteins, as well as GST pulling down p125 antigen from lysates prepared from alone and casein, were used for in vitro kinase assays in the presence cells co-infected with His -Plk3 and p125 baculoviruses, of purified His6-Plk3. Kinase buffer alone was also used as a 6 negative control. Reaction mixtures were fractionated on denatur- but not from parental cells with no viral infection or ing SDS–polyacrylamide gels, followed by autoradiography. Each cells infected with p125 baculovirus alone (Figure 2a). number denotes a peptide fragment encompassing the particular Plk3’s kinase activity is high during the S phase region of p125 protein. (d) The phosphorylated band (arrow GST- (Ouyang et al., 1997). Therefore, in vitro kinase assays 1-110) as shown in (c) was excised from the gel and eluted. The eluted protein was subjected to two-dimensional peptide analysis. were performed to examine whether recombinant Plk3 The letters C and E correspond to chromatography and electro- directly phosphorylates p125. We first made a series of phoresis, respectively, and the letter o denotes the origin. (e) Each truncated forms of p125 expressed as glutathione s of five serine/threonine residues in the GST-1-110 fragment was transferase (GST) fusion proteins. Purified proteins were replaced individually with alanine in five mutants through site- subjected tokinase assays in vitro. We observed that a directed mutagenesis. Mutant proteins were expressed and purified 1À365 and the purified proteins were analysed on an SDS–polyacrylamide deletional form of p125 encompassing amino acids gel, which was stained with Coomasie Brilliant blue (f). Roughly 1–365 (of the total 1107 amino acids) is phosphorylated equal amounts of the purified mutant proteins as shown in e, along by His6-Plk3, whereas other truncated forms were not with casein, were used for in vitro kinase assays in the presence of phosphorylated (Figure 2b). Additional deletions re- His6-Plk3 vealed 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 60 is the site targeted by Plk3. The neighboring sequence major phosphorylation site in the p1251À115 fragment of the identified phosphorylation site, L–Q–S*–V–L–E, (Figure 2d), although five serine residues could be partially conforms to the consensus motif, D/E–X–S/ potential targets of Plk3. To identify p125’s Plk3 T*–U–X–D/E (X, any aminoacid; U, a hydrophobic phosphorylation site, we constructed GST expression amino acid), for phosphorylation by vertebrate Plk1. constructs coding for mutant peptide fragments with The significance of physical interaction and phosphor- each of five serine/threonine residues replaced with ylation of Pol d by Plk3 remains tobe elucidated. We alanines, respectively. Various phosphorylation site speculate that the phosphorylation may regulate the mutant proteins of p1251À115 were then expressed and subcellular localization of pol d because the region of purified (Figure 2e). Approximately equal amounts of p1251À115 contains a nuclear localization signal. the purified mutant proteins were used for in vitro kinase Together with various observations reported in the assays in the presence of His6-Plk3. The mutant literature, a simple model is proposed to illustrate the p1251À115 with serine-60 substituted with alanine is not role of vertebrate Plks in an intra-S-phase checkpoint phosphorylated by His6-Plk3, whereas all other mutants (Figure 3). Although downstream of ATM, Plk1 and are phosphorylated (Figure 2f), confirming that serine- Plk3 appear tobe differentially regulated during the

Oncogene Plks and cell cycle checkpoints SXieet al 280 Replication supplemented with claspin mutants with threonine-906 Error and serine-934 residues replaced with alanines are DNA Damage unable toundergoadaptation(Yoo et al., 2004), strongly suggesting a role for Plx1 in alleviating DNA replication checkpoint response by inactivation of

9-1-1 Complex claspin through phosphorylation. For unicellular organ- P Rad7-RCF P isms, continued cell proliferation through adaptation is ATM/ATR a better alternative than being permanently arrested in DNA Repair the presence of extensive DNA damage. From the evolutionary point of view, this mechanism may even offer certain selective advantages. However, the biolo- PlK1 gical signiﬁcance of checkpoint adaptation in multi- Chkl/2 PlK3 Polδ cellular 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. Claspin p53 Cdc25

