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Previous Article | Next Article 
Molecular and Cellular Biology, December 2007, p. 8502-8509, Vol. 27, No. 24
0270-7306/07/$08.00+0 doi:10.1128/MCB.01382-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
,
Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115
Received 1 August 2007/ Returned for modification 25 August 2007/ Accepted 25 September 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A central question in studying the ATM protein has been to determine the mechanism by which ATM's kinase activity is activated by DNA damage. Activation of ATM's kinase activity is associated with increased autophosphorylation of ATM at multiple sites (23), including serine 1981 (2). This autophosphorylation of ATM is proposed to initiate a dimer-monomer transition and the release of active ATM monomers (2), although the exact contribution of ATM autophosphorylation to ATM activation is still under debate (31). Several additional molecular events, including the Mre11-Rad50-Nbs1 (MRN) DNA binding complex (8, 11, 14, 41) and changes in chromatin structure (2, 4), also contribute to activation of ATM's kinase activity. Mutations in the individual protein components of the MRN complex can reduce or abolish the activation of ATM's kinase activity by DNA damage (8, 11, 14, 41). The recent characterization of a conserved C-terminal domain in Nbs1 which is essential for recruitment of ATM to the MRN complex and for the efficient activation of ATM's kinase activity (14, 42) further supports a key role for MRN in ATM activation. Finally, biochemical studies have shown that MRN can activate ATM's kinase activity in vitro (25, 26), suggesting that inactive ATM is recruited to DSBs by MRN prior to MRN-dependent activation of ATM's kinase activity. However, other studies indicate that disruption of chromatin structure in the absence of DNA damage can activate ATM's kinase activity (2) and that kinase-inactive ATM is not recruited to enzymatically induced DSBs (4), suggesting that ATM is activated in the nucleoplasm by DNA damage-induced changes in chromatin structure. In this case, the MRN complex may function as an adaptor complex which maintains ATM in an active form at the DSB (2, 4, 22). However, the mechanism by which interactions between ATM and MRN or structural changes in chromatin activate ATM's kinase activity is not known.
Previously, we demonstrated that the Tip60 histone acetyltransferase (HAT) is essential for the activation of ATM's kinase activity (19, 37). The HAT activity of Tip60 is increased by DNA damage, and the loss of Tip60 blocks the autophosphorylation and subsequent activation of ATM's kinase activity (37). However, although activation of Tip60's HAT activity is associated with an increase in the overall acetylation level of ATM, neither the acetylation site nor the functional significance of ATM acetylation is known. We now demonstrate that Tip60 directly acetylates ATM on lysine 3016 and that this acetylation is required for activation of ATM's kinase activity. The DNA damage-dependent acetylation of ATM on lysine 3016 by Tip60 is therefore the critical step which links the detection of DSBs to the activation of ATM kinase activity.
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MATERIALS AND METHODS |
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Mutagenesis. Two overlapping fragments at the C terminus of ATM were subcloned from pcDNA3.1-ATM into pBluescript, creating pBS-SPBA (containing a 3,582-base-pair internal SpeI-BamHI fragment) and pBS-XBXH (containing an approximately 1,800-base-pair XbaI-XhoI fragment carrying the entire 3' end of the cDNA, with the XhoI site located in pcDNA3.1). Mutagenesis was carried out using QuikChange II XL site-directed mutagenesis kits (Stratagene, CA) and the primers listed in Table S1 in the supplemental material. Following sequencing, mutations were released from pBS-SPBA and pBS-XBXH by digestion with SpeI-BlpI and BlpI-XhoI, respectively. The corresponding fragments were then excised from pcDNA3.1-ATM, and the mutated versions were ligated into the plasmid to create full-length ATM constructs.
Immunoprecipitation, Western blot analysis, HAT assay, and kinase assay.
