Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.     

The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization

The small family of polo-like kinases (Plks) includes Cdc5 from Saccharomyces cerevisiae, Plo1 from Schizosaccharomyces pombe, Polo from Drosophila melanogaster and the four mammalian genes Plk1, Prk/Fnk, Snk and Sak. These kinases control cell cycle progression through the regulation of centrosome maturation and separation, mitotic entry, metaphase to anaphase transition, mitotic exit and cytokinesis. Plks are characterized by an N-terminal Ser/Thr protein kinase domain and the presence of one or two C-terminal regions of similarity, termed the polo box motifs. These motifs have been demonstrated for Cdc5 and Plk1 to be required for mitotic progression and for subcellular localization to mitotic structures. Here we report the 2.0 A crystal structure of a novel domain composed of the polo box motif of murine Sak. The structure consists of a dimeric fold with a deep interfacial cleft and pocket, suggestive of a ligand-binding site. We show that this domain forms homodimers both in vitro and in vivo, and localizes to centrosomes and the cleavage furrow during cytokinesis. The requirement of the polo domain for Plk family function and the unique physical properties of the domain identify it as an attractive target for inhibitor design.     

Structural analysis of the polo‐box domain of human Polo‐like kinase 2

Polo‐like kinases (Plks) are the key regulators of cell cycle progression, the members of which share a kinase domain and a polo‐box domain (PBD) that serves as a protein‐binding module. While Plk1 is a promising target for antitumor therapy, Plk2 is regarded as a tumor suppressor even though the two Plks commonly recognize the S‐pS/T‐P motif through their PBD. Herein, we report the crystal structure of the PBD of Plk2 at 2.7 Å. Despite the overall structural similarity with that of Plk1 reflecting their high sequence homology, the crystal structure also contains its own features including the highly ordered loop connecting two subdomains and the absence of 3₁₀‐helices in the N‐terminal region unlike the PBD of Plk1. Based on the three‐dimensional structure, we furthermore could model its interaction with two types of phosphopeptides, one of which was previously screened as the optimal peptide for the PBD of Plk2. Proteins 2015; 83:1201–1208. © 2015 Wiley Periodicals, Inc.     

The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex

Human polo-like kinase Plk1 localizes to the centrosomes, kinetochores and central spindle structures during mitosis. It plays an essential role in promoting mitosis and cytokinesis through phosphorylation of a number of different substrates. Kinase activity is regulated by a conserved C-terminal domain, termed the polo box domain (PBD), which acts both as an autoinhibitory domain and as a subcellular localization domain. We have determined the crystal structure of Plk1 PBD (residues 367-603) to 2.2 A resolution and the structure of a phospho-peptide-PBD (residues 345-603) complex to 2.3 A resolution. The two polo boxes of the PBD exhibit identical folds based on a six-stranded beta-sheet and an alpha-helix, despite only 12% sequence identity. The phospho-peptide binds at a site between the two polo boxes. It makes a short antiparallel beta-sheet connection and critical contacts to residues Trp414, Leu490, His538 and Lys540. Most of these residues had been shown to be important for biological activity through mutational studies. The results provide an explanation for phospho-peptide recognition and create the basis for new functional studies.     

The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation

Mitosis is coordinated by carefully controlled phosphorylation and ubiquitin-mediated proteolysis. Polo-like kinase 1 (Plk1) plays a central role in regulating mitosis and cytokinesis by phosphorylating target proteins. Yet, Plk1 is itself a target for posttranslational modification by phosphorylation and ubiquitination. We developed a chemical-genetic complementation assay to evaluate the functional significance of 34 posttranslational modifications (PTMs) on human Plk1. To do this, we used human cells that solely express a modified analog-sensitive Plk1 (Plk1^AS) and complemented with wildtype Plk1. The wildtype Plk1 provides cells with a functional Plk1 allele in the presence of 3-MB-PP1, a bulky ATP-analog inhibitor that specifically inhibits Plk1^AS. Using this approach, we evaluated the ability of 34 singly non-modifiable Plk1 mutants to complement Plk1^AS in the presence of 3-MB-PP1. Mutation of the T-loop activating residue T210 and adjacent T214 are lethal, but surprisingly individual mutation of the remaining 32 posttranslational modification sites did not disrupt the essential functions of Plk1. To evaluate redundancy, we simultaneously mutated all phosphorylation sites in the kinase domain except for T210 and T214 or all sites in the C-terminal polo-box domain (PBD). We discovered that redundant phosphorylation events within the kinase domain are required for accurate chromosome segregation in anaphase but those in the PBD are dispensable. We conclude that PTMs within the T-loop of Plk1 are essential and nonredundant, additional modifications in the kinase domain provide redundant control of Plk1 function, and those in the PBD are dispensable for essential mitotic functions of Plk1. This comprehensive evaluation of Plk1 modifications demonstrates that although phosphorylation and ubiquitination are important for mitotic progression, many individual PTMs detected in human tissue may have redundant, subtle, or dispensable roles in gene function.     

