Phosphorylation Site Interdependence of Human p53 Post-translational Modifications in Response to Stress
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305135200
ISSN1083-351X
AutoresShinichi Saito, Hiroshi Yamaguchi, Yuichiro Higashimoto, Connie Chao, Yang Xu, Albert J. Fornace, Ettore Appella, Carl W. Anderson,
Tópico(s)DNA Repair Mechanisms
ResumoModification-specific antibodies were used to characterize the phosphorylation and acetylation of human p53 in response to genotoxic (UV, IR, and adriamycin) and non-genotoxic (PALA, taxol, nocodazole) stress in cultured human cells at 14 known modification sites. In A549 cells, phosphorylation or acetylation was induced at most sites by the three DNA damage-inducing agents, but significant differences between agents were observed. IR-induced phosphorylation reached a maximum 2 h after treatment and returned to near pretreatment levels by 72 h; UV light and adriamycin induced a less rapid but more robust and prolonged p53 phosphorylation, which reached a maximum between 8 and 24 h, but persisted (UV) even 96 h after treatment. Ser33, Ser37, Ser46, and Ser392 were more efficiently phosphorylated after exposure to UV light than after IR. The non-genotoxic agents PALA, taxol and nocodazole induced p53 accumulation and phosphorylation at Ser6, Ser33, Ser46, and Ser392. Some phosphorylation at Ser15 also was observed. Modifications occurred similarly in the HCT116 human colon carcinoma cell line. Analysis of single site mutant p53s indicated clear interdependences between N-terminal phosphorylation sites, which could be classified in four clusters: Ser6 and Ser9; Ser9, Ser15, Thr18 and Ser20; Ser33 and Ser37; and Ser46. We suggest that p53 phosphorylation is regulated through a double cascade involving both the activation of secondary, effector protein kinases as well as intermolecular phosphorylation site interdependencies that check inappropriate p53 inactivation while allowing for signal amplification and the integration of signals from multiple stress pathways. Modification-specific antibodies were used to characterize the phosphorylation and acetylation of human p53 in response to genotoxic (UV, IR, and adriamycin) and non-genotoxic (PALA, taxol, nocodazole) stress in cultured human cells at 14 known modification sites. In A549 cells, phosphorylation or acetylation was induced at most sites by the three DNA damage-inducing agents, but significant differences between agents were observed. IR-induced phosphorylation reached a maximum 2 h after treatment and returned to near pretreatment levels by 72 h; UV light and adriamycin induced a less rapid but more robust and prolonged p53 phosphorylation, which reached a maximum between 8 and 24 h, but persisted (UV) even 96 h after treatment. Ser33, Ser37, Ser46, and Ser392 were more efficiently phosphorylated after exposure to UV light than after IR. The non-genotoxic agents PALA, taxol and nocodazole induced p53 accumulation and phosphorylation at Ser6, Ser33, Ser46, and Ser392. Some phosphorylation at Ser15 also was observed. Modifications occurred similarly in the HCT116 human colon carcinoma cell line. Analysis of single site mutant p53s indicated clear interdependences between N-terminal phosphorylation sites, which could be classified in four clusters: Ser6 and Ser9; Ser9, Ser15, Thr18 and Ser20; Ser33 and Ser37; and Ser46. We suggest that p53 phosphorylation is regulated through a double cascade involving both the activation of secondary, effector protein kinases as well as intermolecular phosphorylation site interdependencies that check inappropriate p53 inactivation while allowing for signal amplification and the integration of signals from multiple stress pathways. When normal mammalian cells are exposed to genotoxic agents, including ionizing radiation (IR) 1The abbreviations used are: IR, ionizing radiation; ADR, adriamycin; ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; ALLN, N-acetyl-Leu-Leu-Nle-CHO; Chk, checkpoint kinase; CK, casein kinase; ELISA, enzyme-linked immunoadsorbent assay; GST, glutathione S-transferase; Gy, gray; Hprt, hypoxanthine phosphoribosyl transferase; MEF, mouse embryo fibroblasts; PALA, N-phosphonacetyl-l-aspartate; PCAF, p300/CBP-associated factor; Plk3, Polo-like kinase 3; SUMO, small ubiquitin-related modifier; TSA, trichostatin A; UV, ultraviolet.1The abbreviations used are: IR, ionizing radiation; ADR, adriamycin; ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; ALLN, N-acetyl-Leu-Leu-Nle-CHO; Chk, checkpoint