ATM Mediates Phosphorylation at Multiple p53 Sites, Including Ser46, in Response to Ionizing Radiation

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 Ser15phosphorylation 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. 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 Ser15phosphorylation 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. ataxia telangiectasia ataxia telangiectasia mutated ATM-related CREB-binding protein histone acetyltransferase histone deacetylase homeodomain-interacting protein kinase 2 ionizing radiation mitogen-activated protein kinase mouse double minute p300/CBP-associated factor 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.Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (925) Google Scholar, 2.Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (328) Google Scholar). As a transcription factor, p53 induces or represses several genes that regulate cell cycle arrest, DNA repair or apoptosis, including p21WAF1,MDM2, GADD45, p53R2, and p53AIP1. Recent studies suggest that specific p53 phosphorylation events are important for the activation or repression of specific promoters (3.Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Crossref PubMed Scopus (255) Google Scholar, 4.Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (391) Google Scholar, 5.Jabbur J.R. Huang P. Zhang W. Oncogene. 2000; 19: 6203-6208Crossref PubMed Scopus (45) Google Scholar, 6.Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar). Optimal induction and activation of p53 after exposure to IR requires phosphorylation by the ATM protein kinase (1.Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (925) Google Scholar, 2.Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (328) Google Scholar, 7.Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1691) Google Scholar). ATM is thought to directly phosphorylate Ser15in vivo (8.Banin S. Moyal L. Shieh S.-Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1722) Google Scholar, 9.Canman C.E. Lim D.-S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1722) Google Scholar) and also is required for phosphorylation of Ser20 through activation of the Chk2 protein kinase, which phosphorylates Ser20in vitro (10.Chehab N.H. Malikzay A. Stavridi E.S. Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13777-13782Crossref PubMed Scopus (468) Google Scholar, 11.Hirao A. Kong Y.-Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1063) Google Scholar, 12.Shieh S.-Y. Taya Y. Prives C. EMBO J. 1999; 18: 1815-1823Crossref PubMed Scopus (269) Google Scholar). However, the potential role for ATM in regulating p53 modifications at other sites has not previously been explored. 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 nmglutamine 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. Asynchronously growing cultures in 75-cm2 flasks were irradiated using a Shepherd Mark I137Cs 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 μm4 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 β-glycerolphosphate, 1 mm sodium orthovanadate, 1 mm sodium molybdate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 μg/ml pepstatin, 0.5 mmphenylmethylsulfonyl fluoride). Immunoprecipitation and Western blot analyses were performed as described (13.Higashimoto Y. Saito S. Tong X.-H. Hong A. Sakaguchi K. Appella E. Anderson C.W. J. Biol. Chem. 2000; 275: 23199-23203Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14.Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). 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. 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.Higashimoto Y. Saito S. Tong X.-H. Hong A. Sakaguchi K. Appella E. Anderson C.W. J. Biol. Chem. 2000; 275: 23199-23203Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14.Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15.Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1036) Google Scholar). 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. Wild-type and the L22Q/W23S mutant p53 sequences were subcloned from the p53 expression vectors pC53-SN3 (16.Baker S.J. Markowitz S. Fearon E.R. Willson J.K. Vogelstein B. Science. 1990; 249: 912-915Crossref PubMed Scopus (1613) Google Scholar) or pCB6+p53–22/23 (17.Ludwig R.L. Bates S. Vousden K.H. Mol. Cell. Biol. 1996; 16: 4952-4960Crossref PubMed Scopus (253) Google Scholar) into the pCAGGS expression plasmid behind the CAG (cytomegalovirus enhancer-chicken β-actin hybrid) promoter (18.Niwa H. Yamamura K.-i. Miyazaki J.-i. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4662) Google Scholar). 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. 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. 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.Higashimoto Y. Saito S. Tong X.-H. Hong A. Sakaguchi K. Appella E. Anderson C.W. J. Biol. Chem. 2000; 275: 23199-23203Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14.Sakaguchi K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 15.Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1036) Google Scholar, 19.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (715) Google Scholar). 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.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (715) Google Scholar), 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.Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.-Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (876) Google Scholar), and phosphorylation of Ser37was 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.Siliciano J.D. Canman C.E. Taya Y. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (715) Google Scholar). Phosphorylation at Ser20 also was largely abrogated in A-T cells as reported previously (21.Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar), although some Ser20phosphorylation was observed by 2 h after IR. 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.Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (602) Google Scholar), 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.D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (587) Google Scholar, 24.Hofmann T.G. Möller A. Sirma H. Zentgraf H. Taya Y. Dröge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus Google Scholar). The kinase that phosphorylates Ser46 after IR has not been To the of ATM in mediating phosphorylation of Ser46, we the of wortmannin, an inhibitor of ATM J.N. R.S. Abraham R.T. 1998; Google Scholar), and PD169316, a p38 inhibitor S. D. J.M. R.S. A.J. S. Chem. 1997; PubMed Scopus Google Scholar), on Ser46 phosphorylation in A549 cells in response to IR. 2 that of inhibited IR-induced phosphorylation at both Ser15 and Ser46, consistent with on ATM, of PD169316 no on phosphorylation at either were obtained in normal lymphoblasts (data not Phosphorylation at each of the sites in 1 after exposure to IR also was in two other of normal and A-T lymphoblasts, and and and with similar in A-T cell line IR phosphorylation of Ser46, phosphorylation at site was observed in normal lymphoblasts (data not the p53 in A-T cells is of phosphorylated in response to DNA was by the response in and cells exposed to UV In no in p53 phosphorylation the normal and A-T lymphoblasts was observed (data not shown), indicating that phosphorylation at Ser9, Ser15, Ser20, and Ser46 was to be in A-T cells. These previous that ATM in the to IR R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1691) Google Scholar). the N-terminal p53 phosphorylation sites can be into two with to on ATM for phosphorylation in response to IR. of Ser6, Ser33, Ser315, and Ser392, is of ATM and phosphorylated at low the other of Ser9, Ser15, Ser20, and Ser46, is for a response to IR-induced damage, DNA double we and that acetylation of human p53 after DNA may be through phosphorylation at N-terminal sites N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (391) Google Scholar, 15.Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1036) Google Scholar, J.N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). To in we first the time course of p53 acetylation at Lys320 and Lys382 after exposure to 8 Gy IR in normal and A-T lymphoblasts (Fig. In normal lymphoblasts, acetylation at Lys320 was observed within 1 h after IR and at Lys382 by 2 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 at 4 h after IR compared with normal lymphoblasts (Fig. in the acetylation of these sites was observed normal and A-T lymphoblasts after UV radiation (data not To determine which N-terminal phosphorylation are important for acetylation, we mutant in which N-terminal phosphorylation sites were changed to by of H1299 cells with the indicated p53 expression vectors (Fig. p53 protein levels from each were as by staining with the p53-specific antibody and wild-type p53 was acetylated at both sites with or exposure to IR. Acetylation IR was not the a response in human as reported previously K. Saito S. Higashimoto Y. Roy S. Anderson C.W. Appella E. J. Biol. Chem. 2000; 275: 9278-9283Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (602) Google Scholar). In to wild-type p53, acetylation of Lys382 in the p53 as well as the L22Q/W23S double was acetylation of Lys320 was reduced Acetylation at Lys382 also was significantly reduced by that changed Ser6, Ser9, or Thr18 to In contrast, Ser20, Ser33, Ser37, or Ser46 to on acetylation at for the Ser15 to the of phosphorylation site mutants on the acetylation of Lys320 were less in to of the studies that phosphorylation of p53 at Ser15 and Ser20 in response to IR is by the ATM protein kinase and that these modifications are important for and p53 as a transcription show for the first that phosphorylation of Ser9 and Ser46 also are dependent on the ATM kinase 1 and Phosphorylation of Ser46 was to be important for the induction of apoptosis in response to by exposure of cell lines to UV light K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Abstract Full Text Full Text PDF PubMed Scopus (1031) Google Scholar, D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (602) Google Scholar), and two protein of Ser46, p38 MAPK (22.Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (602) Google Scholar) and (23.D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (587) Google Scholar, 24.Hofmann T.G. Möller A. Sirma H. Zentgraf H. Taya Y. Dröge W. Will H. Schmitz M.L. Nat. Cell Biol. 2002; 4: 1-10Crossref PubMed Scopus Google Scholar), both of which are activated after exposure of cells to UV have been In to UV activation of p53 after exposure of cells to IR primarily induces cell cycle arrest in However, human and cells are to IR-induced apoptosis, and both p53 and ATM are required for its induction J. D. Y. S. T. Science. 1999; PubMed Scopus Google G. T. 2001; PubMed Scopus Google Scholar). suggest that in activation of ATM in response to DNA double activation of an protein kinase that phosphorylates p53 at Ser46, possibly through of the product of a recently gene that p53 phosphorylation at Ser46 in response to IR S. Arakawa H. Tanaka T. H. Taya Y. M. Nakamura Y. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). protein kinase is to be p38 which phosphorylates Ser46 in response to PD169316, a p38 inhibitor, to phosphorylation of Ser46 (Fig. also that ATM is required for phosphorylation of p53 at previously reported that and Ser9 phosphorylated in response to both and DNA and that Ser9 may be phosphorylated by in response to phosphorylation of (13.Higashimoto Y. Saito S. Tong X.-H. Hong A. Sakaguchi K. Appella E. Anderson C.W. J. Biol. Chem. 2000; 275: 23199-23203Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In vitro phosphorylates serines and two residues to a phosphorylated serine or The data suggest that in response to IR, phosphorylation of Ser9 may be of phosphorylation at and dependent upon activation of an protein kinase by as for Ser20, the kinase that phosphorylates Ser9 in response to IR is to be activated by the that of Ser9 by its kinase requires either Ser15 or its The of phosphorylation at and Ser9 are either serine to alanine on the of to activate transcription of a in assays N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (391) Google Scholar), consistent with a report that these and other N-terminal phosphorylations are not essential for p53 M. Vousden K.H. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). However, coupled with previous K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1036) Google Scholar) and of N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (391) Google Scholar, J.N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) that phosphorylation of Ser15 to p53, data (Fig. a in which phosphorylation at several N-terminal sites, of which phosphorylation of Ser15 to be acetylation at C-terminal sites (Fig. data suggest that the N-terminal important for regulating p53 with the first 18 residues of human p53, of phosphorylation by Ser20, Ser33, Ser37, and Ser46 to not C-terminal the double mutant L22Q/W23S also C-terminal acetylation as the in mouse p53 C. Saito S. J. Anderson C.W. Appella E. Y. EMBO J. 2000; 19: PubMed Scopus Google Scholar), these the of the acids that is to be important for with both and S. B. J. A.J. Science. 1996; PubMed Scopus Google Scholar). phosphorylation of and Ser9, with phosphorylation of may to a by the phosphorylation of Ser15 that is important for to that of mouse p53, the of human Ser15, to not the acetylation of mouse p53 at and to human Lys320 and Lys382, respectively, in cells C. Saito S. Anderson C.W. Appella E. Y. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). of p53 acetylation through phosphorylation may not be in and K. H. Vousden for the pCB6+p53–22/23 M. for the and cell and J. Miyazaki and I. Saito for the pCAGGS

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