DNA damage-induced acetylation of lysine 3016 of ATM activates ATM kinase activity

Abstract

The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-lysine 3016 demonstrate rapid (within 5 min) in vivo acetylation of ATM following exposure to bleomycin. Furthermore, lysine 3016 of ATM is a substrate in vitro for the Tip60 histone acetyltransferase. Mutation of lysine 3016 does not affect unstimulated ATM kinase activity but does abolish upregulation of ATM's kinase activity by DNA damage, inhibits the conversion of inactive ATM dimers to active ATM monomers, and prevents the ATM-dependent phosphorylation of the p53 and chk2 proteins. These results are consistent with a model in which acetylation of lysine 3016 in the FATC domain of ATM activates the kinase activity of ATM. The acetylation of ATM on lysine 3016 by Tip60 is therefore a key step linking the detection of DNA damage and the activation of ATM kinase activity.

FIG. 1.

Lysine 3016 of ATM is acetylated after DNA damage. (A) 293T cells were transfected with siRNA targeting GFP or Tip60. Extracts were immunoprecipitated with either IgG (lane 1), Tip60 antibody (lanes 2 and 3, upper panel), or ATM antibody (lanes 2 and 3, lower panel). ATM and Tip60 were detected by Western blotting (WB). (B) 293T cells were transiently transfected with siRNA targeting either GFP (siGFP) or Tip60 (siTip60). Forty-eight hours later, cells were exposed to solvent (−) or bleomycin (5 μM for 30 min). Cell extracts were immunoprecipitated with IgG (lane 1) or anti-ATM antibody PC116 (lanes 2 to 5), and ATM protein levels (ATM), ATM acetylation (AcLys), and autophosphorylation (pS1981) of ATM were measured by Western blot analysis. (C) ATM-deficient GM5849 A-T cells transfected with vector, ATM, or ATM mutant K2 (K1772/1773A), K6 (K2204/2207A), or K17 (K3016/3018A) were exposed to bleomycin (+; 5 μM) for 30 min. ATM was immunoprecipitated with ATM antibody PC116, and ATM and ATM acetylation (AcLys) were measured by Western blotting. (D and E) GM5849 A-T cells transfected with vector, ATM, or ATMs with the indicated point mutations were exposed to bleomycin (+; 5 μM) for 30 min. ATM was immunoprecipitated, and Western blot analysis was used to monitor ATM levels, ATM acetylation (AcLys), and ATM autophosphorylation on serine 1981 (pS1981). (F) Location and sequence comparison of ATM acetylation sites.

FIG. 2.

Lysine 3016 of ATM is acetylated in vivo. (A) Tip60 was immunopurified from HeLa cells expressing either HA-Tip60 (Tip60) or HAT-inactive HA-Tip60 (Tip60^HD). Cell extracts were incubated with either HA antibody or IgG and washed as described in Materials and Methods to immunopurify Tip60. The ability of immunopurified Tip60 to acetylate peptides derived from either ATM or the N terminus of histone H4 was determined. Peptides used were as follows: amino acids 3007 to 3024 of ATM (atm^wt; ERVLMRLQEKLKGVEEGT); amino acids 3007 to 3024 of ATM, with lysine 3016 replaced with arginine (atm^KR; ERVLMRLQERLKGVEEGT); or amino acids 1 to 21 of histone H4 (SGRGKGGKGLGKGGAKRHRKV). All peptides contained a C-terminal biotin label. Cells were treated for 30 min with bleomycin (+; 5 μM), where indicated. Results are means ± standard deviations (SD) (n = 3). (B) GM5849 A-T cells transfected with vector, ATM, or ATM^K3016A were exposed to bleomycin (+; 5 μM) for the indicated times (minutes). ATM was immunoprecipitated, and Western blot analysis (WB) was used to monitor ATM levels, ATM autophosphorylation on serine 1981 (pS1981), and ATM acetylation, using the K3016Ac-specific antiserum AcK6. (C) 293T cells were irradiated (2 Gy) and allowed to recover for the indicated times (minutes). ATM was immunoprecipitated, and Western blot analysis was used to monitor ATM levels, ATM-pS1981, and ATM acetylation, using the K3016Ac-specific antiserum AcK6.

FIG. 3.

Mutation of lysine 3016 of ATM blocks activation of ATM's kinase activity. (A) GM5849 A-T cells transfected with vector, ATM, or ATM^K3016A were left untreated (−) or exposed to bleomycin (+; 5 μM) for 30 min, and ATM was immunoprecipitated. The HAT activity of the Tip60 associated with ATM was measured using histone H4 peptide as the substrate. Results are means ± SD (n = 3). (B) GM5849 A-T cells transfected with vector, ATM, ATM^K3016R, or ATM^K3018R were exposed to bleomycin (+; 5 μM) for 20 min. p53, phospho-p53 (pS15-p53), chk2, phospho-chk2 (pT68-chk2), and β-actin were detected by Western blotting (WB) of whole-cell extracts. (C) GM5849 A-T cells transfected with vector, ATM, ATMkd, or ATM^K3016R were left untreated (−) or exposed to bleomycin (+; 5 μM) for 30 min, and ATM was immunoprecipitated. The intrinsic kinase activity of ATM was then measured using a p53 peptide (containing serine 15) as the substrate. Results are means ± SD (n = 3). (D) GM5849 A-T cells expressing ATM or ATM^K3016R were preincubated with OA (0.5 μM) or dimethyl sulfoxide (−) for 20 min and then irradiated (IR; 2 Gy), and cell extracts were prepared 40 min later. ATM was immunoprecipitated, and the levels of ATM, acetylated ATM (AcLys), and ATM autophosphorylated on serine 1981 (pS1981) were determined. In addition, whole-cell extracts were probed by Western blot analysis to determine the levels of p53 and p53 phosphorylated on serine 15 (pS15-p53).

FIG. 4.

Lysine 3016 of ATM is required for ATM to regulate radiosensitivity and dimer-monomer transition. (A) GM5849 A-T cells stably transfected with vector (•), ATM (○), ATM^K3016R (▵), or ATM^K3018R (▪) were irradiated, and cell survival was measured using a clonogenic cell survival assay. Results are means ± SD (n = 3). (B) A-T cells were stably cotransfected with HA- and myc-tagged ATM constructs, such that both HA-ATM and myc-ATM were expressed in the same cell. Cell lines coexpressing HA- and myc-tagged ATM, HA- and myc-tagged ATMkd, or HA- and myc-tagged ATM^K3016A were exposed to bleomycin (5 μM for 30 min). Levels of HA-ATM, myc-ATM, pS1981, and acetylation of lysine K3016 (AcK6) were measured. (C) Cells were treated as described above and then immunoprecipitated with HA antibody. Interaction between HA-ATM and myc-ATM was then assessed by Western blot (WB) analysis to detect HA-ATM and coprecipitating myc-ATM.