A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM

Abstract

The ataxia telangiectasia mutant (ATM) protein kinase regulates the cell's response to DNA damage through the phosphorylation of proteins involved in cell-cycle checkpoints and DNA repair. However, the signal-transduction pathway linking DNA strand breaks to activation of ATM's kinase activity is not clearly defined. Here, we demonstrate that DNA damage induces the rapid acetylation of ATM. This acetylation depends on the Tip60 histone acetyltransferase (HAT). Suppression of Tip60 blocks the activation of ATM's kinase activity and prevents the ATM-dependent phosphorylation of p53 and chk2. Further, inactivation of Tip60 sensitizes cells to ionizing radiation. ATM forms a stable complex with Tip60 through the conserved FATC domain of ATM. The interaction between ATM and Tip60 is not regulated in response to DNA damage. Instead, the HAT activity of the ATM-Tip60 complex is specifically activated by DNA damage. Furthermore, this activation of Tip60 by DNA damage and the recruitment of the ATM-Tip60 complex to sites of DNA damage is independent of ATM's kinase activity. The results demonstrate that the Tip60 HAT plays a key role in the activation of ATM's kinase activity in response to DNA damage.

Fig. 1.

Activation of ATM requires Tip60's HAT activity. (a) HeLa cells were exposed to bleomycin (5 μM) or IR (30 min). Cell extracts were immunoprecipitated with IgG (lane 1) or ATM antibody (lanes 2–8). Western blotting (WB) was used to detect ATM, acetylated ATM (AcLys), or autophosphorylated ATM (phospho-Ser 1981). (b) HeLa cells, HeLa cells expressing Tip60^wt, or a HAT-inactive Tip60^mt were examined for acetylation and phosphorylation of ATM after exposure to bleomycin (5 μM for 30 min). (c) ATM kinase activity was measured in HeLa (H), HeLa^Tip60wt (W) or HeLa^Tip60mt (M) cells. Controls using IgG (A) or in which the peptide was omitted (P) were included. Shown are results (±SEM) (n = 6). (d) HeLa cells were irradiated and clonogenic cell-survival assays carried out. In HeLa^ATM601 cells, ATM expression is silenced by stable expression of ATM-specific short hairpin RNAi (18). ▪, HeLa^Tip60wt; •, HeLa^Tip60mt; ○, HeLa^ATM601. Shown are results (±SEM) (n = 6).

Fig. 2.

Depletion of Tip60 by siRNA attenuates ATM activation. (a) The 293T cells were untreated (-), transfected with siRNA targeting GFP, or Tip60 (T3 and T4). ATM and Tip60 were detected by Western analysis. (b) The 293T cells were transfected with GFP siRNA or Tip60 siRNAs T3 or T4. At 48 h, cells were exposed to bleomycin (5 μM for 30 min), immunoprecipitated with ATM antibody and ATM acetylation (AcLys), and autophosphorylation (phospho-Ser 1981) was measured. (c) The 293T cells were transiently transfected with GFP or Tip60 siRNA. At 48 h, cells were exposed to solvent (-) or bleomycin (5 μM). p53, chk2, or their phosphorylated isoforms were detected by Western analysis. (d) HeLa cell extracts were immunodepleted of Tip60 by three consecutive immunoprecipitations with Tip60 antibody (T, lanes 1–3), followed by immunoprecipitation with ATM antibody (A, lane 4) to detect unbound ATM. The opposite experiment, in which extracts were immunodepleted of ATM (A, lanes 5–7), followed by immunoprecipitation with Tip60 antibody (T, lane 8) to detect unbound Tip60, was also carried out. (e Left) Cell extracts were immunoprecipitated with IgG or TRRAP antibody, and levels of TRRAP and Tip60 were detected by Western blot analysis. (Right) Cell extracts were immunodepleted with IgG (-) or TRRAP antibody and immunoprecipitated with IgG (-), ATM antibody, or TRRAP antibody. ATM was measured by Western blot analysis.

Fig. 3.

DNA damage activates Tip60's HAT activity. (a) To obtain total Tip60 HAT activity, HeLa cells were immunoprecipitated with Tip60 antibody. To obtain bound Tip60 HAT activity, cells were immunoprecipitated with ATM antibody. Free Tip60 was then obtained by immunoprecipitating ATM-depleted extracts with Tip60 antibody. Washed immunoprecipitates were incubated with a peptide derived from the N terminus of histone H4 and acetylation monitored by using a modified ELISA. Controls in which IgG was substituted for Tip60 antibody or the peptide was omitted from the assay are included. (b) ATM-negative GM5849 cells expressing vector, ATM, or ATMkd were exposed to solvent (-) or bleomycin (5 μM for 30 min) and ATM protein levels, ATM acetylation, and ATM autophosphorylation were measured. (c) GM5849 cells expressing vector, ATM, or ATMkd were exposed to bleomycin and ATM immunoprecipitated with ATM antibody. ATM-associated HAT activity was measured as above. Shown are results (±SEM) (n = 6).

Fig. 4.

Formation of the ATM–Tip60 complex requires the FATC domain of ATM. (a) FATC domains of ATM, Atr, TRRAP, and DNA-PKcs, comprising the last 33 amino acids at the C terminus of each of the proteins. Mutations inserted into the FATC domain are shown. blue box, identical; red box, similar. (b Upper) GM5849 cells expressing mutations in the FATC domain were immunoprecipitated with ATM antibody and the levels of ATM and associated Tip60 protein measured. (Lower) GM5849 cells expressing the indicated construct were exposed to solvent (-) or bleomycin (5 μM for 30 min). ATM protein, ATM acetylation, and ATM autophosphorylation were detected by Western blot analysis. (c) To obtain total Tip60 HAT activity, cells were immunoprecipitated with Tip60 antibody. To obtain bound Tip60 HAT activity, cells were immunoprecipitated with ATM antibody. Controls using IgG or omitting the peptide are shown. (d) GM5849 cells expressing the indicated construct were irradiated and colony formation assay carried out. ○, vector; •, ATM; □,= ATM^FATC#1; ▿, ATM^FATC#2. Shown are results (±SEM) (n = 3). Error bars are shown when larger than symbol size.

Fig. 5.

ATM and Tip60 colocalize to IRIF. (a) HeLa cells expressing HA-Tip60 were irradiated and allowed to recover for 20 min. Cells were fixed and immunostained with HA antibody (to detect Tip60) or phospho-Ser 1981 antibody to detect activated ATM. (b) GM5849 AT cells expressing ATM, ATMkd, ATM^FATC#1 (FATC1), or ATM^FATC#2 (FATC2) were irradiated (0.5 Gy) and allowed to recover for 20 min. Cells were fixed and immunostained with HA antibody (to detect Tip60) and phospho-Ser 1981 antibody (to detect activated ATM).