Structure and Function of Polo-Like Kinases

Structure and function of Polo-like kinases

Drew M Lowery1, Daniel Lim1 and Michael B Yaffe*,1

1Center for Cancer Research, E18-580, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

Polo-like kinases play critical roles during multiple stages Plks are defined by two conserved features – an N- of cell cycle progression. All Polo-like kinases contain an terminal Ser/Thr kinase domain and a C-terminal N-terminal Ser/Thr kinase catalytic domain and a C- duplicated Polo-box region (Figure 1).The kinase terminal region that contains one or two Polo-boxes. For domains are very highly conserved among all Plks, Polo-like kinase 1, 2, and 3, and their homologs, the entire and most closely resemble those in Aurora kinases and C-terminal region, including both Polo-boxes, functions as calcium/calmodulin-dependent kinases.The region that a single modular phosphoserine/threonine-binding domain encompasses the two C-terminal Polo-boxes is much less known as the Polo-box domain (PBD). In the absence of a conserved, with only B70 amino acids considered to bound substrate, the PBD inhibits the basal activity of the form the ‘core’, which constitutes less than half the full kinase domain. Phosphorylation-dependent binding of the length of the C-terminal domain.This entire region has PBD to its ligands releases the kinase domain, while recently been renamed the Polo-box domain (PBD) due simultaneously localizing Polo-like kinases to specific to the fact that it functions as a single unit, as described subcellular structures. These observations suggest two in detail below. different models for how the PBD integrates signals In humans, Plk1 is expressed primarily during late G2 arising from other mitotic kinases to target the activated and M phases, where it appears to regulate much of the kinase towards distinct substrates. The recent X-ray machinery involved in mitosis (Golsteyn et al., 1994; crystal structures of the PBD provide insights into the Barr et al., 2004). Plk2 is expressed primarily in early G1, structural basis for PBD function and kinase regulation. where it may control entry into S phase (Simmons et al., Molecular modelling of the structure of the isolated kinase 1992; Liby et al., 2001; Ma et al., 2003). Plk3 appears to domain reveals a potential basis for motif-dependent be expressed at constant levels throughout the cell cycle, substrate specificity. and plays a role in several stress response pathways, Oncogene (2005) 24, 248–259.doi:10.1038/sj.onc.1208280 including those activated by DNA damage and spindle disruption (Donohue et al., 1995; Xie et al., 2001; Keywords: Polo-like kinase; polo-box domain; mitosis; Bahassi el et al., 2002; Xie et al., 2002). Plk2 and Plk3 phosphorylation-dependent binding; modular signalling may also have overlapping roles with Plk1 in several domain mitotic functions, most notably Golgi fragmentation and cytokinesis (Conn et al., 2000; Wang et al., 2002; Burns et al., 2003; Ruan et al., 2004; Xie et al., 2004). In addition, Plk2 and Plk3 have roles in remodelling postmitotic neurons (Kauselmann et al., 1999; Pak and Roles and evolution of Polo-like kinases Sheng, 2003).Yeast and flies probably accomplish the same set of functions using only their single Plk family Polo-like kinases (Plks) play key roles during multiple members, although the mitotic functions of these stages of mitosis including prophase, metaphase, ana- kinases have been most extensively studied.Similarly, phase, and cytokinesis, in addition to less well under- there has been only limited study of worm Plks, and the stood roles during G1/S and in response to DNA data so far suggest that these genes have only limited damage (Barr et al., 2004). Originally named after the overlapping functions with other Plk1 orthologs and phenotype in flies, polo was found to encode a protein paralogs (Barr et al., 2004). kinase whose mutation resulted in abnormal spindle Each of the three Plks in humans, mice, and frogs poles (Sunkel and Glover, 1988; Llamazares et al., appear to be orthologous between species and para- 1991).Flies, budding yeast, and fission yeast contain a logous within a species, since phylogenetic analysis of single Plk family member (Polo, Cdc5, and Plo1, their sequences reveals that each group clusters sepa- respectively), while humans, mice, frogs, and worms rately (Figure 2a).Thus, the expansion from one to have three Plk family members, denoted Plk1/Plx1/Plc1, three Plks must have been an early evolutionary event. Plk2/Plx2/Plc2 (originally known as Snk), and Plk3/ However, this expansion appears to have occurred at Plx3/Plc3 (originally known as Fnk or Prk). least twice because the three worm Plk paralogs cluster together separately from the Plks of other organisms. *Correspondence: MB Yaffe; E-mail: [email protected] This is consistent with the individual worm Plks having Structure and function of Polo-like kinases DM Lowery et al 249 less functional overlap with the other Plk family its kinase domain closely resembles all other Plks.At the members.Plk genes from organisms with single family extreme C-terminus Plk4 contains a single ‘Polo-box’ members are slightly more evolutionarily conserved with instead of the tandem ‘Polo-box’ repeats seen in all the Plk1 class than any other, belying their conserved other Plks.This single ‘Polo-box’ is the most divergent mitotic function.(Figure 2b) in sequence and has been shown to mediate Sak There are no identified proteins with homology to dimerization in vitro and when overexpressed in vivo Plks in bacteria, archae, or plants.This is somewhat (Leung et al., 2002). Like all other Plks, Sak has been surprising for plants because plants have cyclin-depen- implicated in orchestrating cytokinesis (Hudson et al., dent kinases and much of the same cell cycle machinery 2000; Hudson et al., 2001; Leung et al., 2002), but its as yeast and animals.However, plants also show many function remains largely unknown.The large (>500 novel features of cell cycle control (Dewitte and Murray, amino acids) region between the kinase and the single 2003).With several plant genome sequencing projects Polo-box in Sak is a complete mystery as it has no complete or nearly complete, it seems increasingly obvious strong homology to any known proteins and no unlikely that plant Plks are still awaiting discovery. function has been assigned to it. A fourth family member has also been characterized How Plks are regulated and how they select their in humans and mice, and sequenced in frogs and flies, substrates, many of which are unknown, to control so called Sak or Plk4.Plk4 has a highly divergent many distinct aspects of cell division has become the C-terminus from the other Plk family members though recent subject of intense investigation.

