Cytokinesis and cancer： Polo loves ROCK＇n＇ Rho（A）

Cytokinesis is the last step of the M (mitosis) phase, yet it is crucial for the faithful division of one cell into two. Cytokinesis failure is often associated with cancer. Cytokinesis can be morphologically divided into four steps: cleavage furrow initiation, cleavage furrow ingression, midbody formation and abscission. Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis. At the same time, Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis, including cytokinesis. Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks. More specifically, Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1, thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains. Ect2 itself can be phosphorylated by Plk1 in vitro. Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity. Once activated, RhoA-GTP will activate downstream effectors, including ROCK1 and ROCK2. ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen, and Plk1 can phosphorylate ROCK2 in vitro. We review current understandings of the interplay between Plk1, RhoA proteins and other proteins (e.g., NudC, MKLP2, PRC1, CEP55) involved in cytokinesis, with particular emphasis of its clinical implications in cancer.     

Cytokinesis and cancer:Polo loves ROCK'n' Rho(A)

Cytokinesis is the last step of the M (mitosis) phase,yet it is crucial for the faithful division of one cell into two.Cytokinesis failure is often associated with cancer.Cytokinesis can be morphologically divided into four steps:cleavage furrow initiation,cleavage furrow ingression,midbody formation and abscission.Molecular studies have revealed that RhoA as well as its regulators and effectors are important players to ensure a successful cytokinesis.At the same time,Polo-like kinase 1 (Plk1) is an important kinase that can target many substrates and carry out different functions during mitosis,including cytokinesis.Recent studies are beginning to unveil a closer tie between Plk1 and RhoA networks.More specifically,Plk1 phosphorylates the centralspindlin complex Cyk4 and MKLP1/CHO1,thus recruiting RhoA guanine nucleotide-exchange factor (GEF) Ect2 through its phosphopeptide-binding BRCT domains.Ect2 itself can be phosphorylated by Plk1 in vitro.Plk1 can also phosphorylate another GEF MyoGEF to regulate RhoA activity.Once activated,RhoA-GTP will activate downstream effectors,including ROCK1 and ROCK2.ROCK2 is among the proteins that associate with Plk1 Polo-binding domain (PBD) in a large proteomic screen,and Plk1 can phosphorylate ROCK2 in vitro.We review current understandings of the interplay between Plk1,RhoA proteins and other proteins (e.g.,NudC,MKLP2,PRC1,CEP55) involved in cytokinesis,with partitular emphasis of its clinical implications in cancer.     

Dynamic and Multi-Pharmacophore Modeling for Designing Polo-Box Domain Inhibitors

The polo-like kinase 1 (Plk1) is a critical regulator of cell division that is overexpressed in many types of tumors. Thus, a strategy in the treatment of cancer has been to target the kinase activity (ATPase domain) or substrate-binding domain (Polo-box Domain, PBD) of Plk1. However, only few synthetic small molecules have been identified that target the Plk1-PBD. Here, we have applied an integrative approach that combines pharmacophore modeling, molecular docking, virtual screening, and in vitro testing to discover novel Plk1-PBD inhibitors. Nine Plk1-PBD crystal structures were used to generate structure-based hypotheses. A common pharmacophore model (Hypo1) composed of five chemical features was selected from the 9 structure-based hypotheses and used for virtual screening of a drug-like database consisting of 159,757 compounds to identify novel Plk1-PBD inhibitors. The virtual screening technique revealed 9,327 compounds with a maximum fit value of 3 or greater, which were selected and subjected to molecular docking analyses. This approach yielded 93 compounds that made good interactions with critical residues within the Plk1-PBD active site. The testing of these 93 compounds in vitro for their ability to inhibit the Plk1-PBD, showed that many of these compounds had Plk1-PBD inhibitory activity and that compound Chemistry_28272 was the most potent Plk1-PBD inhibitor. Thus Chemistry_28272 and the other top compounds are novel Plk1-PBD inhibitors and could be used for the development of cancer therapeutics.     

