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ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway

Abstract

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation1,2,3,4. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs5,6, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks3,7. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

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Figure 1: Activation of ATM kinase and the p53 pathway by ionizing radiation occurs in NBS cells.
Figure 2: Ionizing radiation (IR) induces ATM-dependent serine/threonine phosphorylation of p95/nbs1.
Figure 3: Phosphorylation of p95/nbs1 by ATM.
Figure 4: ATM-dependent phosphorylation of p95/nbs1 on Ser 343 in vivo after ionizing radiation.
Figure 5: Mutation of the ATM phosphorylation site in p95/nbs1 results in an S-phase checkpoint defect (RDS).

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Acknowledgements

We thank T. M. Gilmer and K. Kilpatrick (Glaxo-Wellcome) for the mouse ATM monoclonal antibodies; Y. Shiloh for AT complemented cell lines; C. Bakkenist, K. McLure and C. Canman for helpful comments on the manuscript; and D. Woods and A. Justman for technical assistance. This work was supported by grants from the NIH (M.B.K, the St. Jude Cancer Centre Core Grant, J.H.J.P. and J.L.). M.B.K. was also supported by Glaxo-Wellcome and the American Lebanese Syrian Associated Charities (ALSAC) of the St. Jude Children's Research Hospital, and J.H.J.P. by the Milwaukee Foundation.

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Correspondence to Michael B. Kastan.

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Lim, DS., Kim, ST., Xu, B. et al. ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature 404, 613–617 (2000). https://doi.org/10.1038/35007091

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