Polo-Like Kinase 1 in the Life and Death of Cancer Cells

The polo-like kinase family plays a vital role in many cell cycle related events. The family includes mammalian Plk1, Snk (Plk2), and Fnk/Prk (Plk3), Xenopus laevis Plx1, Drosophila polo, fission yeast Plo1, and budding yeast Cdc5. These enzymes, in addition to a conserved kinase domain at the N-terminus, have highly conserved sequences called polo-box(s) in the non-catalytic C-terminal domain.1 Genetic and biochemical experiments with several different organisms have documented that polo-like kinases are involved in many aspects of the cell cycle, such as activation of Cdc2, centrosome assembly and maturation, activation of the anaphase-promoting complex (APC) during the metaphase-anaphase transition, and cytokinesis.(1-3)     

Polo-Like Kinase 1 Depletion Induces DNA Damage in Early S Prior to Caspase Activation

Polo-like kinase 1 (Plk1) plays several roles in mitosis, and it has been suggested to have a role in tumorigenesis. We have previously reported that Plk1 depletion results in cell death in cancer cells, whereas normal cells survive similar depletion. However, Plk1 depletion together with p53 depletion induces cell death in normal cells as well. This communication presents evidence on the sequence of events that leads to cell death in cancer cells. DNA damage is detected at the first S phase following Plk1 depletion and is more severe in Plk1-depleted p53-null cancer cells. As a consequence of Plk1 depletion using lentivirus-based small interfering RNA techniques, prereplicative complex (pre-RC) formation is disrupted at the G₁/S transition, and DNA synthesis is reduced during S phase of the first cycle after depletion. The levels of geminin, an inhibitor of DNA pre-RC, and Emi1, an inhibitor of anaphase-promoting complex/cyclosome, are elevated in Plk1-depleted cells. The rate of cell cycling is slower in Plk1-depleted cells than in control cells when synchronized by serum starvation. Plk1 depletion results in disrupted DNA pre-RC formation, reduced DNA synthesis, and DNA damage before cells display severe mitotic catastrophe or apoptosis. Our data suggest that Plk1 is required for cell cycle progression not only in mitosis but also for DNA synthesis, maintenance of DNA integrity, and prevention of cell death.Progression of the cell cycle is tightly regulated in eukaryotic cells by coordinated control of phosphorylation and proteolytic events. Duplication of genetic information for the next cell generation requires the precise coordination of numerous proteins (2). To ensure the accurate division of duplicated DNA, cells require condensed chromosomes, a mitotic spindle, and correct attachment of duplicated chromosomes to the spindle. Errors in DNA replication and mitosis may lead to cell death through apoptosis or result in mutations that lead to cancer (3). Polo-like kinase 1 (Plk1) is essential for several steps in mitosis and is highly expressed in proliferating cells. Expression of Plk1 increases in S phase and peaks during M phase (8). In addition, at the G₂/M boundary, Plk1 is activated by phosphorylation and promotes mitotic entry. Its primary role in mammalian cells appears to be control of mitotic progression, particularly in the metaphase/anaphase transition, and mitotic exit (37). At the G₂/M transition, Plx1, a counterpart of Plk1 in Xenopus, activates cyclin B1/Cdk1 by phosphorylation of Cdc25C (14) or of cyclin B1 (29). During mitotic entry, Plk1 is required for recruitment of the γ-tubulin ring complex (7). Phosphorylation of Emi1 by Plk1 leads to its destruction, release of Cdc20, and activation of the anaphase-promoting complex/cyclosome (APC/C) (10, 22, 26). Active APC/C mediates the degradation of proteins such as cyclin A, cyclin B1, securin, and geminin to promote exit from mitosis (6, 26). The multiple roles of Plk1 from the entry to and exit from mitosis indicate its importance as a regulator of these events.Recently, several reports suggest that Plk1 may play a role in other phases of the cell cycle. Plk1 interacts with prereplicative complex (pre-RC) proteins, such as Mcm2 and Orc2, in yeast two-hybrid studies (32), and coimmunoprecipitates with Mcm2 to Mcm7 and Orc2 (32, 35). Orc2, Mcm4, Mcm6, and Mcm7 proteins colocalize in the centrosome with Plk1 (25, 32). In addition, ectopic expression of Plk1-S137D arrests HeLa cells at the G₁/S boundary (12). Moreover, microinjection of in vitro-transcribed sense mRNA of Plk1 into serum-starved NIH 3T3 cells induced thymidine incorporation, whereas microinjection of antisense mRNA into growing NIH 3T3 cells that were stimulated with serum blocked thymidine incorporation (9). This observation suggests that Plk1 is required for DNA synthesis and that overexpression of Plk1 appears to be sufficient for induction of DNA synthesis. These data raise the possibility that Plk1 might have a required function in DNA replication.Depletion of Plk1 activity by microinjection of neutralizing anti-Plk1 antibody impairs centrosome maturation in HeLa cells (15). When Plk1 function is blocked by dominant-negative Plk1, several human tumor cells undergo mitotic catastrophe independent of Cdc25C (1). In Plk1-deficient human cancer cells, centrosomes do not separate to form bipolar spindles. The cells undergo prometaphase arrest and cell death caused by mitotic catastrophe (18, 33, 38). These effects are more severe in p53-deficient cancer cells. Cells codepleted for p53 and Plk1 undergo cell death as a consequence of mitotic defects (17). However, it is unclear how Plk1 depletion induces cell death or what the sequence of events is prior to cell death.Here, we provide evidence that Plk1 depletion induces DNA damage at G₁/S before cell death responses, such as caspase activation, are initiated.     

Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment

Polo-like kinases (Plks) are a family of serine-threonine kinases that regulate multiple intracellular processes including DNA replication, mitosis, and stress response. Plk1, the most well understood family member, regulates numerous stages of mitosis and is overexpressed in many cancers. Plk inhibitors are currently under clinical investigation, including phase III trials of volasertib, a Plk inhibitor, in acute myeloid leukemia and rigosertib, a dual inhibitor of Plk1/phosphoinositide 3-kinase signaling pathways, in myelodysplastic syndrome. Other Plk inhibitors, including the Plk1 inhibitors GSK461364A, TKM-080301, {"type":"entrez-nucleotide","attrs":{"text":"GW843682","term_id":"295327265","term_text":"GW843682"}}GW843682, purpurogallin, and poloxin and the Plk4 inhibitor CFI-400945 fumarate, are in earlier clinical development. This review discusses the biologic roles of Plks in cell cycle progression and cancer, and the mechanisms of action of Plk inhibitors currently in development as cancer therapies.     

Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirus-based RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy.Hepatitis C virus (HCV) is the major causative agent of non-A/non-B hepatitis (26). More than 170 million people, or 3% of the population in the world, are infected with HCV (29). It establishes chronic infection in at least 85% of infected individuals and is associated with liver cirrhosis and hepatocellular carcinoma. Current treatment, which combines polyethylene glycol-interferon (PEG-IFN) and ribavirin, is ineffective in 22% of patients with non-genotype 1 and in 45% of patients with genotype 1 HCV (1, 16, 23, 55). Therefore, identification of new targets for HCV therapy is an important issue, and cellular genes involved in the HCV life cycle may serve as good candidates.HCV is a positive-strand RNA virus and the only known member of Hepacivirus genus in the family Flaviviridae. Its genome has a length of about 9,600 nucleotides coding for a single polyprotein. The long polyprotein is further processed into at least 10 different products, including four structural proteins (core, E1, E2, and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Nonstructural proteins NS3-NS5B are components of the membrane-associated HCV replication complex (8, 13, 36, 45). NS3 is a bifunctional protein containing an N-terminal protease domain and a C-terminal helicase/NTPase domain, and NS4A serves as a cofactor for NS3 protease. NS4B protein is known to induce intracellular membrane changes that probably serve as the site for viral RNA replication (8). NS5A is required for RNA replication, but little is known about its function. NS5B is the RNA-dependent RNA polymerase (reviewed in reference 47).NS5A is phosphorylated on multiple serine and threonine residues and exists in basal phosphorylated (p56) and hyperphosphorylated (p58) forms (49). Increasing evidence suggests that the regulation of NS5A phosphorylation is important for HCV RNA replication. Adaptive mutations or kinase inhibitors, which reduce NS5A hyperphosphorylation, increased the replication of an HCV replicon in cell culture (HCVcc) systems (2, 4, 38). However, when an adaptive replicon with reduced p58 was further treated with the same kinase inhibitor or introduced with a second adaptive mutation, RNA replication was completely blocked (32, 38). Furthermore, the mutations that reduce NS5A hyperphosphorylation and promote RNA replication in cell culture, paradoxically, prevented productive replication in the chimpanzee model (6). These results imply that the tight control of the p58/p56 ratio is important for HCV replication. The detailed mechanism is still not clear, but a clue was provided by the finding of differential association of NS5A phospho-forms with the host vesicle-associated membrane protein-associated protein A (VAP-A) protein, which is an essential molecule for HCV replicase (9, 12). On the other hand, NS5A phosphorylation was recently found to regulate the production of infectious virus (34, 50). Alanine substitutions in the C-terminal domain III of NS5A impaired NS5A phosphorylation, leading to a decrease in NS5A-core protein interaction, disturbance of subcellular localization of NS5A, and disruption of virion production (3, 34, 50). In summary, phosphorylation on NS5A is not only important for HCV RNA replication but also critical for infectious virus production.Since the phosphorylation state of NS5A is correlated with HCV RNA replication and virion production, cellular kinases responsible for NS5A phosphorylation may serve as good candidates for drug targets. Several kinases have been shown to target NS5A in vitro, including casein kinase I (CKI), CKII, MEK1, MKK6, MKK7, AKT, and p70S6K (7, 24). Among these proteins, CKI and CKII are better characterized for NS5A phosphorylation. CKIα has been identified as the target of kinase inhibitors which decrease the hyperphosphorylation of NS5A and was further confirmed as a direct kinase of NS5A (41, 42). CKI requires prephosphorylation of residues near the predicted phosphorylation site in NS5A for effective modification, suggesting that other kinases are also involved in this process (42). CKII has been shown to bind to the C-terminal domain of NS5A and phosphorylate NS5A in vitro (24). Inhibition of CKII with chemical compounds or small interfering RNA (siRNA) did not significantly affect HCV RNA replication but severely disrupted virus production (50).In this study, using lentivirus-based RNA interference (RNAi) screening, we identified a serine/threonine kinase, Polo-like kinase 1 (Plk1), which is involved in HCV replication. Expression of short hairpin RNAs (shRNAs) targeting Plk1 decreased HCV replication and virus production. Moreover, silencing of Plk1 decreased the hyperphosphorylated form of NS5A. In cells treated with a Plk1-specific kinase inhibitor, HCV replication and NS5A hyperphosphorylation were significantly reduced, indicating that Plk1 kinase activity is required for this process. Further studies showed that Plk1 was coimmunoprecipitated and partially colocalized with NS5A, suggesting NS5A as a possible substrate for Plk1. Finally, NS5A is hyperphosphorylated by Plk1 in vitro, supporting the proposition that Plk1 regulates HCV replication through hyperphosphorylation of NS5A.     

