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J. Biol. Chem., Vol. 277, Issue 15, 12491-12494, April 12, 2002

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ACCELERATED PUBLICATION
ATM Mediates Phosphorylation at Multiple p53 Sites, Including Ser⁴⁶, in Response to Ionizing Radiation^*

Shin'ichi Saito^§¶, Aaron A. Goodarzi^**, Yuichiro Higashimoto^§, Yuka Noda^§, Susan P. Lees-Miller^**, Ettore Appella^§, and Carl W. Anderson^¶

From the  Biology Department, Brookhaven National Laboratory, Upton, New York 11973, the ^§ Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, and the  Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Received for publication, February 13, 2002, and in revised form, March 1, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The p53 tumor suppressor protein preserves genome integrity by regulating growth arrest and apoptosis in response to DNA damage. In response to ionizing radiation (IR), ATM, the gene product mutated in ataxia telangiectasia, stabilizes and activates p53 through phosphorylation of Ser¹⁵ and (indirectly) Ser²⁰. Here we show that phosphorylation of p53 on Ser⁴⁶, a residue important for p53 apoptotic activity, as well as on Ser⁹, in response to IR also is dependent on the ATM protein kinase. IR-induced phosphorylation at Ser⁴⁶ was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, but not PD169316, a p38 MAPK inhibitor. p53 C-terminal acetylation at Lys³²⁰ and Lys³⁸², which may stabilize p53 and activate sequence-specific DNA binding, required Ser¹⁵ phosphorylation by ATM and was enhanced by phosphorylation at nearby residues including Ser⁶, Ser⁹, and Thr¹⁸. These observations, together with the proposed role of Ser⁴⁶ phosphorylation in mediating apoptosis, suggest that ATM is involved in the initiation of p53-dependent apoptosis after IR in human lymphoblastoid cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In response to DNA damage, the p53 tumor suppressor protein is phosphorylated on each of the seven serines and one threonine the in the first 50 amino acids of its N-terminal transactivation domain as well as at several sites in its carboxyl (C)-terminal tetramerization/regulatory domain (1, 2). As a transcription factor, p53 induces or represses several genes that regulate cell cycle arrest, DNA repair or apoptosis, including p21^WAF1, MDM2, GADD45, p53R2, and p53AIP1. Recent studies suggest that specific p53 phosphorylation events are important for the activation or repression of specific promoters (3-6). Optimal induction and activation of p53 after exposure to IR requires phosphorylation by the ATM protein kinase (1, 2, 7). ATM is thought to directly phosphorylate Ser¹⁵ in vivo (8, 9) and also is required for phosphorylation of Ser²⁰ through activation of the Chk2 protein kinase, which phosphorylates Ser²⁰ in vitro (10-12). However, the potential role for ATM in regulating p53 modifications at other sites has not previously been explored.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Cultures and Inhibitors-- Epstein-Barr virus immortalized normal (GM02254) and A-T¹ (GM01526) human lymphoblast cultures were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). H1299 (ATCC CRL-5803), a human lung carcinoma cell line that is null for both TP53 alleles, and A549 (ATCC CCL-185), a human lung carcinoma cell line that expresses wild-type p53, were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in Dulbecco's modified minimal essential medium (Invitrogen) supplemented with 15% (lymphoblasts) or 10% (H1299) fetal bovine serum, 100 nM glutamine and penicillin/streptomycin in a humidified atmosphere with 5% CO₂. Wortmannin (Sigma) was prepared as a 10 mM stock and PD169316 (Calbiochem, Inc.) as a 1 mM stock in Me₂SO; both were stored at 20 °C and diluted into the cell media immediately before use.

Induction of DNA Damage, Immunoprecipitation, and Western Immunoblot Analysis-- Asynchronously growing cultures in 75-cm² flasks were irradiated using a Shepherd Mark I ¹³⁷Cs irradiator at a dose rate of 3.2 Gy/min. To detect p53 acetylation, the deacetylase inhibitor trichostatin A (Wako, Osaka, Japan) was added at a final concentration of 5 µM 4 h before harvesting. Cultures were harvested at the indicated times after treatment, washed twice with ice-cold phosphate-buffered saline, and lysed in ice-cold lysis buffer (50 mM Tris-HCl at pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM sodium pyrophosphate, 25 mM -glycerolphosphate, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 5 µg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride). Immunoprecipitation and Western blot analyses were performed as described (13, 14). Anti-p53 monoclonal antibody DO-1 was purchased from Santa Cruz Biotechnology Inc.; anti-p53 polyclonal antibody Ab-7 was from Calbiochem, Inc. In all Western blot analyses, uniform protein loading was confirmed by Coomassie Brilliant Blue staining of the SDS-polyacrylamide gels after transfer to the polyvinylidene difluoride membranes.

