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.     

Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate

Plk1 (Polo-like kinase 1), an evolutionarily conserved serine/threonine kinase, is crucially involved in multiple events during the M phase. Here we have identified a consensus phosphorylation sequence for Plk1, by testing the ability of systematically mutated peptides derived from human Cdc25C to serve as a substrate for Plk1. The obtained results show that a hydrophobic amino acid at position +1 carboxyl-terminal of phosphorylated Ser/Thr and an acidic amino acid at position -2 are important for optimal phosphorylation by Plk1. We have then found that Myt1, an inhibitory kinase for MPF, has a number of putative phosphorylation sites for Plk1 in its COOH-terminal portion. While wild-type Myt1 (Myt1-WT) served as a good substrate for Plk1 in vitro, a mutant Myt1 (Myt1-4A), in which the four putative phosphorylation sites are replaced by alanines, did not. In nocodazole-treated cells, Myt1-WT, but not Myt1-4A, displayed its mobility shift in gel electrophoresis, due to phosphorylation. These results suggest that Plk1 phosphorylates Myt1 during M phase. Thus, this study identifies a novel substrate for Plk1 by determining a consensus phosphorylation sequence by Plk1.     

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.     

SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis

Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.     

Site recognition and substrate screens for PKN family proteins

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr??? in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr??? is shown to be modulated by PKN in vivo.     

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.     

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).     

Plk3 phosphorylates topoisomerase IIalpha at Thr(1342), a site that is not recognized by Plk1

The Plk (polo-like kinase) family is involved in cell-cycle machinery. Despite the possible overlapping involvement of Plk1 and Plk3 in cell-cycle distribution, the precise role of each Plk might be different. To investigate mechanisms that may differentiate their physiological roles, we compared the substrate specificities of Plk1 and Plk3 using synthetic peptides. Among these substrate peptides, topoisomerase IIalpha EKT(1342)DDE-containing synthetic peptide was strongly phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIalpha peptide, we identified residues at positions +1, +2 and +4 as determinants of differential substrate recognition between Plk1 and Plk3. Acidic residues at positions +2 and +4 appear to be a positive determinant for Plk3 but not Plk1. Variation at position +1 appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. The direct phosphorylation of Thr(1342) of topoisomerase IIalpha by Plk3 was demonstrated with an in vitro kinase assay, and overexpression of Plk3 induced the phosphorylation of Thr(1342) in cellular topoisomerase IIalpha. Furthermore, the physical interaction between Plk3 and topoisomerase IIalpha was also demonstrated in cells in addition to phosphorylation. These data suggest that topoisomerase IIalpha is a novel physiological substrate for Plk3 and that Plk1 and Plk3 play different roles in cell-cycle regulation.     

Identification of β-catenin as a novel substrate of polo-like kinase 1

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified β-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of β-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between β-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that β-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of β-catenin in M phase.     

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.     

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.     

DSas-6 and Ana2 coassemble into tubules to promote centriole duplication and engagement

Centrioles form cilia and centrosomes, organelles whose dysfunction is increasingly linked to human disease. Centriole duplication relies on a few conserved proteins (ZYG-1/Sak/Plk4, SAS-6, SAS-5/Ana2, and SAS-4), and is often initiated by the formation of an inner "cartwheel" structure. Here, we show that overexpressed Drosophila Sas-6 and Ana2 coassemble into extended tubules (SAStubules) that bear a striking structural resemblance to the inner cartwheel of the centriole. SAStubules specifically interact with centriole proximal ends, but extra DSas-6/Ana2 is only recruited onto centrioles when Sak/Plk4 kinase is also overexpressed. This extra centriolar DSas-6/Ana2 induces centriole overduplication and, surprisingly, increased centriole cohesion. Intriguingly, we observe tubules that are structurally similar to SAStubules linking the engaged centrioles in normal wild-type cells. We conclude that DSas-6 and Ana2 normally cooperate to drive the formation of the centriole inner cartwheel and that they promote both centriole duplication and centriole cohesion in a Sak/Plk4-dependent manner.     

Pim kinase substrate identification and specificity

The Pim family of Ser/Thr kinases has been implicated in the process of lymphomagenesis and cell survival. Known substrates of Pim kinases are few and poorly characterized. In this study we set out to identify novel Pim-2 substrates using the Kinase Substrate Tracking and Elucidation (KESTREL) approach. Two potential substrates, eukaryotic initiation factor 4B (eIF4B) and apoptosis inhibitor 5 (API-5), were identified from rat thymus extracts. Sequence comparison of the Pim-2 kinase phosphorylation sites of eIF4B and mouse BAD, the only other known Pim-2 substrate, revealed conserved amino acids preceding the phosphorylated serine residue. Stepwise replacement of the conserved residues produced a consensus sequence for Pim kinase recognition: RXRHXS. Pim-1 and Pim-2 catalyzed the phosphorylation of this recognition sequence 20-fold more efficiently than the original (K/R-K/R-R-K/R-L-S/T-a; a = small chain amino acid) Pim-1 phosphorylation site. The identification of the novel Pim kinase consensus sequence provides a more sensitive and versatile peptide based assay for screening modulators of Pim kinase activity.     

Activation loop phosphorylation of ERK3/ERK4 by group I p21-activated kinases (PAKs) defines a novel PAK-ERK3/4-MAPK-activated protein kinase 5 signaling pathway

Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway.     

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.     

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.     

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.