Understanding the Polo Kinase Machine

REVIEW Understanding the Polo Kinase machine

V Archambault, G Lépine and D Kachaner

The Polo Kinase is a central regulator of cell division required for several events of mitosis and cytokinesis. In addition to a kinase domain (KD), Polo-like kinases (Plks) comprise a Polo-Box domain (PBD), which mediates protein interactions with targets and regulators of Plks. In all organisms that contain Plks, one Plk family member fulﬁlls several essential functions in the regulation of cell division, and here we refer to this conserved protein as Polo Kinase (Plk1 in humans). The PBD and the KD are capable of both cooperation and mutual inhibition in their functions. Crystal structures of the PBD, the KD and, recently, a PBD–KD complex have helped understanding the inner workings of the Polo Kinase. In parallel, an impressive array of molecular mechanisms has been found to mediate the regulation of the protein. Moreover, the targeting of Polo Kinase in the development of anti-cancer drugs has yielded several molecules with which to chemically modulate Polo Kinase to study its biological functions. Here we review our current understanding of the protein function and regulation of Polo Kinase as a fascinating molecular device in control of cell division.

Oncogene (2015) 34, 4799–4807; doi:10.1038/onc.2014.451; published online 26 January 2015

THE POLO KINASE CONTROLS CELL DIVISION specific cell types, including in the nervous system.36,37 Interest- The Polo Kinase was identified almost 30 years ago from genetic ingly, Plk5 has lost kinase activity during evolution and it is 38,39 screens in Drosophila.1,2 It was quickly recognized as an important nevertheless required during development in mammals. regulator of cell division as polo mutants were defective in both Several reviews have provided a comprehensive picture of the meiosis and mitosis. Polo is well conserved between species, from cellular roles of Polo Kinase and other Plks in the cell cycle and 3,4,9,40 yeasts to humans.3,4 Its budding yeast equivalent, Cdc5, was beyond. The implication of Plk1 in cancer has also been at 41–43 independently identified in early screens for genes required for the center of recent reviews. Here we discuss the significant cell cycle progression.5 Plk family members are characterized by a progress made recently in understanding how the conserved Polo C-terminal Polo-Box domain (PBD), which mediates protein Kinase protein functions and is regulated. The level of complexity interactions and regulates the N-terminal serine/threonine kinase and sophistication achieved by this protein is fascinating. domain (KD; Figure 1a).6–8 In humans, five Polo-like kinases (Plks) Incidentally, a fine understanding of the Polo Kinase machine have been identified.9 Of note, Plks have not been found in could help develop better cancer treatments as Plk1 is an Achilles’ plants.10 heel of cancer cells. In all organisms that have Plks, there is one Plk family member that helps coordinate several functions in cell division (Figure 1b). In humans, Plk1 is responsible for an impressive number of OVERVIEW OF THE POLO KINASE MACHINE functions. It is necessary for centrosome maturation,11 Golgi As the cell cycle progresses, the Polo Kinase acts as a hub. It fragmentation,12 spindle assembly and function,13,14 kinetochore receives several signals at specific times and places. In turn, it must function,15,16 centromere assembly17 and cytokinesis.18–22 It also react and transmit multiple signals to the effector proteins that it promotes DNA replication,23 mitotic entry,24 removal of sister regulates (Figure 1c). How is this complex task achieved? chromatid cohesion25 and chromosome condensation.26 These The two domains of the Polo Kinase are capable of both functions involve a complex coordination of Polo Kinase with cooperation and mutual antagonism. Early mutational analysis other major cell cycle regulators, most notably mitotic kinases indicated that the PBD of Plk1 is essential for subcellular including Cdk1, Aurora A and Aurora B. Many or all of these localization to discrete structures (Figure 1d).6,44 Subsequently, it functions are controlled by Polo in Drosophila, Cdc5 in budding was discovered that the PBD is a phosphopeptide-binding yeast and the orthologous Plk in a wide variety of organisms. Here domain.7,8,45 This was an appealing concept as priming phosphor- we globally refer to this conserved Plk family member across ylation of a target protein could induce its direct recognition by species as the Polo Kinase. Polo Kinase, thereby facilitating its phosphorylation by Polo During evolution, relatively few cell cycle functions have been Kinase, or more globally raising the concentration of Polo Kinase delegated to other Plks. The duplication of centrioles requires Plk4 at its sites of action46 (Figure 2 right). In some cases, the KD of Polo in humans as in Drosophila 27,28 and it additionally requires Plk2 in Kinase is responsible for creating the phosphorylated site that is humans.29 Plk3 is required for DNA replication and also regulates bound by the PBD.47 In addition, the KD of Polo Kinase contains a CDK activity at the level of Cdc25 phosphatases.30,31 Plk2 and Plk3 nuclear localization sequence that helps Plk1 access the nucleus also contribute to the DNA damage response and cell cycle before nuclear envelope breakdown.48 Thus, the PBD and the KD checkpoints.32–35 Specific Plks can have more specialized roles in can facilitate each other’s functions.