Plk1 docking to GRASP65 phosphorylated by Cdk1 suggests a mechanism for Golgi checkpoint signalling

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. We show that Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and that phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense.     

Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.     

Interaction of chromatin-associated Plk1 and Mcm7

Plk1 is a multifunctional protein kinase involved in regulation of mitotic entry, chromosome segregation, centrosome maturation, and mitotic exit. Plk1 is a target of DNA damage checkpoints and aids resumption of the cell cycle during recovery from G2 arrest. The polo-box domain (PBD) of Plk1 interacts with phosphoproteins and localizes Plk1 to some mitotic structures. In a search for proteins that interact with the PBD of Plk1, we identified two of the minichromosome maintenance (MCM) proteins, Mcm2 and Mcm7. Co-immunoprecipitation and immunoblot analysis showed an interaction between full-length Plk1 and all other members of the MCM2-7 protein complex. Endogenous Plk1 co-immunoprecipitates with basal forms of Mcm7 as well as with slower migrating forms of Mcm7, induced in response to DNA damage. The strongest interaction between endogenous Plk1 and Mcm7 was detected in a soluble chromatin fraction. These findings suggest a new function for Plk1 in coordination of DNA replication and mitotic events.     

Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1

Polo-like kinase 1 (Plk1) is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. Here, we investigate the mechanism by which Plk1 itself is recruited to kinetochores. We show that Plk1 binds to budding uninhibited by benzimidazole 1 (Bub1) in mitotic human cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by cyclin-dependent kinase 1 (Cdk1)-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNA interference (RNAi) diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.     

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.     

The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain

Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain. The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface. Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.     

Dynactin helps target Polo‐like kinase 1 to kinetochores via its left‐handed beta‐helical p27 subunit

Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)‐based motor. Dynactin binds both dynein and MTs via its p150^Glued subunit, but little is known about the ‘pointed‐end complex’ that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin‐dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo‐like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore–MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left‐handed β‐helix domain, with the phosphorylation site located within a disordered, C‐terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.     

Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays

The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be yen-[Ile/Leu/Val]-Ser/Thr-phi-phi-X- yen/Pro (where phi denotes a large hydrophobic residue, yen is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.     

Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.     

Plk1 activation by Ste20-like kinase (Slk) phosphorylation and polo-box phosphopeptide binding assayed with the substrate translationally controlled tumor protein (TCTP)

The mitotic protein kinase Plk1 catalyzes events associated with centrosome maturation, kinetocore function, spindle formation, and cytokinesis and is a target for anticancer drug design. It is composed of a N-terminal kinase domain and a C-terminal polo-box domain (PBD). The PBD domain serves to localize the kinase on cognate phosphorylated substrates, and this binding relieves the inhibition of the kinase by the PBD. Similar to many protein kinases, Plk1 is activated by phosphorylation on a threonine residue, Thr210, in the activation segment. In this work, we describe expression in Escherichia coli cells and purification of full-length Plk1 in quantities suitable for structural studies and use this material for quantitative characterization of the activation events with the substrate translationally controlled tumour protein (TCTP). The presence of the PBD-binding phosphopeptide enhances phosphorylation by the activating Ste20-like kinase (Slk). Native Plk1 exhibits a basal catalytic efficiency k cat/ K(M) of 9.9 x 10 (-5) s (-1) microM (-1). Association with a polo-box-binding phosphopeptide increased the catalytic efficiency by 11x largely through an increase in k(cat) with no change in K(M). Phosphorylation by Slk increases catalytic efficiency by 202x with a 2.3-fold reduction in K(M) and 88-fold increase in k(cat). Phosphorylation and the presence of the PBD-binding phosphopeptide result in an increase in catalytic efficiency of 1515x with a 2.3-fold decrease in K(M) and a 705-fold increase in k(cat) over the unmodified Plk1. Knowledge of kinase regulatory mechanisms and the structures of the Plk1 individual domains has allowed for a model to be proposed for these activatory events.     

