Abstract
Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition.
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Acknowledgements
We thank Drs Michael B Kastan and Cheryl L Walker for providing Flag-tagged ATM plasmid and Dr Jennifer L Hsu for editing the manuscript. This study was funded in part by the following: National Institutes of Health (CA109311, CA099031, and CCSG CA16672); The University of Texas MD Anderson-China Medical University and Hospital Sister Institution Fund (to M-C H); Ministry of Health and Welfare, China Medical University Hospital Cancer Research Center of Excellence (MOHW103-TD-B-111-03); Program for Stem Cell and Regenerative Medicine Frontier Research (NSC102-2321-B-039-001); International Research-Intensive Centers of Excellence (NSC103-2911-I-002-303); Center for Biological Pathways; Competitive Medical Research Fund (CMRF) of the University of Pittsburgh Medical Center (to LL); and National Institutes of Health (AG045545-01 to LL). This work is in memoriam of Mr Tiong Loi Ang for his courageous fight against cancer.
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Supplementary information, Figure S1
ATM is tyrosine phosphorylated at residue 370. (PDF 367 kb)
Supplementary information, Figure S2
Schematic of DR-GFP reporter integrated in U2OS cell and schematic of the KillerRed system in U2OS TRE cells. (PDF 87 kb)
Supplementary information, Figure S3
EGFR co-localizes with γ-H2AX at laser microirradiation-induced DSBs and is required for ATM S1981 autophosphorylation upon IR stimulation. (PDF 340 kb)
Supplementary information, Figure S4
ATM Y370 is a major EGFR-mediated phosphorylation site. (PDF 1043 kb)
Supplementary information, Figure S5
ATM Y370 phosphorylation regulates radiosensitivity. (PDF 255 kb)
Supplementary information, Figure S6
Proposed model showing the role of EGFR in ATM-mediated DNA damage response. (PDF 49 kb)
Supplementary information, Data S1
Materials and Methods (PDF 123 kb)
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Lee, HJ., Lan, L., Peng, G. et al. Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response. Cell Res 25, 225–236 (2015). https://doi.org/10.1038/cr.2015.8
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DOI: https://doi.org/10.1038/cr.2015.8
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