Function of the PBD Plk activity is controlled by intracellular localization in addition to being regulated at the level of protein abundance.During prophase and metaphase, Plk1 and related family members localize to centrosomes and spindle pole bodies (Lee et al., 1998; Logarinho and Sunkel, 1998; Shirayama et al., 1998; Qian et al., 1998a; Moutinho-Santos et al., 1999; Qian et al., 1999), where Figure 1 Domain structure and key residues of Plks.Plks contain they are required for spindle assembly and centrosome an amino-terminal kinase domain and a carboxy-terminal PBD maturation, presumably by phosphorylating largely connected by a short linker.Plks are activated by phosphorylation unknown centrosome-associated targets (Lane and of a conserved Thr residue within the T-loop (Thr-210), and bind phosphopeptides through a pocket in the PBD containing the key Nigg, 1996; do Carmo Avides et al., 2001). Plk1 family residues Trp-414, His-538, and Lys-540.Numbering is for human members then relocalize to the spindle midzone during Plk1 late anaphase (Golsteyn et al., 1994, 1995; Glover et al.,

Figure 2 Phylogenetic analysis of Plk family members.( a) Full-length sequences of known Plk family members were aligned using ClustalW (Thompson et al., 1994). A rooted tree was constructed using PHYLIP (Felsenstein, 1997) showing one model of evolutionary descent.( b) An unrooted tree shows the distance relationship between Plks from organisms containing only a single Plk family member (Polo, Plo1, Cdc5) from other members of the Plk family

Oncogene Structure and function of Polo-like kinases DM Lowery et al 250 1998; Logarinho and Sunkel, 1998; Qian et al., 1999), other mitotic kinases might generate ‘priming’ phos- perhaps facilitating microtubule sliding or some aspect phorylations on substrates or docking proteins to of kinetochore dynamics (Lee et al., 1995), and localize Plks in the vicinity of their substrates.An eventually come to flank the central portion of the optimal PBD-binding phosphopeptide displaces the cytokinetic bridge (i.e. the midbody) during telophase PBD from binding to centrosomes in experiments using and cytokinesis (Golsteyn et al., 1994; Lee et al., 1995; permeabilized cells (Elia et al., 2003a), and mutations in Moutinho-Santos et al., 1999; Qian et al., 1999; Song the PBD that prevent phospho-peptide binding et al., 2000). At least for Cdc5, a portion of the protein (Figure 1) also prevent centrosomal localization (Elia is localized at the future site of cytokinesis (the bud et al., 2003b). Thus, the phosphopeptide-binding func- neck in budding yeast) from late G2 onward (Sakchaisri tion of the PBD is intricately linked to how Plks control et al., 2004), indicating that Plks may be involved cell cycle progression through mitosis. in organizing the structures that will eventually result in There are two alternative, although not mutually cytokinesis. exclusive, models for how the PBD might function to The PBD is critical for both Plk localization and direct Plk kinase activity (Figure 3).In one model, which function, since mutations in the conserved residues of we refer to as the ‘processive phosphorylation’ model, human Plk1 result in loss of Plk1 localization to spindle the PBD first binds to a site on a protein that has been poles and the bud neck, and an inability to support previously phosphorylated by a mitotic priming kinase. mitosis at the restrictive temperature in cdc5-ts com- This positions the kinase domain of Plk to phosphor- plementation experiments (Lee et al., 1998). Over- ylate the same protein at another site.This type of expression of the isolated PBD causes a failure to processive phosphorylation is seen for the Tyr kinase complete cytokinesis in budding yeast (Song and Lee, Src, which contains a pTyr-binding SH2 domain (Mayer 2001), and preanaphase arrest in mammalian cells et al., 1995; Pellicena et al., 1998; Scott and Miller, (Seong et al., 2002). The molecular basis of PBD 2000), and for the Ser/Thr kinase GSK-3, whose kinase function was clarified by the recent finding that the domain contains a separate pSer/pThr-binding pocket in entire PBD, including both Polo-boxes, the region the substrate binding cleft (Frame and Cohen, 2001). between them, and a portion of the linker between the Plk1 substrates should contain both PBD-binding end of the kinase domain and the first Polo-box, sites and kinase phosphorylation motifs if the processive function as a single phosphoserine/threonine-binding model is correct.Therefore, the model can be examined module (Elia et al., 2003a). Intriguingly, the optimal by looking at known Plk substrates (Table 1).Verified sequence motif recognized by the PBD is Ser-[pSer/ in vivo Plk1 substrates include Cdc25C (Toyoshima- pThr]-[Pro/X], suggesting that Cdks, MAP kinases, and Morimoto et al., 2002), Brca2 (Lin et al., 2003), Myt1

Figure 3 Two proposed models for PBD-mediated substrate targeting.In the distributive model (upper), Plks are targeted to speciﬁc subcellular locales through binding of the PBD to a single protein.Additional substrates of the kinase domain are then encountered in these locations.In this model, Plks function as spatial integrators of mitotic signalling.In the processive model (lower), PBD binding to a protein targets that protein for subsequent phosphorylation by Plks.In this model, Plks function as temporal integrators of mitotic phosphorylation events