Polo-Like Kinase 2-Dependent Phosphorylation of NPM/B23 on Serine 4 Triggers Centriole Duplication

Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2) is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin) as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.     

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.     

Finding Plk3

Polo-like kinases (Plks) are a highly conserved family of kinases found in flies, yeast and vertebrates. Plks derive their name from homology to the gene product of polo, a protein kinase first identified in Drosophila. Three polo-like kinases have been identified in vertebrates: Plk1, Plk2 and Plk3. Studies on Plk1 have revealed a great deal of information on its multiple functions, however Plk2 and Plk3 functions have not been fully explored. In this perspective we discuss recent work on Plk3 expression, function and localization in the context of previous reports on Plk3 and in terms of its relationship to Plk1.     

Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes

Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.     

The Polo kinase Plk4 functions in centriole duplication

The human Polo-like kinase 1 (PLK1) and its functional homologues that are present in other eukaryotes have multiple, crucial roles in meiotic and mitotic cell division. By contrast, the functions of other mammalian Polo family members remain largely unknown. Plk4 is the most structurally divergent Polo family member; it is maximally expressed in actively dividing tissues and is essential for mouse embryonic development. Here, we identify Plk4 as a key regulator of centriole duplication. Both gain- and loss-of-function experiments demonstrate that Plk4 is required--in cooperation with Cdk2, CP110 and Hs-SAS6--for the precise reproduction of centrosomes during the cell cycle. These findings provide an attractive explanation for the crucial function of Plk4 in cell proliferation and have implications for the role of Polo kinases in tumorigenesis.     

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.     

Cotargeting Polo-Like Kinase 1 and the Wnt/β-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of β-catenin by enhancing binding between glycogen synthase kinase 3β (GSK3β) and β-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.     

Calcium- and integrin-binding protein 1 regulates microtubule organization and centrosome segregation through polo like kinase 3 during cell cycle progression

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype.     

Mice lacking both mixed-lineage kinase genes Mlk1 and Mlk2 retain a wild type phenotype

The mitogen-activated protein kinase kinase kinases of the mixed-lineage kinase (MLK) family have been shown to activate the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway, and to regulate the other two principal MAPK cascades, p38 and extracellular signal-regulated kinase (ERK). Although there is growing evidence for their involvement in neuronal cell death leading to neurodegenerative disorders, little in vivo data is available for the members of this family of kinases. Here, we report that the inactivation of mouse Mlk1 and Mlk2 genes. Mlk1-/- and Mlk2-/- mice were found to be viable and healthy. Surprisingly, mice carrying the compound Mlk1/Mlk2 null mutations were also found to be viable, fertile and to have a normal life span. The nervous system, testis and kidney, the major sites of MLK1 and 2 expression, all appear normal, as do other organs where these kinases were found to be more weakly expressed. Surprisingly, developmental neuronal programmed cell death, another potential target for MLK family members, was also found to be unaffected. Our results suggest that there is extensive functional redundancy between MLK1/MLK2 and the other member of the family, MLK3, which is also not required for survival in mouse.     

Polo-Like Kinase 1 as a Target for Human Cytomegalovirus pp65 Lower Matrix Protein

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.     

Polo样激酶1在细胞周期及细胞周期监测点中的功能

Plk1（Polo-like kinase 1）是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶,是真核细胞有丝分裂的重要调控因子.Plk1随有丝分裂进程定位于不同位点,调节分裂期进入、纺锤体形成和胞质分裂等过程.Plk1能够与磷酸化的停靠蛋白结合,从而在不同空间被激活以满足其在细胞周期中的不同功能.Plk1还参与G2和M期DNA损伤监测点的调节,对于DNA损伤恢复后重新进入有丝分裂期是必须的.目前,Plk1的重要功能尤其是在DNA损伤监测点中发挥的重要功能正在被广泛研究.Plk1在多种恶性肿瘤中存在过表达且与肿瘤发生密切相关,对于Plk1功能的深入研究为以Plk1为靶的肿瘤治疗提供理论依据     

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.