General protocols for lysis buffers, immunoprecipitation, and Western blot analysis were done as described previously (19, 37). ATM immunoprecipitation was routinely performed using ATM antibody PC116 (EMD Biosciences, CA). Cells were lysed in ATM lysis buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.2% Tween 20, 1.5 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 50 mM NaF, 500 µM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 0.1 µg/ml aprotinin, 0.1 µg/ml leupeptin) and cleared by centrifugation. For kinase assays, ATM was immunoprecipitated and washed sequentially in ATM lysis buffer followed by ATM kinase buffer (10 mM HEPES, pH 7.4; 10 mM MgCl2; 50 mM NaCl; 10 mM MnCl2). Immunoprecipitates were incubated in kinase buffer (50 µl) supplemented with 50 µM ATP, p53 peptide (2 µg; EPPLSQEAFADLWKK), and 10 µCi [
-32P]ATP for 15 min at 30°C (37). For HAT assays, ATM or Tip60 was immunoprecipitated and washed sequentially in ATM lysis buffer followed by HAT assay buffer (50 mM Tris, pH 8; 10% glycerol; 0.1 mM EDTA; 1 mM dithiothreitol). Immunoprecipitates were then incubated in HAT assay buffer (60 µl) supplemented with acetyl-coenzyme A (100 µM) and either biotinylated histone H4 peptide, biotinylated atmwt peptide (ERVLMRLQEKLKGVEEGT), or atmKR peptide (ERVLMRLQEALKGVEEGT) (0.5 µg) for 30 min at 30°C. An aliquot of the reaction mix was immobilized on a streptavidin plate, and acetylation was detected using a HAT enzyme-linked immunosorbent assay according to the manufacturer's instructions (Upstate Biotechnology, NY). For both kinase and HAT assays, each datum point represents the average for three separate immunoprecipitations, with each derived from approximately 3 x 107 cells and containing equivalent amounts of protein. Antibodies used were as follows: ATM antibodies PC116 (EMD Biosciences, CA), 5C2, 2C1 (both from Genetex, TX), and pS1981 ATM (Rockland Immunochemicals, PA) and antibodies to ß-actin, chk2, hemagglutinin (HA), myc, phospho-chk2 (all from Cell Signaling, MA), Tip60, acetylated lysine (AcLys; Upstate Biotech, NY), p53, and pS15-p53 (EMD Biosciences, CA). A rabbit polyclonal antibody (AcK6) was raised against peptide CRVLMRLQE[KAc]LKG and affinity purified using immobilized acetylated peptide followed by absorption against immobilized nonacetylated peptide. The resulting antiserum was used at a 1:50 dilution for Western blot analysis.
Immunofluorescence. Cells were seeded on LabTek II chamber slides (Nunc, NY). Cells were fixed as previously described (15) and stained with primary antibody ATM 5C2 (Genetex, TX) followed by immunoglobulin G (IgG)-fluorescein isothiocyanate (Santa Cruz, CA). Slides were mounted with Fluoromount-G (Southern Biotech, AL) and visualized with a Nikon Eclipse TE 2000 microscope.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we demonstrated that the acetylation of ATM (detected using the pan-specific acetyl-lysine antibody used for Fig. 1) was abolished when Tip60 was inactivated using either siRNA (Fig. 1B) (37) or dominant-negative Tip60 proteins (37). Figure 1 demonstrates that this Tip60-dependent acetylation of ATM occurs on lysine 3016. To determine if Tip60 could directly acetylate lysine 3016 of ATM, a peptide corresponding to amino acids 3007 to 3024 of ATM (atmwt) and a control peptide in which lysine 3016 was replaced with arginine (atmKR) were synthesized. HA-Tip60 and HA-Tip60 with point mutations in the HAT domain (Tip60HD) were immunopurified from HeLa cells as described previously (37, 38). As shown in Fig. 2A, Tip60 acetylated both the histone H4 and atmwt peptides, and the ability of Tip60 to acetylate these peptides was increased following exposure to bleomycin (Fig. 2A). The higher levels of acetylation of the H4 peptide reflect the presence of three Tip60 acetylation sites in the histone H4 peptide (21), in contrast to one in the atmwt peptide. When lysine 3016 was replaced with arginine, the atmKR peptide was minimally acetylated by Tip60 and displayed no detectable increase in HAT activity in response to bleomycin. Furthermore, when a Tip60 protein with inactivating mutations in the HAT domain (Tip60HD) was used (18), minimal acetylation of either the H4, atmwt, or atmKR peptide was seen (Fig. 2A). These results demonstrate that Tip60 can directly acetylate a peptide containing lysine 3016 of ATM in vitro. In a further series of experiments, immunopurification of the ATM-Tip60 complex from cells also demonstrated that Tip60 acetylated lysine 3016 in vitro (see Fig. S1 in the supplemental material). Figure 2A therefore demonstrates that Tip60 can directly acetylate peptides containing lysine 3016 of ATM in vitro.
