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NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint

Nature Cell Biology volume 11, pages 1247–1253 (2009)Cite this article

Abstract

DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability1,2. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression3,4,5. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for β-TrCP (β-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.

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Figure 1: Identification and validation of genes required for the G2/M checkpoint.
Figure 2: NEK11 is required for the G2/M DNA damage checkpoint.
Figure 3: NEK11 enhances CDC25A degradation and directly phosphorylates CDC25A.
Figure 4: CHK1 directly activates NEK11 through phosphorylation of Ser 273.
Figure 5: Sequential actions of NEK11 and CHK1 accelerate CDC25A degradation after DNA damage.

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Acknowledgements

We thank R. Bernards for the NKI shRNA library, M. Donzelli and L. Busino for reagents and helpful discussions, G. Ossolengo for purification of the antibodies, S. Ronzoni for FACS sorting, S. Bossi for recombinant NEK11 production and P. G. Pelicci for his continuous support. This work was supported by grants from the EU-framework 6 program to INTACT, the Danish Cancer Society, the Novo Nordisk Foundation and the Danish National Research Foundation.

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Authors and Affiliations

  1. Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, Milan, 20135, Italy

    Marina Melixetian & Kristian Helin

  2. Centre for Epigenetics and University of Copenhagen, Ole Maaløes Vej 5,, Copenhagen, 2200, Denmark

    Kristian Helin

  3. Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, Copenhagen, 2200, Denmark

    Ditte Kjærsgaard Klein, Claus Storgaard Sørensen & Kristian Helin

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  1. Marina Melixetian
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  2. Ditte Kjærsgaard Klein
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  3. Claus Storgaard Sørensen
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Contributions

M.M., C.S.S. and K.H. designed the screen. M.M. and C.S.S. performed the screen. D.K.K. performed the experiments shown in Figs 4c and 5b. C.S.S. performed the experiment shown in Fig. 2b. M.M. performed the experiments shown in all the other figures. M.M., C.S.S. and K.H. wrote the manuscript.

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Correspondence to Claus Storgaard Sørensen or Kristian Helin.

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The authors declare no competing financial interests.

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Melixetian, M., Klein, D., Sørensen, C. et al. NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint. Nat Cell Biol 11, 1247–1253 (2009). https://doi.org/10.1038/ncb1969

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