Phosphorylation- and Acetylation-specific p53 Antibodies-- Rabbit polyclonal antibodies specific for p53 phosphorylated at Ser⁶, Ser⁹, Ser¹⁵, Ser²⁰, Ser³³, Ser³⁷, and Thr¹⁸ or acetylated at Lys³²⁰ or Lys³⁸² have been described (13-15). A similar approach was used to generate antibodies specific for p53 phosphorylated at Ser⁴⁶, Ser³¹⁵, or Ser³⁹². Briefly, rabbit antibodies that recognize p53 phosphorylated at Ser⁴⁶ (PAbSer(P)46), Ser³¹⁵ (PAbSer(P)315), or Ser³⁹² (PAbSer(P)392) were elicited against the human p53 sequences Ac-41-51(46P)C, Ac-310-321(315P)C or Ac-C-385-393(392P) coupled to keyhole limpet hemocyanin, respectively. Phosphorylation-specific antibodies were affinity-selected. The specificity of each antibody was confirmed by enzyme-linked immunosorbent assay using synthetically prepared p53 peptides and with immunoblot assays by probing GST-human p53 expressed in Escherichia coli.

Plasmids and cDNA Constructs-- Wild-type and the L22Q/W23S mutant p53 sequences were subcloned from the p53 expression vectors pC53-SN3 (16) or pCB6+p53-22/23 (17) into the pCAGGS expression plasmid behind the CAG (cytomegalovirus enhancer-chicken -actin hybrid) promoter (18). The serine codons at amino acid positions 6, 9, 15, 20, 33, 37, and 46, and the threonine codon at 18, were changed to alanine by site-directed mutagenesis. The entire p53 sequence in each vector was confirmed by DNA sequencing.

Transient Transfections-- The day prior to transfection, 10⁶ H1299 cells were seeded in each 10-cm tissue culture plate. On the following day, the cultures were transfected with the expression vectors for wild-type p53 or p53 mutants using LipofectAMINE PLUS Reagent (Invitrogen) as recommended by the manufacturer. Cells were exposed to 8 Gy IR 18 h after transfection, and the cultures were harvested for immunoprecipitation and Western blot analysis 2 h after IR.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of ATM-dependent p53 Phosphorylation-- To determine whether other p53 posttranslational modifications depend on ATM, we prepared a panel of polyclonal antibodies that, respectively, recognize p53 modified at each of 12 sites at which it is phosphorylated or acetylated (13-15, 19). These antibodies were then used to examine the time course of phosphorylation at each site after exposure of normal (GM02254) and A-T (GM01526) human lymphoblasts to 8 Gy IR (Fig. 1). Acetylation at two C-terminal sites, Lys³²⁰ and Lys³⁸², also was examined. p53 accumulated rapidly and reached a maximum at 4 h after irradiation in normal lymphoblasts, as detected with the p53-specific monoclonal antibody DO-1. As expected (19), delayed p53 accumulation was seen in A-T lymphoblasts. p53 increased slowly and was less that half that in normal cells before 2 h after IR; p53 then increased to ~80% of that in normal lymphoblasts by 4 h after irradiation. Although low levels of constitutive phosphorylation were observed at Ser⁶, Ser³³, Ser³¹⁵, and Ser³⁹², phosphorylation increased rapidly at each of these sites and at Ser⁹, Ser¹⁵, Ser²⁰, and Ser⁴⁶, after exposure of normal lymphoblasts to IR. Phosphorylation at Thr¹⁸ may be cell line-dependent, and no IR-induced increase in phosphorylation was observed at Thr¹⁸ in either normal or A-T lymphoblasts. Likewise, phosphorylation at Ser³⁷ is thought to occur primarily through activation of ATR after exposure to e.g. UV light (20), and phosphorylation of Ser³⁷ was not observed in either the normal or A-T lymphoblasts. A very similar pattern of IR-induced p53 phosphorylation was observed in A549 lung and MCF7 breast carcinoma cell lines (data not shown), indicating that these modifications are not specific to lymphoblasts. In normal lymphoblasts, increased phosphorylation at Ser⁶, Ser⁹, Ser¹⁵, Ser²⁰, Ser³³, Ser⁴⁶, Ser³¹⁵, and Ser³⁹² was observed within 15 min after IR. In contrast, in A-T lymphoblasts, phosphorylation at Ser¹⁵ was significantly delayed (until ~2 h after IR) and reduced compared with normal lymphoblasts, consistent with a previous report (19). Phosphorylation at Ser²⁰ also was largely abrogated in A-T cells as reported previously (21), although some Ser²⁰ phosphorylation was observed by 2 h after IR.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Phosphorylation of p53 on multiple N-terminal serines in response to ionizing radiation requires ATM. A, normal human lymphoblasts (GM02254) and A-T lymphoblasts (GM01526) were treated with 8 Gy IR and harvested at the indicated times. Extracts were immunoprecipitated using anti-p53 antibodies and analyzed for p53 and phosphorylation by Western immunoblot analysis; phosphorylation site-specific antibodies or monoclonal antibody to p53 (DO-1) are indicated (right). Extracts of SHEP human neuroblastoma cells, treated with adriamycin at 0.2 µg/ml for 8 h, served as a positive control (PC) (35). Phosphorylation at Ser9, Ser15, Ser20, and Ser46 was attenuated in the A-T lymphoblasts. B, the Western blot in A was scanned to compare the amount of phosphorylation at individual residues in A-T cells (GM01526) with the same residue in normal lymphoblasts (GM02254). The amount of phosphorylation at each residue was first normalized by dividing its value by the value for the amount of p53 at that time point. The normalized ratio was then multiplied by 100. Shown are data for 5 residues: Ser6, Ser15, Ser20, Ser46, and Ser392. The results show that phosphorylation at Ser15, Ser20, and Ser46 (and Ser9, data not shown) is reduced and delayed in A-T cells compared with normal cells. The amount of p53 in A-T cells was less than half of that in normal cells up to 0.5 h after exposure and then increased to ~80% of the level in normal cells at 4 h.