Institut de recherche en immunologie et en cancérologie, Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec, Canada. Correspondence: Dr V Archambault, Institute for Research in Immunology and Cancer, University of Montreal, Pavillon Marcelle-Coutu, C.P. 6128, Succursale Centre-ville, Montréal H3C 3J7, Québec, Canada. E-mail: [email protected] Received 2 September 2014; revised 27 November 2014; accepted 28 November 2014; published online 26 January 2015 Polo Kinase V Archambault et al 4800

Hinge phos. T-loop phos. Activation site Activation site P (S137 in Plk1) (T210 in Plk1) (D-Box) Polo-Box Domain (PBD) KD X P P P Y 603 a. a. Polo Kinase Domain (KD) PC PB1 PB2 in Plk1 IDL Kinase PBD PBD target

Inter-Domain Loop 2 Figure 2. Basic mechanisms of Polo Kinase activity. Right: the PBD Linker (IDL) Switch binds to target proteins (X) that have been pre-phosphorylated at a speciﬁc motif. The KD can then phosphorylate the same target at a different site, or another protein (Y) in the local environment, to S.c. D.m. modify its function (dotted arrows). By this mechanism, the Polo M Input Polo Kinase regulates several substrates at different locations during cell Cdc5 Polo Kinase Plk4 division. Left: the PBD and KD are capable of reciprocal inhibition Plo1 KD through an intramolecular interaction. The PBD inhibits the KD by S.p. making it less ﬂexible. The KD stabilizes a conformation of the PBD Plk1 IDL that is less capable of interactions with phosphorylated targets. Plx1 Plk2 PBD Phosphopeptide binding or KD phosphorylation at the T-loop Plx2 Plk3 relieve the inhibitory intramolecular interaction (right). T-loop Plx3 Plk4 S Output X.l. Plx4 Plk5 phosphorylation also increases the catalytic activity of the KD. H.s. Red: inhibited domain; green: activated domain.

ProphaseMetaphase Anaphase Telophase domains with a fragment of Map205, a Drosophila protein that stabilizes the complex.51 This is the first structure showing how * the PBD and the KD are positioned relative to each other. It shows that there is no competitive inhibition or steric hindrance by one * domain on the other. Instead, the two domains regulate each other’s activities allosterically. The KD binds the PBD at a distant Cytokinesis site from the substrate binding site. But when bound to the KD, Figure 1. Introduction to the Polo Kinase. (a) Primary structure of the the PBD adopts a conformation that is different from its Polo Kinase. The serine/threonine kinase domain (KD) is N-terminal conformation when in complex with a phosphopeptide.8,45 and the Polo-Box domain (PBD) is C-terminal. The two main Conversely, the PBD binds the KD in the hinge region between activating phosphorylation sites of the KD are shown. The PBD is the two KD lobes, and does not obstruct access to the kinase composed of two similar Polo Boxes (PB1 and PB2) separated by the active site. No major difference in conformation is seen between Loop 2 switch region, and preceded by the Polo Cap (PC). The two the structure of the PBD-bound KD of zebrafish Plk1 and the domains are separated by the Inter-Domain Linker, which contains a fl Destruction Box (D-Box) in several species. (b) Plk family members in structure of an active KD of human Plk1. Instead, the exibility of different organisms: Saccharomyces cerevisiae (S. c.), Schizosacchar- the hinge region between the N-terminal and C-terminal lobes of omyces pombe (S. p.), Drosophila melanogaster (D. m.), Xenopus Leavis the KD is predicted to be reduced when the KD is bound to the (X. l.) and Homo sapiens (H. s.). Orthologs corresponding to the Polo PBD. This crystal structure greatly facilitates the integration of a Kinase are in green. (c) The Polo Kinase is a crucial hub for the wealth of information regarding the function and regulation of coordination of the cell division cycle. It receives several signals as Polo Kinase obtained over the years by several laboratories. cell cycle events progress (Input), and it regulates several events of the cell cycle (Output). (d) The Polo Kinase localizes to several discrete structures via the PBD including kinetochores (foci on THE KD IS REGULATED BY MULTIPLE PHOSPHORYLATION chromosomes), centrosomes (asterisks) and the spindle midzone, EVENTS which resolves into the midbody (arrow). HeLa cells stained for Plk1 (green), α-Tubulin (red) and DNA (blue). Scale bar = 5 μm. The KD of Plks resembles Aurora kinases and calcium/calmodulin- dependent kinases, in both sequence and tertiary structure.46,52 A preferred consensus phosphorylation motif has been suggested Nevertheless, biochemical studies suggested that the PBD and fl KD can also inhibit each other (Figure 2 left). The KD alone was for Plk1 as (D/E)-X-(S/T), but Plk1 appears exible in this respect.53,54 Like several other kinases, Polo Kinase can be more active than full-length Plk1 in in vitro kinase assays, and activated by phosphorylation in its T-loop (Figures 1a, 2, 3a and addition of the PBD to the KD inhibited its kinase activity. Indeed, 4a).55,56 In animals, the T-loop phosphorylation site in Polo Kinase the PBD was capable of binding the KD, and interaction correlated – (Thr210 in human Plk1) matches an Aurora family consensus.57 59 with inhibition.8,44,49 In addition, the PBD alone could bind In humans, Aurora A phosphorylates Plk1 at this site during the G2 phosphopeptides more efficiently than full-length Plk1, suggest- 24,60 8 phase. Importantly, this event requires Bora, a co-factor that ing that the KD could inhibit the PBD. The molecular mechanism facilitates the reaction. Although Bora is largely degraded in for this mutual antagonism between domains was unclear. For mitosis, a low level of Bora persists and is sufficient to maintain a example, one could imagine that the phosphopeptide-binding pool of active Plk1 in mitosis.61 In Drosophila, Polo is activated by site on the PBD could have also mediated its inhibitory interaction Aurora B at centromeres in mitosis.62 Although Aurora B is with the KD. Consistent with this model, adding PBD-binding homologous to Aurora A, it behaves very differently. Aurora B is a 8 phosphopeptides to Plk1 increased its kinase activity. In this part of the chromosomal passenger complex, which localizes to model, the KD would have inhibited the PBD by competing with chromosomes in early mitosis and to the central spindle during its targets. Alternatively, the intramolecular PBD–KD interaction cytokinesis, while Aurora A is concentrated on spindle poles.63,64 could have caused steric hindrance toward target recognition. We have recently shown that Aurora B also activates Polo Kinase Other models were possible.46 Everyone in the field was eagerly during cytokinesis in Drosophila.65 Whether Aurora B contributes wishing for a crystal structure showing the relative positioning of to Plk1 activation in mitosis and in cytokinesis in humans is the PBD and the KD. unclear. Other kinases could contribute to Polo Kinase T-loop Recently, Xu et al.50 were able to obtain a co-crystal structure activation, depending on the species, the physiological situation between the separate PBD and KD of Plk1 from zebrafish or the cell type. In budding yeast, Cdc5 has been shown to be (Figure 3). The trick was to co-crystallize both zebrafish Plk1 activated by Cdk1, which phosphorylates the T-loop at a different