Plks’ role in the G2/M checkpoint PlK2

G2 M The G2/M checkpoint prevents inappropriate initiation of mitosis when there exist abnormalities in the genome. Figure 3 Major pathways where Plks may play a role in intra-S- One major mechanism of this checkpoint in vertebrates phase checkpoint in mammalian systems. Arrows and bars denote the positive and negative regulations, respectively. Dotted lines involves regulation of the activity of Cdc2/cyclin B, denote where the regulatory role in vivo between these proteins whose activation requires the combined action of the remains tobe elucidated upstream phosphatase Cdc25 and a Cdk-activating kinase. Plks have been reported to phosphorylate and regulate two key molecular players involved in control- DNA damage response. Whereas Plk3 positively reg- ling mitotic onset, although their biological signiﬁcance ulates p53 activity, Plk1 functions to inactivate p53 remains tobe elucidated. Early workhas shownthat during DNA damage response. Plk2 is a transcriptional Plx1 indirectly regulates Cdc2/cyclin B, the activity of target of p53 and its function in the checkpoint response which is required for initiation of mitosis (Abrieu et al., remains unclear. Both Plk1 and Plk3 interact and 1998). Recombinant Plx1 is capable of phosphorylation phosphorylate Chk2 in vitro, although their regulatory of Cdc25, an activator of Cdc2, and stimulation of its hierarchy in vivo remains tobe elucidated. Plk3 may also activity in vitro; Cdc25 phosphorylated by Plx1 exhibits participate in DNA damage repair by regulating Pol d strong MPM-2 epitopes (Qian et al., 2001). The activity through phosphorylation. activation of Cdc25 by Plk1 also occurs in a mammalian system. Both Plk1 immunoprecipitates obtained from Checkpoint adaptation G2/M-arrested human cells and recombinant human Plk1 phosphorylate recombinant Cdc25C in vitro and Adaptation to an intra-S-phase checkpoint is the this phosphorylation leads to the activation of the molecular process that alleviates the cell cycle arrest phosphatase (Roshak et al., 2000). Plk3 alsointeracts even in the presence of a damaged genome or replication and phosphorylates Cdc25C in vitro (Ouyang et al., errors. It is best described in the budding yeast as the 1999; Bahassi et al., 2004) although the sites phosphory- response to DNA strand breaks. CDC5, a Polo lated by Plk3 appear to be different from those of Plk1 homologue in the budding yeast, is required for the (Toyoshima-Morimoto et al., 2002). The biological adaptation (Toczyski et al., 1997). A recent study shows consequence of phosphorylation of Cdc25C by Plk1 or that a similar molecular mechanism also exists in Plk3 in vivo remains largely unknown. However, a vertebrates and Polo-like kinase is involved in this couple of studies suggest that Plk-mediated phosphor- process (Yoo et al., 2004). Plx1, the Xenopus counterpart ylation of Cdc25C may promote its nuclear transloca- of mammalian Plk1, physically interacts with the tion (Toyoshima-Morimoto et al., 2002; Bahassi et al., checkpoint protein claspin that is phosphorylated on 2004). For example, Plk1 phosphorylates Cdc25C on threonine-906; this interaction leads to additional serine-198, which is located in a nuclear export signal phosphorylation of claspin on serine-934 by Plx1 (Yoo sequence (Toyoshima-Morimoto et al., 2002). Constitu- et al., 2004). Xenopus egg extracts are capable of tively active Plk1 promotes the nuclear localization of adaptation after a long interphase delay and the this dual-speciﬁc protein phosphatase in vivo, whereas a adaptation is characterized by having incompletely mutant Cdc25C in which serine-198 is replaced by replicated DNA, dissociation of claspin from chromatin, alanine, but not the wild-type one, remains in the and inactivation of Chk1. Interestingly, egg extracts cytoplasm during prophase (Toyoshima-Morimoto treated with the DNA replication poison aphidicolin et al., 2002). Interestingly, a recent study shows that

