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Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage

Oncogene volume 31, pages 1086–1094 (2012)Cite this article

Abstract

Here, we show that activation of the checkpoint effector kinase Chk1 in response to irradiation-induced DNA damage is minimal in G1, maximal during S-phase and diminishes as cells enter G2. In addition, formation of irradiation-induced replication protein A (RPA)-coated single-stranded DNA (RPA–ssDNA), a structure required for ATM and Rad3-related (ATR)–Chk1 activation, occurs in a broadly similar pattern. Cyclin-dependent kinase (Cdk) activity is thought to promote RPA–ssDNA formation by stimulating DNA strand resection at double-strand breaks (DSBs), providing one possible mechanism of imposing cell cycle dependence on DNA damage signaling. However, it has recently been shown that Chk1 itself is also subject to Cdk-mediated phosphorylation at serines 286 and 301 (S286 and 301). We show that Chk1 S301 phosphorylation increases as cells progress through S and G2 and that both Cdk1 and Cdk2 are likely to contribute to this modification in vivo. We also find that substitution of S286 and S301 with non-phosphorylatable alanine residues strongly attenuates DNA damage-induced Chk1 activation and G2 checkpoint proficiency, but does not eliminate the underlying cell cycle dependence of Chk1 regulation. Taken together, these data indicate that Cdk activity regulates multiple steps in the DNA damage response pathway including full activation of Chk1 and checkpoint proficiency.

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Acknowledgements

We wish to thank Cancer Research UK (CR-UK) and the Medical Research Council of the United Kingdom (MRC) for financial support. N Xu and Y Lao were the recipients of CR-UK China Fellowships, S Libertini was the recipient of a Marie Curie Fellowship. We thank Helfrid Hochegger for many stimulating discussions.

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Authors and Affiliations

  1. Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK

    N Xu, S Libertini, E J Black, Y Lao, M Walker & D A Gillespie

  2. Division of Life Science, Tsinghua University, Graduate School at Shenzhen, Shenzhen, PR China

    N Xu

  3. Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton East Sussex, UK

    N Hegarat

  4. College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, UK

    D A Gillespie

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  1. N Xu
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  2. S Libertini
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  3. E J Black
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  6. M Walker
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Correspondence to N Xu or D A Gillespie.

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Xu, N., Libertini, S., Black, E. et al. Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage. Oncogene 31, 1086–1094 (2012). https://doi.org/10.1038/onc.2011.310

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