Oncogene (2015) 4799 – 4807 © 2015 Macmillan Publishers Limited Polo Kinase V Archambault et al 4801 T-loop of Plk1 (T210D), far from the interface between the two domains, also inhibited their interaction. Consistent with this observation, we have recently shown that phosphorylation of Drosophila Polo at the equivalent T-loop site inhibited the PBD interaction with Map205, and that this dissociation is required for Polo recruitment to the midbody and for successful cytokinesis.65 Therefore, T-loop phosphorylation of Polo does not only increase its kinase activity, it also regulates PBD interactions allosterically. This effect may require the presence of the Inter-Domain Linker (IDL) in Polo Kinase.50 Phosphorylation of Ser99 in the KD of Plk1 was recently shown to regulate its activity by yet another completely different mechanism.69 This site is located in the α-helix of the N-terminal lobe of the KD (Figure 3a). Its phosphorylation depends on Bora and on the PI3K-Akt pathway, and creates a binding site for 14-3- 3γ (Figure 4c). Results suggest that this interaction is required for the full activity of Plk1 in mitosis and for the metaphase- to-anaphase transition. Ser99 of Plk1 is conserved between vertebrate Polo Kinases, but not invertebrate orthologs. Phosphor- ylation of the KD at Ser49 in Plk1 was also proposed to contribute to its full activation in mitosis, the mechanism of this regulation remains to be defined.70 Constitutive Polo Kinase activity can cause delays or defects in the events of mitosis, cytokinesis and the centriole cycle.71–75 Thus, Polo Kinase phosphorylation is reversible. Plk1 activity in the centromere/kinetochore region is restricted by the Mypt1-bound form of protein phosphatase 1 that dephosphorylates Thr210, the main activation site of Plk1. Mypt1 is a regulatory subunit of protein phosphatase 1, which is primed for binding to the PBD of Plk1 by Cdk1 phosphorylation.71 Failure of this mechanism results in excessive phosphorylation of centromeric cohesins by Plk1, leading to precocious loss of sister chromatid cohesion and chromosome segregation defects.76 The importance of Plk1 dephosphorylation at other subcellular locations, other times in Figure 3. Sites of activity and regulation in Plk1. The structure of a the cell cycle and other residues have been little explored yet. PBD–KD complex of zebrafish Plk1 is shown (PDB accession 4J7B; a Moreover, additional mechanisms must mediate Polo Kinase segment of Map205 was co-crystallized in the complex and is not inactivation as Mypt1 is not conserved in invertebrates. shown for clarity). The PBD is in green and the KD is in cyan. (a) The Like many kinases, Polo Kinase depends on the Hsp90 ATP-binding site lies between the N-lobe and C-lobe of the KD, near chaperone system for its function.77 The Cdc37 and Sgt1 proteins a PBD contact with the KD. The T-loop, which is the major site of have been shown to function in this regulation of Plk1 in humans activation by phosphorylation, is not visible in the structure, and is and Polo in flies.78–80 Failures in this system result in a partial loss represented by a dashed line. Phosphorylation in the hinge region interferes with binding of the PBD with the KD. (b) The structure was of Polo Kinase function that leads to various mitotic defects. Hsp90 rotated 90 degrees toward the viewer as to bring the KD N-lobe in and its co-chaperones could help in the folding, the stabilization the front. The canonical binding site for phospho-proteins in PB1 is and/or the regulation of Polo Kinase. Their precise modes of close to a hydrophobic binding groove which can also contribute to interaction and functions are not completely defined. interactions. Loop 2 can switch positions upon protein binding thereby modulating the PBD–KD interaction. The mono- ubiquitylation site, which interferes with phospho-protein binding THE PBD IS CAPABLE OF SEVERAL TYPES OF INTERACTIONS is positioned near the binding site and at the top of Loop 2. The PBD of the Polo Kinase is made of two subdomains called Polo Boxes (PB1 and PB2) which adopt a similar fold, and of the Polo residue from the Aurora site.66 Although the precise connectivity Cap that wraps around PB2 (Figures 1a and 3b). Both Polo Boxes obviously varies between systems, in all cases there is cooperation contribute to form a binding pocket that can accommodate between the main mitotic kinases that are Polo Kinase, Aurora A, phosphoserine or phosphothreonine residues immediately pre- Aurora B and Cdk1.67 ceded by a serine residue (Figures 3b and 4d).8,45 In many cases, Phosphorylation events at other sites in Polo Kinase have been the phosphorylated residue is followed by a proline residue, and proposed to contribute to its activation. Years ago, phosphoryla- the phosphorylation is attributable to Cdk1, a proline-directed tion of Plk1 at Ser137 was detected and the phosphomimetic mitotic kinase.46,47,81 Phospho-priming by Cdk1 occurs for several S137D mutation was found to increase kinase activity.58 A Plk1 targets including BubR1 at kinetochores and Wee1, a kinase subsequent study reported that Plk1 was phosphorylated at that regulates Cdk1 activity.15,82 This mechanism allows a coupling Ser137 during a short time window in mitosis, and suggested that of Cdk1 and Plk1 in the regulation of their targets in mitosis. this activating event helps control Plk1 function in the spindle However, the proline residue is not an absolute requirement for a checkpoint.68 From the position of Ser137 near the hinge region in PBD phospho-binding site. Several PBD interactors are phosphory- the KD, it was unclear how the enzymatic activity of Plk1 could be lated by Polo Kinase itself or by other kinases, especially when enhanced by phosphorylation at this site. The recent crystal Cdk1 activity is low. These substrates include the central spindle structure of the PBD–KD complex revealed that phosphorylation microtubule-associated protein PRC1, a target of Plk1 in cytokin- at Ser137 interferes with the inhibitory interaction of the PBD on esis, and the centromeric protein PBIP1, a target of Plk1 in G2.18,83 the KD (Figures 1a, 3a and 4b).50 Intriguingly, Jang et al.58 showed The PBD is also capable of binding proteins that are unpho- in their early study that a phosphomimetic substitution in the sphorylated. Here we discuss the most firmly established of such

