Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G(1) phase to G(2) and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G(1) cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.

Department of Genome Integrity Institute of Molecular Genetics v v i Academy of Sciences of the Czech Republic Prague Czech Republic

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Cell Cycle. 2013 Feb 1;12(3):390. doi: 10.4161/cc.23554. PubMed

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Cell Cycle. 2013 Feb 1;12(3):391. doi: 10.4161/cc.23555. PubMed

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Shiloh Y. ATM and related protein kinases: safeguarding genome integrity. Nat Rev Cancer. 2003;3:155–68. doi: 10.1038/nrc1011. PubMed DOI

Lukas J, Lukas C, Bartek J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance. Nat Cell Biol. 2011;13:1161–9. doi: 10.1038/ncb2344. PubMed DOI

Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 2005;123:1213–26. doi: 10.1016/j.cell.2005.09.038. PubMed DOI

Huen MSY, Grant R, Manke I, Minn K, Yu X, Yaffe MB, et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell. 2007;131:901–14. doi: 10.1016/j.cell.2007.09.041. PubMed DOI PMC

Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, et al. RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell. 2007;131:887–900. doi: 10.1016/j.cell.2007.09.040. PubMed DOI

Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH, Pepperkok R, et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell. 2009;136:435–46. doi: 10.1016/j.cell.2008.12.041. PubMed DOI

Bekker-Jensen S, Rendtlew Danielsen J, Fugger K, Gromova I, Nerstedt A, Lukas C, et al. HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nat Cell Biol. 2010;12:80–6, 1-12. doi: 10.1038/ncb2008. PubMed DOI

Moudry P, Lukas C, Macurek L, Hanzlikova H, Hodny Z, Lukas J, et al. Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage. Cell Cycle. 2012;11:1573–82. doi: 10.4161/cc.19978. PubMed DOI

Chapman JR, Taylor MR, Boulton SJ. Playing the end game: DNA double-strand break repair pathway choice. Mol Cell. 2012;47:497–510. doi: 10.1016/j.molcel.2012.07.029. PubMed DOI

Lukas C, Savic V, Bekker-Jensen S, Doil C, Neumann B, Pedersen RS, et al. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress. Nat Cell Biol. 2011;13:243–53. doi: 10.1038/ncb2201. PubMed DOI

Harrigan JA, Belotserkovskaya R, Coates J, Dimitrova DS, Polo SE, Bradshaw CR, et al. Replication stress induces 53BP1-containing OPT domains in G1 cells. J Cell Biol. 2011;193:97–108. doi: 10.1083/jcb.201011083. PubMed DOI PMC

Giunta S, Belotserkovskaya R, Jackson SP. DNA damage signaling in response to double-strand breaks during mitosis. J Cell Biol. 2010;190:197–207. doi: 10.1083/jcb.200911156. PubMed DOI PMC

Giunta S, Jackson SP. Give me a break, but not in mitosis: the mitotic DNA damage response marks DNA double-strand breaks with early signaling events. Cell Cycle. 2011;10:1215–21. doi: 10.4161/cc.10.8.15334. PubMed DOI PMC

Harris DR, Bunz F. Protein phosphatases and the dynamics of the DNA damage response. Cell Cycle. 2010;9:861–9. doi: 10.4161/cc.9.5.10862. PubMed DOI

Shreeram S, Demidov ON, Hee WK, Yamaguchi H, Onishi N, Kek C, et al. Wip1 phosphatase modulates ATM-dependent signaling pathways. Mol Cell. 2006;23:757–64. doi: 10.1016/j.molcel.2006.07.010. PubMed DOI

Fujimoto H, Onishi N, Kato N, Takekawa M, Xu XZ, Kosugi A, et al. Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase. Cell Death Differ. 2006;13:1170–80. doi: 10.1038/sj.cdd.4401801. PubMed DOI

Lu X, Ma O, Nguyen T-A, Jones SN, Oren M, Donehower LA. The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop. Cancer Cell. 2007;12:342–54. doi: 10.1016/j.ccr.2007.08.033. PubMed DOI