Unexpectedly, phosphorylation at Ser⁴⁶ also was defective in A-T lymphoblasts after exposure to IR, and phosphorylation at Ser⁹ was reduced and delayed (Fig. 1, A and B). These data suggest that Ser⁹ and Ser⁴⁶ also may be phosphorylated by an ATM-activated protein kinase (or possibly ATM itself); alternatively, in response to IR, these phosphorylations may depend upon prior phosphorylation of Ser¹⁵ or Ser²⁰. In response to UV irradiation, Ser⁴⁶ can be phosphorylated by the p38 MAPK (22), and recently Ser⁴⁶ was reported to be phosphorylated by homeodomain-interacting protein kinase 2 (HIPK2), which also is activated by exposure of cells to UV light (23, 24). The kinase that phosphorylates Ser⁴⁶ after IR has not been identified.

To further establish the importance of ATM in mediating phosphorylation of Ser⁴⁶, we examined the effect of wortmannin, an inhibitor of ATM (25), and PD169316, a p38 MAPK-specific inhibitor (26), on Ser⁴⁶ phosphorylation in A549 cells in response to IR. Fig. 2 shows that increasing concentrations of wortmannin inhibited IR-induced phosphorylation at both Ser¹⁵ and Ser⁴⁶, consistent with dependence on ATM, while increasing concentrations of PD169316 had no effect on phosphorylation at either site. Similar results were obtained in BT normal lymphoblasts (data not shown). Phosphorylation at each of the sites shown in Fig. 1 after exposure to IR also was examined in two other pairs of normal and A-T lymphoblasts, C3ABR and AT24RM, and BT (normal) and L3 (A-T), with similar results; in neither A-T cell line did IR induce phosphorylation of Ser⁴⁶, while robust phosphorylation at this site was observed in normal lymphoblasts (data not shown). That the p53 in A-T cells is capable of being phosphorylated in response to DNA damage was shown by examining the response in GM02254 and GM01526 cells exposed to UV light. In this case, no significant difference in p53 phosphorylation between the normal and A-T lymphoblasts was observed (data not shown), indicating that phosphorylation at Ser⁹, Ser¹⁵, Ser²⁰, and Ser⁴⁶ was unlikely to be masked in A-T cells. These findings fit previous observations that ATM acts specifically in the cellular responses to IR (7). Thus, the N-terminal p53 phosphorylation sites can be grouped into two categories with respect to dependence on ATM for rapid phosphorylation in response to IR. One group, consisting of Ser⁶, Ser³³, Ser³¹⁵, and Ser³⁹², is independent of ATM and constitutively phosphorylated at low levels; the other group, consisting of Ser⁹, Ser¹⁵, Ser²⁰, and Ser⁴⁶, is ATM-dependent for a rapid response to IR-induced damage, presumably DNA double strand breaks.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Phosphorylation at Ser15 and Ser46 is inhibited by wortmannin. Wortmannin (WM), an inhibitor of phosphatidylinositol-3-like kinases including ATM, or PD169316 (PD), a p38 MAPK-specific inhibitor, were added to A549 cultures as indicated, and 30 min later cultures were irradiated with 10 Gy. Two hour postirradiation cultures were harvested for Western immunoblot analysis as described in the legend to Fig. 1. A, wortmannin was added at 10 (lane 3), 50 (lane 4), or 100 (lane 5) µM. B, PD169316 was added at 2.5 (lane 3), 5 (lane 4), or 10 (lane 5) µM.