kinase; CK, casein kinase; ELISA, enzyme-linked immunoadsorbent assay; GST, glutathione S-transferase; Gy, gray; Hprt, hypoxanthine phosphoribosyl transferase; MEF, mouse embryo fibroblasts; PALA, N-phosphonacetyl-l-aspartate; PCAF, p300/CBP-associated factor; Plk3, Polo-like kinase 3; SUMO, small ubiquitin-related modifier; TSA, trichostatin A; UV, ultraviolet. or ultraviolet (UV) light, they exhibit a number of responses including a transient inhibition of DNA and RNA synthesis, activation of several kinase-mediated signaling pathways, and the transcriptional induction or repression of several hundred genes, which result in the arrest of cell cycle progression in late G1, in S, or in G2 or in the induction of apoptosis. These responses are mediated, in part, by the p53 tumor suppressor gene, a transcription factor that integrates a variety of stress response signals (reviewed in Refs. 1Anderson C.W. Appella E. Bradshaw R.A. Dennis E. Handbook of Cell Signaling. Vol. 3. Academic Press, New, York2003: 237-247Google Scholar, 2Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (912) Google Scholar, 3Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2715) Google Scholar, 4Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (325) Google Scholar)). Exposing cells to genotoxic agents and some non-genotoxic stresses inhibits the degradation of p53 protein through the ubiquitin-mediated pathway and also activates p53 as a DNA-binding, site-specific transcription factor. However, several studies point to important differences in the cellular responses to agents that produce primarily DNA strand breaks (e.g. IR) compared with those that primarily cause base damage such as UV light (reviewed in Refs. 5Schwartz D. Rotter V. Semin. Cancer Biol. 1998; 8: 325-336Crossref PubMed Scopus (178) Google Scholar and 6Gottlieb T.M. Oren M. Semin. Cancer Biol. 1998; 8: 359-368Crossref PubMed Scopus (221) Google Scholar). Thus, while p53 protein increases in cells exposed to either IR or UV light, the latter induces much greater p53 transcriptional activity, measured with reporter constructs, than does IR. In fibroblasts and epithelial cells, UV light is a more effective activator of apoptosis than IR (5Schwartz D. Rotter V. Semin. Cancer Biol. 1998; 8: 325-336Crossref PubMed Scopus (178) Google Scholar, 6Gottlieb T.M. Oren M. Semin. Cancer Biol. 1998; 8: 359-368Crossref PubMed Scopus (221) Google Scholar, 7Meyer K.M. Hess S.M. Tlsty T.D. Leadon S.A. Oncogene. 1999; 18: 5795-5805Crossref PubMed Scopus (35) Google Scholar). p53 accumulation and activation also occurs in response to several physiological processes that are not associated with frank DNA damage, including nucleotide deprivation, exposure to microtubule inhibitors, osmotic shock, and senescence potentiated by oncogene activation (see Ref. 1Anderson C.W. Appella E. Bradshaw R.A. Dennis E. Handbook of Cell Signaling. Vol. 3. Academic Press, New, York2003: 237-247Google Scholar). p53 accumulation and activation are believed to be regulated through protein phosphorylations and acetylations (reviewed in Refs. 1Anderson C.W. Appella E. Bradshaw R.A. Dennis E. Handbook of Cell Signaling. Vol. 3. Academic Press, New, York2003: 237-247Google Scholar and 2Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (912) Google Scholar). At least 20 sites in the human p53 protein, located primarily in the N-terminal transactivation domains or in the C-terminal regulatory domain proximal or distal to the tetramerization domain, are modified in response to the activation of different stress signaling pathways (1Anderson C.W. Appella E. Bradshaw R.A. Dennis E. Handbook of Cell Signaling. Vol. 3. Academic Press, New, York2003: 237-247Google Scholar, 2Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (912) Google Scholar). For example, at the N terminus of human p53, phosphorylation of Ser15, Ser20, and Ser37, after either IR or UV light, was reported to stabilize p53 (8Chehab N.H. Malikzay A. Stavridi E.S. Halazonetis T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13777-13782Crossref PubMed Scopus (460) Google Scholar, 9Shieh S.-Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1752) Google Scholar); at the C terminus, phosphorylation of Ser315 and Ser392 was implicated in regulating the oligomerization state of p53 (10Sakaguchi K. Sakamoto H. Lewis M.S. Anderson C.W. Erickson J.W. Appella E. Xie D. Biochemistry. 