Lu X, Nannenga B, Donehower LA. PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev. 2005;19:1162–74. doi: 10.1101/gad.1291305. PubMed DOI PMC

Macůrek L, Lindqvist A, Voets O, Kool J, Vos HR, Medema RH. Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. Oncogene. 2010;29:2281–91. doi: 10.1038/onc.2009.501. PubMed DOI

Moon S, Lin L, Zhang X, Nguyen T, Darlington Y, Waldman A. Wildtype p53-induced phosphatase 1 dephosphorylates histone variant {gamma}-H2AX and suppresses DNA double strand break repair. J Biol Chem. 2010;23:12935–47. doi: 10.1074/jbc.M109.071696. PubMed DOI PMC

Cha H, Lowe JM, Li H, Lee J-S, Belova GI, Bulavin DV, et al. Wip1 directly dephosphorylates γ-H2AX and attenuates the DNA damage response. Cancer Res. 2010;70:4112–22. doi: 10.1158/0008-5472.CAN-09-4244. PubMed DOI PMC

Lu X, Nguyen TA, Moon SH, Darlington Y, Sommer M, Donehower LA. The type 2C phosphatase Wip1: an oncogenic regulator of tumor suppressor and DNA damage response pathways. Cancer Metastasis Rev. 2008;27:123–35. doi: 10.1007/s10555-008-9127-x. PubMed DOI PMC

Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer WE, et al. Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc Natl Acad Sci USA. 1997;94:6048–53. doi: 10.1073/pnas.94.12.6048. PubMed DOI PMC

Zhang X, Wan G, Mlotshwa S, Vance V, Berger FG, Chen H, et al. Oncogenic Wip1 phosphatase is inhibited by miR-16 in the DNA damage signaling pathway. Cancer Res. 2010;70:7176–86. doi: 10.1158/0008-5472.CAN-10-0697. PubMed DOI PMC

Lindqvist A, de Bruijn M, Macurek L, Brás A, Mensinga A, Bruinsma W, et al. Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression. EMBO J. 2009;28:3196–206. doi: 10.1038/emboj.2009.246. PubMed DOI PMC

Nannenga B, Lu X, Dumble M, Van Maanen M, Nguyen T-A, Sutton R, et al. Augmented cancer resistance and DNA damage response phenotypes in PPM1D null mice. Mol Carcinog. 2006;45:594–604. doi: 10.1002/mc.20195. PubMed DOI

Bulavin DV, Phillips C, Nannenga B, Timofeev O, Donehower LA, Anderson CW, et al. Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway. Nat Genet. 2004;36:343–50. doi: 10.1038/ng1317. PubMed DOI

Shreeram S, Hee WK, Demidov ON, Kek C, Yamaguchi H, Fornace AJ, Jr., et al. Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase. J Exp Med. 2006;203:2793–9. doi: 10.1084/jem.20061563. PubMed DOI PMC

Rauta J, Alarmo E-L, Kauraniemi P, Karhu R, Kuukasjärvi T, Kallioniemi A. The serine-threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours. Breast Cancer Res Treat. 2006;95:257–63. doi: 10.1007/s10549-005-9017-7. PubMed DOI

Bulavin DV, Demidov ON, Saito S, Kauraniemi P, Phillips C, Amundson SA, et al. Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity. Nat Genet. 2002;31:210–5. doi: 10.1038/ng894. PubMed DOI

Li J, Yang Y, Peng Y, Austin RJ, van Eyndhoven WG, Nguyen KCQ, et al. Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23. Nat Genet. 2002;31:133–4. doi: 10.1038/ng888. PubMed DOI

Castellino RC, De Bortoli M, Lu X, Moon S-H, Nguyen T-A, Shepard MA, et al. Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D. J Neurooncol. 2008;86:245–56. doi: 10.1007/s11060-007-9470-8. PubMed DOI PMC