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Previously, we demonstrated that ATM and Tip60 interact and that this interaction requires the FATC domain at the C terminus of ATM (19). Because lysine 3016 is located within the FATC domain of ATM (Fig. 1F), we determined if the mutation of lysine 3016 affected either ATM-Tip60 interactions or activation of Tip60's HAT activity by DNA damage. Immunoprecipitation studies demonstrate that mutation of lysine 3016 does not alter the interaction between ATM and Tip60 (see Fig. S3A in the supplemental material). As shown in Fig. 3A, ATM was immunoprecipitated from ATM-deficient A-T cells complemented with either vector, ATM, or ATMK3016A, and the associated Tip60-dependent HAT activity was measured. In the absence of ATM, minimal Tip60 HAT activity was measured, and this was not increased by bleomycin (Fig. 3A, vector). However, the HAT activities of Tip60 associated with both ATM and ATMK3016A were increased to similar levels in response to bleomycin treatment (Fig. 3A). Mutation of lysine 3016 therefore does not affect either the activation of Tip60's HAT activity or the interaction between ATM and Tip60.
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The inability of ATMK3016R to phosphorylate p53 or chk2 suggests a defect in the kinase activity of ATMK3016R. The kinase activity of ATM was measured by immunoprecipitating ATM and then incubating it with a substrate peptide containing the phosphorylation site of the p53 protein. Immunoprecipitation with IgG or from cells expressing a kinase-inactive ATM (ATMkd) yielded minimal levels of ATM kinase activity (Fig. 3C). ATM had significant basal levels of ATM kinase activity, which were increased threefold following exposure to bleomycin. In contrast, whereas ATMK3016R had the same levels of basal kinase activity as ATM, the kinase activity of ATMK3016R was not increased by bleomycin exposure (Fig. 3C). This implies that the function of acetylation is to activate ATM's kinase activity. To further explore the functional properties of ATMK3016R, we took advantage of previous work demonstrating that ATM and protein phosphatase 2A exist as a complex in the cell (17). Inhibition of protein phosphatase 2A by okadaic acid (OA) leads to rapid accumulation of ATM autophosphorylated on serine 1981, a process requiring ATM's kinase activity. However, this form of ATM does not exhibit elevated kinase activity and is functionally inactive (17). Therefore, we examined if OA treatment of ATMK3016R could induce autophosphorylation of ATM on serine 1981. Figure 3D demonstrates that OA induced the autophosphorylation of both ATM and ATMK3016R on serine 1981. However, only wild-type ATM showed inducible acetylation and autophosphorylation in response to ionizing radiation or combined DNA damage and OA treatment. Mutation of lysine 3016 of ATM therefore does not affect the ability of ATM to undergo autophosphorylation on serine 1981, indicating that substitution of arginine for lysine at amino acid 3016 does not affect the intrinsic kinase activity of ATM. Autophosphorylation of ATM is postulated to be a key step in the production of active ATM (2, 23). This raises the possibility that sequential exposure of ATMK3016R-expressing cells to OA (to induce autophosphorylation of serine 1981) followed by ionizing radiation may bypass the need for acetylation, leading to the generation of an active ATM molecule able to phosphorylate key target proteins, including p53. A-T cells expressing wild-type ATM did not exhibit p53 phosphorylation following OA treatment but displayed robust p53 phosphorylation after either ionizing radiation or ionizing radiation plus OA (Fig. 3D), as previously shown (17). However, OA treatment of ATMK3016R cells, either alone or in combination with ionizing radiation, did not generate an active ATM molecule able to phosphorylate p53 in vivo (Fig. 3D). Figure 3D therefore clearly shows that ATMK3016R retains the ability to undergo autophosphorylation on serine 1981 in response to OA exposure but does not undergo autophosphorylation in response to DNA damage signals.