1997; 36: 10117-10124Crossref PubMed Scopus (226) Google Scholar) and its ability to bind DNA in a sequence-specific manner (11Hao M. Lowy A.M. Kapoor M. Deffie A. Liu G. Lozano G. J. Biol. Chem. 1996; 271: 29380-29385Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 12Wang Y. Prives C. Nature. 1995; 376: 88-91Crossref PubMed Scopus (326) Google Scholar). Phosphorylation of p53 at N-terminal serines also may enhance interactions with the transcriptional coactivators p300/CBP and PCAF (13Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar, 14Lambert P.F. Kashanchi F. Radonovich M.F. Shiekhattar R. Brady J.N. J. Biol. Chem. 1998; 273: 33048-33053Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 15Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (653) Google Scholar, 16Sakaguchi 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 (1021) Google Scholar, 17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), while acetylation of C-terminal lysines was reported to activate sequence-specific DNA binding and to impair ubiquitination (16Sakaguchi 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 (1021) Google Scholar, 18Li M. Luo J. Brooks C.L. Gu W. J. Biol. Chem. 2002; 277: 50607-50611Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). However, the picture of p53 post-translational modifications in response to genotoxic and non-genotoxic stresses is incomplete. To characterize the roles of post-translational modifications in mediating p53 function, we prepared antibodies that recognize 14 of the known p53 specific modification sites only when they have been modified (16Sakaguchi 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 (1021) Google Scholar, 17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Higashimoto 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 (102) Google Scholar, 20Sakaguchi 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 (235) Google Scholar). Here we describe the phosphorylation of p53 at ten different sites in response to two different classes of DNA damaging agents, UV light and IR, in A549 cells, a commonly used line with wild-type p53 that was derived from a human lung carcinoma. We also examined phosphorylation of these sites after treatment with four non-genotoxic agents, ALLN, an inhibitor of ubiquitin-mediated degradation by the 26 S proteasome, taxol and nocodazole, which disrupt microtubules (21Stewart Z.A. Tang L.J. Pietenpol J.A. Oncogene. 2001; 20: 113-124Crossref PubMed Scopus (59) Google Scholar, 22Damia G. Filiberti L. Vikhanskaya F. Carrassa L. Taya Y. D'incalci M. Broggini M. Neoplasia. 2001; 3: 10-16Crossref PubMed Scopus (71) Google Scholar), and N-phosphonacetyl-l-aspartate (PALA), which causes depletion of ribonucleotides without detectable DNA damage (23Linke S.P. Clarkin K.C. Di Leonardo A. Tsou A. Wahl G.M. Genes Dev. 1996; 10: 934-947Crossref PubMed Scopus (480) Google Scholar). p53 in the HCT116 colon carcinoma cell line, which also expresses wild-type p53, was phosphorylated in essentially an identical manner in response to each agent. p53 acetylation was examined at two sites, Lys320 and Lys382. We furthermore examined the site interdependence of p53 phosphorylation in transient transfection assays by use of mutant p53 expression vectors that had individual serines changed to alanines at most of the reported phosphorylation sites. Together, our results demonstrate the existence of complex signaling cascades that modulate the modification state of the human p53 molecule in cells exposed to stress. Phosphorylation- and Acetylation-specific p53 Antibodies—Rabbit polyclonal antibodies specific for human p53 phosphorylated at Ser6, Ser9, Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, Ser315, or Ser392 or acetylated at Lys320 or Lys382 have been described (16Sakaguchi 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 (1021) Google Scholar, 17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Higashimoto 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 (102) Google Scholar, 20Sakaguchi 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 (235) Google Scholar). Rabbit polyclonal antibodies specific for mouse p53 phosphorylated at Ser23 and Ser37 also have been described (24Takai H. Naka K. Okada Y. Watanabe M. Harada N. Saito S. Anderson C.W. Appella E. Nakanishi M. Suzuki H. Nagashima K. Sawa H. Ikeda K. Motoyama N. EMBO J. 2002; 21: 5195-5205Crossref PubMed Scopus (351) Google Scholar, 25Wu Z. Earle J. Saito S. Anderson C.W. Appella E. Xu Y. Mol. Cell Biol. 2002; 22: 2441-2449Crossref PubMed Scopus (88) Google Scholar). Phosphorylation site-specific antibodies were affinity-purified from