Saito-Ohara F, Imoto I, Inoue J, Hosoi H, Nakagawara A, Sugimoto T, et al. PPM1D is a potential target for 17q gain in neuroblastoma. Cancer Res. 2003;63:1876–83. PubMed

Tan DSP, Lambros MBK, Rayter S, Natrajan R, Vatcheva R, Gao Q, et al. PPM1D is a potential therapeutic target in ovarian clear cell carcinomas. Clin Cancer Res. 2009;15:2269–80. doi: 10.1158/1078-0432.CCR-08-2403. PubMed DOI

Bolderson E, Scorah J, Helleday T, Smythe C, Meuth M. ATM is required for the cellular response to thymidine induced replication fork stress. Hum Mol Genet. 2004;13:2937–45. doi: 10.1093/hmg/ddh316. PubMed DOI

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132:487–98. doi: 10.1016/j.cell.2007.12.033. PubMed DOI

Song JY, Han H-S, Sabapathy K, Lee B-M, Yu E, Choi J. Expression of a homeostatic regulator, Wip1 (wild-type p53-induced phosphatase), is temporally induced by c-Jun and p53 in response to UV irradiation. J Biol Chem. 2010;285:9067–76. doi: 10.1074/jbc.M109.070003. PubMed DOI PMC

Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, et al. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA. 2006;103:10660–5. doi: 10.1073/pnas.0600447103. PubMed DOI PMC

Skaar JR, Pagano M. Control of cell growth by the SCF and APC/C ubiquitin ligases. Curr Opin Cell Biol. 2009;21:816–24. doi: 10.1016/j.ceb.2009.08.004. PubMed DOI PMC

Koepp DM, Schaefer LK, Ye X, Keyomarsi K, Chu C, Harper JW, et al. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase. Science. 2001;294:173–7. doi: 10.1126/science.1065203. PubMed DOI

Chan EH, Santamaria A, Silljé HH, Nigg EA. Plk1 regulates mitotic Aurora A function through betaTrCP-dependent degradation of hBora. Chromosoma. 2008;117:457–69. doi: 10.1007/s00412-008-0165-5. PubMed DOI PMC

Wolthuis R, Clay-Farrace L, van Zon W, Yekezare M, Koop L, Ogink J, et al. Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A. Mol Cell. 2008;30:290–302. doi: 10.1016/j.molcel.2008.02.027. PubMed DOI

den Elzen N, Pines J. Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase. J Cell Biol. 2001;153:121–36. doi: 10.1083/jcb.153.1.121. PubMed DOI PMC

Geley S, Kramer E, Gieffers C, Gannon J, Peters J-M, Hunt T. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J Cell Biol. 2001;153:137–48. doi: 10.1083/jcb.153.1.137. PubMed DOI PMC

Yamada S, Nakamura H, Kinoshita E, Kinoshita-Kikuta E, Koike T, Shiro Y. Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis. Anal Biochem. 2007;360:160–2. doi: 10.1016/j.ab.2006.10.005. PubMed DOI

Dinkel H, Michael S, Weatheritt RJ, Davey NE, Van Roey K, Altenberg B, et al. ELM--the database of eukaryotic linear motifs. Nucleic Acids Res. 2012;40(Database issue):D242–51. doi: 10.1093/nar/gkr1064. PubMed DOI PMC

Moon S-H, Nguyen T-A, Darlington Y, Lu X, Donehower LA. Dephosphorylation of γ-H2AX by WIP1: an important homeostatic regulatory event in DNA repair and cell cycle control. Cell Cycle. 2010;9:2092–6. doi: 10.4161/cc.9.11.11810. PubMed DOI PMC

Ichijima Y, Sakasai R, Okita N, Asahina K, Mizutani S, Teraoka H. Phosphorylation of histone H2AX at M phase in human cells without DNA damage response. Biochem Biophys Res Commun. 2005;336:807–12. doi: 10.1016/j.bbrc.2005.08.164. PubMed DOI

