A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Less is known about the mechanisms that allow resumption of the cell cycle once checkpoint signaling is silenced. Here we show that while in undamaged cells several redundant pathways can promote the onset of mitosis, this redundancy is lost in cells recovering from a DNA damage-induced arrest. We demonstrate that Plk1 is crucial for mitotic entry following recovery from DNA damage. However, Plk1 is no longer required in cells depleted of Wee1, and we could show that Plk1 is involved in the degradation of Wee1 at the onset of mitosis. Thus, our data show that the cell cycle machinery is reset in response to DNA damage and that cells become critically dependent on Plk1-mediated degradation of Wee1 for their recovery.     

Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis

Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to separate their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addition, co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degradation of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis.     

Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair

Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.     

ATM/Wip1 activities at chromatin control Plk1 re‐activation to determine G2 checkpoint duration

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM‐/ATR‐dependent signaling that inhibits mitosis‐promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR‐dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET‐based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re‐activation. These phosphorylations are rapidly counteracted by the chromatin‐bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.     

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

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

Polo-like kinase-1 in DNA damage response

Polo-like kinase-1 (Plk1) belongs to a family of serine-threonine kinases and plays a critical role in mitotic progression. Plk1 involves in the initiation of mitosis, centrosome maturation, bipolar spindle formation, and cytokinesis, well-reported as traditional functions of Plk1. In this review, we discuss the role of Plk1 during DNA damage response beyond the functions in mitotsis. When DNA is damaged in cells under various stress conditions, the checkpoint mechanism is activated to allow cells to have enough time for repair. When damage is repaired, cells progress continuously their division, which is called checkpoint recovery. If damage is too severe to repair, cells undergo apoptotic pathway. If damage is not completely repaired, cells undergo a process called checkpoint adaptation, and resume cell division cycle with damaged DNA. Plk1 targets and regulates many key factors in the process of damage response, and we deal with these subjects in this review. [BMB Reports 2014; 47(5): 249-255]     

Liaisons between Survivin and Plk1 during Cell Division and Cell Death

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser²⁰ by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.     

Regulation of Polo-like kinase 1 by DNA damage in mitosis. Inhibition of mitotic PLK-1 by protein phosphatase 2A

DNA damage triggers multiple checkpoint pathways to arrest cell cycle progression. Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. In addition to Plk1 functions in cell cycle, Plk1 is involved in DNA damage check-point in G2 phase. Normally, ataxia telangiectasia-mutated kinase (ATM) is a key enzyme involved in G2 phase cell cycle arrest following DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in a ATM/ATR-dependent manner. However, it is still unclear how Plk1 is regulated in response to DNA damage in mitosis in which Plk1 is already activated. Here, we show that treatment of mitotic cells with doxorubicin and gamma-irradiation inhibits Plk1 activity through dephosphorylation of Plk1, and cells were arrested in G2 phase. Treatments of the phosphatase inhibitors and siRNA experiments suggested that PP2A pathway might be involved in regulating mitotic Plk1 activity in mitotic DNA damage. Finally, we propose a novel pathway, which is connected between ATM/ATR/Chk and protein phosphatase-Plk1 in DNA damage response in mitosis.     

Checkpoint Adaptation and Recovery: Back with Polo after the Break

S. cerevisiae cells that are unable to repair a double strand break ultimatelyescape the DNA damage checkpoint arrest and enter mitosis. This process called'adaptation' depends on functional Cdc5, a Polo-like kinase, and was long thoughtto be limited to single-cell organisms. However, the recent finding that Xenopusextracts can adapt to a long-lasting stall in DNA replication indicates thatcheckpoint adaptation does also occur in multicellular organisms. Interestingly, theXenopus Polo-like kinase (Plx1) plays an important role in this adaptation. To addto this, data from our laboratory have shown that the human Polo-like kinase (Plk1)is also required for cell cycle re-entry following a DNA damage-induced arrest. Buthere, Plk1 was shown to be required for bona-fide checkpoint recovery, rather thanadaptation. That is, Plk1 is required to restart the cell cycle once all of the damage isrepaired and checkpoint signaling is turned off. While the target of Plx1 duringadaptation is a component of the checkpoint machinery (Claspin), the target of Plk1during recovery turns out to be a mitotic regulator (Wee1). Here, we discuss some ofthe remarkable similarities and subtle differences in the molecular mechanisms thatcontrol checkpoint adaptation and recovery, and the role of Polo-like kinasestherein.     

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.     

