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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
Bartek J, Lukas J . (2003). Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3: 421–429.
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.
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.
Chen Y, Sanchez Y . (2004). Chk1 in the DNA damage response: conserved roles from yeasts to mammals. DNA Repair (Amst) 3: 1025–1032.
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.
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.
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.
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.
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.
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.
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.
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.
Katsuragi Y, Sagata N . (2004). Regulation of Chk1 kinase by autoinhibition and ATR-mediated phosphorylation. Mol Biol Cell 15: 1680–1689.
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.
Lee JH, Paull TT . (2005). ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex. Science 308: 551–554.
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.
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.
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.
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.
Myers JS, Cortez D . (2006). Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J Biol Chem 281: 9346–9350.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Zhao H, Piwnica-Worms H . (2001). ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol 21: 4129–4139.
Zou L, Elledge SJ . (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542–1548.
Acknowledgements
We thank Dr Frank Kozielski for his comments on the manuscript. This work was supported by Cancer Research UK (CR-UK).
Author information
Authors and Affiliations
Corresponding author
Additional information
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2009.102
Keywords
This article is cited by
-
Quantitative phosphoproteomics to unravel the cellular response to chemical stressors with different modes of action
Archives of Toxicology (2020)
-
SPRTN protease and checkpoint kinase 1 cross-activation loop safeguards DNA replication
Nature Communications (2019)
-
Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation
Scientific Reports (2018)
-
KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage
Scientific Reports (2015)
-
Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors
Oncogene (2015)