Identification and Characterization of a Novel p300-mediated p53 Acetylation Site, Lysine 305*

Yan-Hsiung Wang {ddagger}, Yeou-Guang Tsay {ddagger}, Bertrand Chin-Ming Tan {ddagger}, Wen-Yi Lo {ddagger} and Sheng-Chung Lee {ddagger} § ¶ ||

From the Institutes of {ddagger}Molecular Medicine and §Clinical Medicine, College of Medicine, National Taiwan University, and the Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan

Received for publication, December 10, 2002 , and in revised form, April 30, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Post-translational modifications serve as important regulatory elements in modulating the transcriptional activity of the tumor suppressor protein p53. We have previously reported a tandem mass spectrometry-based method (viz. selected ion tracing analysis) that can be applied to the identification of phosphopeptides as well as exact mapping of the phosphorylated residues within. In this study, we describe the application of the same strategy for the identification of p300 acetyltransferase-mediated acetylation sites on p53. Consistent with the previous finding, lysines 370, 372, 373, 381, and 382 were detected by this modified selected ion tracing method as the target sites of p300 in vitro. Moreover, two novel acetylation sites, Lys-292 and Lys-305, were also found. Immunoblotting using anti-acetyl-Lys-305 antibody confirmed this discovery and demonstrated that Lys-305 could be acetylated by p300 both in vitro and in vivo. We also show that an alanine or glutamine substitution at Lys-305 (K305A or K305Q) suppressed the transcriptional activity of p53, whereas an arginine mutation (K305R) increased the transcriptional activity. Thus, p300 may further regulate the transcriptional activity of p53 through a novel acetylation site, Lys-305.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The p53 tumor suppressor protein is a sequence-specific, DNA-binding transcription factor. In response to a wide variety of stress signals, it acts to regulate processes such as the cell cycle, cell death, and DNA repair. Loss of p53 activity has been identified in 60% of the human cancers examined, and >90% of the p53 missense mutations are clustered within the sequence-specific DNA-binding domain (1, 2). The tumor suppressor functions of p53 are directly linked to its ability to mediate transcriptional activation (3).

The regulation of p53 transcriptional activity involves several mechanisms including post-translational modifications such as phosphorylation, acetylation, and ubiquitination (49). Most of these modifications occur in the N- and C-terminal regions of p53. Stress-induced N-terminal phosphorylation increases p53 stability by dissociating MDM2, a negative regulator. For example, Ser-15, Thr-18, and Ser-20, located in the MDM2-binding site, are phosphorylated in response to DNA damage. This phosphorylation is probably induced by protein kinases such as ATM, ATR, Chk2, CKI, and DNA-PK (1012). Acetylation is another type of p53 post-translational modification shown to be affected by DNA damage signals (9, 13). Two histone acetyltransferases (HATs)1 are known to acetylate p53, although on different sites: p300/CBP acetylates the C terminus of p53 at lysines 372, 373, 381, and 382, whereas p300/CBP-associated factor acetylates at Lys-320 (5, 9, 13). The acetylated residues of p53 are located in the regulatory domains adjacent to the tetramerization domain (amino acids 252–355). Acetylation of p53 may be an important regulatory mechanism of p53 function because p53 deacetylation by overexpression of histone deacetylase-associated proteins compromises its ability to induce cell cycle arrest and apoptosis (14). Inhibition of p53 deacetylation also increases the p53 half-life, suggesting that acetylation may play important roles in the turnover of the p53 protein (15).