Oncogene Plks and cell cycle checkpoints SXieet al 281 Plk3 phosphorylates Cdc25C mainly on serine-191, of cells with ICRF-193, an inhibitor of topoisomerase II which is also located within the nuclear exclusion motif that does not cause DNA damage, suppresses Plk1 (Bahassi et al., 2004). Further analysis of ectopically kinase activity, which is correlated with a reduction in expressed mutant forms of Cdc25 as well as Plk3 leads cyclin B phosphorylation; the ICRF-193-mediated authors to conclude that Plk3 mediates Cdc25C nuclear suppression of Plk1 appears to be ATR-dependent translocation by phosphorylating it primarily on serine- (Deming et al., 2002). 191 (Bahassi et al., 2004). Plk1 and its orthologue are reported to regulate the nuclear translocation of cyclin B, resulting in activation Plks’ role in spindle checkpoint of Cdc2 as well. Plx1 appears to be the primary enzyme that phosphorylates serine-147 in vitro within the The requirement of Plks in metaphase/anaphase transi- nuclear export signal sequence of cyclin B1, because tion is evident from the study of RNAi. Silencing Plk1 antibody depletion abolishes the activity of Plx1 via RNAi alone induces a significant increase in the 4N towards cyclin B1 (Toyoshima-Morimoto et al., 2001). cell population, as shown by the analysis of flow Serine-147 is involved in both retention of cyclin B in the cytometry (Figure 4a and b). A further examination nucleus and maintenance of the activity of Cdc2, by fluorescence microscopy reveals that a majority of because a phospho-specific antibody to this residue cells are arrested at metaphase when they are transfected detects cyclin B1 only during the G2/M phase and with siRNA targeting Plk1, but not luciferease (Figure because a mutant cyclin B1 with serines 133 and 147 4c and d). Similar observations are also reported in replaced with alanines remains in the cytoplasm several independent studies (Liu and Erikson, 2002; van (Toyoshima-Morimoto et al., 2001). Consistently, Yuan Vugt et al., 2004). Silencing of Plk1 by RNAi causes the et al. (2002) demonstrate that human cyclin B1 contains accumulation of almost half of the cells with incomple- four major serine residues (serines 126, 128, 133, and tely separated chromatids (Liu and Erikson, 2002). 147), phosphorylation of which is important for its What is intriguing is that silencing of Plk1 apparently nuclear translocation. Moreover, Plk1 is primarily does not cause G2/M arrest because these cells exhibit a responsible for phosphorylation of serine-133 (Yuan high level of Cdc2/cyclin B activity (Liu and Erikson, et al., 2002). On the other hand, Walsh et al. (2003) 2002). In addition, a separate study shows that Plk1 report that cyclin B1 is phosphorylated by Plx1 on depletion via RNAi leads to prometaphase arrest (van serine-101 (equivalent toserine-133 ofhuman cyclin Vugt et al., 2004). A detailed analysis of the timing of B1), but not on serine-113 (equivalent to serine-147 of mitotic entry indicates that the majority of cells with human cyclin B1), within the cytoplasmic retention Plk1 deletion initiate mitotic entry with kinetics similar sequence. Furthermore, Jackman et al. (2003) demon- to that of control cells, indicating that Plk1 is not strate that human Plk1 neither phosphorylates cyclin B1 required for mitotic entry (van Vugt et al., 2004). in the nuclear export sequence nor causes translocation Furthermore, overexpression of kinase-dead Plk1 or into the nucleus; using phospho-specific antibodies to Plk1 with an amino-terminal deletion also causes pre- cyclin B1, these authors show that centrosome-localized anaphase arrest, which is characterized by an elevated cyclin B is first phosphorylated during prophase, Cdc2/cyclin B activity with DNA content X4N (Seong suggesting that Cdc2/cyclin B1 may be first activated et al., 2002). Together, these observations indicate that, in the cytoplasm and that the centrosomes may be the whereas Plk1 is essential for progression of mitosis, its primary sites for activation of proteins that are essential activity may not be essential for the onset of mitosis in for mitotic entry. mammalian cells. Together, these studies all indicate the importance of Plk1 or its orthologue in promoting mitotic onset by Bipolar mitotic spindle controlling the activity of Cdc2/cyclin B. Plk1 activity in turn is partly regulated through ubiquitination by Chfr The spindle checkpoint (also known as the spindle (Kang et al., 2002), an E3 ubiquitin ligase commonly assembly/integrity checkpoint or mitotic checkpoint) referred to as a checkpoint protein. Chfr contains a ring- ensures that cells with a defective spindle or with finger domain which auto-ubiquitinates and is required defective spindle–kinetochore interactions do not initi- for the ligase activity towards substrates such as Plk1; ate anaphase entry. The accurate segregation of Chfr-mediated ubiquitination of Plk1 impairs both chromosomes during mitosis is a crucial cellular process activation of Cdc25C and inactivation of Wee1, result- that depends on the formation of intact bipolar spindles. ing in a delayed activation of Cdc2 (Kang et al., 2002). Faithful transmission of chromosomes is at least partly Chfr activity is negatively regulated through phosphor- regulated by the spindle checkpoint, a mechanism ylation by PKB both in vivo and in vitro; a phosphor- preventing the cell from prematurely entering anaphase ylation mutant of Chfr that cannot be phosphorylated until all replicated and condensed chromosomes have by PKB causes significant reduction of Plk1 as well as attached to functional bipolar spindles. Early studies inhibition of mitotic entry in the presence or absence of have demonstrated the importance of Plks in the DNA damage (Shtivelman, 2003). In addition to DNA formation of bipolar spindle (Sunkel and Glover, damage and incompletion of DNA replication, insuffi- 1988; Ohkura et al., 1995). In fission yeast, either cient chromatid decatenation also triggers the activation disruption of the Plo1 or its overexpression results in the ofaG2/M checkpoint (Deming et al., 2002). Treatment formation of monopolar spindles due to the failure of

