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Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity

Abstract

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.

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References

  • Adams KE, Medhurst AL, Dart DA, Lakin ND . (2006). Recruitment of ATR to sites of ionising radiation-induced DNA damage requires ATM and components of the MRN protein complex. Oncogene 25: 3894–3904.

    Article  CAS  Google Scholar 

  • Bartek J, Lukas J . (2003). Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3: 421–429.

    Article  CAS  Google Scholar 

  • Bonilla CY, Melo JA, Toczyski DP . (2008). Colocalization of sensors is sufficient to activate the DNA damage checkpoint in the absence of damage. Mol Cell 30: 267–276.

    Article  CAS  Google Scholar 

  • Chen P, Luo C, Deng Y, Ryan K, Register J, Margosiak S et al. (2000). The 1.7 A crystal structure of human cell cycle checkpoint kinase Chk1: implications for Chk1 regulation. Cell 100: 681–692.

    Article  CAS  Google Scholar 

  • Chen Y, Sanchez Y . (2004). Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst) 3: 1025–1032.

    Article  CAS  Google Scholar 

  • Cross DA, Smythe C . (1998). PD 98059 prevents establishment of the spindle assembly checkpoint and inhibits the G2-M transition in meiotic but not mitotic cell cycles in Xenopus. Exp Cell Res 241: 12–22.

    Article  CAS  Google Scholar 

  • Dart DA, Adams KE, Akerman I, Lakin ND . (2004). Recruitment of the cell cycle checkpoint kinase ATR to chromatin during S-phase. J Biol Chem 279: 16433–16440.

    Article  CAS  Google Scholar 

  • Feijoo C, Hall-Jackson C, Wu R, Jenkins D, Leitch J, Gilbert DM et al. (2001). Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol 154: 913–923.

    Article  CAS  Google Scholar 

  • Gatei M, Sloper K, Sorensen C, Syljuasen R, Falck J, Hobson K et al. (2003). Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation. J Biol Chem 278: 14806–14811.

    Article  CAS  Google Scholar 

  • Guo Z, Kumagai A, Wang SX, Dunphy WG . (2000). Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev 14: 2745–2756.

    Article  CAS  Google Scholar 

  • Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J et al. (2006). ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol 8: 37–45.

    Article  CAS  Google Scholar 

  • Jiang K, Pereira E, Maxfield M, Russell B, Goudelock DM, Sanchez Y . (2003). Regulation of Chk1 includes chromatin association and 14-3-3 binding following phosphorylation on Ser-345. J Biol Chem 278: 25207–25217.

    Article  CAS  Google Scholar 

  • Kaneko YS, Watanabe N, Morisaki H, Akita H, Fujimoto A, Tominaga K et al. (1999). Cell-cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18: 3673–3681.

    Article  CAS  Google Scholar 

  • Katsuragi Y, Sagata N . (2004). Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation. Mol Biol Cell 15: 1680–1689.

    Article  CAS  Google Scholar 

  • Kramer A, Mailand N, Lukas C, Syljuasen RG, Wilkinson CJ, Nigg EA et al. (2004). Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase. Nat Cell Biol 6: 884–891.

    Article  Google Scholar 

  • Lee JH, Paull TT . (2005). ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308: 551–554.

    Article  CAS  Google Scholar 

  • Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K et al. (2000). Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev 14: 1448–1459.

    Article  CAS  Google Scholar 

  • Lupardus PJ, Byun T, Yee MC, Hekmat-Nejad M, Cimprich KA . (2002). A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev 16: 2327–2332.

    Article  CAS  Google Scholar 

  • Mailand N, Bekker-Jensen S, Bartek J, Lukas J . (2006). Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress. Mol Cell 23: 307–318.

    Article  CAS  Google Scholar 

  • Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge SJ . (2000). Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci USA 97: 10389–10394.

    Article  CAS  Google Scholar 

  • Myers JS, Cortez D . (2006). Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J Biol Chem 281: 9346–9350.

    Article  CAS  Google Scholar 

  • Niida H, Katsuno Y, Banerjee B, Hande MP, Nakanishi M . (2007). Specific role of Chk1 phosphorylations in cell survival and checkpoint activation. Mol Cell Biol 27: 2572–2581.

    Article  CAS  Google Scholar 

  • Oe T, Nakajo N, Katsuragi Y, Okazaki K, Sagata N . (2001). Cytoplasmic occurrence of the Chk1/Cdc25 pathway and regulation of Chk1 in Xenopus oocytes. Dev Biol 229: 250–261.

    Article  CAS  Google Scholar 

  • Peschiaroli A, Dorrello NV, Guardavaccaro D, Venere M, Halazonetis T, Sherman NE et al. (2006). SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response. Mol Cell 23: 319–329.

    Article  CAS  Google Scholar 

  • Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L et al. (2005). Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell 7: 193–204.

    Article  CAS  Google Scholar 

  • Shimada M, Niida H, Zineldeen DH, Tagami H, Tanaka M, Saito H et al. (2008). Chk1 is a histone H3 threonine 11 kinase that regulates DNA damage-induced transcriptional repression. Cell 132: 221–232.

    Article  CAS  Google Scholar 

  • Smith GC, Cary RB, Lakin ND, Hann BC, Teo SH, Chen DJ et al. (1999). Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM. Proc Natl Acad Sci USA 96: 11134–11139.

    Article  CAS  Google Scholar 

  • Smits VA, Reaper PM, Jackson SP . (2006). Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response. Curr Biol 16: 150–159.

    Article  CAS  Google Scholar 

  • Suzuki K, Kodama S, Watanabe M . (1999). Recruitment of ATM protein to double strand DNA irradiated with ionizing radiation. J Biol Chem 274: 25571–25575.

    Article  CAS  Google Scholar 

  • Traven A, Heierhorst J . (2005). SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins. Bioessays 27: 397–407.

    Article  CAS  Google Scholar 

  • Wilsker D, Petermann E, Helleday T, Bunz F . (2008). Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control. Proc Natl Acad Sci USA 105: 20752–20757.

    Article  CAS  Google Scholar 

  • Zachos G, Rainey MD, Gillespie DA . (2003). Chk1-deficient tumour cells are viable but exhibit multiple checkpoint and survival defects. EMBO J 22: 713–723.

    Article  CAS  Google Scholar 

  • Zachos G, Rainey MD, Gillespie DA . (2005). Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures. Mol Cell Biol 25: 563–574.

    Article  CAS  Google Scholar 

  • Zhang YW, Otterness DM, Chiang GG, Xie W, Liu YC, Mercurio F et al. (2005). Genotoxic stress targets human Chk1 for degradation by the ubiquitin-proteasome pathway. Mol Cell 19: 607–618.

    Article  CAS  Google Scholar 

  • Zhao H, Piwnica-Worms H . (2001). ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 21: 4129–4139.

    Article  CAS  Google Scholar 

  • Zou L, Elledge SJ . (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542–1548.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr Frank Kozielski for his comments on the manuscript. This work was supported by Cancer Research UK (CR-UK).

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Correspondence to M T Scott.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Walker, M., Black, E., Oehler, V. et al. Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity. Oncogene 28, 2314–2323 (2009). https://doi.org/10.1038/onc.2009.102

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