Next, we determined if the failure to activate the kinase activity of ATMK3016R was reflected in a loss of functional activity. First, the ability of ATMK3016R to complement the increased radiosensitivity of A-T cells was examined. As shown in Fig. 4A, the expression of ATM or ATMK3018R complemented the radiosensitivity of A-T cells, whereas expression of ATMK3016R had no effect, consistent with the lack of activation of the kinase activity of ATMK3016R and its inability to phosphorylate key target proteins. Autophosphorylation of ATM is proposed to initiate a dimer-monomer transition, releasing active ATM monomers (2, 23). To determine the contribution of acetylation to the dimer-monomer transition, the ability of ATM, ATMkd, and ATMK3016A to undergo dimer-monomer conversion was examined. A-T cells were stably transfected with equimolar ratios of HA-ATM and myc-ATM expression vectors, and the dimer-monomer transition was monitored by examining dissociation of the HA-ATM/myc-ATM dimer by coimmunoprecipitation techniques. Figure 4B demonstrates the coexpression of HA-ATM and myc-ATM in A-T cells. In some experiments, a nonspecific band of 340 kDa was detected for the ATM-negative A-T cells by the HA and myc antibodies (Fig. 4B and C); however, this band was not detected with ATM antibodies (Fig. 1 and 2). Following exposure to bleomycin, wild-type ATM, but not ATMkd or ATMK3016A, was autophosphorylated on serine 1981. Similarly, wild-type ATM, but not ATMK3016A, was acetylated. Interestingly, ATMkd, which does not possess kinase activity and does not autophosphorylate (40), was also acetylated. Immunoprecipitation with HA antibody brought down both HA-ATM and myc-ATM in all unstimulated cells (Fig. 4C). Following exposure to bleomycin, wild-type HA-ATM no longer interacted with myc-ATM, whereas both ATMkd and ATMK3016A remained in the dimeric form. Importantly, since ATMkd was still acetylated on lysine 3016, this indicates that acetylation on its own is not sufficient for initiating the dimer-monomer transition of the ATM protein, suggesting that additional events, such as autophosphorylation of ATM, may also be required (2, 23). The AcK6 anti-acetyl K3016 antiserum was not competent for immunofluorescence, precluding its use for monitoring recruitment of the acetylated ATM to DSBs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results indicate that mutation of lysine 3016 blocks the activation of ATM's kinase activity and subsequent autophosphorylation and dimer-monomer transition. Furthermore, ATMK3016R does not phosphorylate downstream target proteins or complement the increased radiosensitivity of A-T cells. These results are consistent with an essential role for acetylation in the activation of ATM kinase activity. A potentially confounding problem is that mutation of lysine 3016, within the FATC domain of ATM, may have a negative impact on the correct function of the kinase domain independent of the loss of acetylation. However, we carried out several controls which suggest that this is not the case, as follows: (i) ATMK3016R retains normal basal kinase activity and lacks only inducible activation; (ii) unlike many mutations in the kinase domain identified in A-T patients, substituting lysine 3016 for arginine does not destabilize ATM or alter its intrinsic kinase activity; (iii) introducing a lysine-to-arginine mutation at an adjacent lysine (amino acid 3018) does not affect ATM function, indicating that the region of ATM containing lysine 3016 can tolerate small changes in protein architecture; and (iv) ATMK3016R can still undergo autophosphorylation on serine 1981 following the addition of OA, indicating that mutation of lysine 3016 to arginine does not disrupt the critical intermolecular protein-protein interactions within the ATM dimer which are required for the autophosphorylation of ATM by the kinase domain (2). Taken together, we interpret these results to support a model in which the failure of ATMK3016R to increase its kinase activity in response to DNA damage is due to a loss of acetylation of lysine 3016 rather than to nonspecific effects related to the lysine-to-arginine substitution. Thus, the primary function of acetylation of lysine 3016 is to upregulate ATM's kinase activity.