Dependence of C-terminal Acetylation on N-terminal Phosphorylation-- Previously, we and others suggested that acetylation of human p53 after DNA damage may be mediated through phosphorylation at N-terminal sites (4, 15, 27). To address this issue in vivo, we first examined the time course of p53 acetylation at Lys³²⁰ and Lys³⁸² after exposure to 8 Gy IR in normal and A-T lymphoblasts (Fig. 3A). In normal lymphoblasts, acetylation at Lys³²⁰ was observed within 1 h after IR and at Lys³⁸² by 2 h; both sites were well acetylated at 4 h after IR. In contrast, in A-T lymphoblasts, acetylation at both sites was significantly delayed and reduced (by 20-40%) at 4 h after IR compared with normal lymphoblasts (Fig. 3B). No differences in the acetylation of these sites was observed between normal and A-T lymphoblasts after UV radiation (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Efficient acetylation of p53 on C-terminal lysine residues in response to ionizing radiation requires phosphorylation at multiple N-terminal residues. A, normal lymphoblasts (GM02254) and A-T lymphoblasts (GM01526) were treated with 8 Gy IR and analyzed as described in the legend to Fig. 1 except that immunoblot analysis was with antibodies specific for acetylation at Lys320 (PAbLys(Ac)320) or Lys382 (PAbLys(Ac)382) as indicated. A549 human lung carcinoma cells, harvested 8 h after exposure to 25 J/m2 UV-C, served as a positive control (PC) (15). B, the ratio of acetylation at Lys320 and Lys382 in A-T to that in normal lymphoblasts (×100) was calculated after normalization for p53 amounts as described for phosphorylation in the legend to Fig. 1. The data show that acetylation in A-T cells was dramatically delayed before approaching levels in normal cells by 4 h after irradiation. C, H1299 cells (TP53/) were transiently transfected with vectors that expressed wild-type or p53 mutants with single amino acid substitutions (serine/threonine to alanine) at the indicated sites and were exposed or not to 8 Gy IR. Extracts were prepared 18 h after transfection and 2 h after irradiation for immunoprecipitation and Western blot analysis as described (see "Experimental Procedures"). Ab-7 is a p53-specific polyclonal antibody.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Model for activation of p53 by IR-induced DNA double strand breaks. Exposure of cells to IR produces DNA double strand breaks, which leads to activation of ATM as well as other unidentified protein kinases that phosphorylate p53 at Ser6 and Ser33 in its N-terminal transactivation domain. ATM directly phosphorylates p53 on Ser15 and induces phosphorylation at Ser9, Ser20, and Ser46 through activation of additional protein kinases, including Chk2. In human lymphoblastoid cells, Thr18 and Ser37 were not phosphorylated in response to IR. Phosphorylation of p53 on Ser15 recruits HATs, including p300/CBP and PCAF, which acetylate Lys320 and Lys382 in the C-terminal domain. Phosphorylation at Ser6, Ser9, and Thr18 helps recruit HATs to p53. Posttranslational modifications also contribute to the stabilization of p53 and its activation as a transcription factor.