the resulting serum by use of each phosphorylated peptide coupled with Sulfolink (Pierce Chemical Co.). The purified antibodies then were passed through a column coupled with the respective unphosphorylated peptide to deplete antibodies that react with unphosphorylated p53. The specificity of each antibody was confirmed by enzyme-linked immunosorbent assay (ELISA) and dot blot assay using synthetically prepared p53 peptides, and immunoblot assay by probing GST-human p53 expressed in Escherichia coli. Cell Cultures—A549 (ATCC CCL-185), a human lung carcinoma cell line which express wild-type p53, was obtained from the American Type Culture Collection (Manasas, VA) as was H1299 (ATCC CRL-5803), a human lung carcinoma cell line that is null for both p53 alleles. HCT116, a human colon carcinoma cell line that expresses wild-type p53, was kindly provided by B. Voglstein. A549 and H1299 cells were grown in Dulbecco's modified minimal essential medium (DMEM) and HCT116 cells were in McCoy's 5A medium (Invitrogen Corp., Carlsbad, CA), both supplemented with 10% fetal bovine serum, 100 nm glutamine and penicillin/streptomycin in a humidified atmosphere with 5% CO2. Wild-type and p53S18A/S18A mouse embryonic fibroblasts (MEFs) were prepared from p53 wild-type and p53S18A/S18A embryos. p53S23A/– MEFs were developed from corresponding mutant ES cells using the Hprt-deficient blastocyst complementation approach (25Wu Z. Earle J. Saito S. Anderson C.W. Appella E. Xu Y. Mol. Cell Biol. 2002; 22: 2441-2449Crossref PubMed Scopus (88) Google Scholar). All MEFs were cultured in DMEM supplemented with 10% fetal bovine serum, 100 nm glutamine, penicillin/streptomycin, and 50 μm β-mercaptoethanol in a humidified atmosphere with 5% CO2. In the case of PALA treatment, dialyzed fetal bovine serum was used. Treatment of Cell Cultures, Immunoprecipitation, and Western Immunoblot Analysis—Cells were seeded in plastic dishes 24 h prior to treatment and were 60–70% confluent at the time of treatment. Cultures were exposed to UV light (UV-C, 254 nm) using a Stratagene UV Stratalinker 2400 or to ionizing radiation at a dose rate of 3.2 Gy per minute using a Shepherd Mark I 137Cs irradiator as described (17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The proteasome inhibitor ALLN (Calbiochem, La Jolla, CA) was added to cells at a final concentration of 20 μm. Adriamycin, taxol, nocodazole (Sigma Chemical Co.) and PALA (NSC224131, obtained from National Cancer Institute, Bethesda, MD) were added to cultures and remained in the cultures until they were harvested. Final concentrations of these agents were chosen after measuring IC50 values using the WST-1 cell proliferation reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol (Table I). The deacetylase inhibitor trichostatin A (TSA) (WAKO, Osaka, Japan) was added at a final concentration of 5 μm 4 h before harvest. Cultures were harvested at the indicated times after treatment and processed for immunoprecipitation and Western immunoblot analyses as described (17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Anti-human p53 monoclonal antibody DO-1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-human p53 polyclonal antibody Ab-7 (Calbiochem), and anti-mouse p53 polyclonal antibody CM-5 (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) were obtained as indicated. GST-tagged full-length human p53 protein was purchased from Santa Cruz Biotechnology Inc.Table IGrowth suppression of cell lines by genotoxic and non-genotoxic agentsCell lineIC50aIC50 values were measured using the WST-1 reagent, see "Experimental Procedures."ADRPALATaxolNocodazolenmA54965151.848HCT116178.91.347a IC50 values were measured using the WST-1 reagent, see "Experimental Procedures." Open table in a new tab Plasmids, cDNA Constructs, and Transient Transfection—All plasmids and cDNA constructs have been described (17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The day prior to transfection, 106 H1299 cells were seeded in each 10 cm tissue culture dish. On the following day, the cells were transfected with 4 μg of the indicated wild-type or mutant p53 expression vector using LipofectAMINE PLUS Reagent (Invitrogen) following the manufacturer's protocol. Cells were exposed to 8 Gy ionizing radiation or 25 J/m2 UV 18 h after transfection, harvested 2 h or 8 h after exposure to IR or UV light, respectively, then lysed and subjected to immunoprecipitation and Western blot analyses. Phosphorylation of p53 in A549 Cells in Response to DNA Damage-inducing