Recently, we employed a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify residues bearing phosphorylation modifications (16). Based on this technique, moderately phosphorylated peptides can be identified by close examination of specific ion chromatograms (16). This selected ion tracing (SIT) method has a major advantage in analyzing modified peptides of low abundance in a complex peptide mixture. More importantly, we have proposed that this method can be generally employed to identify peptides of various modifications. Here, we illustrate how this approach is adapted to identify acetylated peptides and to map the modified residues. Although we successfully verified the previously reported acetylated residues of p53, we also uncovered two novel acetylation targets using this approach. We also present evidence that acetylation of p53 at Lys-305, like that at Lys-382, is elevated by different stress signals. In addition, we found that acetylation at Lys-305 is important for regulating p53 transcriptional activity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs and Reagents—Plasmid pcDNA3-p53w, which contains human wild-type p53, was kindly provided by Dr. Ming-Yang Lai (National Taiwan University). The mammalian expression vector for hemagglutinin-tagged p300 and the bacterial expression vector containing the His-tagged HAT domain (amino acids 1195–1673) of p300 were gifts from Dr. Li-Jung Juan (National Health Research Institutes, Taipei, Taiwan). The p50-2 reporter, which contains the p53-responsive promoter, was obtained from Dr. Young-Sun Lin (Academia Sinica). C-terminally FLAG-tagged p53 was produced by PCR amplification with a pair of primers (forward T7 oligonucleotide, 5'-TAATACGACTCACTATAGGG-3'; and reverse C-terminal FLAG primer, 5'-TCACTTATCGTCGTCATCCTTGTAATCGTCTGAGTCAGGCCCTTCTGTCT-3', where the underlined sequence corresponds to the FLAG tag sequence). PCR was carried out by 30 cycles at 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 1.5 min, followed by denaturation at 94 °C for 2 min and elongation at 72 °C for 5 min. The PCR fragment was then cloned into the pCR2.1-TOPO plasmid using the TA cloning kit (Invitrogen). C-terminally FLAG-tagged p53 cDNA was excised by EcoRI digestion and cloned into the corresponding site of pcDNA3 (Invitrogen). C-terminally FLAG-tagged p53 was also subcloned into the baculovirus expression vector pVL1393 (Pharmingen). The expression plasmid pVL-p53-FLAG was introduced into Sf21 insect cells using a BaculoGold transfection kit (Pharmingen). The FLAG-p53 protein was purified using an anti-FLAG (M2) immunoaffinity column. The His-tagged HAT domain (amino acids 1195–1673) of the p300 protein (HAT-p300) was expressed in Escherichia coli BL21(Lys) cells transformed with a bacterial expression vector. The protein was purified from the clarified lysate on nickel-nitrilotriacetic acid-agarose (QIAGEN Inc.) according to the manufacturer's instructions and then dialyzed against storage buffer (20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 100 mM KCl, 20% glycerol, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) before storage at –80 °C in aliquots.

Site-directed Mutagenesis—Mutants of FLAG-tagged p53 were constructed by site-directed mutagenesis using a two-step PCR technique as described (17, 18). Briefly, mutants were prepared by introducing the desired mutation into overlapping oligonucleotide primers. In the first round of PCR, the mutagenic primer and an outside primer were used to amplify a partial fragment of the region in which the mutation would be created. This mutagenized fragment was then purified and used as a "megaprimer," together with the second outside primer, in a second round of PCR to amplify the entire region to be subcloned. Conditions for the first round of PCR were 30 cycles at 94 °C for 30 s, 47 °C for 30 s, and 72 °C for 1 min. Conditions for the second round of PCR were as follows: a denaturation step at 94 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 46.5 °C for 30 s, and 72 °C for 1.5 min and an elongation step at 72 °C for 10 min.

Plasmid pcDNA3 containing FLAG-tagged p53 was used as the template in the PCR. The outside primers used for all of the mutants in this study were as follows: the forward T7 oligonucleotide and SP6 oligonucleotide (5'-ATTTAGGTGACACTATAG-3'). Mutations were introduced into wild-type p53 by two-step PCR mutagenesis with oligonucleotides containing base alterations to convert lysines 305 and 382 to alanine, arginine, or glutamine. The oligonucleotides for the mutants were as follow (where underlined regions correspond to direct amino acid substitutions): FP53-K305R, 5'-AGGGAGCACTCGGCGAGCACTGC-3'; RP53-K305R, 5'-GCAGTGCTCGCCGAGYGCTCCCT-3'; FP53-K305A, 5'-AGGGAGCACTGCGCGAGCACTGC-3'; RP53-K305A, 5'-GCAGTGCTCGCGCAGTGCTCCCT-3'; FP53-K305Q, 5'-CCCCCAGGGAGCACTCAGCGAGCACTGCCCAAC-3'; RP53-K305Q, 5'-GTTGGGCAGTGCTCGCTGAGTGCTCCCTGGGGG-3'; FP53-K382R, 5'-CTCCCGCCATAAACGACTCATGT-3'; RP53-K382R, 5'-ACATGAGTCGTTTATGGCGGGAG-3'; FP53-K382A, 5'-CTCCCGCCATAAAGCACTCATGT-3'; RP53-K382A, 5'-ACATGAGTGGTTTATGGCGGGAG-3'; FP53-K382Q, 5'-CCTCCCGCCATAAACAACTCATGTTCAAGACAG-3'; and RP53-K382Q, 5'-CTGTCTTGAACATGAGTTGTTTATGGCGGGAGG-3'. The mutated PCR fragments were cut with BamHI and XhoI and subcloned into pcDNA3.