Oncogene Structure and function of Polo-like kinases DM Lowery et al 251 Table 1 Potential polo-box domain (PBD)-binding motifs in known Plk1 substrates Protein Plk1 phosphorylation Potential polo-box Interacts with polo-box Phosphorylated by CDKs site(s) domain binding site domain of Plkl

Brca2 DMS193WSS, DES239LKK ST77P, SS1818P,ST2310P ND + ST3193P, SS3219P, ST3242P Cdc25C EFS198LKD ST130P + + Cyclin B ETS133GCA,AFS147DVI ST414LP ND ND Emil EDS145GYS or YSS149FSL SS41TL, several others ND + Grasp65 ND SS122P++ MKLP1 RRS911STV,RSS912TVA SS719IS, several others + ND MKLP2 EHS528LQV ST255SF, several others + + Mytl DSS426LSS, DDS435LGP ST455PND+ DLS469DIN, EDT495LDP Nlp EDS87S88SLE, ST167KEA SS213GH, several others ND ND EKS686QEV 274 326 232 260 NudC ENS KLS, DFS KAK SS WL, SS DP + ND TCTP DDS46LIG, TES64TVI ST65VI + ND Weel EDS53AFQ SS123PND+ Claspin SSS934FLT ST906Q + ND

ND, not determined.Sequences of proteins with mapped Plkl phosphorylation sites were examined for matches to the optimal Polo-box domain- binding motif.Also indicated is the presence or absence of experimental evidence for Polo-box domain binding and for CDK phosphorylation.The Polo-box-binding site for Cdc25C has been identiﬁed.Claspin is known to both interact and be phosphorylated by Plxl at the sites indicated. References for the mapped phosphorylation and interaction sites are given in the body of the text

(Nakajima et al., 2003), cyclin B (Toyoshima-Morimoto PBD binding in cyclin B phosphorylation by Plk1. et al., 2001; Yuan et al., 2002; Jackman et al., 2003), Another protein, Emi1, was recently identified as a Plk1 NudC (Zhou et al., 2003), Nlp (Casenghi et al., 2003), substrate in vitro (Moshe et al., 2004). Emi1 could be TCTP (Yarm, 2002), MKlp1 (Lee et al., 1995; Liu et al., phosphorylated by nonphysiological concentrations of 2004), MKlp2 (Neef et al., 2003), Grasp65 (Lin et al., Plk1 that were B20-fold higher than those found in 2000; Sutterlin et al., 2001), and Wee1 (Sakchaisri et al., mitotic extracts.However, if Emi1 was first phosphory- 2004; Watanabe et al., 2004). All of these contain lated by Cdk1, then the amount of Plk1 that was potential PBD-binding sites, but only half match the required was reduced to physiological levels.Quantifica- phosphorylation motif of the only known priming tion of this effect revealed that incubation of Emi1 with kinases for PBD binding, CDK/MAPK.Also, Cdc25C, Cdk1 prior to incubation with Plk1 increased the MKlp1, MKlp2, TCTP, Grasp65, and NudC have been phosphorylation of Emi1 by Plk1 by threefold (Moshe definitively shown to interact with the Plk1-PBD (Lin et al., 2004). Unfortunately the sites of Cdk1 phosphor- et al., 2000; Sutterlin et al., 2001; Yarm, 2002; Elia et al., ylation on Emi1 or the ability of Emi1 to associate with 2003a, b; ; Neef et al., 2003; Zhou et al., 2003; Liu et al., the PBD of Plk1 were not examined.Nevertheless, this 2004).(Interactions with the other substrate proteins data clearly agrees with a processive phosphorylation have either not been examined or have only been model.Another recent study demonstrated that Wee1 assessed with full-length Plk1.) In addition, Cdc25C, phosphorylation by Plk1 was stimulated by phosphor- Brca2, MKlp2, Grasp65, Wee1, and Myt1 are known to ylation of Wee1 by Cdk1 (Watanabe et al., 2004). The be phosphorylated by Cdk1; and NudC, cyclin B, and site of phosphorylation by Cdk1 perfectly matched a TCTP are similarly phosphorylated by mitotic kinases consensus site for PBD binding again indicating a role other than Plk1, although the identity of these kinases for the processive model though this interaction was not and the sites and timing of phosphorylation are not directly addressed in the study.A separate study known.Taken together, these data suggest that the demonstrated that these same events likely occur for processive phosphorylation model may apply to at least the budding yeast homolog of Wee1 and Swe1, although some of these substrates. the priming kinase was suggested to be Cla4 instead of In vitro, however, Plk1 can phosphorylate bacterially Cdk1 (Sakchaisri et al., 2004). produced (and therefore unprimed) Cdc25C (Kumagai Very recently a novel substrate of Plx1, claspin, was and Dunphy, 1996), TCTP (Yarm, 2002), Myt1 described (Kumagai and Dunphy, 2000).Claspin binds (Nakajima et al., 2003), cyclin B (Jackman et al., to the PBD of Plx1 through a phospho-dependent 2003), and NudC (Zhou et al., 2003), suggesting that interaction and is subsequently phosphorylated on a priming phosphorylation is not absolutely required nearby site (Yoo et al., 2004). The timing of these two under conditions of high substrate and enzyme con- events was clearly delineated, and the necessity of PBD centration.The kinetics and efficiency of phosphoryla- binding for the subsequent phosphorylation event was tion, before and after a priming phosphorylation, were demonstrated.Thus, claspin is the first Plk substrate to not investigated for most of these substrates.Phosphor- definitively use the processive phosphorylation model, ylation of cyclin B by Erk, however, was found to although cyclin B, Emi1, and Wee1 are very strong dramatically enhance its subsequent phosphorylation by candidates for additional Plk substrates that use this Plk1 (Yuan et al., 2002), suggesting a potential role of process.