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.     

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.     

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.     

A high-throughput assay based on fluorescence polarization for inhibitors of the polo-box domain of polo-like kinase 1

The serine/threonine kinase polo-like kinase 1 (Plk1) is critically involved in multiple mitotic processes and has been established as an adverse prognostic marker for tumor patients. Plk1 localizes to its substrates and its intracellular anchoring sites via its polo-box domain (PBD), which is unique to the family of polo-like kinases. Therefore, inhibition of the Plk1 PBD has been suggested as an approach to the inhibition of Plk1 that circumvents specificity problems associated with the inhibition of the conserved adenosine triphosphate (ATP) binding pocket. Here we report on the development of a high-throughput assay based on fluorescence polarization that allows the discovery of small-molecule inhibitors of the Plk1 PBD. The assay is based on binding of the Plk1 PBD to a phosphothreonine-containing peptide comprising its optimal binding motif with a K_d of 26 ± 2 nM. It is stable with regard to dimethyl sulfoxide (DMSO) and time, and it has a Z′ value of 0.73 ± 0.06 in a 384-well format.     

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.     

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.     

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.     

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.     

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.     

Design and Synthesis of a Cell-Permeable,Drug-Like Small Molecule Inhibitor Targeting the Polo-Box Domain of Polo-Like Kinase 1

Background

Polo-like kinase-1 (Plk1) plays a crucial role in cell proliferation and the inhibition of Plk1 has been considered as a potential target for specific inhibitory drugs in anti-cancer therapy. Several research groups have identified peptide-based inhibitors that target the polo-box domain (PBD) of Plk1 and bind to the protein with high affinity in in vitro assays. However, inadequate proteolytic resistance and cell permeability of the peptides hinder the development of these peptide-based inhibitors into novel therapeutic compounds.

Methodology/Principal Findings

In order to overcome the shortcomings of peptide-based inhibitors, we designed and synthesized small molecule inhibitors. Among these molecules, bg-34 exhibited a high binding affinity for Plk1-PBD and it could cross the cell membrane in its unmodified form. Furthermore, bg-34-dependent inhibition of Plk1-PBD was sufficient for inducing apoptosis in HeLa cells. Moreover, modeling studies performed on Plk1-PBD in complex with bg-34 revealed that bg-34 can interact effectively with Plk1-PBD.

Conclusion/Significance

We demonstrated that the molecule bg-34 is a potential drug candidate that exhibits anti-Plk1-PBD activity and possesses the favorable characteristics of high cell permeability and stability. We also determined that bg-34 induced apoptotic cell death by inhibiting Plk1-PBD in HeLa cells at the same concentration as PEGylated 4j peptide, which can inhibit Plk1-PBD activity 1000 times more effectively than bg-34 can in in vitro assays. This study may help to design and develop drug-like small molecule as Plk1-PBD inhibitor for better therapeutic activity.     

Mechanisms of mammalian polo-like kinase 1 (Plk1) localization: Self-versus non-self-priming

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key element in regulating diverse mitotic events during M-phase progression. Plk1 is spatially regulated through the targeting activity of the conserved polo-box domain (PBD) present in the C-terminal non-catalytic region. Over the years, studies have demonstrated that the PBD forms a phospho-epitope binding module and the PBD-dependent interaction is critical for proper subcellular localization of Plk1. The current prevailing model is that the PBD binds to a phospho-epitope generated by Cdc2 or other Pro-directed kinases. Here we discuss a recent finding that Plk1 also self-promotes its localization by generating its own PBD-docking site.     

A role for Plk1 phosphorylation of NudC in cytokinesis

Polo-like kinase 1 (Plk1) plays essential roles at multiple events during cell division, yet little is known about its physiological substrates. In a cDNA phage display screen using Plk1 C-terminal affinity columns, we identified NudC (nuclear distribution gene C) as a Plk1 binding protein. Here, we characterize the interaction between Plk1 and NudC, show that Plk1 phosphorylates NudC at conserved S274 and S326 residues in vitro, and present evidence that NudC is also a substrate for Plk1 in vivo. Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, which are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites. These results suggest that Plk1 phosphorylation of NudC may influence cytokinesis.     