T-loop Phosphorylation (Thr210 in Plk1) Phosphorylation Hinge Phosphorylation and 14-3-3γ binding (Ser137 in Plk1) P (Ser99 in Plk1) P P

Loop2 KD P IDL P Phosphopeptide PBD binding IDL Phos- phorylation

Map205 binding Ub- (other proteins?) IDL poly-ubiquitylation Mono-ubiquitylation and degradation Figure 4. Multiple ways to regulate Polo Kinase protein activity. The PBD (left) and KD (right) are linked by the IDL (dotted line) and they also engage in an intramolecular interaction. Yellow: basal state of the protein with basal domain activities; green: domain is known to be activated by an event; red: domain is known to be inactivated by an event; gray: effect on the domain is not known. All reactions are reversible except degradation. See text for discussion of each mechanism. The combination of these different modes of regulation in vivo results in a highly complex spectrum of possible mechanisms and ﬁne-tuned activities.

instances, although more cases likely remain to be uncovered. if they invariably induce either the fully active (KD-dissociated) or Some interactions are enhanced by phosphorylation but can inactive (KD-bound) conformation of the PBD. Additional struc- already form without phosphorylation. A phosphopeptide from tures of a full-length Polo Kinase in an inactive or active form will the substrate Cdc25C binds the PBD of Plk1 with an affinity five be required before we can fully understand the structural basis of times higher than the corresponding unphosphorylated peptide.84 Polo Kinase activation and inhibition. In other words, unphosphorylated Cdc25C is capable of significant Drosophila Map205 is not closely conserved in vertebrates, but binding to Plk1. This can be explained by the fact that PBD human Plk1 is capable of a strong interaction with it. This residues that recognize the S-p(S/T) minimal motif engage in observation suggests that Plk1 may interact with some of its multiple contacts, some of them independent from the phosphate targets or regulators in similar ways to Map205, by natural group in this motif.8 phosphomimetism at the canonical binding pocket and/or using In addition, the PBD knows other tricks. Map205 is a the two additional binding sites on the PBD. Such proteins remain microtubule-associated protein that interacts with Polo in to be clearly identified. Bora, the co-factor that facilitates Drosophila.51,85 Map205 binds the PBD of Polo at the same phosphorylation of Plk1 at the T-loop by Aurora A, may be one binding site as canonical, phosphorylated targets (Figures 3b and of them. Biochemical results indicate that Bora can interact with 4e). This is achieved partly by a naturally phosphomimetic both domains of Plx1, and that its interaction with the PBD does glutamic acid residue, which substitutes for a phosphate group not require priming phosphorylation.24 in the canonical binding pocket of the PBD, as seen in the crystal In budding yeast, the Polo Kinase Cdc5 interacts with Dbf4 structure.50 This type of binding is not unique to Drosophila Polo without a requirement for phosphorylation.88 Dbf4 is well-known as Plk1 from humans and zebrafish can also bind Map205.50,51 as a regulatory subunit of the Cdc7 kinase, and together they are Mutation of this Glu residue strongly abrogates Map205 binding required for DNA replication.89 In the Dbf4–Cdc5 interaction, to zebrafish Plk1.50 In addition, the Map205-PBD-KD crystal unlike for the Map205–Polo interaction, the canonical binding site structure reveals that Map205 interacts with the PBD at two more on the PBD is not required. Instead, the motif RSIEGA of Dbf4 was sites, which contribute to the high affinity of Map205 for Polo shown to bind the PBD of Cdc5 on a distinct, independent and Kinase. One of them is a hydrophobic groove adjacent to the undefined site.88 Whether this site corresponds to one of the two phosphopeptide-binding site (Figure 3b).50 This binding groove non-canonical Map205 binding sites on the PBD of Polo is has also been identified in two other studies. In one of them, it unknown. Genetic results indicate that Dbf4 inhibits the function was found that a peptide binding the classical site on the PBD had of Cdc5, but the mechanism is unclear.88,90 Biochemical experi- a much higher affinity when it was alkylated, which allowed its ments have failed to detect direct inhibition of Cdc5 kinase additional binding in the hydrophobic groove.86 In the other activity by Dbf4. It has been suggested that Dbf4 could titrate study, the same site was found to promote crystal packing of the Cdc5 away from other interacting partners, but it was found that PBD.87 Which other physiological partners or targets of Polo Dbf4 does not compete for the same binding site as canonical Kinase bind via this hydrophobic groove should be explored. phosphotargets.88 More experiments are required to dissect the As a result of Map205 binding, the PBD is stabilized in a regulation of Cdc5 by Dbf4. conformation that binds and stabilizes the KD, thereby inhibiting Another interesting case of non-canonical PBD interaction is the the KD (Figure 3).50,65 This structure of the PBD is different from regulation of Drosophila Polo by a protein named matrimony those obtained for the PBD bound to phosphopeptides derived (Mtrm) during female meiosis.91 Mtrm appears to sequester and from canonical targets, which are known to lead to KD activation inhibit Polo until late prophase, when Mtrm is degraded and Polo rather than inhibition.8,45 In these cases, the Loop 2 segment is freed.92,93 Mtrm interacts with Polo via its PBD, but it does not joining the PB1 and PB2 in the PBD (Figure 3b), adopts a position require phosphorylation at a canonical PBD-binding motif for this which is incompatible with an interaction with the KD interaction.94 Instead, Mtrm requires phosphorylation at three (Figure 4d).50 Clearly, protein interactions on the PBD are capable phosphorylation sites that do not match this motif, as well as a of modulating the activity of the KD, both positively and functional SAM domain. The interaction does not require known negatively. It will be interesting to investigate if different PBD- phosphopeptide-binding residues of the PBD. Thus, the Polo– interacting proteins are used to fine-tune the activity of the KD, or Mtrm complex could inform on a potential alternative mode of

