Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

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 G1 phase to G2 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 G1 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.

[1]  A. Fornace,et al.  Wip1 directly dephosphorylates gamma-H2AX and attenuates the DNA damage response. , 2010, Cancer research.

[2]  S. Jackson,et al.  The mitotic DNA damage response marks DNA double strand breaks with early signaling events , 2011 .

[3]  Akira Nakagawara,et al.  PPM1D is a potential target for 17q gain in neuroblastoma. , 2003, Cancer research.

[4]  J. Pines,et al.  Cyclin a Is Destroyed in Prometaphase and Can Delay Chromosome Alignment and Anaphase , 2001, The Journal of cell biology.

[5]  C. Smythe,et al.  ATM is required for the cellular response to thymidine induced replication fork stress. , 2004, Human molecular genetics.

[6]  Xiongbin Lu,et al.  The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop. , 2007, Cancer cell.

[7]  Tim Hunt,et al.  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 , 2001, The Journal of cell biology.

[8]  Stephen T. C. Wong,et al.  Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint. , 2011, Molecular cell.

[9]  Richard J. Edwards,et al.  ELM—the database of eukaryotic linear motifs , 2011, Nucleic Acids Res..

[10]  Jeffrey R. Marks,et al.  Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23 , 2002, Nature Genetics.

[11]  S. Jackson,et al.  Give me a break, but not in mitosis , 2011, Cell cycle.

[12]  R. Medema,et al.  Wip1 phosphatase is associated with chromatin and dephosphorylates γH2AX to promote checkpoint inhibition , 2010, Oncogene.

[13]  Albert J. Fornace,et al.  Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity , 2002, Nature Genetics.

[14]  Michael B. Yaffe,et al.  RNF8 Transduces the DNA-Damage Signal via Histone Ubiquitylation and Checkpoint Protein Assembly , 2007, Cell.

[15]  Hongmao Sun,et al.  Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Marketa Zvelebil,et al.  PPM1D Is a Potential Therapeutic Target in Ovarian Clear Cell Carcinomas , 2009, Clinical Cancer Research.

[17]  Albert J. Fornace,et al.  Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase , 2006, The Journal of experimental medicine.

[18]  R. Wolthuis,et al.  Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression , 2007, PLoS biology.

[19]  P. Reaper,et al.  Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. , 2011, Nature chemical biology.

[20]  Päivikki Kauraniemi,et al.  The serine-threonine protein phosphatase PPM1D is frequently activated through amplification in aggressive primary breast tumours , 2006, Breast Cancer Research and Treatment.

[21]  I. Hardcastle,et al.  Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. , 2004, Bioorganic & medicinal chemistry letters.

[22]  M. Fiscella,et al.  Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[23]  N. Curtin,et al.  Identification and Characterization of a Novel and Specific Inhibitor of the Ataxia-Telangiectasia Mutated Kinase ATM , 2004, Cancer Research.

[24]  L. Donehower,et al.  Augmented cancer resistance and DNA damage response phenotypes in PPM1D null mice , 2006, Molecular carcinogenesis.

[25]  J. Ellenberg,et al.  RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins , 2009, Cell.

[26]  Yunhua Zhu,et al.  Wip1 Regulates the Generation of New Neural Cells in the Adult Olfactory Bulb through p53‐Dependent Cell Cycle Control , 2009, Stem Cells.

[27]  D. Bulavin,et al.  WIP1 phosphatase at the crossroads of cancer and aging. , 2010, Trends in biochemical sciences.

[28]  L. Donehower,et al.  Reversal of the ATM/ATR-Mediated DNA Damage Response by the Oncogenic Phosphatase PPM1D , 2005, Cell cycle.

[29]  K. McManus,et al.  ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells. , 2005, Molecular biology of the cell.

[30]  M. Pagano,et al.  Control of cell growth by the SCF and APC/C ubiquitin ligases. , 2009, Current opinion in cell biology.

[31]  Lawrence A. Donehower,et al.  Mice Deficient for the Wild-Type p53-Induced Phosphatase Gene (Wip1) Exhibit Defects in Reproductive Organs, Immune Function, and Cell Cycle Control , 2002, Molecular and Cellular Biology.

[32]  E. Kinoshita,et al.  Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis. , 2007, Analytical biochemistry.