Oncogene Plks and cell cycle checkpoints SXieet al 282 NIp, ninein-like protein, and the phosphorylation disrupts the ability of NIp to be associated with a˜- tubulin, 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; twoserine residues ofTCTP 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 tostabilize microtubules, leading toincreased microtubule dynamics needed during mitosis. (iii) Plx1 is capable of phosphorylating stathmin (alsotermed Op18), a microtubule-destabiliz- ing 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 phosphoryla- Figure 4 Silencing Plk1 results in metaphase arrest. (a) A549 or tion by Plks plays a key role in mitotic progression. 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 Spindle checkpoint machinery lysates prepared from the transfected and untransfected parental cells were blotted for Plk1 and tubulin. Recombinant flag-tagged Extensive research in the past several years has Plk1 was used as a positive control for blotting. (b) HeLa cells were delineated the molecular pathway by which spindle transfected with siRNA duplex toPlk1 (Plk1-siRNA) orto luciferase (Luc-siRNA, 50-CUUACGCUGAGUACUUCGA-30) checkpoint activation leads to metaphase arrest for 3 days. Transfected cells, as well as the untransfected cells, (Figure 5). Spindle checkpoint is fully activated when were analysed for DNA content by flow cytometry. (c) HeLa cells there exists a defective spindle or a defective spindle– seeded on chamber slides were transfected with or without siRNA kinetochore interaction (Musacchio and Hardwick, duplex to Plk1 or to luciferase for 3 days. Cells were then fixed, 2002; Bharadwaj and Yu, 2004). Activated checkpoint stained with DAPI, and examined for their cell cycle status using fluorescence microscopy. The brightly stained cells are in various components, including Bub1, BubR1, Bub3, Mad1, stages of mitosis. (d) The number of mitotic cells as shown in (c) Mad2, and CENP-E, inhibit anaphase-promoting com- were scored and the percentage of cells in various stages of mitosis plex/cyclone (APC/C) via direct interaction with Cdc20, was summarized. P, M, and A denote prophase, metaphase, and an APC/C activator (Bharadwaj and Yu, 2004). Besides, anaphase, respectively. (e) HeLa cells were fixed and stained with DAPI (blue), and the antibody with BubR1 (green). Representative it has been shown that early mitotic inhibitor 1 (Emi1) cells of various cell cycle stages (interphase, prophase and also functions to block the activity of APC/C (Moshe metaphase) are shown. Arrows indicate the location of spindle et al., 2004). When the checkpoint is inactivated, APC/ poles. Bar, 5 mM C, a ubiquitin E3 ligase, poly-ubiquitinates securin (a separase inhibitor) for degradation by the proteasomal pathway. After dissociation from securin, separase (a the spindle pole body to complete either its duplication caspase-like protease) becomes activated, which cleaves or separation (Ohkura et al., 1995). Vertebrate Plks are cohesin subunit Scc1/hRad21 that connects sister also involved in regulating bipolar spindle formation. In chromatids, thus allowing mitotic progression from HeLa cells, neutralizing Plk1 activity by antibody metaphase into anaphase. In other words, the cohesin injection leads to a mitotic arrest, with a monopolar complex must be removed by the proteolytic action of spindle formed around a centrosome that is reduced in separase to allow the onset of anaphase. Both occu- size (Lane and Nigg, 1996). Moreover, human cells with pancy at the kinetochore and tension generated between Plk1 deficiency are incapable of formation of a bipolar the kinetochores and the spindle poles are enough to spindle, resulting in mitotic arrest (van Vugt et al., satisfy (inactivate) the spindle checkpoint (Musacchio 2004). and Hardwick, 2002). Plks appear toregulate the Although the molecular mechanism by which Plk1, or spindle checkpoint at least at four different levels its orthologue, regulates mitotic spindle formation (Figure 5). (i) Plk1 facilitates the degradation of Emi1 remains nebulous, several proteins appear to play a by SCFbÀTrCP ubiquitin ligase. This may be mediated central role in this process (Budde et al., 2001; Yarm, through phosphorylation of a sequence in Emi1 that is 2002; Casenghi et al., 2003). (i) Plk1 phosphorylates required for polyubiquitin conjugation (Moshe et al.,

Oncogene Plks and cell cycle checkpoints SXieet al 283

BubR1 Mad2 these two mitotic kinases may cooperate to promote Plks Bub3 Cdc20 mitotic progression.

Emi 1 Ub APC/C Ub Plk1 Ub Plks’ role in the cytokinesis checkpoint Ub Securin Securin Degradation Separase In low eukaryotes

Cohesin The essential role of Plks in cytokinesis is ﬁrst

