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Enhanced Phosphorylation of p53 by ATM in Response to DNA Damage

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Science  11 Sep 1998:
Vol. 281, Issue 5383, pp. 1674-1677
DOI: 10.1126/science.281.5383.1674

Abstract

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase–related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

Strand breaks in cellular DNA occur continuously as a consequence of normal processes such as recombination or the infliction of DNA damage. DNA damage triggers several signal transduction pathways that lead either to damage repair coupled with attenuation of cell cycle progression, or to programmed cell death (apoptosis). A junction of such pathways is controlled by the transcription factor p53. After DNA damage, the amount of p53 in cells is increased through posttranscriptional mechanisms and its transactivation activity is enhanced, leading to the activation of downstream genes (1).

The genetic disorder A-T results in genome instability, cerebellar and thymic degeneration, immunodeficiency, gonadal dysgenesis, radiation sensitivity, and predisposition to cancer. A-T cells exhibit acute sensitivity to ionizing radiation and radiomimetic chemicals, and their cell cycle checkpoints fail to be activated after treatment with these agents (2). The responsible gene, ATM, encodes a 370-kD protein with a COOH-terminal domain similar to the catalytic subunit of phosphoinositide 3-kinases (PI 3-kinases) (3). This similarity places ATM within a family of proteins that share the PI 3-kinase–related domain and function in maintenance of genome stability, in cell cycle control, and in cellular responses to DNA damage (4). These proteins appear to be protein kinases rather than lipid kinases, as evidenced by the DNA-dependent protein kinase (DNA-PK) and the mammalian target of rapamycin (mTOR) (5).

To study ATM's catalytic activity, we raised monoclonal antibodies (mAbs) to two ATM-derived peptides, ATM132 (spanning ATM positions 819 to 844) and ATML2 (positions 2581 to 2599). Both mAbs recognize a 370-kD protein in normal cells that is not detected in A-T cells but reappears upon ectopic expression of ATM in them. These mAbs also recognize endogenous, recombinant, and in vitro translated ATM immunoprecipitated by other antibodies to ATM (6). Protein kinase activity was revealed in immunoprecipitates obtained with these antibodies (7) from a normal lymphoblastoid cell line (L-40) when the translation repressor PHAS-I (8) was used as a substrate (Fig. 1A). This activity was not detected with an unrelated antibody, and was retained when ATM was eluted from the beads using an excess of the peptide to which the antibody was raised (9). Phosphorylation of PHAS-I was considerably more intense with immunoprecipitates obtained from G361, a melanoma cell line in which the amount of ATM is greater than that in lymphoblasts (Fig. 1A); it was not observed in immunoprecipitates obtained from an A-T cell line, L-6, that lacks the ATM protein (Fig. 1A). Immunoprecipitates containing recombinant ATM expressed in A-T cells (10) showed kinase activity similar to that of the endogenous protein (Fig. 1B). Unlike DNA-PK, this activity was strictly dependent on manganese ions and was not enhanced by the addition of 200 μg of sheared double-stranded DNA, which considerably stimulated DNA-PK activity (9).

Figure 1

Kinase activity of ATM with PHAS-I as a substrate. (A) Activity of ATM immunoprecipitated from the melanoma cell line G361, the normal lymphoblastoid cell line L-40, and an A-T lymphoblastoid cell line, L-6, lacking the ATM protein. Total cellular extracts (total) or ATM immunoprecipitates (IP) obtained from equal amounts of cells with ATM132 mAb were immunoblotted with ATML2 mAb. Bottom panel: autoradiography of phosphorylated PHAS-I (7). (B) Activity of recombinant ATM stably expressed in an immortalized A-T cell line, AT22IJE-T (10). The cells were transfected with full-length cDNA in the episomal expression vector pEBS7, or with an empty vector. Top panel: Protein immunoblotting analysis of ATM immune complexes. Bottom panel: Kinase activity of ATM (7). (C) Kinase activity of ATM immunoprecipitated from a normal cell line (L-40), an A-T cell line lacking the ATM protein (L-6), an A-T cell line homozygous for the truncation mutation R3047X (9RM), and an A-T cell line homozygous for the missense mutation E2904G (41RM). (D) Effect of wortmannin on ATM-associated kinase activity. ATM immune complexes were incubated with various concentrations of the drug and washed with the reaction buffer before kinase activity was measured. Activity was normalized to the amount of ATM protein obtained from immunoblots.

We also measured ATM-associated kinase activity in two A-T cell lines containing structural ATM mutations that allow expression of the mutant proteins. The cell line 41RM is homozygous for a missense mutation, E2904G (11), that replaces a highly conserved glutamic acid residue with glycine within the PI 3-kinase–related domain that is expected to contain the catalytic site of the kinase. The cell line 9RM is homozygous for a nonsense mutation, R3047X, that removes the 10 COOH-terminal residues of ATM (12). Little or no kinase activity was detected in immunoprecipitates from either cell line (Fig. 1C). Our results indicate that ATM has an intrinsic protein kinase activity.