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.     

Several inhibitors of the Plk1 Polo-Box Domain turn out to be non-specific protein alkylators

For almost a decade, there has been much interest in the development of chemical inhibitors of Polo-like kinase 1 (Plk1) protein interactions. Plk1 is a master regulator of the cell division cycle that controls numerous substrates. It is a promising target for cancer drug development. Inhibitors of the kinase domain of Plk1 had some success in clinical trials. However, they are not perfectly selective. In principle, Plk1 can also be inhibited by interfering with its protein interaction domain, the Polo-Box Domain (PBD). Selective chemical inhibitors of the PBD would constitute tools to probe for PBD-dependent functions of Plk1 and could be advantageous in cancer therapy. The discovery of Poloxin and thymoquinone as PBD inhibitors indicated that small, cell-permeable chemical inhibitors could be identified. Other efforts followed, including ours, reporting additional molecules capable of blocking the PBD. It is now clear that, unfortunately, most of these compounds are non-specific protein alkylators (defined here as groups covalently added via a carbon) that have little or no potential for the development of real Plk1 PBD-specific drugs. This situation should be minded by biologists potentially interested in using these compounds to study Plk1. Further efforts are needed to develop selective, cell-permeable PBD inhibitors.     

Centralspindlin assembly and 2 phosphorylations on MgcRacGAP by Polo-like kinase 1 initiate Ect2 binding in early cytokinesis

Cytokinesis is the final step of cell division which partitions genetic and cytosolic content into daughter cells. Failed cytokinesis causes polyploidy, genetic instability, and cancer. Kinases use phosphorylation to regulate the timing and location of the cytokinetic furrow. Polo-like kinase 1 (Plk1) is an essential mitotic kinase that triggers cytokinesis by phosphorylating MgcRacGAP to create a docking site for Ect2 at the central spindle. Ect2 binds to MgcRacGAP via its N-terminal BRCT domain (BRCA1 C-terminal), which docks at specific phosphorylated residues. Here we investigate the minimal Plk1-dependent phosphorylation sites required for cytokinesis onset. We demonstrate that phosphorylation of the major MgcRacGAP site, S157, is necessary but not sufficient to bind the Ect2 BRCT domain. Phosphorylation of an additional residue on MgcRacGAP at S164 is also required to elicit efficient binding. Surprisingly, BRCT binding additionally requires MKLP1 and its cognate interacting N-terminal domain of MgcRacGAP. Our findings indicate that central spindle assembly and 2 Plk1-dependent phosphorylations are required to establish efficient binding of the Ect2 BRCT in early cytokinesis. We propose that these requirements establish a high threshold to restrain premature or ectopic cytokinesis.     

Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation

The polo-box domain (PBD) of mammalian polo-like kinase 1 (Plk1) is essential in targeting its catalytic activity to specific subcellular structures critical for mitosis. The mechanism underlying Plk1 recruitment to the kinetochores and the role of Plk1 at this site remain elusive. Here, we demonstrate that a PBD-binding protein, PBIP1, is crucial for recruiting Plk1 to the interphase and mitotic kinetochores. Unprecedentedly, Plk1 phosphorylated PBIP1 at T78, creating a self-tethering site that specifically interacted with the PBD of Plk1, but not Plk2 or Plk3. Later in mitosis, Plk1 also induced PBIP1 degradation in a T78-dependent manner, thereby enabling itself to interact with other components critical for proper kinetochore functions. Absence of the p-T78-dependent Plk1 localization induced a chromosome congression defect and compromised the spindle checkpoint, ultimately leading to aneuploidy. Thus, Plk1 self-regulates the Plk1-PBIP1 interaction to timely localize to the kinetochores and promote proper chromosome segregation.