Insights into how acetylation may regulate the intrinsic kinase activity of ATM are provided by recent structural studies on ATM and the structurally related DNA-PKcs protein. PIKK protein family members, including ATM and DNA-PKcs, contain a conserved FAT domain/kinase domain/FATC domain structure (6). Low-resolution structural modeling of the DNA-PKcs and ATM proteins indicates that the FAT and FATC domains protrude from either side of the kinase domain (27, 33, 36). The FAT domain is weakly conserved between family members, whereas the kinase and FATC domains are highly conserved (1). FATC domains are highly flexible 
- helical structures (10, 33) and are required for the kinase activities of the mTor (39), DNA-PKcs (3, 32), and ATM (19) proteins. Studies indicate that the interaction between DNA-PK and DNA induces conformational changes in the FATC and FAT domains (33, 36), and these conformational changes are predicted to influence the catalytic activity of DNA-PK and ATM (33). Acetylation of lysine 3016 within the flexible FATC domain of ATM may therefore influence ATM kinase activity. This is further supported by reports that a tumor-derived mutation near the kinase domain of the ATM-like p110 phosphatidylinositol 3-kinase subunit results in constitutive kinase activity (20, 34). Sequence alignment of ATM and p110 indicates that one of these p110 mutations (at amino acid 1054) occurs in the same relative position as lysine 3016 of ATM. This implies that the C-terminal regions of p110 and ATM may be essential for regulating kinase activity. Based on the results presented here and the results of structural studies (10, 33, 36), we propose that acetylation of lysine 3016, which is located at the junction between the FATC and kinase domains, alters the conformation of the FATC domain. This altered conformation of the FATC domain could allow substrate proteins access to the kinase domain as well as positively regulating the intrinsic kinase activity of the kinase domain. Further structural studies are required to address this point.
A key question is to address the mechanistic links between ATM acetylation and autophosphorylation. The autophosphorylation of serine 1981 of ATM was originally proposed to be essential for ATM activation and to play a key role in initiating the dimer-monomer transition and the release of active ATM monomers (2). However, recent biochemical studies have shown that in vitro activation of ATM kinase activity can be achieved in the absence of significant serine 1981 autophosphorylation (12, 25). In addition, a mouse model expressing a mutation in the mouse equivalent of serine 1981 of ATM (serine 1987) displays normal ATM function. These observations indicate that autophosphorylation of serine 1981 is dispensable for ATM function under some conditions (31). Recently, several additional ATM autophosphorylation sites were identified (23). Mutation of these sites impaired the ability of ATM to phosphorylate downstream targets and to correct the radiosensitive phenotype of A-T cells (23). Therefore, although some studies demonstrate that autophosphorylation of serine 1981 is dispensable for ATM activity (12, 25, 31, 42), the identification of additional ATM autophosphorylation sites raises the possibility that, similar to the case for DNA-PKcs (5, 9), multiple ATM autophosphorylation sites contribute to ATM activation. The results presented here indicate that the main function of ATM acetylation is to activate ATM's kinase activity. In this model, acetylation of ATM precedes and is required for the activation of ATM's kinase activity. Once activated, ATM kinase activity induces autophosphorylation of ATM at multiple sites, including serine 1981 (2, 23), and subsequent conversion of ATM dimers to monomers. Thus, while ATM autophosphorylation and subsequent dimer-monomer transitions may be important for achieving full ATM activation, the results presented in this report indicate that acetylation of ATM, rather than autophosphorylation, is the key step in upregulating ATM's kinase activity.
The results reported here and in previous works from us (19, 37) and other groups (13) demonstrate that Tip60 plays a key role in the acetylation and activation of ATM and indicate a central role for Tip60 in the early events leading to activation of ATM's kinase activity. However, other significant contributors to ATM activation include both the MRN complex (8, 11, 22, 25, 41) and changes in chromatin structure (2). A wide range of experimental approaches have established that the MRN complex plays a key role in several aspects of ATM activation, including the recruitment of ATM to DSBs (14, 42) and modulation of the activation of ATM's kinase activity by DSBs (8, 22, 25, 26, 41). This raises the possibility that MRN may regulate ATM activation by influencing the HAT activity of Tip60, either through direct interactions between MRN and Tip60 or through positioning of the ATM-Tip60 complex at DSBs. Tip60 also contains a chromodomain, a protein domain with the potential to interact with methylated lysine residues on histones (7). Changes in chromatin structure following ionizing radiation (either localized to the DSB or generated over large chromatin domains) may expose methylated histones as potential binding sites for Tip60's chromodomain. Such interactions between Tip60 and altered chromatin structures adjacent to DSBs may be essential for upregulating Tip60's HAT activity and for the acetylation and activation of the ATM protein. Tip60 may therefore provide the key step which links previous observations on the relative contributions of the MRN complex, chromatin structural changes, and ATM acetylation with the activation of the ATM protein kinase by DNA damage.
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ACKNOWLEDGMENTS |
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We thank Susan Lees-Miller and Sheng-Chung Lee for helpful discussions.
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FOOTNOTES |
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Published ahead of print on 8 October 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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REFERENCES |
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), ATMK3016R (
), or ATMK3018R (
) 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 ATMK3016A 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.