Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Polo-like kinase 1 inhibits DNA damage response during mitosis

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.     

Inhibition of Polo-like kinase 4 induces mitotic defects and DNA damage in diffuse large B-cell lymphoma

Polo-like kinase 4 (PLK4), a key regulator of centriole biogenesis, has recently been shown to play key roles in tumorigenesis. Blocking PLK4 expression by interference or targeted drugs exhibits attractive potential in improving the efficacy of chemotherapy. Nevertheless, the role of PLK4 in diffuse large B-cell lymphoma (DLBCL) is still undefined. In this study, we discover that PLK4 is a potential target for the treatment of DLBCL, and demonstrate the efficacy of a PLK4 inhibitor when used in combination with doxorubicin. Pharmaceutical inhibition of PLK4 with CFI-400945 inhibited DLBCL cell proliferation and induced apoptotic cell death. The anti-tumor effects were accompanied by mitotic defects, including polyploidy and cytokinesis failure. Activation of p53 and Hippo/YAP tumor suppressor signaling pathway was identified as the potential mechanisms driving CFI-400945 activity. Moreover, CFI-400945 treatment resulted in activation of DNA damage response. Combining CFI-400945 with doxorubicin markedly delayed tumor progression in DLBCL xenografts. Finally, PLK4 was increased in primary DLBCL tissues and cell lines. High levels of PLK4 expression were associated with poor survival in the patients receiving CHOP-based treatment, implicating PLK4 as a predictive biomarker of DLBCL chemosensitivity. These results provide the therapeutic potential of CFI-400945 both as monotherapy or in combination with doxorubicin for the treatment of DLBCL.Subject terms: B-cell lymphoma, Cytokinesis, DNA damage response     

Regulation of protein kinase cascades by protein phosphatase 2A.

Many protein kinases themselves are regulated by reversible phosphorylation. Upon cell stimulation, specific kinases are transiently phosphorylated and activated. Several of these protein kinases are substrates for protein phosphatase 2A (PP2A), and PP2A appears to be the major kinase phosphatase in eukaryotic cells that downregulates activated protein kinases. This idea is substantiated by the observation that some viral proteins and naturally occurring toxins target PP2A and modulate its activity. There is increasing evidence that PP2A activity is regulated by extracellular signals and during the cell cycle. Thus, PP2A is likely to play an important role in determining the activation kinetics of protein kinase cascades.     

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.     

Promotion of mitosis by activated protein kinase B after DNA damage involves polo-like kinase 1 and checkpoint protein CHFR

The role of the protein kinase B (PKB/Akt) in the regulation of cell survival and proliferation is well established. PKB is a key effector in the phosphatidylinositol 3-kinase pathway and plays a role in the initiation of S phase and in the G(2)-M transition. I report here that activated PKB shortens the G(2) arrest induced by DNA damage and promotes early entry into mitosis. Activated PKB supports high levels of expression and activity of the polo-like kinase 1 (Plk1) after DNA damage as cells accumulate in G(2). The checkpoint protein CHFR implicated in degradation of Plk1 is involved in the regulation of Plk1 by PKB. PKB phosphorylates CHFR in vitro and in vivo. Expression of a mutant form of CHFR that cannot be phosphorylated by PKB results in reduction of levels of Plk1 and inhibition of mitotic entry under normal conditions and after DNA damage. Results of this study support a model in which PKB facilitates mitotic resolution of DNA damage-induced G(2) arrest by inhibiting the checkpoint function of CHFR. The deregulated activation of PKB that occurs frequently in tumors might inhibit CHFR activity after DNA damage and therefore promote Plk1 accumulation leading to the disruption of the DNA damage checkpoint.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Regulation of Mih1/Cdc25 by protein phosphatase 2A and casein kinase 1

The Cdc25 phosphatase promotes entry into mitosis by removing cyclin-dependent kinase 1 (Cdk1) inhibitory phosphorylation. Previous work suggested that Cdc25 is activated by Cdk1 in a positive feedback loop promoting entry into mitosis; however, it has remained unclear how the feedback loop is initiated. To learn more about the mechanisms that regulate entry into mitosis, we have characterized the function and regulation of Mih1, the budding yeast homologue of Cdc25. We found that Mih1 is hyperphosphorylated early in the cell cycle and is dephosphorylated as cells enter mitosis. Casein kinase 1 is responsible for most of the hyperphosphorylation of Mih1, whereas protein phosphatase 2A associated with Cdc55 dephosphorylates Mih1. Cdk1 appears to directly phosphorylate Mih1 and is required for initiation of Mih1 dephosphorylation as cells enter mitosis. Collectively, these observations suggest that Mih1 regulation is achieved by a balance of opposing kinase and phosphatase activities. Because casein kinase 1 is associated with sites of polar growth, it may regulate Mih1 as part of a signaling mechanism that links successful completion of growth-related events to cell cycle progression.     