PI 3-kinases are inhibited by wortmannin, which covalently modifies conserved lysine residues at their catalytic sites (13). ATM-associated kinase activity was inhibited in immune complexes exposed to wortmannin for 30 min (Fig. 1D). The sensitivity of ATM to this drug (median inhibitory concentration IC50 = 100 nM) is comparable to that of other PI 3-kinase–related protein kinases (5).

A known target of DNA damage–induced phosphorylation is the p53 protein (1). Ser15 of p53 undergoes phosphorylation in cells after DNA damage (14,15), and this response is decreased in A-T cells (14). ATM also interacts directly with p53 in vivo (16). Full-length recombinant p53 was phosphorylated in vitro by ATM immunoprecipitates (Fig. 2A). Phosphorylation of p53 fragments and full-length p53 in which both Ser15 and Ser37 had been substituted by alanine residues indicated that Ser15 was the predominant, if not the only, site phosphorylated by ATM (Fig. 2B). Analysis with antibodies raised to p53-derived phosphopeptides containing phosphorylated Ser15, Ser33, or Ser37 (14, 15, 17) (Fig. 2C) confirmed that only Ser15 was phosphorylated by ATM. The effect of wortmannin on this reaction was similar to that obtained with PHAS-I as substrate (Fig. 2D).

Figure 2

Phosphorylation of Ser15 on p53. (A) Kinase activity of ATM immunoprecipitates from L-40 and L-6 cells. Full-length p53 (top panel) and two NH2-terminal fragments of p53 [see (B)] were used as substrates. (B) Phosphorylation of various p53 derivatives by ATM. Numbers denote the position of the terminal residues of each fragment. A15A37, Ser15 and Ser37 substituted by alanines; (P), phosphorylation; +, phosphorylation of the substrate; –, signal undetectable or <10% of that obtained with full-length p53. (C) Protein immunoblotting of the reaction products obtained by phosphorylating the p53-N47 fragment. Antibodies to phosphorylated Ser15, Ser33, and Ser37 were used in the top panel. Antibody DO1 to an epitope spanning amino acids 2 to 25 of p53 was used to detect the N47 fragment (bottom panel). +, samples taken after a phosphorylation reaction; –, the same substrate exposed to a mock reaction. (D) Effect of wortmannin on phosphorylation of p53-N47 by ATM immunoprecipitates.

These results and the observation that damage-induced phosphorylation of Ser15 of p53 is slower in A-T cells (14) suggest that this residue might be a physiological target of ATM after damage caused by ionizing radiation. This amino acid is not phosphorylated in untreated cells (15), so the responsible kinase must be expressed or stimulated by DNA damage. The amount of ATM remains unaltered after treatment of cells with ionizing radiation or radiomimetic drugs (16, 18). However, the activity of ATM immunoprecipitated from normal lymphoblastoid cells that had been treated with various doses of the radiomimetic drug neocarzinostatin (NCS) was increased (Fig. 3A). NCS belongs to a family of enediyne antibiotics that intercalate into the DNA and induce double-strand breaks by free radical attack on the deoxyribose moieties in both DNA strands. The biological effects of these compounds are similar to those of ionizing radiation (19). This enhancement was observed in five different cell lines (9). ATM was activated within minutes after treatment (Fig. 3B). In nine experiments, the maximal enhancement of ATM's activity was 4.4 ± 0.8 times its basal activity. Activated ATM still phosphorylated p53 only on Ser15(9).

Figure 3

Increased kinase activity of ATM immunoprecipitates from cells treated with NCS. Kinase reactions were incubated for 3 min. The concentration of nonradioactive ATP in the reaction mix (5 μM) was not rate-limiting. (A) ATM was immunoprecipitated from cells 20 min after treatment with various concentrations of NCS. Kinase activity was assayed using the p53-N47 substrate. The histogram presents quantitation of the data from the top panel. Phosphorylation signals were normalized against ATM amounts. (B) ATM's kinase activity at various time points after treatment of the cells with NCS (50 ng/ml). U, untreated cells. The curve presents quantitation of the data from the top panel.

Associated protein kinase activity was shown in immunoprecipitates obtained with other antibodies to ATM (20). Our experiments with mutant ATM indicate that the observed activity is an intrinsic property of the ATM molecule. The biological significance of Ser15 phosphorylation on p53 is highlighted by its de novo occurrence in vivo after DNA damage, and by its delay in A-T cells (14, 15). Mutation of Ser15leads to a reduction in the ability of p53 to arrest cell growth (21), but p53 phosphorylated at this position maintains its ability to bind to DNA (15). However, the interaction of p53 with its negative regulator, MDM2, is reduced when Ser15 and Ser37 are phosphorylated by DNA-PK, and there is a reduced interaction of p53 with MDM2 after DNA damage in vivo (15). MDM2 can both inhibit p53-mediated transcription (22) and target p53 for proteasome-mediated degradation (23). Phosphorylation of p53 at Ser15 may relieve either or both of these effects of MDM2 on p53. Our finding thus point to another mechanism by which mammalian cells maintain the stability and integrity of their genome.

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