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.     

Purification and proteomic identification of putative upstream regulators of polo-like kinase-1 from mitotic cell extracts

Polo-like kinase-1 (Plk1) is phosphorylated on Thr210 for activation during mitosis. Here, we investigated the question of which kinase(s) is the specific upstream kinase of mitotic Plk1. Upstream kinases of Plk1 were purified from mitotic cell extracts through column chromatography procedures, and identified by mass spectrometry. Candidates for Plk1 kinase included p21-activated kinase, aurora A, and mammalian Ste20-like kinases. Immunoprecipitates of these proteins from mitotic cell extracts phosphorylated Plk1 on Thr210. Even if the activity of Aurora A was blocked with a specific inhibitor, Plk1 phosphorylation still occurred, suggesting that function of Plk1 could be controlled by these kinases for proper mitotic progression, as well as by Aurora A in very late G2 phase for the beginning of mitosis.

Structured abstract

MINT-7996332: PAK1 (uniprotkb:Q13153) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)MINT-7996345: PAK3 (uniprotkb:O75914) physically interacts (MI:0915) with PLK1 (uniprotkb:P53350) by pull down (MI:0096)     

Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210 phosphorylation precedes Ser-137 phosphorylation in vivo, the latter occurring specifically in late mitosis. We show that expression of two activating mutants of these residues, S137D and T210D, results in distinct mitotic phenotypes. Whereas expression of both phospho-mimicking mutants as well as of the double mutant leads to accelerated mitotic entry, further progression through mitosis is dramatically different: the T210D mutant causes a spindle assembly checkpoint-dependent delay, whereas the expression of the S137D mutant or the double mutant results in untimely activation of the anaphase-promoting complex/cyclosome (APC/C) and frequent mitotic catastrophe. Using nonphosphorylatable Plk1-S137A and Plk1-T210A mutants, we show that both sites contribute to proper mitotic progression. Based on these observations, we propose that Plk1 function is altered at different stages of mitosis through consecutive posttranslational events, e.g., at Ser-137 and Thr-210. Furthermore, our data show that uncontrolled Plk1 activation can uncouple APC/C activity from spindle assembly checkpoint control.     

Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase

The mammalian polo-like kinase (Plk) plays a critical role in M-phase progression. Plk is phosphorylated and activated by an upstream kinase(s), which has not yet been identified in mammalian cells. Phosphopeptide mapping and phosphoamino acid analyses of Plk labeled in vivo and phosphorylated in vitro by Xenopus polo-like kinase kinase-1 (xPlkk1) or by lymphocyte-oriented kinase, its most closely related mammalian enzyme, indicate that Thr-210 is a major phosphorylation site in activated Plk from mitotic HeLa cells. Although the amino acid sequence surrounding Ser-137 is similar to that at Thr-210 and is conserved in Plk family members, Ser-137 is not detectably phosphorylated in mitotic mammalian cells or by xPlkk1 in vitro. Nevertheless, the substitution of either Thr-210 or Ser-137 with Asp (T210D or S137D) elevates the kinase activity of Plk. The kinase activity of the double mutant S137D/T210D is not significantly different from that of T210D or S137D, demonstrating that substitution of both residues does not have an additive effect on Plk activity. Expression of the S137D mutant construct arrested HeLa cells in early S-phase with slightly separated centrosomes, whereas cells expressing wild type and T210D were arrested or delayed in M-phase. These data indicate that the Ser-137 may have an unexpected and novel role in the function of Plk.     

Genetic depletion of Polo‐like kinase 1 leads to embryonic lethality due to mitotic aberrancies

Polo‐like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non‐mitotic arrest in early embryos, we show here that the bi‐allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi‐ and mono‐polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid‐gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1‐heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small‐molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

Mitotic entry: The interplay between Cdk1, Plk1 and Bora

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Förster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.     

PARP1 and CHK1 coordinate PLK1 enzymatic activity during the DNA damage response to promote homologous recombination-mediated repair

Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1–PLK1–RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.     