Oncogene Structure and function of Polo-like kinases DM Lowery et al 252 A second alternative model is the ‘distributive interphase baseline at the beginning of the next G1 phase phosphorylation’ model in which the ligand to which due to degradation of these Plks by the APC/C the PBD binds is distinct from the substrates which the (Hamanaka et al., 1995; Charles et al., 1998; Cheng Plk kinase domain phosphorylates (Figure 3).In this et al., 1998; Shirayama et al., 1998; Lindon and Pines, case, PBD-binding to a phosphorylated scaffold or 2004). docking protein localizes the kinase domain to specific Studies of Plk1, Plx1, Polo, and Cdc5 have established subcellular structures, vicinal to substrates of the kinase that activation of these kinases requires their direct domain that could even be bound to the same scaffold phosphorylation (Hamanaka et al., 1995; Tavares et al., molecule.The movement of Plk1 from centrosomes to 1996; Mundt et al., 1997; Cheng et al., 1998; Qian et al., kinetochores and the spindle midzone, and then to the 1998b).Plk1 and Plx1 activation have been the most septin ring as cells progress from G2 through mitosis is extensively examined in mechanistic and structural consistent with this model.Most of the putative scaffold detail, although activation of other family members is molecules are yet to be defined, but there are at least two believed to occur through the same mechanisms.Several potential examples of such Plk1 scaffolds.One of the conserved Ser and Thr residues are present within the roles of Plk1-related family members is the activation of kinase domains of Plks.Mutation of two of these, T210 the anaphase promoting complex (APC), and Cdc23/ and S137 in Plk1 (or the corresponding residues T201 Cut23 is responsible for localizing Plk/Plo1 to the APC, and S128 in Plx1), to Asp as a mimic of phosphorylation where additional Plk1/Plo1 substrates are found (Golan results in the formation of activated Plk1 and Plx1 (Lee et al., 2002; May et al., 2002). The kinesin-like proteins and Erikson, 1997; Qian et al., 1999; Jang et al., 2002b). Mklp1/2, which seem to carry Plk1 to the central spindle Mutation of these residues results in multiple mitotic during anaphase and telophase (Neef et al., 2003; Liu phenotypes as discussed below. et al., 2004), may be the scaffolds for Plk1’s role in T210/201 lies within the T-loop of the Plk kinase cytokinesis, which presumably requires multiple sub- domain, and has been confirmed to be an in vivo strates.Both of the Plk1 scaffolds, Cut23 and Mklp1/2, phosphorylation site through phosphopeptide mapping are themselves substrates of Plk1, suggesting that they of Plk1 immunoprecipitated from nocodazole-arrested function as more than simple docking sites. lysates and Plx1 immunoprecipitated from mitotic Both models will surely turn out to be correct for Xenopus egg extracts (Jang et al., 2002b; Kelm et al., specific Plk substrates, although which model will 2002).Phosphorylation in T loops is a common predominate is an open question.In either case, the mechanism of activation for many protein kinases PBD appears to function as an ‘AND’ gate in the lingo including Aurora A/B, PKA, Cdk1/2, Erk1/2, Rsk1/2, of systems biology, by ‘integrating’ the activity of other Akt/PKB, and Mek1 (Johnson et al., 1996). Unfortu- mitotic kinases with that of Plks.By functioning as a nately, the lack of a phospho-specific antibody against pSer/pThr-binding module, the PBD ensures that Plk- the Plk1 T210/201 site has prevented detailed studies mediated progression through M phase and sequential correlating phosphorylation at this site with the activity activation of discrete mitotic events occurs if, and only of Plks or specific phenotypes.Still it is generally if, a prior CDK/MAPK or other mitotic kinase believed that phosphorylation at T210 is a requirement phosphorylation has occurred.In the processive phos- for Plk activation because the phosphomimic mutations phorylation model, the PBD functions as an ‘integrator’ mentioned earlier activate the kinase, and T210-V in time – the priming phosphorylation event has to mutations prevent activation (Lee and Erikson, 1997; occur at the same time that Plk kinase is present and Qian et al., 1999; Jang et al., 2002b). The corresponding active, or else the PBD-binding site will be lost through phosphomimic mutation in human Plk2 also activates the rapid action of phosphatases.In the distributive the kinase (Ma et al., 2003), suggesting that this is a phosphorylation model, the PBD functions as an conserved mechanism of Plk activation. ‘integrator’ in space – substrates must be localized in Production of a T201-D mutant form of Plx1 in a close proximity to the phosphorylated scaffold molecule Xenopus egg extract system resulted in acceleration of to which the PBD has bound. Cdc25C activation during mitotic entry and delayed Cdc25C deactivation during mitotic exit, although Activation and phosphorylation of Plks T201D expression was not sufficient to induce the first mitotic cell cycle.Additionally, there was a delay in The cell cycle dynamics of Plks has been examined cyclin B2 degradation during anaphase and a corre- primarily for Cdc5 and Plk1.The levels of these Plks rise sponding delay in the loss of Cdc2 kinase activity (Qian dramatically in the G2 phase of the cell cycle starting et al., 1999). On the other hand, overexpression of a from undetectable levels during G1 and beginning their T210-D mutant form of Plk1 in HeLa cells caused an rise in mid S phase (Golsteyn et al., 1994; Hardy and increased G2/M population, but only slighty more so Pautz, 1996; Cheng et al., 1998). With this rise in protein than overexpression of wild-type Plk1 or even kinase- levels there is a concordant increase in kinase activity, dead Plk1 (Jang et al., 2002b). At later times, over- but the activity rises much more dramatically than the expression of wild-type or kinase-dead Plk1 delayed levels, implicating an activation event (Golsteyn et al., mitotic exit, leading these cells to ultimately exit mitosis 1995; Hamanaka et al., 1995; Cheng et al., 1998). Both without undergoing cytokinesis (Mundt et al., 1997). the protein levels and the activity peak at the There have been at least two proposals for the metaphase–anaphase transition before returning to the upstream kinase that phosphorylates T210/201 – Xeno-