Separase established from the study in yeast. Several studies Plks report that CDC5 in the budding yeast and Plo1 in the p Cohesin Cohesin fission yeast are involved in controlling mitotic exit (Heitz et al., 2001; Hu et al., 2001). (i) CDC5 is one of Metaphase Anaphase 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 Figure 5 A model illustrating Plks’ role in the spindle checkpoint alsopart ofthe FEAR (Cdc fourteen early anaphase response pathway. Four potential levels of regulation of the pathway by Plks are indicated 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 2004). Thus, Plk1 promotes mitotic progression through anaphase, whereas the MEN maintains Cdc14 in the removal of an APC/C inhibitor; (ii) Plks may directly released state during late anaphase and telophase regulate the activity of spindle checkpoint components. (Stegmeier et al., 2002). In fact, Cdc5 is capable of For example, Plk3 and Plk1 interact with BubR1 and affecting the phosphorylation state of Net1, a nucleolar Plk1 is capable of phosphorylating BubR1 in vitro inhibitor of Cdc14, thus reducing its affinity with Cdc14 (unpublished results). We have alsoobservedthat (Yoshida and Toh-e, 2002). Given that Cdc14 is BubR1 exhibits spindle pole subcellular localization required for the timely activation of MEN, Cdc14 during metaphase but not in prophase or prometaphase releasing through the FEAR network is an important (Figure 4e). Plks are known to reside at centrosomes and step for initiating mitotic exit. This notion is supported spindle poles (Arnaud et al., 1998; Wang et al., 2002). It by several lines of additional evidence obtained from the is possible that the transit of BubR1 through spindle study in low eukaryotes. By using temperature-sensitive poles is a necessary step for its inactivation at the onset mutants of CDC5 with carboxyl-terminal defects, Park of anaphase. (iii) Plk1 is associated with APC compo- et al. (2004) show that, at semi-permissive temperatures, nents at the metaphase–anaphase transition, thus, certain mutants exhibit chained cell morphology and indirectly regulating its ubiquitination activity (Kotani shared cytoplasms between connected cell bodies; a et al., 1998; Golan et al., 2002). Mouse Plk1 specifically further examination reveals that septin assembly at the phosphorylates at least three components of APC incipient bud site is delayed with a loosely organized (subunits Cdc16, Cdc27, and Tsg24) and activates septin ring at the neck of the mother bud, indicating a APC/C in vitro (Kotani et al., 1998). In fission yeast, defect in cytokinesis. A similar requirement for Plk in Plo1 physically interacts with Cut23, a subunit of APC/ the fission yeast has also been reported. Plo1 is a key C, through its noncatalytic domain; the physical regulator of cytokinetic actomyosin ring (CAR) forma- interaction is compromised by a Cut23 mutation that tion and recruitment of Plo1 to the spindle pole body, as is accompanied by a metaphase arrest, while over- well as a functional spindle checkpoint, is required for expression of Plo1 can rescue this phenotype (Golan CAR formation and septation (Mulvihill and Hyams, et al., 2002). (iv) A Plk1 homologue in yeast plays an 2002). important role in degradation of a cohesin subunit. In Overexpression of wild-type Cdc5, or a catalytically Saccharomyces cerevisiae, Cdc5 phosphorylates serine inactive form, results in the formation of multinucleated residues adjacent tothe cleavage site ofScc1, a subunit cells in the budding yeast, which is apparently due to of cohesin, thereby enhancing its cleavage by separase at negative regulation of Swe1, a Wee1 kinase, by Cdc5 the onset of anaphase (Alexandru et al., 2001). (Bartholomew et al., 2001). Moreover, ectopic expres- Similarly, Cdc5 is also required for phosphorylation sion of wild-type mammalian Plk1 complements the cell and removal of cohesin from chromosomes during division defect associated with the CDC5-1 mutation in meiosis (Lee and Amon, 2003). In Xenopus, Plx1 S. cerevisiae and the degree of complementation destabilizes the linkage of sister chromatids because, correlates closely with the Plk1 activity measured in when Plx1 is depleted from Xenopus egg extracts, the vitro; expression of an activated Plk1 (Plk1T210D) also release of cohesin during prophase is blocked (Losada induces a class of cells with unusually elongated buds et al., 2002). Interestingly, this blockage is more which develop multiple septal structures (Lee and pronounced when both Plx1 and aurora B are simulta- Erikson, 1997). In addition, a loss of S. pombe Plo1 neously depleted (Losada et al., 2002), suggesting that function leads to the failure of septation both in the

