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An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

Nature Cell Biology volume 11, pages 278–285 (2009)Cite this article

Abstract

Activation of the protein kinases ATM and ATR following chromosomal breakage prevents initiation of DNA replication and entry into mitosis. However, the effects of ATM and ATR activation in cells already progressing through mitosis are poorly understood. Here we report that ATM and ATR activation induced by DNA double-strand breaks (DSBs) inhibits centrosome-driven spindle assembly in Xenopus laevis mitotic egg extract and somatic cells, delaying mitotic progression. Using a cDNA expression library to screen for ATM and ATR substrates, we identified centrosomal protein CEP63 as an ATM and ATR target required for normal spindle assembly. ATM and ATR phosphorylate Xenopus CEP63 (XCEP63) on Ser 560 and promote its delocalization from the centrosome. Suppression of ATM and ATR activity or mutation of XCEP63 Ser 560 to Ala prevented spindle assembly defects. Consistently, inactivation of the CEP63 gene in avian DT40 cells impaired spindle assembly and prevented ATM- and ATR-dependent effects on mitosis. These data indicate that ATM and ATR control mitotic events in vertebrate cells by targeting CEP63 and centrosome dependent spindle assembly.

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Figure 1: Effects of ATM and ATR activation on spindle assembly.
Figure 2: Effects of ATM and ATR activation on mitotic kinases and anastral spindles.
Figure 3: Identification and characterization of the ATM and ATR target XCEP63.
Figure 4: The function of XCEP63 in spindle assembly and in the ATM- and ATR-dependent checkpoint.
Figure 5: Characterization of CEP63 in DT40 cells.

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Acknowledgements

We thank Tim Hunt, members of Clare Hall Laboratories and of the Genome Stability Unit for their comments. We thank H Mahbubani and J Kirk for technical support with Xenopus laevis. This work was funded by Cancer Research UK. V. Costanzo is also supported by the Lister Institute of Preventive Medicine, the European Research Council (ERC) start up grant and the EMBO Young Investigator Program (YIP). A. Vindigni thanks AIRC for its support.

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Authors and Affiliations

  1. Genome Stability Unit, London Research Institute, Clare Hall Laboratories, South Mimms, London, EN6 3LD, UK

    Eloise Smith, Donniphat Dejsuphong, Alessia Balestrini & Vincenzo Costanzo

  2. Department of Radiation Genetics, Kyoto University Graduate, School of Medicine, Yoshidakonoe, Sakyo-ku, Kyoto, 606-8501, Japan

    Donniphat Dejsuphong & Shunichi Takeda

  3. ICGEB (International Centre for Genetic Engineering and Biotechnology) Padriciano, Trieste, 9934012, Italy

    Martin Hampel & Alessandro Vindigni

  4. Applied Biosystems, Frankfurter Darmstadt, Strasse 129 B, 64293, Germany

    Christof Lenz

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Contributions

E.S, D.D. and A.B. performed the experiments and analysed the data in the Xenopus system and in avian DT40 cells; M.H., C.L. and A.V. provided technical and conceptual advice with mass spectrometry; S.T. provided technical and conceptual advice with avian DT40 cells; V.C. planned the experiments and wrote the manuscript.

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Correspondence to Vincenzo Costanzo.

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Smith, E., Dejsuphong, D., Balestrini, A. et al. An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63. Nat Cell Biol 11, 278–285 (2009). https://doi.org/10.1038/ncb1835

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