Cell Culture, Transient Transfections, and Reporter Assays—H1299 human lung carcinoma cells and MCF-7 human breast cancer cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone Laboratories) at 37 °C in a 5% CO2 humidified atmosphere. Approximately 1 x 106 cells were seeded in each 100-mm culture dish 12–24 h before transfection. Calcium phosphate-mediated DNA transfection was performed as described previously (19). DNA was prepared by the Clontech procedure and adjusted to 12 µg/transfection with the pcDNA3 plasmid. Chloramphenicol acetyltransferase (CAT) assays were performed with two-thirds of the total cell extract as described (17, 20). p50-2, which contains two copies of the p53-binding motif in its promoter region, was used as the p53-responsive CAT reporter plasmid (21). A 1-µg aliquot of a Rous sarcoma virus/{beta}-galactosidase vector was included as an internal control in all transfections. Quantification of acetylated [14C]chloramphenicol was determined with a PhosphorImager (Amersham Biosciences). The {beta}-galactosidase assays (Promega) were performed according to the manufacturer's protocols. A total of 10 µg of plasmid were used for each experiment.

Antibodies—Antisera against acetylated p53 peptides were generated in rabbits using keyhole limpet hemocyanin-conjugated acetylated peptides (p53-(300–310), PPGSTK*RALPN, acetylated at Lys-305; and p53-(376–387), STSRHKK*LMFKT, acetylated at Lys-382) (22). The antisera from immunized rabbits were affinity-purified successively with non-acetylated and acetylated peptide columns (10). Monoclonal antibody DO-1 was obtained from Oncogene Research Products. Anti-FLAG monoclonal antibody M2 was obtained from Sigma.

Western Blot and Dot-blot Analyses—To verify the antibody specificity, we used dot-blot analysis as described previously (23). Equal amount of peptides (20 µg) were spotted onto strips of Hybond-C membrane (Amersham Biosciences). After blocking with 5% nonfat milk in phosphate-buffered saline and 0.1% Tween 20, membranes were probed with the purified rabbit polyclonal antibodies. The blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) and visualized using the enhanced chemiluminescence system (ECL, Amersham Biosciences).

Western blotting was performed as described previously (24). Briefly, equal amounts of cell extracts were separated on 10% SDS-polyacrylamide gel and blotted onto membranes. The membranes were blocked with 5% nonfat milk in phosphate-buffered saline and 0.1% Tween 20 and probed with anti-p53 (DO-1), anti-FLAG, anti-acetyl-Lys-305 (Ab-165), or anti-acetyl-Lys-382 (Ab-166) antibody.

In Vitro Acetylation Assay—Acetylation assays were carried out primarily according to published methods (1, 6). Four micrograms of recombinant FLAG-p53 were acetylated in vitro by incubation with the indicated amount of HAT-p300 in the presence of a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 10% (v/v) glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and 18 µM [14C]acetyl-CoA (Amersham Biosciences). The mixture was incubated at 30 °C for 60 min and analyzed by SDS-PAGE, followed by Coomassie Blue staining. 14C-Labeled p53 was detected by autoradiography and quantified using a PhosphorImager.

In-gel Digestion—The gel piece containing p53 polypeptides was first reduced and pyridylethylated as described previously (16). Up to 0.2 µg of enzyme (Arg-C or Asp-N (Roche Applied Science)) were added to the dried gel. After overnight incubation, the supernatant was removed, and the gel was extracted twice with an adequate amount of 0.1% formic acid. The supernatant and extracts were combined together and dried in a SpeedVac. The digests were kept at –20 °C and suspended in 0.1% formic acid immediately before use.

LC-MS/MS Analysis—Electrospray mass spectrometry was performed using a Finnigan MAT LCQ ion trap mass spectrometer interfaced with an ABI 140D HPLC apparatus (PerkinElmer Life Sciences). A 150 x 0.5-mm PE Brownlee C18 column (5-mm particle diameter, 300-Å pore size; PerkinElmer Life Sciences) with mobile phases of 0.1% formic acid in water and 0.085% formic acid in acetonitrile were used. The peptides were eluted using the acetonitrile gradient and analyzed in a "triple-play" experiment as described (16). The samples were analyzed by two runs of LC-MS/MS experiments. In the first analysis, the most abundant ion in a mass spectrum was selected for a collision-induced dissociation (CID) experiment; in the latter analysis, only the ions with m/z values corresponding to the potential acetylated peptides were selected for a CID experiment. The acquired CID spectra were interpreted using a Finnigan MAT software package, SEQUEST Browser, which correlated the tandem mass spectrum with the amino acid sequence of the human p53 protein. Enzyme was not specified in the search parameters, which increased the confidence of identification when the matched peptides had appropriate cleavage sites. A 105.14-Da tag was constitutively assigned to the Cys residue, which was modified with 4-vinylpyridine in these experiments. Those MS/MS scans that matched the peptide sequences with proper cleavage sites were considered significant and also were subjected to manual evaluation to confirm the SEQUEST results.