Oncogene Plks and cell cycle checkpoints SXieet al 284 formation of an F-actin ring and in the deposition of that EGFP–Plk3 fusion protein concentrates at the septal material, suggesting that Plo1 function is required midbody and is associated with the cellular cortex in the regulatory cascade that controls septation (Conn et al., 2000), suggesting a potential role for (Schwab et al., 2001). The overexpression of Plo1 also Plk3 during mitotic exit. Our independent study induces the formation of multiple septa without nuclear confirms that ectopic expression of kinase-active Plk3, division (Schwab et al., 2001). A more detailed analysis but not the kinase-defective one, causes a defect in on Plo1 indicated that it plays a role in the positioning cytokinesis, eventually resulting in apoptosis (Wang of division sites by regulating Mid1p, a known gene et al., 2002). product regulating cytokinesis in the fission yeast The exact molecular mechanism by which mammalian (Bahler et al., 1998). A recent study on CAR has Plks regulate cytokinesis remains to be elucidated. revealed that recruitment of Plo1 to the spindle pole However, a series of recent studies begin to shed light body is substantially reduced in the presence of the on the possible role of Plk1 in the regulation of microtubule-depolymerizing agent thiabendazole, caus- cytokinesis. (i) Plk1 colocalizes with Pavarotti, a ing an extensive delay in CAR formation (Mulvihill and kinesin-related motor protein that is required for the Hyams, 2002). organization of the central spindle, the formation of a contractile ring, and cytokinesis (Adams et al., 1998). In high eukaryotes Consistent with a role in regulating microtubule motor proteins, Plk1 also phosphorylates mitotic kinesin-like Cytokinesis in animal cells is achieved by the formation protein 2 (MKlp), the phosphorylation of which is of an actin ring that contracts to divide the cytoplasm. essential for cytokinesis in mammalian cells (Neef et al., Polo and vertebrate Plks are essential for this process. A 2003). It is proposed that Plk1-mediated phosphoryla- Drosophila genetic study reveals the requirement of Polo tion of MKlp2 may facilitate its spatial restriction to the in the regulation of cytokinesis (Carmena et al., 1998). spindle region during late mitosis, a necessary step for In fruit flies harboring hypomorphic alleles of the Polo cytokinesis (Neef et al., 2003). Together, these studies gene, a number of defects in cytokinesis are observed, suggest that motor proteins are targets of Plk1 during which include a failure to form the correct mid-zone and exit from mitosis. (ii) Plk1 physically interacts with mid-body structures at telophase and the frequent nuclear distribution gene C (NudC) (Zhou et al., 2003). formation of binuclear or tetranuclear spermatids In fact, Plk1 phosphorylates NudC at conserved serine- during meiosis. There are also defects in correct 274 and serine-326 both in vivo and in vitro; silencing localization of the Pavarotti kinesin-like protein to NudC via RNAi causes multiple defects in mitosis, function in cytokinesis, and incorporation of the septin including arresting cells at the midbody stage, which can Peanut and actin molecules into a contractile ring be rescued by wild-type NudC, but not NudC with (Carmena et al., 1998). In Xenopus, it has been serines 274 and 326 mutated intoalanines (Zhou et al., demonstrated that the addition of a catalytically inactive 2003). As NudC, as well as Plk1, concentrates at the Plx1 mutant toM-phase-arrested Xenopus egg extracts midbody during cytokinesis (Tsvetkov et al., 2003; Zhou inhibits the proteolytic destruction of several APC/C et al., 2003), it is possible that NudC phosphorylated by targets, the inactivation of Cdc2 protein kinase activity, Plk1 may generate signals that are essential for and the entry intointerphase induced by Ca 2 þ (Brassac completion of cell division. (iii) Some recent studies on et al., 2000). the role of Plks in Golgi dynamics may provide new Degradation of mammalian Plks such as Plk1 insights into the mechanism of action of these kinases in contributes to inactivation of this kinase, a necessary cytokinesis (Lin et al., 2000; Litvak et al., 2004; Ruan step for normal mitotic exit (Lindon and Pines, 2004), et al., 2004; Xie et al., 2004). Plk1 is shown to interact which may explain why overexpression of kinase-active with Golgi-specific protein GRASP65 and participate in Plk3 causes the accumulation of cells with cytokinetic regulation of fragmentation of the Golgi apparatus failure (Wang et al., 2002). Putative destruction boxes during mitosis (Lin et al., 2000). Similarly, human Plk3 have been identified in Plk1 (Lindon and Pines, 2004) as is Golgi-localized during interphase and distributes in a well as in the N-terminus of Cdc5 (Golsteyn et al., 1994), manner similar to that of Golgi components during indicating that at least some Plks may function as the mitosis; moreover, during telophase, Plk3 and Golgi activator as well as the target of APC/C. Interestingly, components exhibit two clusters, with one localized at Plk1 alsointeracts with 20S and 26S proteasome the spindle pole and the other close to the midbody subunits and phosphorylates the 20S proteasome, (Ruan et al., 2004). Furthermore, Nir2, a peripheral leading to its enhanced proteolytic activity (Feng et al., Golgi protein essential for cytokinesis (Litvak et al., 2001). Thus, Plk1 functions as a mitotic regulator of 2002), is phosphorylated by Cdk1 during mitosis and proteolytic activities through its effect on both APC/C this phosphorylation provides a binding site for Plk1 and proteasomes. (Litvak et al., 2004). Given that phosphorylation of Nir2 Several mammalian Plks are found at the midbody during mitosis is an essential modification mediating region during late mitosis (Conn et al., 2000; Tsvetkov normal cytokinesis (Litvak et al., 2004), it is tempting to et al., 2003; Ruan et al., 2004). It has been shown speculate that Plk1 and perhaps other Plks may play a that the functional Polo box domain of Plk4 localizes major role in coordinating Golgi fragmentation and the enzyme to the cleavage furrow during cytokinesis assembly, mitotic spindle assembly and disassembly, (Hudson et al., 2001). It has alsobeen demonstrated and chromosome segregation during mitosis, deregula-