To determine which N-terminal phosphorylation site(s) are important for acetylation, we examined mutant p53s in which individual N-terminal phosphorylation sites were changed to Ala by transient transfection of p53-negative H1299 cells with the indicated p53 expression vectors (Fig. 3C). p53 protein levels from each transfection were comparable as shown by staining with the polyclonal, p53-specific antibody Ab-7, and wild-type p53 was acetylated at both sites with or without exposure to IR. Acetylation without IR was not unexpected, since the transfection procedure elicits a strong "stress-like" response in human cells, as reported previously (14, 22). In contrast to wild-type p53, acetylation of Lys³⁸² in the p53 S15A mutant, as well as the L22Q/W23S double mutant, was virtually abrogated, while acetylation of Lys³²⁰ was reduced significantly. Acetylation at Lys³⁸² also was significantly reduced by mutations that changed Ser⁶, Ser⁹, or Thr¹⁸ to Ala. In contrast, changing Ser²⁰, Ser³³, Ser³⁷, or Ser⁴⁶ to Ala had little, if any, effect on acetylation at Lys³⁸². Except for the Ser¹⁵ to Ala change, the effects of phosphorylation site mutants on the acetylation of Lys³²⁰ were less clear, in part, due to weakness of the Ac-Lys³²⁰ signal.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies showed that phosphorylation of p53 at Ser¹⁵ and Ser²⁰ in response to IR is mediated by the ATM protein kinase and that these modifications are important for stabilizing and activating p53 as a transcription factor. We show here, for the first time, that phosphorylation of Ser⁹ and Ser⁴⁶ also are dependent on the ATM kinase (Figs. 1 and 4). Phosphorylation of Ser⁴⁶ was shown to be important for the induction of apoptosis in response to damage caused by exposure of epithelial-derived cell lines to UV light (6, 22), and two protein kinases capable of phosphorylating Ser⁴⁶, p38 MAPK (22) and HIPK2 (23, 24), both of which are activated after exposure of cells to UV light, have been described. In contrast to UV light, activation of p53 after exposure of epithelial cells to IR primarily induces cell cycle arrest in G₁. However, human lymphoid and neuronal cells are much more sensitive to p53-dependent, IR-induced apoptosis, and both p53 and ATM are required for its induction (28, 29). We suggest that in lymphoid cells, activation of ATM in response to DNA double strand breaks mediates activation of an unidentified protein kinase that phosphorylates p53 at Ser⁴⁶, possibly through recruitment of p53DINP1, the product of a recently identified p53-inducible gene that facilitates p53 phosphorylation at Ser⁴⁶ in response to IR (30). This protein kinase is unlikely to be p38 MAPK, which phosphorylates Ser⁴⁶ in response to UV, since PD169316, a p38 kinase-specific inhibitor, failed to block phosphorylation of Ser⁴⁶ (Fig. 2).

We also found that ATM is required for phosphorylation of p53 at Ser⁹. We previously reported that Ser⁶ and Ser⁹ became strongly phosphorylated in response to both IR- and UV-induced DNA damage and suggested that Ser⁹ may be phosphorylated by CK1 in response to phosphorylation of Ser⁶ (13). In vitro CK1 phosphorylates serines and threonines two residues distal to a phosphorylated serine or threonine. The data presented here suggest that in response to IR, phosphorylation of Ser⁹ may be independent of phosphorylation at Ser⁶ and dependent upon activation of an unknown protein kinase by ATM. Thus, as for Ser²⁰, the kinase that phosphorylates Ser⁹ in response to IR is likely to be activated by ATM. We cannot, however, rule out the possibility that recognition of Ser⁹ by its kinase requires either Ser¹⁵ or its phosphorylation.

The functional consequences of phosphorylation at Ser⁶ and Ser⁹ are unknown. Changing either serine to alanine had little effect on the ability of chimeric Gal4-p53(1-42) to activate transcription of a reporter in transient transfection assays (4), consistent with a report that these and other N-terminal phosphorylations are not essential for p53 activity (31). However, coupled with our previous results (15) and those of others (4, 27) showing that phosphorylation of Ser¹⁵ recruits CBP/p300 to p53, our current data (Fig. 3C) supports a cascade model in which phosphorylation at several N-terminal sites, of which phosphorylation of Ser¹⁵ appears to be most important, promotes acetylation at C-terminal sites (Fig. 4). Our data further suggest that the N-terminal region important for regulating p53 interactions with HATs includes the first 18 residues of human p53, since abrogation of phosphorylation by changing Ser²⁰, Ser³³, Ser³⁷, and Ser⁴⁶ to Ala did not affect C-terminal acetylation. While the double mutant L22Q/W23S also blocked C-terminal acetylation as did the equivalent mutations in mouse p53 (32), these changes most likely profoundly affect the structure of the amphipathic helical region (amino acids 17-28) that is likely to be important for interactions with both HATs and MDM2 (33). Thus, phosphorylation of Ser⁶ and Ser⁹, along with phosphorylation of Thr¹⁸, may serve to amplify a primary signal initiated by the ATM-mediated phosphorylation of Ser¹⁵ that is important for recruiting HATs to p53. We note, however, that changing Ser¹⁸ of mouse p53, the equivalent of human Ser¹⁵, to Ala did not block the acetylation of mouse p53 at Lys³¹⁷ and Lys³⁷⁹, equivalent to human Lys³²⁰ and Lys³⁸², respectively, in ES cells (34). Thus, regulation of p53 acetylation through phosphorylation may not be equivalent in mice and humans.