Oncogene Structure and function of Polo-like kinases DM Lowery et al 253 pus Plkk1 (equivalent to human SLK or Stk10) and phase-arrested or asynchronous cells, and were therefore PKA.xPlkk1 was purified as the Plx1 T201 phosphor- better suited to identify stable phosphorylation sites, ylating factor in Xenopus oocyte extracts (Qian et al., perhaps explaining the failure to observe Ser-137/128 1998b).A recent study demonstrated that xPlkk1 may phosphorylation to date.Since Plks show striking actually be downstream of Plx1 and the two kinases changes in subcellular localization during anaphase could function in a positive feedback loop (Erikson et al., and telophase, and participate in different processes at 2004).Immunodepletion of xPlkk1 did not prevent these times than during earlier stages of mitosis (Barr activation of Plx1 or alter the kinetics of mitotic entry in et al., 2004), the possibility for additional post-transla- Xenopus egg extracts as monitored by H1 kinase tional modifications of Plks, such as Ser-137 phosphor- activity, implying that this loop is not required. ylation, during the postmetaphase stages remains Activation of Plx1 by phosphorylation was still promising.Demonstration that Ser-137/128 is phos- required, implying that there must be another kinase phorylated in vivo, and identifying an upstream kinase that can phosphorylate Plx1 T201 in the xPlkk1- responsible for this event, would constitute a seminal depleted extracts.Both human SLK and Stk10 have advance in the field. been shown to phosphorylate human Plk1 at T210 in vitro and are able to stimulate Plk1 activation in vivo (Ellinger-Ziegelbauer et al., 2000; Walter et al., 2003). It Structure of the Sak Polo-box was previously believed that Stk10 could not be the Plk1-activating kinase because the mouse homolog, Structural analysis of the Polo-box first emerged from LOK, was only expressed in lymphatic tissues.However, the crystal structure of the single Polo-box of murine more complete expression profiling has shown that Sak (residues 845–919) (Leung et al., 2002). The Stk10 is expressed in most tissues, so both SLK and structure revealed that the Polo-box had crystallized as Stk10 remain viable candidates or partners for Plk1 an intermolecular dimer with a clam-like fold, consist- T210 phosphorylation.PKA has also been shown to be ing, in total, of two a-helices and two six-stranded able to phosphorylate T201 and activate Plx1 activity in antiparallel b-sheets (Figure 4a).Each b-sheet contained vitro (Kelm et al., 2002). In this same study, xPlkk1 was four contiguous strands from one Polo-box (b-strands 6, not able to phosphorylate Plx1 T201 creating a currently 1, 2, and 3), with the remaining two strands coming unresolved discrepancy in the field. from the other Polo-box.The association of b-strand In contrast to T210/201, there is no direct evidence for 3 from both Polo-boxes formed a hinge region, oppo- the in vivo phosphorylation of S137/128.At least two site to which was an interfacial cleft and pocket studies searched directly for S137/128 phosphorylation (B17 Â 8 Â 12 A˚ ).The presence of nine of 19 conserved using tryptic phosphopeptide analysis combined with hydrophobic residues lining the interior of the pocket mass-spectrometry in mitotic Xenopus extracts (Kelm hinted at a possible ligand-binding function, although et al., 2002), and phosphopeptide mapping and phos- actual ligands of the Sak Polo-box dimer remain to be phoamino acid analysis in mammalian cells (Jang et al., identified. 2002b), and both came up short.Although these and The dimeric structure of the Sak Polo-box seen in the other studies did identify a number of other phospho- crystals was also shown to occur within cells using serine residues (Hamanaka et al., 1995; Qian et al., coimmunoprecipitation assays of differentially tagged 1998b; Kelm et al., 2002; Wind et al., 2002), none of Sak Polo-box constructs (Leung et al., 2002). Interest- these sites has been shown to have functional signifi- ingly, a C-terminal construct of Sak containing residues cance.Furthermore, mutation of S137 -A failed to 596–836 (Sak241) but lacking the Polo-box region was prevent activation of Plk1/Plx1 and did not produce any also observed to dimerize, indicating the presence of one noticeable phenotypes when overexpressed, suggesting or more dimerization domains in addition to the Polo- that phosphorylation of this site is not critical in vivo box within Sak.The subcellular localization of enhanced (Qian et al., 1999; Jang et al., 2002b). Still the green fluorescence protein (EGFP) fusion constructs of phenotypes seen with an S137-D mutant form of the Polo-box region alone and of full-length Sak in NIH Plk1 are striking.When overexpressed in HeLa cells, the 3T3 cells suggested that the Polo-box may play a role in S137D mutant Plk1 caused the cells to arrest as a G1/S targeting Sak to centrosomes and the cleavage furrow. population rather than the G2/M population seen with However, in contrast to loss of localization observed for T210D or wild type, and the double mutant S137D/ Polo-box truncations in other members of the Plk T210D also shows the G1/S block (Jang et al., 2002b). family, subcellular localization of Sak was not disrupted The equivalent S128D/T201D double mutant in Plx1 by deletion of the Polo-box.Significant loss of proper caused cleavage arrest resulting in large cells when subcellular localization of Sak was only observed if both injected into a Xenopus two cell embryo, and was able to the C-terminal region following the kinase domain but induce the first mitotic cycle in a Xenopus egg extract preceeding the Polo-box (residues 596–836) and the system unlike wild-type or single mutants of Plx1 (Qian Polo-box itself were deleted.Given the ability of the et al., 1999). Together, these data suggest that if S137/ Polo-box and Sak241 constructs to dimerize, it is unclear 128 is phosphorylated in vivo, it is probably only a if the observed subcellular localizations of these transient event during late mitosis. domains result from their interaction with components The systems that have been used to map phosphor- of centrosomes and the cleavage furrow or from ylation sites in Plks have focused primarily on meta- association with endogenous Sak.