Agents—To characterize the post-translational modifications to p53 induced by different DNA damage-producing agents, we prepared individual polyclonal antibodies for different sites that recognize p53 only when it has been modified at that site. The specificity of each antibody was confirmed by ELISA with synthetically prepared p53 peptides or by probing an unmodified human p53 GST-fusion protein expressed in E. coli with purified antibodies. None of the antibodies reacted with the unphosphorylated, wild-type GST-p53 fusion protein (Fig. 1A and data not shown). We then used these antibodies to follow the phosphorylation of individual sites in response to treatment with three DNA damage-inducing agents, IR, UV light, or adriamycin (ADR) (17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). We also examined acetylation at two sites in the C terminus, Lys320 and Lys382, as previously described (16Sakaguchi 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 (1021) Google Scholar). Samples were harvested before treatment and at different times after treatment and then p53 was captured by immunoprecipitation and analyzed by Western immunoblotting (see "Experimental Procedures"). We first examined the dose response of phosphorylation and acetylation to treatment with the three DNA damage-inducing agents. Fig. 1B shows the phosphorylation response after IR for doses between 2 and 32 Gy, and after UV for doses between 7 and 50 J/m2 at six representative sites: Ser6, Ser15, Ser20, Ser37, Ser46, and Ser315. Also shown is the induction of p53 protein, measured with a p53-specific monoclonal antibody, DO-1. For all sites except perhaps Ser20, phosphorylation was induced by 2 h after exposure to as little as 2 Gy IR; however, phosphorylation was significantly greater above 8 Gy. Similar results were seen for UV light at 8 h. Increased phosphorylation was clearly seen at Ser6 with as little as 7 J/m2; maximum induction of phosphorylation required ∼25 J/m2. Pilot studies showed that 2 h after IR and 8 h after UV were appropriate for assessing phosphorylation responses (see Fig. 2). p53 protein accumulation was maximal after exposure to 8 Gy IR or 25 J/m2 UV; higher doses did not result in significant additional accumulation. Similarly, addition of ADR to 345 nm (0.2 μg/ml) was chosen for subsequent analyses (data not shown). We next examined the time course of modifications at each of 12 p53 sites between 0.5 and 96 h after exposure to 8 Gy IR, 25 J/m2 UV light, or 345 nm ADR (Fig. 2). As expected, each treatment induced p53 accumulation, detected with the DO-1 monoclonal antibody. One set of cultures also was treated with 20 μm ALLN, which induced p53 accumulation to similar amounts (16Sakaguchi 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 (1021) Google Scholar). With ALLN or IR, significant accumulation was observed by one h after treatment; for UV or ADR, significant p53 accumulation clearly was observed by 4 h. Accumulation was persistent after IR, UV, and ADR, and high p53 levels remained even 4 days after exposure. After treatment with ALLN or adriamycin, p53 levels declined between 8 and 24 h after treatment. Fig. 2 shows exposure to 8 Gy IR induced a clear increase in phosphorylation at Ser6 and Ser15 by 0.5 h after treatment (17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Higashimoto 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 (102) Google Scholar), and by 2 h maximum phosphorylation was observed at all sites examined except Ser376, Ser378 (data not shown), and Ser392. For these three sites, little increase in phosphorylation was observed in response to IR at any time examined. We and others reported increased phosphorylation at Ser6 and Ser15 in response to IR as early as 15 min after treatment (16Sakaguchi 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 (1021) Google Scholar, 17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 19Higashimoto 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 (102) Google Scholar). In general, IR-induced phosphorylation declined at most sites 24 to 48 h after treatment and had returned to near pretreatment levels by 72 h (Fig. 2). In contrast, UV light induced a less rapid but more prolonged and robust increase in p53 phosphorylation. Phosphorylation increased at most sites between 2 and 4 h after UV treatment and reached a maximum between 8 and 24 h; at 96 h, significant phosphorylation was still apparent. Phosphorylation in response to ADR resembled the pattern produced by exposure to UV