The hypothetical m/z values of peptides used for generation of ion tracing chromatograms were derived using the PEPSTAT algorithm in the SEQUEST package. The EXPLORE program (Finnigan MAT) was used to plot the ion tracings.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determination of p300-mediated in Vitro Acetylation Sites on p53 by LC-MS/MS—In our previous report (16), we established the SIT method to identify phosphorylated peptides. Here, we set out to test whether the SIT method could be adapted to identify acetylated peptides. Previous studies have demonstrated that p53 can be acetylated by p300 in vitro (5). Therefore, we analyzed acetylated peptides derived from recombinant FLAG-tagged p53 protein that was acetylated in vitro by p300. Baculovirus-expressed recombinant FLAG-tagged p53 was immobilized on antibody M2-agarose beads and served as the substrate for the in vitro acetylation reactions. Various amounts of the HAT domain from the p300 acetyltransferase (HAT-p300) were incubated with a fixed amount of FLAG-p53 in the presence of [14C]acetyl-CoA (Fig. 1A). After in vitro acetylation, the reaction mixtures were analyzed by SDS-PAGE, followed by autoradiography (Fig. 1B). FLAG-tagged p53 was acetylated by the enzyme in a dose-dependent fashion. Quantitative determination of the autoradiogram showed that the acetylation was not saturated.



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FIG. 1.
In vitro acetylation of recombinant FLAG-p53 by the HAT domain of p300 acetyltransferase. A, 4 µg of FLAG-p53 were incubated with various amounts of HAT-p300 (0 ng (lane 1), 3 ng (lane 2), 10 ng (lane 3), 30 ng (lane 4), and 100 ng (lane 5)) in the presence of [14C]acetyl-CoA for 1 h at 37 °C. The reaction mixtures were subjected to SDS-PAGE, followed by staining with Coomassie Blue. The dried gel was then subjected to autoradiography. The numbers on the left indicate the migration positions of molecular mass markers (in kilodaltons). B, shown are the results from the quantitative analysis of the autoradiogram in A for acetylated p53 polypeptides.

 

Acetylated p53 resolved on the gel was excised and divided into two portions. One was treated with Arg-C, and the other was digested with Asp-N. An aliquot of each digested peptide mixture was analyzed by LC-MS/MS. The raw data were then interpreted using the SEQUEST software with the optional addition of 44 Da to the Lys residues. This analysis yielded 15 Arg-C peptides and 6 Asp-N peptides (Table I). These two peptide groups were estimated to account for 87% (350/401) of the amino acid residues of FLAG-p53 and 95% (21/22) of the lysine residues (Fig. 2A). Two acetylated residues were found directly using the SEQUEST software, Lys-373 and Lys-381/382. Subsequently, we used the SIT approach to examine all of these peptides, and their putative acetylated analogs were chromatographed. We found that three Arg-C peptides and one Asp-N peptide were probably acetylated (Table I).


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TABLE I
Characteristics of acetylated residues of the p53 protein

 


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FIG. 2.
Identification of p300-mediated acetylation sites of FLAG-p53 in vitro. A, peptide coverage of the p53 protein by automated LC-MS/MS analysis. Boldface letters represent the peptides identified by LC-MS/MS analysis, and gray letters represent the residues that were not covered. The hatched underlines denote the sequence coverage of Arg-C peptides, and the gray underlines indicate the residues corresponding to Asp-N peptides. The identified acetylated lysine residues are indicated by closed arrowheads, whereas these acetylated residues are marked by open arrowheads. B, chromatographic tracing (SIT) of the ions at m/z 600.4 (upper panel), 615.0 (middle panel), and 462.5 (lower panel). The retention times are noted above the major peaks. C, CID spectrum of the m/z 615 ion at 35.6 min. D, CID spectrum of the m/z 615 ion at 39.8 min. NL, normalization, amplitude magnitude for a better view; aK, acetylated lysine residue.

 

In particular, we found that the triply charged, unmodified peptide-(291–306) with m/z 600.4 had a retention time of 35.66 min in our analysis (Fig. 2B). The +3 ion tracing for the singly acetylated peptide has multiple peaks migrating closely with the unmodified peaks (Fig. 2B, middle panel). To determine whether these contained the acetylated peptide ions, the +4 ion tracing was also chromatographed (Fig. 2B, lower panel). The results reveal that there were two major peaks (at 35.6 and 39.8 min) that were commonly seen in the two tracings. On the other hand, many signals found between 50~60 min in the +3 ion tracing were not observed in the +4 ion tracing. This is consistent with the notion that these peaks contain ions that may have different molecular masses but that have similar m/z values compared with the triply charged peptide-(291–306). Also, based on their relative abundances, these lysine residues appeared to be modified at a lower efficiency (Fig. 2B).

The CID spectrum of the 615 m/z ion in the peak at 35.6 min was acquired for identification of the acetylation sites (Fig. 2C). The presence of y4 and y5 ions argues against the possibility that Lys-305 is acetylated, whereas the masses of the b9 and b10 ions indicate that the acetyl group is on the lysine residue at the N-terminal end. The observation of a y15 ion indicates that Lys-292 is the acetylated residue.