McManus KJ, Hendzel MJ. ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells. Mol Biol Cell. 2005;16:5013–25. doi: 10.1091/mbc.E05-01-0065. PubMed DOI PMC

Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NMB, Orr AI, et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004;64:9152–9. doi: 10.1158/0008-5472.CAN-04-2727. PubMed DOI

Leahy JJJ, Golding BT, Griffin RJ, Hardcastle IR, Richardson C, Rigoreau L, et al. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett. 2004;14:6083–7. doi: 10.1016/j.bmcl.2004.09.060. PubMed DOI

Reaper PM, Griffiths MR, Long JM, Charrier J-D, Maccormick S, Charlton PA, et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011;7:428–30. doi: 10.1038/nchembio.573. PubMed DOI

Le Guezennec X, Bulavin DV. WIP1 phosphatase at the crossroads of cancer and aging. Trends Biochem Sci. 2010;35:109–14. doi: 10.1016/j.tibs.2009.09.005. PubMed DOI

Lu X, Nguyen T-A, Donehower LA. Reversal of the ATM/ATR-mediated DNA damage response by the oncogenic phosphatase PPM1D. Cell Cycle. 2005;4:1060–4. doi: 10.4161/cc.4.8.1876. PubMed DOI

Zhu Y-H, Zhang C-W, Lu L, Demidov ON, Sun L, Yang L, et al. Wip1 regulates the generation of new neural cells in the adult olfactory bulb through p53-dependent cell cycle control. Stem Cells. 2009;27:1433–42. doi: 10.1002/stem.65. PubMed DOI

Choi J, Nannenga B, Demidov ON, Bulavin DV, Cooney A, Brayton C, et al. Mice deficient for the wild-type p53-induced phosphatase gene (Wip1) exhibit defects in reproductive organs, immune function, and cell cycle control. Mol Cell Biol. 2002;22:1094–105. doi: 10.1128/MCB.22.4.1094-1105.2002. PubMed DOI PMC

Petermann E, Caldecott KW. Evidence that the ATR/Chk1 pathway maintains normal replication fork progression during unperturbed S phase. Cell Cycle. 2006;5:2203–9. doi: 10.4161/cc.5.19.3256. PubMed DOI

Yang C, Tang X, Guo X, Niikura Y, Kitagawa K, Cui K, et al. Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint. Mol Cell. 2011;44:597–608. doi: 10.1016/j.molcel.2011.09.016. PubMed DOI PMC

Hubackova S, Novakova Z, Krejcikova K, Kosar M, Dobrovolna J, Duskova P, et al. Regulation of the PML tumor suppressor in drug-induced senescence of human normal and cancer cells by JAK/STAT-mediated signaling. Cell Cycle. 2010;9:3085–99. doi: 10.4161/cc.9.15.12521. PubMed DOI

Lindqvist A, van Zon W, Karlsson Rosenthal C, Wolthuis RMF. Cyclin B1-Cdk1 activation continues after centrosome separation to control mitotic progression. PLoS Biol. 2007;5:e123. doi: 10.1371/journal.pbio.0050123. PubMed DOI PMC

Kosar M, Bartkova J, Hubackova S, Hodny Z, Lukas J, Bartek J. Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16(ink4a) Cell Cycle. 2011;10:457–68. doi: 10.4161/cc.10.3.14707. PubMed DOI

CK1-mediated phosphorylation of FAM110A promotes its interaction with mitotic spindle and controls chromosomal alignment

WIP1 Promotes Homologous Recombination and Modulates Sensitivity to PARP Inhibitors

Tubulin is actively exported from the nucleus through the Exportin1/CRM1 pathway

Residual Cdk1/2 activity after DNA damage promotes senescence

WIP1 phosphatase as pharmacological target in cancer therapy

Inhibition of WIP1 phosphatase sensitizes breast cancer cells to genotoxic stress and to MDM2 antagonist nutlin-3

Polo-like kinase 1 inhibits DNA damage response during mitosis