in robustness and timing; note, however, that ADR remained in the culture media until the cells were harvested. Remarkably, increased phosphorylation was observed at most sites in response to each of the three DNA damage-inducing agents; nevertheless, clear differences were observed (Fig. 2). Phosphorylation at most sites was more strongly induced by UV light and ADR than by IR, especially at Ser33, Ser37, Ser46, and Ser392. All three agents also promoted acetylation of Lys382, while acetylation of Lys320 was induced most strongly after UV light. The time course of acetylation was similar to phosphorylation but occurred slightly later, consistent with a role for phosphorylation in mediating p53 acetylation (14Lambert P.F. Kashanchi F. Radonovich M.F. Shiekhattar R. Brady J.N. J. Biol. Chem. 1998; 273: 33048-33053Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 16Sakaguchi 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 (1021) Google Scholar, 17Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Four sites, Ser6, Ser33, Ser315, and Ser392 appeared to be constitutively phosphorylated at a relatively low level in the absence of DNA damage (Fig. 2). For these sites, phosphorylation increased after adding ALLN in parallel with the increase in p53 protein, but the levels did not approach those observed after treatment with DNA damage-inducing agents. Constitutive phosphorylation was confirmed by showing that the antibodies did not cross-react with a larger amount of an unphosphorylated, human, GST-p53 fusion protein made in E. coli (Fig. 1A). Phosphorylation of p53 in A549 and HCT116 Cells in Response to Genotoxic and Non-genotoxic Agents—We next examined HCT116 human colon carcinoma cells (Fig. 3, right) and two normal human fibroblast cell strains to clarify whether the modification patterns seen in A549 cells (Fig. 3, left) were common in other cells after exposure to IR, UV, and ADR. We also examined the effects of three non-genotoxic agents known to activate p53, PALA (Fig. 3, lane 6), an inhibitor of the l-aspartate transcarbamylase component of the CAD protein that is required for pyrimidine biosynthesis, and two microtubule active drugs, taxol (Fig. 3, lane 7) and nocodazole (Fig. 3, lane 8). Cultures were treated with ADR or these three agents at three times the concentration required for the inhibition of growth by 50% (IC50, see Table I) for 8, 24, or 48 h. Cultures also were treated with 20 μm ALLN for 4 h. Extracts then were prepared and subjected to Western blot analysis. Each treatment induced significant p53 accumulation, detected by the p53-specific polyclonal antibody Ab-7. Except for ALLN, p53 protein accumulation was maximal by 24 h after the beginning of treatment (data not shown). With the exception of Thr18, Ser9, and Ser20, the pattern of post-translational modifications in HCT116 cells in response to IR (8 Gy, 2h; Fig. 3, right, lane 3), UV (25 J/m2, 24h; Fig. 3, right, lane 4) and ADR (51 nm,24h; Fig. 3, right, lane 5) were similar to that for A549 cells (Fig. 3, left), suggesting that the signaling pathways producing these modifications in response to DNA damage probably are common to most cell types. No phosphorylation was observed at Thr18 in HCT116 cells, and the signals for Ser9 and Ser20 were much weaker than for A549 cells. We next examined modifications at each of the 12 p53 sites after exposure for 24 h to the three non-genotoxic agents PALA, taxol, and nocodazole, each at three times their IC50 (Fig. 3). Each induced p53 accumulation to levels similar to those obtained after UV or ADR; however, only a subset of the sites showed strongly enhanced phosphorylation. Phosphorylation was induced at Ser6, Ser33, Ser46, Ser315, and Ser392 in both cell lines by PALA (Fig. 3, lane 6), taxol (Fig. 3, lane 7), and nocodazole (Fig. 3, lane 8) to levels equivalent or nearly equivalent to those obtained with UV light or ADR. In contrast, phosphorylation at Ser9, Ser15 and Ser20 (in A549 cells) was significantly weaker than with UV light or ADR, although some phosphorylation was observed at Ser15. No phosphorylation was observed at Thr18, nor was acetylation observed at Lys320. Lys382 became acetylated in response to PALA or nocodazole, but acetylation at this site was much weaker than after UV (Fig. 3, lane 4). Similar results to those shown in Fig. 3 also were obtained with WS1 normal human skin fibroblasts and MRC-5 normal human lung fibroblasts (data not shown), although in both of t
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