Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites

Abstract

Ataxia telangiectasia mutated (ATM) plays a critical role in the cellular response to DNA damage. In response to DNA double-strand breaks (DSBs), ATM is autophosphorylated at serine 1981. Although this autophosphorylation is widely considered a sign of ATM activation, it is still not clear if autophosphorylation is required for ATM functions including localization to DSBs and activation of ATM kinase activity. In this study, we show that localization of ATM to DSBs is differentially regulated with the initial localization requiring the MRE11-RAD50-NBS1 complex and sustained retention requiring autophosphorylation of ATM at serine 1981. Autophosphorylated ATM interacts with MDC1 and the latter is required for the prolonged association of ATM to DSBs. Ablation of ATM autophosphorylation or knock-down of MDC1 protein affects the ability of ATM to phosphorylate downstream substrates and confer radioresistance. Together, these data suggest that autophosphorylation at serine 1981 stabilizes ATM at the sites of DSBs, and this is required for a proper DNA damage response.

Figure 1.

The MRN complex is essential for ATM recruitment to laser-induced DNA double-strand breaks. (A) Accumulation of YFP-ATM wt at laser-induced DNA double-strand breaks. Time-lapse imaging of YFP-ATM wt expressing ATM-deficient AT5BIVA (AT5) and HT1080 cells before and after micro-irradiation. (B) Autophosphorylation of YFP-ATM wt at DSBs. YFP-ATM wt–expressing AT5 and HT1080 cells were micro-irradiated, incubated for 10 min, fixed, and co-immunostained with phospho-specific antibodies to ATM (S1981) and γH2AX. (C) Kinetics of relative fluorescence of YFP-ATM wt at the DSBs after micro-irradiation in MRN knock-down cells. YFP-ATM wt–expressing HT1080 cells were transfected with control, MRE11, NBS1, or RAD50 siRNA for 72 h and micro-irradiated. (D) Accumulation of YFP-ATM wt in NBS1-deficient NBS and the NBS1-complemented (NBS+NBS1-FL) cells. YFP-ATM wt–expressing NBS and NBS+NBS1-FL cells were micro-irradiated, incubated for 10 min, fixed, and co-immunostained with phospho-specific antibodies to γH2AX (left). Kinetics of relative fluorescence of YFP-ATM wt at the DSBs after micro-irradiation in NBS and NBS-FL cells (right). Error bars represent the SD. Bars, 10 µm.

Figure 2.

Ablation of autophosphorylation at serine 1981 of ATM accelerates dissociation of ATM from DSBs. (A) Accumulation of autophosphorylation mutant ATM (S1981A) at DSBs. AT5 cells expressing YFP-ATM wt or YFP-ATM S1981A were micro-irradiated, incubated for 10 min, fixed, and immunostained with phospho-specific antibody to ATM (S1981). Initial accumulation kinetics (B) and 2-h time course (C) of YFP-ATM wt or YFP-ATM S1981A at laser-generated DSBs. Error bars represent the SD. (D) Biochemical retention assay in AT5 cells expressing YFP-ATM wt or YFP-ATM S1981A. Cells were irradiated with 10 Gy of γ-ray. At 10 min or 1 h after irradiation, the cells were biochemically fractionated as described in the Materials and methods. Equal amounts of protein from soluble fraction (Fraction I) and chromatin enriched fraction (Fraction III) were separated by 6% SDS-PAGE and immunoblotted for ATM and SMC1. (E) Immunostaining of AT5 cells expressing YFP-ATM wt or YFP-ATM S1981A after 4 Gy of γ-ray. Cells were preextracted and fixed at 10 min (left) or 2 h (right) after irradiation and immunostained with a phospho-specific antibody to γH2AX. Bars, 10 µm.

Figure 3.

Effect of autophosphorylation at serine 1981 on ATM kinase activity. AT5 cells stably expressing YFP-ATM wt or YFP-ATM S1981A were irradiated with 4 Gy of IR and incubated for 10 min or 1 h. Whole-cell extracts were prepared and analyzed by Western blotting for ATM and downstream substrates SMC1, pS966-SMC1, KAP1, pS824-KAP1, and pS15-p53.