The CID spectrum of the 615 m/z ion in the peak at 39.8 min is shown in Fig. 2D. The observation of b7–b12 ions indicates that the acetyl group is on the C-terminal three amino acids. Lys-305 is presumably the acetylation target base on the m/z values of the y1 and y2 ions. This assumption is further supported by the fact that it is the only acetylatable residue in the region. Therefore, we identified two distinct acetylation sites, Lys-292 and Lys-305, in this particular peptide.

Subsequently, we used the SIT approach to analyze peptide-(354–379) and peptide-(380–401). Both peptides could take up as much as two acetyl groups. The three sets of Arg-C peptides were subjected to LC-MS/MS analysis, which was carried out in a mass-specific mode such that the necessary tandem mass spectra were acquired. The tandem mass spectrum of the peptide-(380–401) ion indicated that the acetylation group could be located at either Lys-381 or Lys-382. Additionally, double acetylation at Lys-381 and Lys-382 was also observed (data not shown). These results are in agreement with the previous finding that both Lys-381 and Lys-382 are acetylated. Although the SEQUEST analysis revealed that peptide-(354–379) could be singly acetylated, this region could incorporate as many as two acetyl groups based on SIT analysis. Nevertheless, our current data could not pinpoint the exact modified residues in the four-amino acid stretch from Lys-370 to Lys-373. Thus, p53 could be in vitro acetylated by p300 at lysines 370, 372, and 373 as well as lysines 382 and 383. Moreover, novel p53 sites of acetylation by p300 were also found at lysines 292 and 305.

Detection of in Vitro Acetylated p53 by Antibodies Specific to Acetyl-Lys-305 and Acetyl-Lys-382 of p53—The rabbit antibodies raised against Lys-305 (Ab-165) and Lys-382 (Ab-166) were purified by peptide affinity chromatography. The specificity of the purified antibodies was verified by dot-blot analysis (Fig. 3A). We found that Ab-165 specifically recognized acetylated peptide-(300–310) (Ac-K305), but it did not recognize acetylated peptide-(376–387) (Ac-K382) or an acetylated peptide from the HMG-14 peptide (Ac-HMG) (Fig. 3A, upper panel). Likewise, Ab-166 recognized only acetylated peptide-(376–387), but not acetylated peptide-(300–310) or the acetylated HMG-14 peptide (Fig. 3A, lower panel).



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FIG. 3.
Specificities of Ab-165 and Ab-166. A, dot-blot analysis of purified Ab-165 and Ab-166 against synthetic acetylated peptides. Acetylated peptides (20 µg) were spotted onto Hybond-C membrane. Lane 1, acetylated peptide-(300–310) (Ac-K305); lane 2, acetylated peptide-(376–387) (Ac-K382); lane 3, acetylated HMG-14 peptide (Ac-HMG). The membrane was then subjected to immunoblotting with the indicated antibodies. B, Ab-165 and Ab-166 recognize in vitro acetylated recombinant FLAG-p53. Recombinant FLAG-p53 (0.2 µg) was acetylated by HAT-p300 at 0.1 µg (lanes 3 and 4) or 0.4 µg (lane 5) in the presence of acetyl-CoA (100 pmol; lanes 2, 4, and 5) or in its absence (lanes 1 and 3). Acetylated p53 was detected by immunoblotting with the indicated antibodies. The levels of p53 protein in these reactions were revealed by immunoblotting with anti-p53 monoclonal antibody DO-1.

 

We next tested whether these antibodies can recognize full-length p53 acetylated by p300 in vitro (Fig. 3B). After in vitro acetylation, recombinant FLAG-p53 was analyzed by immunoblotting using Ab-165 and Ab-166. The amount of p53 used for each reaction was about the same (Fig. 3B, lower panel). Ab-165 recognized p53 that was acetylated in the presence of p300 and acetyl-CoA. The acetylated signals increased proportionately to the p300 amounts in the reaction (Fig. 3B, upper panel). Similarly, Ab-166 specifically recognized acetylated (but not non-acetylated) p53 (Fig. 3B, middle panel). Ab-165 and Ab-166 did not recognize p53 in the absence of acetyl-CoA or p300 in the acetylation reactions (Fig. 3B). These results indicate that Ab-165 and Ab-166 specifically recognized acetylated p53. Together with the fact that Ab-165 specifically recognized acetylated Lys-305, these data further corroborate the conclusion that Lys-305 is indeed acetylated in vitro by p300 in a dose-dependent fashion.