Oncogene Plks and cell cycle checkpoints SXieet al 285 tion of which would compromise cytokinesis, resulting the temporal and spatial coordination of cell division. in formation of multinucleated cells. 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 Conclusions identification of new molecular targets for the develop- ment of drugs that are more effective and specific for Cell cycle checkpoints monitor specific cell cycle-related killing tumor cells. processes and block cycle progression until these processes are completed with high fidelity. Plks partici- Acknowledgements pate in almost all known cell cycle checkpoints that We apologize to many authors whose articles could not be protect cells against genetic instability during cell cited in this review due tospace limitations.We thank Dr Frank Traganos for critical reading of the manuscript and the division. Although it remains unclear as to their exact members in Dr Dai’s laboratory for helpful discussions. We mechanism of action in controlling the intra-S-phase also thank Ms Lisa Buerle for editorial and administrative checkpoint, spindle checkpoint, and cytokinesis check- assistance. The work is supported in part by grants from the point, an increasing amount of evidence suggests that National Institutes of Health to WD (CA90658 and CA74229) protein kinases of the polo family are central players in and toML (GM31973).

References

Abrieu A, Brassac T, Galas S, Fisher D, Labbe JC and Doree Elledge SJ. (1996). Science, 274, 1664–1672. M. (1998). J. Cell Sci., 111 (Part 12), 1751–1757. Feng Y, Longo DL and Ferris DK. (2001). Cell Growth Adams RR, Tavares AA, Salzberg A, Bellen HJ and Glover Differ., 12, 29–37. DM. (1998). Genes Dev., 12, 1483–1494. Golan A, Yudkovsky Y and Hershko A. (2002). J. Biol. Ahn JY, Schwarz JK, Piwnica-Worms H and Canman CE. Chem., 277, 15552–15557. (2000). Cancer Res., 60, 5934–5936. Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T and Alexandru G, Uhlmann F, Mechtler K, Poupart MA and Nigg EA. (1994). J. Cell Sci., 107 (Part 6), 1509–1517. Nasmyth K. (2001). Cell, 105, 459–472. Hamanaka R, Maloid S, Smith MR, O’Connell CD, Longo Ando K, Ozaki T, Yamamoto H, Furuya K, Hosoda M, DL and Ferris DK. (1994). Cell Growth Differ., 5, 249–257. Hayashi S, Fukuzawa M and Nakagawara A. (2004). J. Biol. Haracska L, Prakash S and Prakash L. (2003). Mol. Cell. Biol., Chem., 279, 25549–25561. 23, 1453–1459. Arnaud L, Pines J and Nigg EA. (1998). Chromosoma, 107, Heitz MJ, Petersen J, Valovin S and Hagan IM. (2001). J. Cell 424–429. Sci., 114, 4521–4532. Bahassi EM, Conn CW, Myer DL, Hennigan RF, McGowan Hubscher U, Maga G and Spadari S. (2002). Annu. Rev. CH, Sanchez Y and Stambrook PJ. (2002). Oncogene, 21, Biochem., 71, 133–163. 6633–6640. Hudson JW, Kozarova A, Cheung P, Macmillan JC, Bahassi EM, Hennigan RF, Myer DL and Stambrook PJ. Swallow CJ, Cross JC and Dennis JW. (2001). Curr. Biol., (2004). Oncogene, 23, 2658–2663. 11, 441–446. Bahler J, Steever AB, Wheatley S, Wang Y, Pringle JR, Gould Hu F, Wang Y, Liu D, Li Y, Qin J and Elledge SJ. (2001). Cell, KL and McCollum D. (1998). J. Cell Biol., 143, 1603–1616. 107, 655–665. Barr FA, Sillje HH and Nigg EA. (2004). Nat. Rev. Mol. Cell Jackman M, Lindon C, Nigg EA and Pines J. (2003). Nat. Cell Biol., 5, 429–440. Biol., 5, 143–148. Bartholomew CR, Woo SH, Chung YS, Jones C and Hardy Kang D, Chen J, Wong J and Fang G. (2002). J. Cell Biol., CF. (2001). Mol. Cell. Biol., 21, 4949–4959. 156, 249–260. Bharadwaj R and Yu H. (2004). Oncogene, 23, 2016–2027. Kotani S, Tugendreich S, Fujii M, Jorgensen PM, Watanabe Brassac T, Castro A, Lorca T, Le Peuch C, Doree M, Labbe N, Hoog C, Hieter P and Todokoro K. (1998). Mol. Cell, 1, JC and Galas S. (2000). Oncogene, 19, 3782–3790. 371–380. Budde PP, Kumagai A, Dunphy WG and Heald R. (2001). Lane HA and Nigg EA. (1996). J. Cell Biol., 135, 1701–1713. J. Cell Biol., 153, 149–158. Lee BH and Amon A. (2003). Science, 300, 482–486. Burns TF, Fei P, Scata KA, Dicker DT and El Deiry WS. Lee KS and Erikson RL. (1997). Mol. Cell. Biol., 17, (2003). Mol. Cell. Biol., 23, 5556–5571. 3408–3417. Carmena M, Riparbelli MG, Minestrini G, Tavares AM, Li B, Ouyang B, Pan H, Reissmann PT, Slamon DJ, Arceci R, Adams R, Callaini G and Glover DM. (1998). J. Cell Biol., Lu L and Dai W. (1996). J. Biol. Chem., 271, 19402–19408. 143, 659–671. Lin CY, Madsen ML, Yarm FR, Jang YJ, Liu X and Erikson Casenghi M, Meraldi P, Weinhart U, Duncan PI, Korner R RL. (2000). Proc. Natl. Acad. Sci. USA, 97, 12589–12594. and Nigg EA. (2003). Dev. Cell, 5, 113–125. Lindon C and Pines J. (2004). J. Cell Biol., 164, 233–241. Cheng L, Hunke L and Hardy CF. (1998). Mol. Cell. Biol., 18, Litvak V, Argov R, Dahan N, Ramachandran S, Amarilio R, 7360–7370. Shainskaya A and Lev S. (2004). Mol. Cell, 14, 319–330. Conn CW, Hennigan RF, Dai W, Sanchez Y and Stambrook Litvak V, Tian D, Carmon S and Lev S. (2002). Mol. Cell. PJ. (2000). Cancer Res., 60, 6826–6831. Biol., 22, 5064–5075. Dai W and Cogswell JP. (2003). Prog. Cell Cycle Res., 5, Liu X and Erikson RL. (2002). Proc. Natl. Acad. Sci. USA, 99, 327–334. 8672–8676. Dai W, Wang Q and Traganos F. (2002). Oncogene, 21, LosadaA, HiranoM and HiranoT. (2002). Genes Dev., 16, 6195–6200. 3004–3016. Deming PB, Flores KG, Downes CS, Paules RS and Moshe Y, Boulaire J, Pagano M and Hershko A. (2004). Proc. Kaufmann WK. (2002). J. Biol. Chem., 277, 36832–36838. Natl. Acad. Sci. USA, 101, 7937–7942.