The centrosomal kinase Plk1 localizes to the transition zone of primary cilia and induces phosphorylation of nephrocystin-1

Polo-like kinase (Plk1) plays a central role in regulating the cell cycle. Plk1-mediated phosphorylation is essential for centrosome maturation, and for numerous mitotic events. Although Plk1 localizes to multiple subcellular sites, a major site of action is the centrosomes, which supports mitotic functions in control of bipolar spindle formation. In G0 or G1 untransformed cells, the centriolar core of the centrosome differentiates into the basal body of the primary cilium. Primary cilia are antenna-like sensory organelles dynamically regulated during the cell cycle. Whether Plk1 has a role in ciliary biology has never been studied. Nephrocystin-1 (NPHP1) is a ciliary protein; loss of NPHP1 in humans causes nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. We here demonstrate that Plk1 colocalizes with nephrocystin-1 to the transition zone of primary cilia in epithelial cells. Plk1 co-immunoprecipitates with NPHP1, suggesting it is part of the nephrocystin protein complex. We identified a candidate Plk1 phosphorylation motif (D/E-X-S/T-φ-X-D/E) in nephrocystin-1, and demonstrated in vitro that Plk1 phosphorylates the nephrocystin N-terminus, which includes the specific PLK1 phosphorylation motif. Further, induced disassembly of primary cilia rapidly evoked Plk1 kinase activity, while small molecule inhibition of Plk1 activity or RNAi-mediated downregulation of Plk1 limited the first and second phase of ciliary disassembly. These data identify Plk1 as a novel transition zone signaling protein, suggest a function of Plk1 in cilia dynamics, and link Plk1 to the pathogenesis of NPH and potentially other cystic kidney diseases.     

Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.     

Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

Background

Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that has been implicated in the regulation of mitosis. In addition, the activation of mitogen-activated protein kinase (MAPK) is a key event in the early stage of the growth factor response. The role of Plk1 in MAPK phosphorylation in cells has not been investigated.

Methods

Immunoblot analysis was used to evaluate Plk1 and MAPK phosphorylation in cells upon stimulation with platelet-derived growth factor (PDGF). We also generated stable Plk1 knockdown (KD) cells to assess the role of Plk1 in MAPK activation and cell proliferation. Furthermore, we used a non-phosphorylatable Plk1 mutant to determine the function of Plk1 phosphorylation in these processes.

Results

Treatment with PDGF increased Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in human airway smooth muscle cells. Plk1 KD attenuated the PDGF-induced phosphorylation of MEK1/2 and ERK1/2 as well as cell proliferation. However, phosphorylation of Raf-1 and AKT upon stimulation with PDGF was not reduced in Plk1 KD cells. Furthermore, the expression of T210A Plk1 (alanine substitution at Thr-210) inhibited the PDGF-stimulated MEK1/2 phosphorylation, ERK1/2 phosphorylation and cell proliferation.

Conclusions

Together, these findings suggest that Plk1 is activated upon growth factor stimulation, which may control the activation of MEK1/2 and ERK1/2, and smooth muscle cell proliferation.     