KIBRA protein phosphorylation is regulated by mitotic kinase aurora and protein phosphatase 1

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser(539) as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser(539) phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser(539) plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.     

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.     

Inhibition of Polo-like kinase-1 by DNA damage occurs in an ATM- or ATR-dependent fashion

Polo-like kinases play multiple roles in different phases of mitosis. We have recently shown that the mammalian polo-like kinase, Plk1, is inhibited in response to DNA damage and that this inhibition may lead to cell cycle arrests at multiple points in mitosis. Here we have investigated the role of the checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) in DNA damage-induced inhibition of Plk1. We show that inhibition of Plk1 kinase activity is efficiently blocked by the radio-sensitizing agent caffeine. Using ATM(-/-) cells we show that under certain circumstances, inhibition of Plk1 by DNA-damaging agents critically depends on ATM. In addition, we show that UV radiation also causes inhibition of Plk1, and we present evidence that this inhibition is mediated by ATR. Taken together, our data demonstrate that ATM and ATR can regulate Plk1 kinase activity in response to a variety of DNA-damaging agents.     

Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.     

Polo-like kinase 1 inactivation following mitotic DNA damaging treatments is independent of ataxia telangiectasia mutated kinase

Polo-like kinase 1 (Plk1) is an important regulator of several events during mitosis. Recent reports show that Plk1 is involved in both G2 and mitotic DNA damage checkpoints. Ataxia telangiectasia mutated kinase (ATM) is an important enzyme involved in G2 phase cell cycle arrest following interphase DNA damage, and inhibition of Plk1 by DNA damage during G2 occurs in an ATM-/ATM-Rad3-related kinase (ATR)-dependent fashion. However, it is unclear how Plk1 is regulated in response to M phase DNA damage. We found that treatment of mitotic cells with DNA damaging agents inhibits Plk1 activity primarily through dephosphorylation of Plk1, which occurred in both p53 wild-type and mutant cells. Inhibition of Plk1 is not prevented by caffeine pretreatment that inhibits ATM activity and also occurs in ATM mutant cell lines. Furthermore, ATM mutant cell lines, unlike wild-type cells, fail to arrest after mitotic DNA damaging treatments. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, reduces Plk1 dephosphorylation following mitotic DNA damaging treatments, suggesting that the PI3K pathway may be involved in regulating Plk1 activity. Earlier studies showed that inhibition of Plk1 by G2 DNA damage occurs in an ATM-dependent fashion. Our results extend the previous studies by showing that ATM is not required for dephosphorylation and inhibition of Plk1 activity following mitotic DNA damage, and also suggest that Plk1 is not a principal regulator or mediator of the mitotic DNA damage response.     

Deactivation of sphingosine kinase 1 by protein phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.     

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]     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

Activation of type-1 protein phosphatase by cdc2 kinase.

Purified cdc2 or cdc2 obtained from HeLa cells in association with p13suc1 activate inactive type-1 protein phosphatase (PP1) (catalytic subunit.inhibitor-2 complex, purified from skeletal muscle). Likewise in the case of PP1 activation by FA/GSK3, activation by cdc2 is accompanied by phosphorylation of inhibitor-2 (I2) and free I2 can be phosphorylated as well. Correlation between PP1 activation and I2 phosphorylation is suggested by the fact that both activation and phosphorylation (a) increase in parallel during incubation with cdc2, (b) decrease in parallel upon subsequent cdc2 inhibition by EDTA, and (c) are inhibited by the cdc2 inhibitor 5,6-dichlorobenzimidazole riboside. cdc2 also phosphorylates the catalytic subunit of PP1, whether in the complex with I2 or as free molecule. The activation of PP1 by cdc2 and by FA/GSK3 is compared.