PMC

PAbLys(Ac)382 and PAbLys(Ac)320 recognize p53 acetylated by p300 and PCAF, respectively. Recombinant p53 was acetylated by incubation with p300, PCAF, or both, and a Western blot was prepared as described in Fig. . Affinity-purified rabbit polyclonal antibodies were prepared by use of acetylated peptides corresponding to sequences around Lys-382 and Lys-320 as described in Materials and Methods. The blot was probed sequentially with PAbLys(Ac)382, PAbLys(Ac)320, and DO-1, a monoclonal antibody specific for the amino terminus of p53. (Lane 1) Unacetylated p53; (lane 2) p53 incubated with p300 and acetyl–CoA; (lane 3) p53 incubated with PCAF and acetyl–CoA; (lane 4) p53 incubated with both p300 and PCAF and acetyl–CoA.

Acetylation of mutant and phosphorylated p53 carboxy-terminal fragments by PCAF and P300. Chemically synthesized, unmodified p53(319–393), p53(319–393) with phosphoserine incorporated at either Ser-392 or Ser-378, or p53(319–393) with alanine replacing each of the three tetramerization domain residues Leu-323, Tyr-327, and Leu-330 [p53(319–393)AAA] were acetylated with p300. The radioactive image (¹⁴C-Label) is shown in C; the corresponding Coomassie brilliant blue (CBB)-stained image is in A. PCAF acetylation is in D; the corresponding Coomassie brilliant blue-stained image is in B. Histone H1 served as a positive control for acetylation. (Lanes M) Molecular weight markers; (lanes 1) histone H1; (lanes 2) unmodified p53(319–393); (lanes 3) p53(319–393) phosphorylated at Ser-392; (lanes 4) p53(319–393) phosphorylated at Ser-378; (lanes 5) tetramerization mutant p53(319–393)AAA.

Inhibition of in vitro acetylation by PCAF (open bars) and p300 (solid bars) of full-length, wild-type p53 by p53 peptides. Assays were performed as described in Materials and Methods and the radioactivity incorporated into wild-type p53 was quantitated with a PhosphorImager and Imagequant software. Amino-terminal p53 peptides and phosphopeptides were present at 100 μm; the p53 substrate concentration was 0.1 μm. The results shown are an average of three independent assays for p300 (error bars, s.d.) and two independent assays for PCAF (error bars, range). Acetylation of the carboxy-terminal fragment p53(319–393) by p300 was not inhibited by these same amino-terminal peptides (data not shown).

Phosphorylation by DNA–PK potentiates acetylation of human p53 by p300. Recombinant human p53 (250 ng in 20 μl) purified from insect cells was incubated with purified DNA–PK (40 units), DNA, and ATP at 30°C for 1 hr and then with recombinant p300 (∼0.5 ng) and acetyl–CoA at 37°C for 20 min. Western blots of the reaction products were probed sequentially with PAbLys(Ac)382, PAbSer(P)33, PAbSer(P)37, and DO-1. Note that incubation with DNA–PK produced a new p53 isoform (labeled 3) that is phosphorylated on Ser-37 as well as on Ser-33. This isoform was preferentially acetylated by p300.

Phosphorylation and acetylation of p53 in response to UV and γ radiation after ALLN treatment. A549 cells were treated with 20 μm calpain inhibitor I (ALLN) and 4 hr later were exposed to 25 J/m² of UV-C or 8 Gy γ-rays (IR). TSA was added to 5 μm immediately after DNA damage treatment. Samples were harvested at the indicated times after DNA damage treatment (Lanes 0,1,2,4 hr), and extracts were immunoprecipitated and analyzed by western immunobloting as described in Fig. and Materials and Methods. Blots were probed sequentially with PAbLys(Ac)382, PAbSer(P)33, PAbSer(P)37, and DO-1.