Oncogene (2015) 4799 – 4807 © 2015 Macmillan Publishers Limited Polo Kinase V Archambault et al 4803 interaction of the PBD, which could share features with the with both promising and disappointing results.42,43,98 Trials are still Map205–Polo and/or the Dbf4–Cdc5 complexes. underway, including some combining Plk1 inhibitors with other The fact that the PBD-binding proteins Map205, Mtrm and Dbf4 chemotherapeutics, and up to phase III. The most spectacular have all been shown, biochemically or genetically, to inhibit Plk results have been obtained for the treatment of acute myeloid function leads us to hypothesize that they all inhibit the Polo leukemia with the drug volasertib.43 Meanwhile, these molecules Kinase by stabilizing the form of the PBD that binds and inhibits have also provided tools to interfere with the Polo Kinase in efforts the KD. In addition, whether other upstream regulators of Polo to understand its function and the cellular pathways it controls. Kinase and/or some of its phosphorylation targets are bound by Cellular phenotypes obtained upon chemical inhibition of the KD the PBD at non-canonical binding sites remains to be seen. of Plk1 recapitulated those observed when Polo Kinase was Answers to these important questions should come from crystal- inactivated by other means including mutations, RNAi or antibody lography in the coming years. In addition to bringing a better injections.1,11,14,16,102–104 Mutations or RNAi tend to lead pre- understanding of Polo Kinase regulation and function, this dominantly to an arrest in prometaphase, the first stage of cell information could guide the development of novel chemical division where Plk1 activity is absolutely necessary. The ability to modulators of this protein. rapidly inhibit Plk1 with chemicals additionally allowed a The PBD of Polo Kinase is not known to be regulated by dissection of Plk1 functions in cytokinesis by adding the inhibitor phosphorylation. However, there are strong indications that its after the metaphase-to-anaphase transition.20,21,105 ability to interact with phospho-primed targets is regulated by Because kinases are highly similar in structure and sequence, ubiquitylation.95 The ubiquitin ligase CUL3–KLHL22 ubiquitylates especially in their active site, it has been extremely challenging to Plk1 at Lys492 in the PBD, very close to the canonical phospho- develop Plk1-specific inhibitors.106 Some of the most selective binding site (Figures 3b and 4f). This reaction is thought to result inhibitors obtained, including BI 2536 and BI 6727 (volasertib), also in mono-ubiquitylation of Plk1 and it occurs specifically at inhibit Plk2 and Plk3 with similar potency.102,107 As it happens, kinetochores of chromosomes that have reached correct bipolar Plk2 and Plk3 function to protect the cell against DNA damage, attachment at metaphase. As a consequence, ubiquitylated Plk1 which promotes cancer.4 Therefore, molecules capable of inhibit- detaches from its phosphorylated partners and is displaced from ing Plk1 but not Plk2 or Plk3 are desirable. In attempts to inhibit a kinetochores, promoting silencing of the spindle assembly Plk family member specifically, the PBD constitutes an interesting checkpoint. The identity of a putative deubiquitylation enzyme target. This domain is unique to the Plk family and it differs more that could reverse this modification is unknown. Whether than the KD between members.4 Moreover, Plks have been shown ubiquitylation of the PBD contributes to regulate Plk1 in other to differ in their peptide binding specificities.108 Interference with functions should also be explored. the PBD of Plk1 causes mitotic defects, cytokinetic failure and cell death.109–111 As expected, peptides derived from PBD targets are effective PBD competitive inhibitors in vitro.8,84 However, these THE INTER-DOMAIN LINKER IS POISED TO CONTRIBUTE TO molecules cross the plasma membrane very inefficiently. Strate- POLO KINASE REGULATION gies are being developed to facilitate the entry of peptide-like Connecting the KD and the PBD, there is the IDL (Figure 1a). This prodrugs into