Normal cells, but not cancer cells, survive severe Plk1 depletion

We previously reported the phenotype of depletion of polo-like kinase 1 (Plk1) using RNA interference (RNAi) and showed that p53 is stabilized in Plk1-depleted cancer cells. In this study, we further analyzed the Plk1 depletion-induced phenotype in both cancer cells and primary cells. The vector-based RNAi approach was used to evaluate the role of the p53 pathway in Plk1 depletion-induced apoptosis in cancer cells with different p53 backgrounds. Although DNA damage and cell death can occur independently of p53, p53-deficient cancer cells were much more sensitive to Plk1 depletion than cancer cells with functional p53. Next, the lentivirus-based RNAi approach was used to generate a series of Plk1 hypomorphs. In HeLa cells, two weak hypomorphs showed only slight G2/M arrest, a medium hypomorph arrested with 4N DNA content, followed later by apoptosis, and a strong Plk1 hypomorph underwent serious mitotic catastrophe. In well-synchronized HeLa cells, a medium level of Plk1 depletion caused a 2-h delay of mitotic progression, and a high degree of Plk1 depletion significantly delayed mitotic entry and completely blocked cells at mitosis. In striking contrast, normal hTERT-RPE1 and MCF10A cells were much less sensitive to Plk1 depletion than HeLa cells; no apparent cell proliferation defect or cell cycle arrest was observed after Plk1 depletion in these cells. Therefore, these data further support suggestions that Plk1 may be a feasible cancer therapy target.     

Mitotic DNA damage response: Polo-like Kinase-1 is dephosphorylated through ATM-Chk1 pathway

DNA damage during the cell division cycle can activate ATM/ATR and their downstream kinases that are involved in the checkpoint pathway, and cell growth is halted until damage is repaired. As a result of DNA damage induced in mitotic cells by doxorubicin treatment, cells accumulate in a G2-like phase, not in mitosis. Under these conditions, two mitosis-specific kinases, Cdk1 and Plk1, are inhibited by inhibitory phosphorylation and dephosphorylation, respectively. G2-specific phosphorylation of Cdc25 was increased during incubation after mitotic DNA damage. Inhibition of Plk1 through dephosphorylation was dependent on ATM/Chk1 activity. Depleted expression of ATM and Chk1 was achieved using small hairpin RNA (shRNA) plasmid constructs. In this condition, damaged mitotic cells did not accumulated in a G2-like stage, and entered into G1 phase without delay. Protein phosphatase 2A was responsible for dephosphorylation of mitotic Plk1 in response to DNA damage. In knockdown of PP2A catalytic subunits, Plk1 was not dephosphorylated, but rather degraded in response to DNA damage, and cells did not accumulate in G2-like phase. The effect of ATM/Chk1 inhibition was counteracted by overexpression of PP2A, indicated that PP2A may function as a downstream target of ATM/Chk1 at a mitotic DNA damage checkpoint, or may have a dominant effect on ATM/Chk1 function at this checkpoint. Finally, we have shown that negative regulation of Plk1 by dephosphorylation is important to cell accumulation in G2-like phase at the mitotic DNA damage checkpoint, and that this ATM/Chk1/PP2A pathway independent on p53 is a novel mechanism of cellular response to mitotic DNA damage.     

Ectopic Expression of Plk1 Leads to Activation of the Spindle Checkpoint

Polo-like kinase 1 (Plk1), the best characterized member of the mammalian polo-like kinase family, is well regulated throughout the cell cycle, and is inhibited following DNA damage. Chk1 plays a key role in the response to DNA damage. We recently reported that Chk1 is required for mitotic progression through negative regulation of Plk1. Here, we report the phenotypes of cultured cells upon ectopic expression of various forms of Plk1. Epitopic expression of Plk1 led to mitotic arrest, whereas co-expression of Chk1 could release this mitotic block. Moreover, the Plk1 expression-induced mitotic block was also released by inactivation of the spindle-assembly checkpoint.     

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.     

The role of Polo-like kinase 1 in the inhibition of centrosome separation after ionizing radiation

Activation of the G2/M cell cycle checkpoint by DNA damage prevents cells from entering mitosis. Centrosome separation is initiated in G2 phase and completed in M phase. This critical process for cell division is targeted by G2/M checkpoint. Here we show that Plk1 signaling plays an important role in regulation of centrosome separation after DNA damage. Constitutively active Plk1 overrides the inhibition of centrosome separation induced by DNA damage. This inhibition is dependent on ATM, but not on Chk2 or Chk1. Nek2 is a key regulator of centrosome separation and is a target of Plk1 in blocking centrosome separation. We found that Plk1 can phosphorylate Nek2 in vitro and interacts with Nek2 in vivo. Down-regulation of Plk1 with RNA interference prevents Nek2-induced centrosome splitting. DNA damage is known to inhibit Plk1 activity. We propose that the DNA damage-induced inhibition of Plk1 leads to inhibition of Nek2 activity and thus prevents centrosome separation.     

Use of the novel Plk1 inhibitor ZK-thiazolidinone to elucidate functions of Plk1 in early and late stages of mitosis

Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with 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.