Lys-305 Is Acetylated by p300 in Vivo—To address whether Lys-305 of p53 might be acetylated in vivo, we performed an in vivo acetylation assay that previously identified Lys-382 as an acetylation target. H1299 cells were transfected with the plasmid expressing C-terminal p53 (Fig. 4). Without further treatment, no acetylated signal was detected on the Western blot using the indicated antibodies. Upon addition of sodium butyrate, an inhibitor of histone deacetylases, acetylation at Lys-305 as well as at Lys-382 was detected (Fig. 4). Overexpression of hemagglutinin-tagged p300 also enhanced acetylation at Lys-305, similar to Lys-382. This enhancement was correlated with the hemagglutinin-p300 levels (Fig. 4). Synergism between sodium butyrate and p300 overexpression was also observed for the Lys-305 acetylation (Fig. 4). These data indicate that Lys-305, like Lys-382, is an acetylation target of p300.



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FIG. 4.
Sodium butyrate and p300 enhance acetylation at Lys-305 in vivo. H1299 cells were transiently transfected with FLAG-p53-encoded plasmid with or without p300 and subsequently treated or not with sodium butyrate (50 µM) for 6 h. Forty-eight hours after transfection, whole cell lysate from each transient transfection group was prepared and analyzed by SDS-PAGE. The acetylation level at each residue was determined by immunoblotting with the indicated antibodies. The amounts of p53 protein were revealed by immunoblotting with antibody DO-1.

 

Acetylation of p53 at Lys-305 Is Induced by Various Stress Stimuli—Previous studies have shown that various stress stimuli are capable of inducing p53 acetylation (25, 26). To determine whether Lys-305 acetylation is induced in response to these stimuli, we used Ab-165 and Ab-166 to monitor the acetylation levels of p53 in normal and stressed cells. First, we tested whether Lys-305 acetylation is induced by UV or ionization irradiation. MCF-7 cells were exposed to 50 J/m2 UVC or 20-gray {gamma}-rays (IR), and the cell lysates were prepared at various time points after treatment. Before initiation of DNA damage, these cells were treated with sodium butyrate to reduce the rapid deacetylation by endogenous histone deacetylase activity (13).

Without any stimuli, the level of p53 in MCF-7 cells was low, whereas acetylation could not be detected with either Ab-165 or Ab-166 (Fig. 5A, lane 1). Sodium butyrate treatment slightly increased the p53 level, which could be sustained for as long as 8 h after treatment (Fig. 5A, lanes 2–4). After IR, acetylation at Lys-382 was detected as early as 1 h and become prominent at 4 h post-irradiation. A similar course was also seen upon IR treatment for Lys-305 (Fig. 5A, lanes 5–7). Meanwhile, UV irradiation dramatically increased Lys-305 acetylation at 8 h after treatment (Fig. 5A, lanes 8–10). The kinetics of irradiation-induced p53 acetylation at Lys-305 and Lys-382 were very similar. This suggests that Lys-305 and Lys-382 acetylation might be activated through a similar mechanism under these conditions.



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FIG. 5.
Lys-305 acetylation is elevated in response to various stress stimuli. MCF-7 cells were treated with a variety of p53-activating agents. A, the cells were treated with sodium butyrate (50 µM; lanes 2–4), IR irradiation (20 grays; lanes 5–7), and UV (50 J/m2; lanes 8–10), and then the cell lysates were analyzed by Western blotting using the indicated antibodies. B, the cells were treated with H2O2 (1 mM; lanes 2–5) and actinomycin D (ActD; 5 nM; lanes 6–9), and then lysates were analyzed by Western blotting using the indicated antibodies. The cells were harvested at various time points after these treatments.

 

The effects of H2O2 and actinomycin D on p53 acetylation in MCF-7 cells were also investigated. Like irradiation-induced responses, actinomycin D induced accumulation of p53 as well as acetylation at Lys-305 (Fig. 5B). However, H2O2-mediated oxidative stress resulted in a slower accumulation of p53 than that elicited by actinomycin D. Intriguingly, it appears that the kinetics of Lys-305 acetylation was quite different from that of Lys-382 acetylation (Fig. 5B). Lys-305 acetylation reached the highest level within 1 h after treatment, whereas Lys-382 acetylation appeared to be higher at 4–8 h after treatment (Fig. 5B). Hence, the H2O2-induced p53 acetylation at Lys-305 and Lys-382 showed different kinetic patterns, implying that different mechanisms are responsible for acetylation at these two sites under this particular condition. Together, these results confirm that acetylation at Lys-305, like that at Lys-382, is responsive to all p53-activating agents, further suggesting that Lys-305 acetylation may play important roles in p53 activation.

Lys-305 Is Important for p53 Transcriptional Activity—To further determine the function of Lys-305, we tested whether it might affect p53-mediated transcriptional activation. We analyzed the effects of site-specific mutants of Lys-305 on transactivation function. Plasmids expressing various Lys-305 and Lys-382 mutants as well as wild-type proteins were introduced into the p53-null human lung cancer cell line H1299. These proteins were examined for their ability to activate a p53-responsive CAT reporter plasmid (p50-2) that contains two copies of the p53-binding motif in its promoter region (21).