the cells, with promising results.112 In vitro screens part of the protein, thought to be loosely structured, is less using recombinant PBD have also been undertaken to identify conserved in amino-acid sequence between species than the two small molecules capable of blocking the PBD of Plk1. Poloxin and globular domains, but it is positioned to have an important role in its natural analog thymoquinone were first reported as such Polo Kinase function, especially given the bidirectional allosteric molecules.113,114 Crystallography showed that thymoquinone relationship between the two domains. The structure of the IDL competes with targets of the PBD at its phosphopeptide-binding has never been solved in the context of the full protein, with site.115 A separate in vitro screen identified purpurogallin as a PBD either domain or alone. Nevertheless, biochemical results suggest inhibitor, and its binding site could be distinct from that of that the IDL participates in KD inhibition by interfering with the thymoquinone and Poloxin.116,117 Existing PBD inhibitors induce T-loop. It has been suggested that Bora could displace the IDL to various mitotic defects and apoptosis, but their potency is rather facilitate T-loop phosphorylation by Aurora A.50 In addition, the low. Now that proofs of principle have been established, IDL appears to restrain KD activity directly as a KD–IDL truncation additional chemical screens or drug design should yield better is less active than the KD alone in vitro.50 The IDL also comprises a PBD inhibitors. functional destruction box motif, which allows recognition by the The Polo Kinase is an allosterically regulated protein, and APCCdh1, poly-ubiquitylation and degradation of Plk173 (Figure 4g). perturbations on one domain of the Polo Kinase can be Several phosphorylation sites have been identified in the IDL communicated to its other domain. Depending on their mode region of Plk1 and Drosophila Polo, but their functions have not of action, different inhibitors of one domain can have different yet been explored.96 They could contribute to modulate the effects on the activity of the other domain. PBD-binding activity of the KD and/or the PBD (Figure 4h). In fission yeast, Plo1 phosphopeptides induce a conformation of the PBD which can phosphorylation in the IDL at Ser402 promotes its recruitment to no longer interact with the KD. As a result, the KD is relieved from the spindle polo bodies and mitotic entry.97 Phosphorylation in its intramolecular inhibition.8,44,50 It is therefore possible that PBD the IDL could also regulate recognition of the destruction box by inhibition at the classical binding site increases the phosphoryla- the APCCdh1. tion of potential KD substrates that do not require recognition by the PBD. Conversely, there exists at least one protein, Map205, of which the binding to the PBD reinforces PBD-dependent CHEMICAL MODULATION INFORMS ON POLO KINASE inhibition of the KD.50 Thus, chemical inhibitors of the PBD may FUNCTION AND REGULATION be developed that will allosterically inhibit the KD in the same Human Plk1 is overexpressed in numerous types of cancer cells, way. Similarly, different KD-binding inhibitors could have different both solid tumors and leukemias.41,98 Moreover, cancer cells are effects on the activity of the PBD. Inhibitors that lock the KD in a often addicted to an elevated activity of Plk1.99–101 Consequently, conformation that cannot bind the PBD would activate the PBD. it is possible to kill these cancer cells whilst keeping healthy cells As a result the activated PBD could have a dominant-negative alive. These and other observations have established Plk1 as a effect on the functions of Plk1, which could contribute to the promising target for the development of cancer drugs. Potent inhibitor’s mechanism of inhibition. We have recently found that ATP-competitive inhibitors of the Plk1 KD have been developed. the position of the ATP-competitive KD inhibitor BI 2536 in the Some of these molecules have been submitted to clinical trials, Plk1 structure clashes with the position of the PBD when it is