Oncogene Structure and function of Polo-like kinases DM Lowery et al 254 the Sak Polo-box homodimer correspond to residues on the exterior faces of the b-sandwich in the Plk1 PBD. The PBD also contained a Polo cap (Pc) region consisting of an a-helix connected via a linker (L1) to the first b-strand of PB1 and a second linker (L2) connecting the two Polo-box repeats, helping to hold both Polo-boxes in the correct orientation. Peptide library screening to determine the optimal phosphopeptide motifs recognized by the PBDs of human Plk1, Plk2 and Plk3, Xenopus Plx1, and Saccharomyces cerevisiae Cdc5p revealed a strong selection for Ser in the pThr/pSer-1 position and a modest preference for Pro in the pThr/pSer þ 1 position (Elia et al., 2003a; Elia et al., 2003b). Crystal structures of the human Plk1 PBD in complex with its optimal phosphopeptide revealed the structural basis of the phospho-dependent binding and motif recognition.The phosphopeptide-binding site is located at one end of a shallow cleft between the two b-sheets within the only highly conserved region on the PBD surface.The phosphate group is bound via eight hydrogen-bonding interactions involving indirect contacts through a lattice of ordered water molecules and direct contacts arising from the pincer-like arrangement of the His-538 and Lys-540 side chains.The importance of His-538 and Lys-540 in phosphospecific binding was confirmed by mutation of these residues to Ala, which eliminated binding (Elia et al., 2003b). The strong selection for Ser at the pThr-1 position can be accounted for by three hydrogen bond interactions of the Ser-1 side chain Figure 4 Structures of the Sak Polo-box and Plk1 PBD.( a)A hydroxyl to the main chain atoms of Trp-414 and with homodimer of the Sak Polo-box (PDB accession code: 1MBY) is the backbone carbonyl of Leu 491 via a water molecule. shown as a ribbon figure in two orthogonal orientations.The Van der Waals contacts between the Ser-1 Cb atom and individual monomers are colored green and blue.( b) The structure of the Plk1 PBD (PDB accession code: 1UMW) is shown as a the Cd1 of a strictly conserved Trp-414 side chain reveal ribbon figure in two orthogonal views.The optimal phosphopep- that larger hydroxyl-containing side chains such as Thr tide (Pro-Met-Gln-Ser-[phospho-Thr]-Pro-Leu) is shown in stick or Tyr at the pThr/pSer-1 position would result in a representation with yellow carbons and with the phosphate group steric clash.The pThr/pSer þ 1 Pro residue makes a highlighted with transparent spheres.The two Polo-boxes (PB1 and smaller contribution to the binding surface and likely PB2) are colored red and blue, while the Polo cap (Pc) is colored green.Figure created with Molscript (Kraulis, 1991) and Raster3D increases binding affinity by introducing a kink via its (Merritt and Murphy, 1994) trans peptide conformation to direct the residues C- terminal to it back towards the binding surface, thereby reducing the entropic penalty of binding. A mutually inhibitory interaction between the Plk1 Structure of the Plk1 PBD kinase domain and PBD results in both a B3-fold reduction in kinase activity and a B10-fold reduction in Crystal structures of the PBD of human Plk1 (Cheng phosphopeptide binding by the PBD with significantly et al., 2003; Elia et al., 2003b) revealed some similarities reduced phosphospecificity (Lee and Erikson, 1997; and a number of key differences with the structure of the Mundt et al., 1997; Jang et al., 2002a; Elia et al., Sak Polo-box dimer.Each of the individual Polo-box 2003b).Conversely, binding of the optimal phospho- repeats (PB1 and PB2) in Plk1 consisted of a self- peptide to full-length Plk1 stimulated kinase activity by contained six-stranded b-sheet and a-helix (Figure 4b). B2.6-fold and suggests that binding of the PBD to This was similar to the structure of the single a-helix/b- substrates with primed phosphorylation sites simulta- sheets seen in the Sak Polo-box dimer, however in the neously targets Plk1 to its substrates and activates the Sak structure, each of the b-sheets contains contribu- kinase domain.The structural basis for regulatory tions from two Polo-boxes.In contrast, no strand interaction between the kinase and PBD remains swapping was observed in the Plk1 PBD b-sandwich. unclear.In the Src family of protein tyrosine kinases, Furthermore, the association between the Polo-box intramolecular binding of the SH2 domain to a C- repeats in Plk1 differed completely from that of the terminal phosphotyrosine motif positions the N-term- Sak Polo-box dimer.The b-sheets in the Plk1 PBD pack inal SH3 domain for binding to a linker connecting the together with the two a-helices on either side of the SH2 and kinase domains (Sicheri et al., 1997; Xu et al., b-sandwich, such that residues located at the interface in 1999).The SH3 domain and the linker interact with the