Overexpression of wild-type p53 generated an ~50-fold stimulation of the CAT activity (Fig. 6), which is similar to previous reports that p53 is sufficient to stimulate this promoter activity of the p50-2 reporter gene (22, 27). When cells expressed the p53 mutant K305R, the p50-2 promoter activity was ~2-fold higher than that induced by wild-type p53. In contrast, overexpression of the K305A mutant resulted in a 25–30% decrease in p50-2 promoter activity. These mutations had similar effects compared with the Lys-382 mutants (Fig. 6A), further suggesting that Lys-305 may influence the transcriptional activity of p53 through a similar mechanism mediated by Lys-382 acetylation. Unexpectedly, overexpression of the glutamine substitution p53 mutants K305Q and K382Q did not activate the p50-2 promoter, and the CAT activities were similar to those of the mock control (Fig. 6A). These glutamine mutants seem to have completely lost the intrinsic p53 transcriptional activity.



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FIG. 6.
Effects of Lys-305 mutations on the transcriptional activity of p53. A, H1299 cells were transfected with the p50-2 CAT reporter gene together with a plasmid expressing wild-type (p53w) or mutant p53 (K305A, K305R, K305Q, K382A, K382R, or K382Q). B, the transcriptional activities of wild-type p53 and p53 mutants were determined after the transfectants were treated with UV irradiation. After 48 h, {beta}-galactosidase and CAT activities were measured. Relative CAT activities were calculated from two independent experiments, normalized by {beta}-galactosidase expression, and the S.D. values were derived from the activity means. The Western blots show the protein expression levels of wild-type p53 and mutants and were performed using anti-FLAG antibody.

 

To further study the functional significance of Lys-305 acetylation, we tested whether it might affect p53-mediated transcriptional activation when exposed to stress stimuli (e.g. UV irradiation). After UV irradiation, the wild-type p53-mediated transcriptional activity stimulated the CAT activity by up to ~2-fold (Fig. 6B). However, none of p53 mutants could respond to UV irradiation by inducing higher CAT activities. The transcriptional activities of K305Q and K382Q was completely abolished whether the transfectants were treated with UV irradiation or not.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we first illustrated how partially acetylated peptides can be identified using an LC-MS/MS-based SIT approach. Using this approach, we successfully verified the previously reported in vitro p53 sites of acetylation by the acetyltransferase p300/CBP. We further demonstrated the prowess of this method by discovering two additional acetylation targets, Lys-292 and Lys-305. Several lines of evidence indicate that Lys-305 is acetylated both in vivo and in vitro by p300. The acetylation at Lys-305, just like that at Lys-382, may be induced by various stress stimuli that are known to activate p53. Transfection assays also established the important roles of Lys-305 acetylation.

LC-MS/MS analysis is very useful in the identification of residues bearing modifications such as phosphorylation (16). The SIT method is based on the hypothesis that the small modification groups do not change by much the overall physiochemical properties of peptides, and thus, the modified peptides have similar retentive behaviors in chromatographic analyses as the unmodified peptides. Based on the principles we previously discovered, modestly phosphorylated peptides could be identified by close examination of specific ion chromatograms (16). Here, our present data also indicate that this method can be applied to studying protein acetylation. Based on these observations, we surmise that this method should be applied to studies of other protein modifications such as methylation. Recently, we have indeed located dimethylarginine residues on several nuclear proteins using a similar approach.2

The most intriguing finding in this study is two additional novel p53 acetylation sites, Lys-292 and Lys-305. Considering that they have not been reported in the literature, it is quite surprising that acetylation at Lys-292 and Lys-305 was detected only by our system. There are several plausible explanations for our unique observation. The first is that the efficiency of acetylation at Lys-305 is probably much lower than that at other sites (Fig. 2B). The ion counts of the singly acetylated peptide for acetylation at Lys-381 and Lys-382 are about half of those of the unmodified isoform (data not shown). In contrast, the amounts of the acetylated peptides at Lys-292 and Lys-305 are only about one-thirtieth of those of the corresponding unmodified peptides. Therefore, acetylation at Lys-292 and Lys-305 may have been neglected in previous experiments due to their suboptimal labeling under these conditions.

Another possibility is that we used full-length p53 as the substrate rather than the deletion mutants used by other investigators (5). The reduction approach has been frequently used in experiments involving identification of modified residues. However, an important pitfall is that construction of these deletion mutants, either randomly or deliberately, is associated with distortion of important local protein conformations. This can lead to introduction of false targets or obliteration of true ones. As these two sites are positioned over the region between the DNA-binding and tetramerization domains, the context surrounding them may have been disrupted in mutants consisting of only individual domains.