The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.During mitosis, multiple processes, such as mitotic entry, spindle assembly, chromosome segregation, and cytokinesis, must be carefully coordinated to ensure the error-free distribution of chromosomes into the newly forming daughter cells. The physical separation of the chromosomes to opposite poles of the cell is driven by the mitotic spindle, a proteinaceous and highly dynamic microtubule (MT)1-based macromolecular machine. Spindle assembly begins early in mitosis and is completed when the bipolar attachment of microtubules to kinetochore (KT) pairs is achieved (1, 2). Polo-like kinase 1 (Plk1), a serine/threonine-specific kinase first identified in Drosophila (3), is one of the key regulators of this essential mitotic process and has therefore attracted much attention (4–6). In agreement with its diverse functions, the localization of Plk1 during mitosis is dynamic. Plk1 first associates with centrosomes in prophase before it localizes to spindle poles and KTs in prometaphase and metaphase. During anaphase, Plk1 is recruited to the central spindle and finally accumulates at the midbody during telophase. Proteomics studies using oriented peptide libraries have shown that two so-called polo boxes at the C-terminal end of Plk1, the polo box domain (PBD), are crucial for the localization of this kinase to cellular structures (7, 8). This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self-primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space (9–11).Despite the pleiotropic and critical functions of Plk1 during mitosis, only a limited number of target proteins and phosphorylation sites on substrates have so far been identified or studied in detail (4–6, 12). The difficulties in identification of bona fide Plk1 substrates stem from the low abundance of some substrates, technical limitations for determining in vivo phosphorylation sites, the requirement for Plk1 localization for recognition of some substrates, and the possibility that Plk1 may phosphorylate a broader consensus motif than determined previously (13). Recent developments in mass spectrometry (MS)-based proteomics have allowed the identification of a large number of in vivo phosphorylation sites from complex samples (14). However, the nature of the kinase(s) responsible for most of these phosphorylation events is still unclear, and the assignment of phosphorylation sites to individual kinases remains a challenging task. Previously, we explored the human mitotic spindle by MS and successfully identified a large number of novel spindle proteins and phosphorylation sites (15, 16). Now, the development of quantitative methods to monitor in vivo phosphorylation changes in complex samples (17–19) represents a unique opportunity to address the role of individual kinases in spindle function.To study Plk1 function at the mitotic spindle, we combined quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) (20) with the isolation of human mitotic spindles and phosphopeptide enrichment. To expand the experimental coverage of Plk1 substrates and gain further insight into direct and indirect functions of Plk1, we compared the phosphoproteomes of mitotic spindles isolated from cells lacking Plk1 activity with spindles from cells with fully active kinase. Two independent approaches were used to interfere with Plk1 activity: protein depletion using an inducible small hairpin (shRNA) cell line and selective inhibition of the kinase by the small molecule inhibitor ZK-thiazolidinone (TAL) (21). Phosphorylation sites found to be down-regulated after Plk1 inhibition/depletion were subsequently validated using in vitro phosphorylation of synthetic peptide arrays. This approach identified many candidate Plk1 substrates, allowed confirmation of direct phosphorylation by Plk1 of more than 100 sites identified in vivo, and suggested a broader phosphorylation consensus motif for this kinase. Collectively, our data set provides a rich resource for in-depth studies on the spindle-associated Plk1-dependent phosphoproteome. This is illustrated by selective follow-up studies in which we validated the Plk1-dependent localization of substrates to centrosomes and kinetochores. In particular, using a phosphospecific antibody, we confirmed Plk1-dependent CENP-F phosphorylation in vivo and demonstrated that CENP-F localization to kinetochores depends on Plk1 kinase activity. Furthermore, we identified several Aurora A-dependent phosphorylation events that are regulated by Plk1, supporting the emerging view of an intimate functional relationship between Plk1 and Aurora A kinase (22, 23).     

Regulation of the final stage of mitosis by components of the pre-replicative complex and a polo kinase

The accurate division of duplicated DNA is essential for maintenance of genomic stability in proliferating eukaryotic cells. Errors in DNA replication and chromosomal segregation may lead to cell death or genomic mutations that lead to oncogenic properties. Thus, tight regulation of DNA replication and mitosis is essential for maintaining genomic integrity. Cell division cycle 6 (Cdc6) is an essential factor for initiating DNA replication. Recent work shows that phosphorylation of Cdc6 by pololike kinase 1 (Plk1), one of the essential mitotic kinases, regulates mitotic exit mediated by Cdk1 and separase. Here we discuss how pre-replicative complex factors are connected with Plk1 and affect mitotic exit.Key words: Plk1, Cdc6, DNA replication, mitotic exit, chromosomal segregation     

Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1

Background

Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.

Methodology/Principal Findings

Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.

Conclusions/Significance

hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.     

B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1

Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF^−/−, or p53^−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value.