Oncogene Structure and function of Polo-like kinases DM Lowery et al 255 kinase domain to stabilize it in an inactive conforma- 5a, b).This Plk1 model was subsequently used as a tion.This mechanism of intramolecular regulation does template to model the Plk2, Plk3, and Plk4 kinase not appear to be applicable to Plk1, due to the absence domains (Figure 5c–e). of an internal optimal PBD motif.Additionally, Thr-210 of Plk1 is located within the activation mutations of the His-538/Lys-540 pincer residues, which segment, a centrally located loop that contributes a disrupt phosphospecific binding by the PBD, do not significant portion of the peptide substrate-binding site. abrograte binding of the PBD to the isolated kinase Similar to other kinases, phosphorylation of T210 in domain in trans.These observations indicate that the sites of interaction on the PBD with phosphopeptide ligands and with the kinase domain are distinct.Partial overlap of these sites, such that binding at one site sterically occludes binding to the other site, however, would provide one possible mechanism of the mutually inhibitory interaction. An alternative mechanism to regulate catalytic activity via an intramolecular phosphopeptide binding domain is illustrated by the SHP family of phosphatases, in which binding of the N-terminal SH2 domain to the phosphatase domain partially occludes the catalytic cleft and distorts the conformation of the SH2 domain, reducing its phospholigand affinity (Hof et al., 1998). Conversely, phospholigand binding to the SH2 domain results in activation by stabilizing an alternate conformation of the SH2 domain with reduced affinity for the phosphatase domain.A similar ‘molecular switch’ mechanism may be applicable to the PBD domain and would be consistent with its reduced phosphodependent binding in full-length Plk1 and the increased activity of full-length Plk1 upon addition of optimal phosphopeptide.Crystal structures of the PBD in the apo state and in complex with its optimal phosphopeptide reveal that phosphopeptide binding does not induce significant conformational changes in the PBD (Cheng et al., 2003). However, a conformational change in the PBD with distortion of its phosphopeptide-binding site upon binding to the kinase domain remains possible.Additionally, it is not known if the kinase active site is simply occluded as in the SHP phosphatases, or if the kinase domain adopts a distinct inactive conformation when bound to the PBD.A crystal structure of full-length Plk1 or a complex of the isolated kinase domain with the PBD will be needed to observe the kinase domain and the PBD in their mutually inhibited states. Figure 5 Molecular modelling of the Plk kinase domain.( a)A ribbon figure of a model of the Plk1 kinase domain (residues 48– 309) is shown in two orthogonal views and with the N- and C- Model of the Plk kinase domain terminal lobes colored green and blue, respectively.The model of the core kinase domain is based primarily on the structures of No crystal structure is currently available for a Plk Aurora-A and -B (Bayliss et al., 2003; Cheetham et al., 2002; family kinase domain; however, given the high structur- Nowakowski et al., 2002). The activation loop was modelled in an active conformation using the structure of the Akt/PKB : AMP- al conservation of the protein kinase catalytic core in the PNP : GSK3-peptide ternary complex (Yang et al., 2002). ATP and active conformation, molecular models of kinase do- a substrate peptide (Gly-Ala-Ala-Ala-Glu-Ala-Ser-Phe-Ala-Ala) mains based on available crystal structures can be used are shown in stick representation and were also modelled based on to predict structural features of the active site that may the Akt/PKB : AMP-PNP : GSK3-peptide ternary complex struc- be important for regulation and substrate selectivity. ture (Yang et al., 2002). The side chains of Ser-137 and phosphorylated Thr-210 (pT210) are shown in stick representation The kinase domain of human Plk1 was therefore and highlighted with transparent spheres.Figure created with modelled using the SWISS-MODEL server (Guex and Molscript (Kraulis, 1991) and Raster3D (Merritt and Murphy, Peitsch, 1997) based on the crystal structures of Aurora- 1994).( b-e) The surfaces of the substrate binding cleft for Plk1 (b), A (Bayliss et al., 2003) (PDB codes 1Ol5 and 1Ol7), Plk2 (c), Plk3 (d), and Plk4 (e) are shown colored by electrostatic potential.Positively and negatively charged regions are shaded blue (Nowakowski et al., 2002) (PDB code 1MQ4), Aurora-B and red, respectively.ATP and substrate peptide are shown as (Cheetham et al., 2002) (PDB code 1MUO), and Akt/ displayed in panel a.Figure created using GRASP (Nicholls et al., PKB (Yang et al., 2002) (PDB code 1O6K) (Figure 1993)