Figure 4.

Ablation of ATM phosphorylation at serine 1981 results in a reduced interaction with MDC1. (A) ATM interacts with MDC1 after DNA damage. Lysates were prepared from AT5 cells stably expressing YFP-ATM wt after mock or γ-ray irradiation (10 Gy, 1 h) and immunoprecipitated using an anti-FLAG antibody. A nonimmune species-matched antibody was used as a control. Western blot analysis was then performed using antibodies against pS1981-ATM, ATM, MDC1, and RAD50. (B) Interaction between ATM and MDC1 is mediated by autophosphorylation at serine 1981 of ATM. Lysates were prepared from AT5 cells stably expressing YFP-ATM wt or YFP-ATM S1981A and immunoprecipitated as described in A. Expression levels of ATM, MDC1, and RAD50 in cell lysates (bottom) indicate an equal input in each lane. (C) Interaction between ATM and MDC1 is mediated by the FHA domain of MDC1. AT5 cells stably expressing YFP-ATM wt or YFP-ATM S1981A were irradiated with 10 Gy of γ-ray and incubated for 1 h. Nuclear extracts were prepared from the cells and each protein was immunoprecipitated using an anti-FLAG antibody. Immunoprecipitates were untreated or treated with λ-phosphatase and then incubated with either purified GST or the GST-FHA domain of MDC1. Western blot analysis was performed for ATM and GST. (D) Purified ATM was incubated with either purified GST or the GST-FHA domain of MDC1. Western blot analysis was performed for ATM and GST.

Figure 5.

MDC1 is required for ATM retention at the damage sites. (A) Effect of MDC1 on behavior of ATM at DSBs. Kinetics of relative fluorescence of YFP-ATM at the DSBs after micro-irradiation. AT5 cells stably expressing YFP-ATM wt or YFP-ATM S1981A were transfected with control or MDC1 siRNA for 72 h and micro-irradiated. Error bars represent the SD. (B) MDC1 localization in AT5 cells expressing YFP-ATM wt or YFP-ATM S1981A. Cells were preextracted and fixed at 10 or 60 min after micro-irradiation and immunostained with antibodies against MDC1 and γH2AX. Bars, 10 µm.

Figure 6.

ATM retention at DSBs is independent of NBS1 or RNF8. (A) NBS cells complemented with NBS1 full length (NBS-FL) or FHA deletion form of NBS1 (NBS1-ΔFHA) were micro-irradiated. After 1 h incubation, cells were fixed with or without preextraction and immunostained with antibodies against NBS1 or γH2AX. (B) NBS cells complemented with NBS1-FL or NBS1-ΔFHA were transfected with YFP-ATM wt or YFP-ATM S1981A and micro-irradiated. Cells were fixed at the indicated time and immunostained with antibodies against MDC1 or γH2AX. (C) AT5 cells expressing YFP-ATM wt were transfected with control or RNF8 siRNA for 72 h and micro-irradiated. Cells were fixed at the indicated time and immunostained with antibodies against MDC1 or γH2AX. (D) 2-h time course of YFP-ATM wt at laser-generated DSBs in control or RNF8 siRNA-transfected cells. Error bars represent the SD. Bars, 10 µm.

Figure 7.

MDC1 depletion recapitulates the effects of S1981A mutation. (A) Effect of MDC1 on ATM kinase activity. AT5 cells stably expressing YFP-ATM wt were irradiated with 4 Gy of IR and incubated for 10 min or 1 h. Whole-cell extracts were prepared and analyzed by Western blotting for MDC1 and ATM downstream substrates SMC1, pS966-SMC1, KAP1, pS824-KAP1, and pS15-p53. (B) Inability of S1981A mutant ATM to correct radiosensitivity of ATM-deficient cells. Survival assay of AT5 and its derivatives stably expressing YFP-ATM wt and YFP-ATM S1981A. Cells were transfected with control or MDC1 siRNA for 72 h and then exposed to 1, 2, 3, or 5 Gy of γ-ray. Colonies were counted after 14 d incubation. Error bars represent the SD. Each point represents an average of three independent experiments.