Identification of the p53 sites acetylated by PCAF and p300. (A) p53(319–393) acetylated by p300 was digested with trypsin and the peptide products were analyzed by nanospray ion trap mass spectrometry. (Bottom) The fragmentation mass spectrum for the acetylated peptide of m/z 708. The mass of the acetylated tryptic product corresponds to that of monoacetylated p53(382–386). (Top) The sequence and expected m/z values of fragmentation products. Observed ions are underlined. The fragmentation pattern is consistent only with acetylation of Lys-382 and identical to synthetic p53(382–386) with Lys-382 acetylated. (B) p53(319–393) acetylated by PCAF was digested with trypsin and the peptide products were analyzed by nanospray ion trap mass spectrometry. (Bottom) The fragmentation mass spectrum for the acetylated peptide of doubly charged ion of m/z 876. Segments of the profile were expanded in thedirection of the y-axis by 2-, 5-, or 10-fold as indicated. The mass of the acetylated tryptic product corresponds to that of monoacetylated p53(320–333). (Top) The sequence and expected m/z values of fragmentation products. Observed ions are underlined. The fragmentation pattern is consistent only with acetylation of Lys-320 and identical to synthetic p53(320–333) with Lys-320 acetylated.

p300 and PCAF each acetylate a single site in the carboxyl terminus of human p53. E. coli expressed p53(319–393) was acetylated with either p300 or PCAF, and the repurified fragments were analyzed by ion spray mass spectrometry. The mass profiles are shown. The major peak labeled A in each case corresponds to the mass of the unmodified p53 fragment (8707); the major peak labeled B is larger by 42 mass units, corresponding to the addition of one acetate residue. No mass was observed at the position expected for diacetylated or higher acetylated forms. Minor shoulders result from oxidation of methionine to the sulfone (A⁰ and B⁰) and formation of the phosphate salt (A^p and B^p).

Model for the activation of p53 in response to DNA damage. Unmodified p53 interacts with the Mdm2 protein, which targets it for rapid degradation. p53 that enters the nucleus interacts with chromatin through the carboxy-terminal domain that prevents sequence-specific DNA binding. DNA damage induces phosphorylation of amino-terminal p53 residues that increase p53’s affinity for p300 and PCAF, thus promoting acetylation of carboxy-terminal sites thereby allowing sequence-specific DNA binding. The model incorporates recent findings from other studies including the role of phosphorylation in regulating p53’s interaction with mdm2 (), induction of amino-terminal phosphorylation by UV, IR, and other agents (), and the effect of p53’s nonspecific interaction with DNA on sequence-specific DNA binding ().

Induction of p53 acetylation and phosphorylation by UV, γ-rays, and ALLN. A549 cells were exposed to 25 J/m² of UV-C, 8 Gy γ-rays (IR), or treated with 20 μm calpain inhibitor I (ALLN). TSA was added to 5 μm immediately after DNA damage treatment. Samples were harvested at the indicated times after initiating treatment (top of lanes), and extracts were prepared for immunoprecipitation with Pab1801, a monoclonal antibody specific for human p53, followed by Western immunoblot analysis as described in Materials and Methods. The Western blots were probed sequentially with PAbLys(Ac)382, PAbLys(Ac)320, PAbSer(P)37, and DO-1.

Acetylation of p53 and p53 fragments by p300 and PCAF. Wild-type human p53 or truncated p53 fragments were acetylated with either PCAF or p300 at 37°C for 20 min as described in Materials and Methods, and the reaction products were analyzed by SDS-PAGE. P300 acetylation (¹⁴C-Label) is depicted in the radioactive image in C; the corresponding Coomassie brilliant blue-stained image (CBB) is in A. PCAF acetylation is in D; the corresponding Coomassie brilliant blue-stained image is in B. Histone H1 served as a positive control for acetylation (). (Lanes M) Molecular weight markers; (lanes 1) full-length wild-type, baculovirus-produced human p53; (lanes 2) p53(1–355); (lanes 3) p53(283–393); (lanes 4) p53(318–393); (lanes 5) histone H1.

Activation of sequence-specific binding by acetylation of p53 with p300 and PCAF. Baculovirus-produced wild-type p53 was acetylated with p300 or with PCAF as described in Materials and Methods, and the reaction products then were used in electrophoretic mobility shift assays as described by . (A) Radioactive images of the EMSA gels; the ingredients present during the p53 modification reactions are indicated at top. (Lanes 14,15) The order of p300 and PCAF additions are indicated by superscripts; (lane 7,11) unacetylated CoA was added in place of acetyl–CoA (Ac–CoA). The p53-shifted radioactive probe appears as a band near the top of the gel; free probe is at the bottom. (B) Parallel acetylation reactions were performed with ¹⁴C-labeled acetyl–CoA, and the reactions were fractionated by SDS-PAGE. Shown is the radioactive image of the gel. (Lane 1) Reaction with p53 and p300, (lane 2) reaction with p53 and PCAF; (lane 3) reaction with p53 incubated with PCAF and then also with p300; (lane 4) reaction with p53 incubated with p300 and then with PCAF.