Oncogene Plks and cell cycle checkpoints SXieet al 286 Mulvihill DP and Hyams JS. (2002). J. Cell Sci., 115, 3575–3586. Toczyski DP, Galgoczy DJ and Hartwell LH. (1997). Cell, 90, MusacchioA and Hardwick KG. (2002). Nat. Rev. Mol. Cell 1097–1106. Biol., 3, 731–741. Toyoshima-Morimoto F, Taniguchi E and Nishida E. (2002). Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, EMBO Rep., 3, 341–348. Mayer TU and Barr FA. (2003). J. Cell Biol., 162, 863–875. Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A Nigg EA. (1998). Cell Biol., 10, 776–783. and Nishida E. (2001). Nature, 410, 215–220. Ohkura H, Hagan IM and Glover DM. (1995). Genes Dev., 9, Tsvetkov L, Xu X, Li J and Stern DF. (2003). J. Biol. Chem., 1059–1073. 278, 8468–8475. Ouyang B, Li W, Pan H, Meadows J, Hoffmann I and Dai W. van Vugt MA, Smits VA, Klompmaker R and Medema RH. (1999). Oncogene, 18, 6029–6036. (2001). J. Biol. Chem., 276, 41656–41660. Ouyang B, Pan H, Lu L, Li J, Stambrook P, Li B and Dai W. van Vugt MA, Van De Weerdt BC, Vader G, Janssen H, (1997). J. Biol. Chem., 272, 28646–28651. Calafat J, Klompmaker R, Wolthuis RM and Medema RH. Park CJ, Song S, Giddings Jr TH, Ro HS, Sakchaisri K, Park (2004). J. Biol. Chem., 279, 36841–36854. JE, Seong YS, Winey M and Lee KS. (2004). Mol. Biol. Cell, Walsh S, Margolis SS and Kornbluth S. (2003). Mol. Cancer 15, 1711–1723. Res., 1, 280–289. Qian YW, Erikson E, Taieb FE and Maller JL. (2001). Mol. Wang Q, Xie S, Chen J, Fukasawa K, Naik U, Traganos F, Biol. Cell, 12, 1791–1799. Darzynkiewicz Z, Jhanwar-Uniyal M and Dai W. (2002). Roshak AK, Capper EA, Imburgia C, Fornwald J, Scott G Mol. Cell. Biol., 22, 3450–3459. and Marshall LA. (2000). Cell Signal., 12, 405–411. Xie S-Q, Wu H-Y, Wang Q, Kunicki J, Thomas RO, Ruan Q, Wang Q, Xie S, Fang Y, Darzynkiewicz Z, Guan K, Hollingsworth RE, Cogswell J and Dai W. (2002). Cell Jhanwar-Uniyal M and Dai W. (2004). Exp. Cell Res., 294, Cycle, 1, 424–429. 51–59. Xie S, Wang Q, Ruan Q, Liu T, Jhanwar-Uniyal M, Guan K Sancar A, Lindsey-Boltz LA, Unsal-Kaccmaz K and Linn S. and Dai W. (2004). Oncogene, 23, 3822–3829. (2004). Annu. Rev. Biochem., 73, 39–85. Xie S, Wang Q, Wu H, Cogswell J, Lu L, Jhanwar-Uniyal M Sanchez Y, Bachant J, Wang H, Hu F, Liu D, Tetzlaff M and and Dai W. (2001a). J. Biol. Chem., 276, 36194–36199. Elledge SJ. (1999). Science, 286, 1166–1171. Xie S, Wu H, Wang Q, Cogswell JP, Husain I, Conn C, Schwab M, Neutzner M, Mocker D and Seufert W. (2001). Stambrook P, Jhanwar-Uniyal M and Dai W. (2001b). EMBO J., 20, 5165–5175. J. Biol. Chem., 276, 43305–43312. Seong YS, Kamijo K, Lee JS, Fernandez E, Kuriyama R, Miki Xu J and Du W. (2003). FEBS Lett., 545, 209–212. T and Lee KS. (2002). J. Biol. Chem., 277, 32282–32293. Yarm FR. (2002). Mol. Cell. Biol., 22, 6209–6221. Shimizu-Yoshida Y, Sugiyama K, Rogounovitch T, Ohtsuru YooHY,Kumagai A, ShevchenkoA, ShevchenkoA and A, Namba H, SaenkoV and Yamashita S. (2001). Biochem. Dunphy WG. (2004). Cell, 117, 575–588. Biophys. Res. Commun., 289, 491–498. Yoshida S and Toh-e A. (2002). Biochem. Biophys. Res. Shtivelman E. (2003). Mol. Cancer Res., 1, 959–969. Commun., 294, 687–691. Simmons DL, Neel BG, Stevens R, Evett G and Erikson RL. Yuan J, Eckerdt F, Bereiter-Hahn J, Kurunci-CsacskoE, (1992). Mol. Cell. Biol., 12, 4164–4169. Kaufmann M and Strebhardt K. (2002). Oncogene, 21, Smits VA, Klompmaker R, Arnaud L, Rijksen G, Nigg EA 8282–8292. and Medema RH. (2000). Nat. Cell Biol., 2, 672–676. Zeng XR, HaoH, Jiang Y and Lee MY. (1994). J. Biol. Chem., Stegmeier F, Visintin R and Amon A. (2002). Cell, 108, 269, 24027–24033. 207–220. Zhou BB and Elledge SJ. (2000). Nature, 408, 433–439. Sunkel CE and Glover DM. (1988). J. Cell Sci., 89 (Part 1), Zhou T, Aumais JP, Liu X, Yu-Lee LY and Erikson RL. 25–38. (2003). Dev. Cell, 5, 127–138.

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