Different Plk1 functions show distinct dependencies on Polo-Box domain-mediated targeting

Polo-like kinase 1 (Plk1) has multiple important functions during M-phase progression. In addition to a catalytic domain, Plk1 possesses a phosphopeptide-binding motif, the polo-box domain (PBD), which is required for proper localization. Here, we have explored the importance of correct Plk1 subcellular targeting for its mitotic functions. We either displaced endogenous Plk1 through overexpression of the PBD or introduced the catalytic domain of Plk1, lacking the PBD, into Plk1-depleted cells. Both treatments resulted in remarkably similar phenotypes, which were distinct from the Plk1 depletion phenotype. Cells depleted of Plk1 mostly arrested with monoastral spindles, because of inhibition of centrosome maturation and separation. In contrast, these functions were not impaired in cells with mislocalized Plk1. Instead, these latter cells showed a checkpoint-dependent mitotic arrest characterized by impaired chromosome congression. Thus, whereas chromosome congression requires localized Plk1 activity, other investigated Plk1 functions are less dependent on correct PBD-mediated targeting. This opens the possibility that PBD-directed drugs might be developed to selectively interfere with a subset of Plk1 functions.     

Polo-box domains confer target specificity to the Polo-like kinase family

Polo-like kinases (Plks) contain a conserved Polo-box domain, shown to bind to phosphorylated Ser-pSer/pThr-Pro motifs. The Polo-box domain of Plk-1 mediates substrate interaction and plays an important role in subcellular localization. Intriguingly, the major interactions between the PBD and the optimal recognition peptide are mediated by highly conserved residues in the PBD, suggesting there is little target specificity conveyed by the various PBDs. However, here we show that the affinity of the purified Plk1-3 PBDs to both a physiological Cdc25C derived phospho-peptide and an optimal recognition phospho-peptide differs significantly among family members. To decipher the role of the PBDs and kinase domains in inferring Plk specificity, we exchanged the PBD of Plk1 (PBD1) with the PBD of Plk2, 3, or 4 (PBD2-4). The resulting hybrid proteins can restore bipolar spindle formation and centrosome maturation in Plk1-depleted U2OS cells to various degrees. In these experiments PBD2 was most efficient in complementing PBD-function. Using the MPM2 antibody that recognizes a large set of mitotic phospho-proteins, we could show that PBD1 and PBD2 display some limited overlap in target recognition. Thus, PBDs convey a significant deal of target specificity, indicating that there is only a limited amount of functional redundancy possible within the Plk family.     

Interaction of Sororin protein with polo-like kinase 1 mediates resolution of chromosomal arm cohesion

Unlike in budding yeast, sister chromatid cohesion in vertebrate cells is resolved in two steps: cohesin complexes are removed from sister chromatid arms during prophase via phosphorylation, whereas centromeric cohesins are removed at anaphase by Separase. Phosphorylation of cohesin subunit SA2 by polo-like kinase 1 (Plk1) is required for the removal of cohesins at prophase, but how Plk1 is recruited to phosphorylate SA2 during prophase is currently not known. Here we report that Sororin, a cohesin-interacting protein essential for sister chromatid cohesion, plays a novel role in the resolution of sister chromatid arms by direct interaction with Plk1. We identified an evolutionarily conserved motif (ST(159)P) on Sororin, which was phosphorylated by Cdk1/cyclin B and bound to the polo box domain of Plk1. Mutating Thr(159) into alanine prevented the interaction of Plk1 and Sororin and inhibited the resolution of chromosomal arm cohesion. We propose that Sororin is phosphorylated by Cdk1/cyclin B at prophase and acts as a docking protein to bring Plk1 into proximity with SA2, resulting in the phosphorylation of SA2 and the removal of cohesin complexes from chromosomal arms.     

Design,synthesis and evaluation of d-amino acid-containing peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of d-amino acid-containing peptidomimetics were designed, synthesized as novel polo-like kinase 1 (Plk1) polo-box domain (PBD) inhibitors based on the reported peptide Plk1 PBD inhibitor. Their inhibitory activity to Plk1, Plk2, and Plk3 PBD were evaluated using our fluorescence polarization (FP) assay. Compound 18 bound to Plk1 PBD with IC₅₀ of 0.80 μM and showed nearly no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Compound 18 induced Hela cells to undergo apoptosis by increasing the ratio of the cells at the G2/M phase by decreasing the neosynthesized proteins in a dose-dependent manner from 50 to 150 μM. Compound 18 showed improved stability in rat plasma compared to l-peptide inhibitor LHSpTA. These novel d-amino acid modified selective Plk1 PBD inhibitors may provide new lead compounds for further optimization.     

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.