Oncogene Structure and function of Polo-like kinases DM Lowery et al 256 Plk1 and its mutation to Asp has a stimulatory effect on Prospects for structure-based drug design kinase activity, as discussed earlier.Phosphorylation of the equivalent Thr in PKA stabilizes the activation loop Plk1 is highly expressed in proliferating cells and in in an open and extended conformation to allow various human tumors including squamous cell cancers substrate binding (Knighton et al., 1991a, b). Addition- of the head and neck, melanomas, gliomas, esophageal, ally, interactions of the phospho-threonine with an colorectal and lung carcinomas, and endometrial, adjacent cluster of positively charged side chains and ovarian and breast cancers (Holtrich et al., 1994; Wolf backbone nitrogens may play a role in aligning the et al., 1997, 2000; Knecht et al., 1999; Tokumitsu et al., N- and C-lobes of the kinase domain into a catalyti- 1999; Dietzmann et al., 2001; Takai et al., 2001; Kneisel cally competent conformation or in properly orienting et al., 2002; Takahashi et al., 2003). High levels of Plk1 and positioning key catalytic residues (Johnson et al., expression in tumors often correlate with poor prog- 1996). nosis.Constitutive expression of Plk1 in NIH 3T3 cells A comparison of the active sites of the four human causes malignant transformation and induces tumor Plk kinase models suggests that Plk1, Plk2, and Plk3 growth in nude mice (Smith et al., 1997). Importantly, may share a similar substrate phosphorylation motif, antisense oligonucleotides that downregulate Plk1 cause while the sequence motif phosphorylated by Plk4 may decreased cell proliferation in MDA-MB-435 breast differ significantly.Based on the phosphorylation sites cancer cells and A549 non-small-cell lung cancer cells in of known substrates and mutational studies, a consensus vitro and in tumor regression when these cells were recognition motif for Plk1 has been deduced and xenografted onto nude mice (Spankuch-Schmitt et al., consists of Glu/Asp at the À2 position and a hydro- 2002; Elez et al., 2003). Interestingly, the viability of phobic residue in the þ 1 position relative to the nontransformed cells such as amniocytes, mesangial phosphorylated Ser/Thr residue (Toyoshima-Morimoto cells, and skin fibroblasts was unaffected by these types et al., 2001; Toyoshima-Morimoto et al., 2002; Casenghi of Plk1 downregulation (Elez et al., 2003). These et al., 2003; Jackman et al., 2003; Lin et al., 2003; observations strongly indicate that Plk1 is likely to be Nakajima et al., 2003; Neef et al., 2003). A peptide a highly effective target for anticancer drug design. corresponding to this motif was therefore modelled into The Plk1 kinase domain is an obvious target for drug the active site using the crystal structure of Akt/PKB in development, and high-throughput screening and struc- complex with GSK3-peptide and AMP-PNP (Yang ture-based approaches used in the discovery and et al., 2002). The distibution of electrostatic potential optimization of inhibitors of other protein kinases can within the modelled Plk1 active site is consistent with be applied (Fabbro et al., 2002; Noble et al., 2004; Stout this putative substrate phosphorylation motif.A hydro- et al., 2004). The deep cavity of the ATP-binding pocket phobic pocket at the þ 1 position appears to be of kinases provides an excellent binding site for small conserved in all four kinase domains, while a positively molecules, and consequently the majority of available charged region lies in close proximity to the substrate kinase inhibitors are ATP analogs.The lack of Ser/Thr-2 position in Plk1, Plk2, and Plk3 but not in specificity of many such inhibitors is a significant Plk4.In Plk4, Thr-138 substitutes for a Lys residue, problem, however, owing to the highly conserved nature which is conserved in the other three Plks at this of the ATP-binding pocket.In some cases, selectivity position (Lys 178 in Plk1). can be achieved by exploiting unique structural features. Ser-137 of Plk1 shares a similar surrounding sequence For example, deschloro-flavopiridol utilizes an addi- context as Thr-210 and has been proposed to be a tional less-conserved pocket within the ATP-binding site second site of phosphorylation (Jang et al., 2002b), of CDK2 (De Azevedo et al., 1996). The diaryl urea, and although this remains controversial as discussed above. quinazolinone and pyridol-pyrimidine classes of p38 In the Plk1 kinase model, Ser-137 is solvent exposed and MAP kinase inhibitors also exploit novel binding sites located within the peptide-binding cleft near the within the ATP-binding pocket, some of which are predicted substrate Ser/Thr-3 position.Phosphorylation created by inducing subtle conformational changes of Ser-137 would be expected to introduce a strong (Tong et al., 1997; Pargellis et al., 2002; Fitzgerald negative charge and increase selectivity for a positively et al., 2003). High specificity is also achieved by charged residue such as Arg or Lys in the substrate at targeting a distinct inactive conformation of a kinase the À3 position.Interestingly, the crystal structure of as shown by the inhibition of Abelson tyrosine kinase by Akt/PKB in complex with GSK3-peptide shows a STI571/Gleevec (Schindler et al., 2000). Important to negatively charged residue at this position (Glu 236) the development of Plk1 inhibitors that target the kinase forming a bidentate salt bridge to the À3 Arg of GSK3- domain will be the ability to produce sufficient peptide (Yang et al., 2002). A similar interaction is also quantities of either the full-length Plk1 or kinase domain observed in the structure of a PKA–peptide complex for high-throughput screening and crystallographic (Bossemeyer et al., 1993). Thus, phosphorylation of Ser- studies.Crystal structures of the Plk1 kinase domain 137, if it occurs, might be expected to alter Plk substrate in both the active and inactive conformations will be specifity, leading to phosphorylation of alternative extremely valuable for the optimization of lead com- targets than those selected when Ser-137 is not pounds. phosphorylated.Intriguingly, Ser-137 of Plk1 is con- The PBD presents an additional unique target for the served in Plk2 (Ser 166) and Plk3 (Ser 107) but is development of novel and specific Plk inhibitors, since it replaced with glutamate in Plk4 (Glu 96). is essential for normal Plk function.To date, disruption

Oncogene Structure and function of Polo-like kinases DM Lowery et al 257 of protein–protein interactions have been looked upon will facilitate structure-based optimization of any lead unfavorably as targets for drug design, primarily compounds identified in these screens. because such interactions often involve burial of large amounts of hydrophobic surface.However, the identi- fication of a relatively polar, small optimal phosphopeptide ligand for the PBD capable of disrupting Plk- Acknowledgements substrate interactions (Elia et al., 2003a) suggests that We are grateful to Drs Louise N Johnson, Erich A Nigg, Frank Sicheri, Peter Rellos, and Stephen J Smerdon for small molecules should be sufficient to accomplish this discussions and insight into Plk structure and function.DML task.Furthermore, the availability of Plk PBD optimal is supported by a Howard Hughes Medical Institute Pre- phosphopeptides should facilitate development of high doctoral Fellowship.DL is supported by a Jane Coffin Childs throughput assays for small molecule inhibitors that Memorial Fellowship.This work was supported by NIH will disrupt PBD function, while the availability of Grant GM60594 and a Burroughs-Wellcome Career Develop- crystal structures of the PBD–phosphopeptide complex ment Award to MBY.

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