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Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1

Nature Cell Biology volume 2, pages 762–765 (2000)Cite this article

Abstract

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase1. The Cds1 (Chk2) tumour-suppressor protein2 has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to γ-irradiation3,4,5. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon γ-ionizing radiation5,6,7. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo4 and can phosphorylate Cds1 in vitro8, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.

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Figure 1: Damage-induced and ATM-dependent phosphorylation of Cds1.
Figure 2: Mutation at threonine 68 reduces Cds1 activity in vivo but not in vitro.
Figure 3: Mutation of threonine 68 reduces damage-induced activation and phosphorylation of Cds1 in vivo.

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Acknowledgements

We thank T. Halezonetis for advice on transfection of U2OS cells, and S. Elledge and members of the Scripps cell-cycle group for encouragement and advice. This work was funded by the National Cancer Institute.

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Author notes

  1. Alessandra Blasina

    Present address: Agouron Pharmaceuticals, 4245 Sorrento Valley Boulevard, San Diego, California, 92121, USA

Authors and Affiliations

  1. Department of Molecular Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Roberta Melchionna, Xiao-Bo Chen, Alessandra Blasina & Clare H. McGowan

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  1. Roberta Melchionna
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Correspondence to Clare H. McGowan.

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Correspondence and requests for materials should be addressed to C. H. M.

Supplementary information

Figure S1

Phophorylation of Cds1 by DNA-PK in vitro. (PDF 101 kb)

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Melchionna, R., Chen, XB., Blasina, A. et al. Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1. Nat Cell Biol 2, 762–765 (2000). https://doi.org/10.1038/35036406

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