	ACKNOWLEDGEMENTS

We thank K. H. Vousden for the pCB6+p53-22/23 plasmid, M. F. Lavin for the BT and L3 cell lines, and J. Miyazaki and I. Saito for the pCAGGS plasmid.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors were supported in part by a Laboratory Directed Research and Development Grant at the Brookhaven National Laboratory under contract with the United States Department of Energy.

^** These authors were supported by grants from the Alberta Heritage Foundation for Medical Research and the National Cancer Institute of Canada Operating Grant 11053.

To whom correspondence should be addressed: Biology Dept., 50 Bell Ave., Brookhaven National Laboratory, Upton, NY 11973. Tel.: 631-344-3375; Fax: 631-344-6398; E-mail: cwa@bnl.gov.

Published, JBC Papers in Press, March 1, 2002, DOI 10.1074/jbc.C200093200

	ABBREVIATIONS

The abbreviations used are: A-T, ataxia telangiectasia; ATM, ataxia telangiectasia mutated; ATR, ATM-related; CBP, CREB-binding protein; HAT, histone acetyltransferase; HDAC, histone deacetylase; HIPK2, homeodomain-interacting protein kinase 2; IR, ionizing radiation; MAPK, mitogen-activated protein kinase; MDM, mouse double minute; PCAF, p300/CBP-associated factor.

	REFERENCES

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				J.-L. Perfettini, M. Castedo, R. Nardacci, F. Ciccosanti, P. Boya, T. Roumier, N. Larochette, M. Piacentini, and G. Kroemer Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope J. Exp. Med., January 18, 2005; 201(2): 279 - 289. [Abstract] [Full Text] [PDF]

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				T. Thompson, C. Tovar, H. Yang, D. Carvajal, B. T. Vu, Q. Xu, G. M. Wahl, D. C. Heimbrook, and L. T. Vassilev Phosphorylation of p53 on Key Serines Is Dispensable for Transcriptional Activation and Apoptosis J. Biol. Chem., December 17, 2004; 279(51): 53015 - 53022. [Abstract] [Full Text] [PDF]

				E. U. Kurz, P. Douglas, and S. P. Lees-Miller Doxorubicin Activates ATM-dependent Phosphorylation of Multiple Downstream Targets in Part through the Generation of Reactive Oxygen Species J. Biol. Chem., December 17, 2004; 279(51): 53272 - 53281. [Abstract] [Full Text] [PDF]

				S. Caporali, S. Falcinelli, G. Starace, M. T. Russo, E. Bonmassar, J. Jiricny, and S. D'Atri DNA Damage Induced by Temozolomide Signals to both ATM and ATR: Role of the Mismatch Repair System Mol. Pharmacol., September 1, 2004; 66(3): 478 - 491. [Abstract] [Full Text] [PDF]

				H. R. McMurray and D. J. McCance Degradation of p53, Not Telomerase Activation, by E6 Is Required for Bypass of Crisis and Immortalization by Human Papillomavirus Type 16 E6/E7 J. Virol., June 1, 2004; 78(11): 5698 - 5706. [Abstract] [Full Text] [PDF]

				W. Y. Siu, A. Lau, T. Arooz, J. P.H. Chow, H. T.B. Ho, and R. Y.C. Poon Topoisomerase poisons differentially activate DNA damage checkpoints through ataxia-telangiectasia mutated-dependent and -independent mechanisms Mol. Cancer Ther., May 1, 2004; 3(5): 621 - 632. [Abstract] [Full Text] [PDF]

H. K. Sluss, H. Armata, J. Gallant, and S. N.