[33]  N. Onishi,et al.  Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase , 2006, Cell Death and Differentiation.

[34]  Yolanda F. Darlington,et al.  The type 2C phosphatase Wip1: An oncogenic regulator of tumor suppressor and DNA damage response pathways , 2008, Cancer and Metastasis Reviews.

[35]  S. Boulton,et al.  Playing the end game: DNA double-strand break repair pathway choice. , 2012, Molecular cell.

[36]  S. Jackson,et al.  DNA damage signaling in response to double-strand breaks during mitosis , 2010, The Journal of cell biology.

[37]  Zhang,et al.  Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway , 2010 .

[38]  Yolanda F. Darlington,et al.  Wild-type p53-induced Phosphatase 1 Dephosphorylates Histone Variant γ-H2AX and Suppresses DNA Double Strand Break Repair* , 2010, The Journal of Biological Chemistry.

[39]  B. Neumann,et al.  53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress , 2011, Nature Cell Biology.

[40]  C. Bradshaw,et al.  Replication stress induces 53BP1-containing OPT domains in G1 cells , 2011, The Journal of cell biology.

[41]  L. Donehower,et al.  PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints. , 2005, Genes & development.

[42]  Jiri Bartek,et al.  RNF8 Ubiquitylates Histones at DNA Double-Strand Breaks and Promotes Assembly of Repair Proteins , 2007, Cell.

[43]  E. Nigg,et al.  Plk1 regulates mitotic Aurora A function through βTrCP-dependent degradation of hBora , 2008, Chromosoma.

[44]  M. Yaffe,et al.  MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks , 2008, Cell.

[45]  E. Yu,et al.  Expression of a Homeostatic Regulator, Wip1 (Wild-type p53-induced Phosphatase), Is Temporally Induced by c-Jun and p53 in Response to UV Irradiation* , 2010, The Journal of Biological Chemistry.

[46]  Lawrence A. Donehower,et al.  Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D , 2007, Journal of Neuro-Oncology.

[47]  J. Bartek,et al.  Regulation of the PML tumor suppressor in drug-induced senescence of human normal and cancer cells by JAK/STAT-mediated signaling , 2010, Cell cycle.

[48]  R. Medema,et al.  Wip1 confers G2 checkpoint recovery competence by counteracting p53‐dependent transcriptional repression , 2009, The EMBO journal.

[49]  N. Mailand,et al.  HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes , 2010, Nature Cell Biology.

[50]  S. Mizutani,et al.  Phosphorylation of histone H2AX at M phase in human cells without DNA damage response. , 2005, Biochemical and biophysical research communications.

[51]  S. Elledge,et al.  Phosphorylation-Dependent Ubiquitination of Cyclin E by the SCFFbw7 Ubiquitin Ligase , 2001, Science.

[52]  J. Bartek,et al.  Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage , 2012, Cell cycle.

[53]  Y. Shiloh.  ATM and related protein kinases: safeguarding genome integrity , 2003, Nature Reviews Cancer.

[54]  F. Bunz,et al.  Protein phosphatases and the dynamics of the DNA damage response , 2010, Cell cycle.

[55]  R. Wolthuis,et al.  Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A. , 2008, Molecular cell.

[56]  Atsushi Miyawaki,et al.  Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression , 2008, Cell.

[57]  L. Donehower,et al.  Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK–mediated activation of the p16Ink4a-p19Arf pathway , 2004, Nature Genetics.

[58]  Yolanda F. Darlington,et al.  Dephosphorylation of γ-H2AX by WIP1: An important homeostatic regulatory event in DNA repair and cell cycle control , 2010, Cell cycle.

[59]  J. Bartek,et al.  Senescence-associated heterochromatin foci are dispensable for cellular senescence, occur in a cell type- and insult-dependent manner and follow expression of p16ink4a , 2011, Cell cycle.

[60]  E. Appella,et al.  Wip1 phosphatase modulates ATM-dependent signaling pathways. , 2006, Molecular cell.

[61]  E. Petermann,et al.  Evidence That the ATR/Chk1 Pathway Maintains Normal Replication Fork Progression during Unperturbed S Phase , 2006, Cell cycle.

[62]  J. Bartek,et al.  More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance , 2011, Nature Cell Biology.