© 2015 Macmillan Publishers Limited Oncogene (2015) 4799 – 4807 Polo Kinase V Archambault et al 4804 bound to the KD.65 As a result, BI 2536 induces the dissociation of Consistent with these molecular studies, the consequences of Map205 from the PBD of Polo Kinase. We hypothesize that BI 2536 chemical inhibition of the KD versus chemical inhibition of the treatment will promote PBD-dependent interactions of Plk1 with PBD also differ. Chemical inhibition of the KD of Plk1 in mitosis several targets. If it indeed occurs, this effect should be taken into typically results in monopolar spindles, scattered chromosomes consideration when using BI 2536 in cell biological experiments or and immature centrosomes.107 In contrast, several reports showed in chemotherapy. Unrelated KD inhibitors may not induce this that chemical inhibition of the PBD lead to multipolar spindles and effect. fragmented centrosomes, in addition to chromosome congression defects.114,116,119 The molecular basis for these differences could be complex. Experimental results suggested that the centrosome KD AND PBD INHIBITION CAN RESULT IN DIFFERENT fragmentation observed upon PBD inhibition could be attributed CONSEQUENCES to a defect in Plk1 regulation of kizuna, a protein shown to be Although both the KD and the PBD are implicated in the required for the cohesion of the pericentriolar material.114,120 This regulation of several Polo Kinase targets, they do not necessarily protein could be particularly sensitive to a diminution of Plk1 PBD contribute equally for all targets (Figure 5). Several substrates of function. Although kizuna is not conserved in yeast, multiple Polo Kinase are known to be recognized by the PBD before being spindle pole body foci are observed in phosphopeptide-binding- phosphorylated by the KD. Nevertheless, some phosphorylation deficient cdc5 mutants.118 In budding yeast as in animals, the substrates of Polo Kinase may never bind to the PBD or, even if maintenance of spindle pole integrity may critically rely on PBD they do bind, may not require this binding to be phosphorylated function. Phosphoproteomic studies on cells treated with KD or by Polo Kinase (Figure 5, bottom right). Consistent with this idea, PBD inhibitors could help profile the relative dependency of overexpression of the dominant-negative PBD alone, which phosphorylation substrates on the PBD. displaces Plk1 from its subcellular localization, or replacement of The hypomorphic phenotypes resulting from the inactivation of Plk1 with its KD alone, results in only some of the defects obtained PBD interactions could reflect a graded spectrum of PBD with complete Plk1 inactivation.110 Chromosome congression was dependency among the hundreds of Polo Kinase phosphorylation seen to be particularly sensitive to loss of PBD function. Moreover, substrates (Figure 5). Alternatively, it could be attributed to a expression of a phosphopeptide-binding-defective form of Plk1 in decrease in properly localized kinase activity generally felt by all cells where endogenous Plk1 was inactivated, could not rescue Plk1 substrates, with different substrates requiring different kinase normal chromosome congression and segregation, but could activity thresholds. Plk1 inhibition with decreasing doses of KD rescue bipolar spindle formation, implying that some function was inhibitor suggested a hierarchy of functions requiring different retained by the mutant protein.111 Similarly, mutation of critical levels of Plk1 kinase function with, from low to high: bipolar phosphopeptide-binding residues in yeast Cdc5 allows viability, spindle formation, timely mitotic entry and formation of a 111 although defects have been observed in microtubule dynamics, cytokinetic furrow. Consistent with this idea, a mutant form of mitotic progression, spindle pole body function, maintenance of Drosophila Polo which cannot be activated by T-loop phosphor- ploidy and genome integrity.88,118 One possible explanation for ylation but shows a basal activity, could rescue bipolar spindle 62 these results is that the PBD is not required for the regulation of formation but not normal chromosome congression. Similarly, some Cdc5 targets. However, in contrast to point mutations of the phosphorylation site mutants that prevent full activation of human Plk1 (Ser137A or Ser99A) promote bipolar spindle phospho-binding residues, complete truncation of the PBD is 68,69 lethal to yeast cells.118 It is therefore possible that some obligatory formation but fail to rescue later events of mitosis. targets of the Cdc5 PBD do not require priming phosphorylation. Finally, some PBD targets may not require phosphorylation by This explanation appears plausible given the increasing number of Polo Kinase as a part of their regulation (Figure 5, top left). This is non-canonical PBD-dependent interactions with Polo Kinase an intriguing possibility that has been little explored. Interestingly, Plk5, which is an essential protein in mice, lacks kinase activity but presented above. Finally, it is also possible that the lethality 38,39 induced by deletion of the PBD is caused by a gain of Cdc5 KD retains as a functional PBD. Plk5 or any other Plk could bind activity due to a loss of intramolecular inhibition by the PBD.118 target proteins and thereby promote a conformational change or protect them from other protein interactions or modifications, much like 14-3-3 family proteins.121 They could even act as physiological dominant-negative regulators, preventing binding of a different Plk.

CONCLUSION AND PERSPECTIVE The seemingly simple Polo Kinase with its two domains has revealed itself as a complex machine endowed with several PBD KD regulatory mechanisms, all encoded in a single polypeptide. The KD and PBD are capable of reciprocal allosteric inhibition and activation. Protein interactions on the PBD not only mediate substrate recognition and subcellular targeting of Polo Kinase; they can also dictate the activity of the KD. Various sites of phosphorylation and ubiquitylation also regulate the activities and the stability of the protein. Given the large number of cellular events regulated by the Polo Kinase during the cell division cycle, the existence of this complex regulatory network appears necessary. How the various post-translational modiﬁcations and protein interactions are coordinated in time and space, and how Figure 5. Graded dependency of Plk targets on the KD and PBD. While canonical Plk targets depend on both the KD and the PBD for individual regulatory pathways are combined to precisely adjust their regulation, some targets may depend less heavily on the PBD the activities of the two domains of the Polo Kinase for its (bottom right) or on the KD (top left). Each target could be situated functions in the cell cycle are questions that now need to be at a speciﬁc position in this space. See text for discussion. pursued.

Oncogene (2015) 4799 – 4807 © 2015 Macmillan Publishers Limited Polo Kinase V Archambault et al 4805 Basic research has brought a rich understanding of the 17 McKinley KL, Cheeseman IM. Polo-like kinase 1 licenses CENP-A deposition at functions and regulation of the Polo Kinase in its roles in mitosis centromeres. Cell 2014; 158:397–411. and cytokinesis. This knowledge has helped develop chemical 18 Neef R, Gruneberg U, Kopajtich R, Li X, Nigg EA, Sillje H et al. Choice of Plk1 inhibitors of Plk1 that modulate its activity by different mechan- docking partners during mitosis and cytokinesis is controlled by the activation 9 – isms. In turn, these molecules constitute tools with which to state of Cdk1. Nat Cell Biol 2007; : 436 444. further dissect the cellular functions of Polo Kinase. Given our new 19 Burkard ME, Randall CL, Larochelle S, Zhang C, Shokat KM, Fisher RP et al. Chemical genetics reveals the requirement for Polo-like kinase 1 activity in knowledge of the structure/function relationship and the regula- positioning RhoA and triggering cytokinesis in human cells. Proc Natl Acad Sci tion of the protein, it may now be possible to develop new drugs USA 2007; 104: 4383–4388. that will interfere more specifically with some aspects of Polo 20 Brennan IM, Peters U, Kapoor TM, Straight AF. Polo-like kinase controls verte- Kinase function, such as its activation by upstream kinases, its brate spindle elongation and cytokinesis. PLoS One 2007; 2: e409. ubiquitylation or its recognition of a subset of targets. Such 21 Petronczki M, Glotzer M, Kraut N, Peters JM. Polo-like kinase 1 triggers the compounds would allow a finer probing of the multiple cellular initiation of cytokinesis in human cells by promoting recruitment of the RhoGEF functions of Polo Kinase in various systems. They could also lead Ect2 to the central spindle. Dev Cell 2007; 12:713–725. to useful therapeutic agents if specific types of cancer cells show 22 Santamaria A, Neef R, Eberspacher U, Eis K, Husemann M, Mumberg D et al. particular sensitivities to interference with specific Polo Kinase Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis. Mol Biol Cell 2007; 18: 4024–4036. pathways. 23 Wu ZQ, Liu X. Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing. Proc Natl Acad Sci USA 2008; 105:1919–1924. 24 Seki A, Coppinger JA, Jang CY, Yates JR, Fang G. Bora and the kinase Aurora a CONFLICT OF INTEREST cooperatively activate the kinase Plk1 and control mitotic entry.Science 2008; The authors declare no conflict of interest. 320: 1655–1658. 25 Sumara I, Vorlaufer E, Stukenberg PT, Kelm O, Redemann N, Nigg EA et al. The dissociation of cohesin from chromosomes in prophase is regulated by ACKNOWLEDGEMENTS Polo-like kinase. Mol Cell 2002; 9: 515–525. 26 Abe S, Nagasaka K, Hirayama Y, Kozuka-Hata H, Oyama M, Aoyagi Y et al. Research on Polo Kinase in VA’s lab is supported by the Canadian Institutes of Health The initial phase of chromosome condensation requires Cdk1-mediated Research. GL holds a studentship from the Fonds de recherche du Québec –Santé phosphorylation of the CAP-D3 subunit of condensin II. Genes Dev 2011; 25: (FRQS). DK holds a postdoctoral fellowship from the Cole Foundation. IRIC is 863–874. supported in part by the Canada Foundation for Innovation and the FRQS. 27 Bettencourt-Dias M, Rodrigues-Martins A, Carpenter L, Riparbelli M, Lehmann L, Gatt MK et al. SAK/PLK4 is required for centriole duplication and flagella development. Curr Biol 2005; 15:2199–2207. REFERENCES 28 Habedanck R, Stierhof YD, Wilkinson CJ, Nigg EA. The Polo kinase Plk4 functions 1 Sunkel CE, Glover DM. Polo, a mitotic mutant of Drosophila displaying abnormal in centriole duplication. 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