Specific protein cleavage by endoproteinases is very important in the SIT method. One basic premise is that protein digestion is minimally or not affected by protein modification per se. If the moiety prevents the modified residue from being cleaved, it is conceivable that unmodified peptides derived from this region should have lengths different from those of modified peptides. Therefore, it becomes unlikely that modified peptides can be identified using the unmodified counterparts as references. Considering that acetyllysine residues are very poor substrates for both trypsin and Lys-C, either enzyme is not suitable for our experiment. Therefore, we digested substrate proteins with enzymes whose cleavage is minimally affected by acetylation, e.g. Asp-N and Arg-C.

Previous studies have demonstrated that different post-translational modifications of p53 occur under different stress-induced conditions. It may be activated through different signal transduction pathways (28, 29). Our data demonstrate that the acetylation of p53 at Lys-305, like that at Lys-382, is responsive to all p53-activating agents. Intriguingly, the acetylation profiles elicited by H2O2 at Lys-305 and Lys-382 are quite different. The induction of Lys-382 acetylation was slower than that of Lys-305 acetylation (Fig. 5B). It is likely that Lys-305 can be acetylated by a mechanism different from that of Lys-382. This interesting observation remains to be studied.

We have attempted to investigate the mechanism through which Lys-305 acetylation may modulate it transcriptional activity. Alteration of p53 subcellular distribution has been an attractive possibility since Lys-305 has been documented as part of a bipartite nuclear localization signal (30). Nevertheless, we found that both wild-type p53 and mutants K305R and K305A have very similar immunofluorescence staining patterns (data not shown). The results are the same as those previously documented by other investigators (31). Therefore, it seems unlikely that alteration of subcellular/subnuclear distribution may fully account for the stimulatory/inhibitory effects of these mutants. In accordance with these findings, we also found that the subcellular/subnuclear localization pattern of acetyl-Lys-305, as well as that of acetyl-Lys-382, is identical to the that of the overall population of p53 molecules (data not shown). This also supports the idea that Lys-305 acetylation is not directly associated with subcellular/subnuclear targeting of the p53 protein.

To further examine whether glutamine/arginine substitutions may mimic constitutively acetylated/non-acetylated lysine residues, cells were cotransfected with the K305Q or K382Q mutant; the p50-2 promoter activity was markedly decreased in both cases (Fig. 6). The glutamine substitution mutants did not mimic constitutively acetylated lysine. In contrast, the arginine substitution mutants K305R and K382R led to increased transcriptional activity. Normally, arginine substitution would mimic a non-acetylatable lysine and thus could not alter the p53 transcriptional activity, whereas the glutamine substitution would mimic the acetylated lysine and thus could increase the p53 transcriptional activity. Previous studies demonstrated that single and multiple Lys-to-Arg p53 mutations result in higher transcriptional activities (32, 33). The amino acid substitutions may not properly reflect the acetylated lysine and non-acetylated lysine. It is possible that the p53 C-terminal domain is a multifunctional domain, responsible for oligomerization, nuclear translocation, protein interaction, and negative regulation of the specific DNA-binding activity of p53. In addition, the C-terminal domain of p53 may be modified by other post-translational modifications such as phosphorylation and ubiquitination. Even substitution of only one amino acid may still affect several functions of p53. Alternatively, although it may be possible that acetylation is indeed involved in the down-regulation of p53 function, the increased acetylation that occurs under stress conditions reflects a negative autoregulatory loop that serves to limit the extent of p53 activation.

Although the glutamine/arginine substitutions may not mimic constitutively acetylated/non-acetylated lysine residues as we initially presumed, there is a parallel change in p53 transcriptional activities by different amino acid substitutions at Lys-305 and Lys-382. Meanwhile, these p53 mutants did not respond to UV irradiation as efficiently and failed to produce the optimal transcriptional activity that is intrinsic to wild-type p53. These results suggest that Lys-305, like Lys-382, plays an important role in modulating the precise transcriptional activity of p53.

Lys-292 of p53 was also identified as an in vitro p300 acetylation target by the SIT approach (Fig. 2D). Unfortunately, we have not been able to generate the specific anti-acetyl-Lys-292 antibody to test the possibility that Lys-292 can also be acetylated in vivo. However, we have constructed Lys-292 mutants and found that these mutants also have altered p53 transcriptional activity. Interestingly, it was reported that some cancer and cell lines have mutations at Lys-292.3 The function of Lys-292 will be the subject of future investigations.

We have also tested the possibility that Lys-305 acetylation might actually modulate the transcriptional activity of p53 through influencing the modification of other p53 residues. It has been documented that post-translational modification at certain sites can alter the properties of p53 such as protein stability (11, 34), tetramerization (35), and DNA-binding capacity (15, 36). To test this possibility, we investigated how modification states of other residues are changed in the K305A and K305R mutants compared with the wild-type protein. Our preliminary data seem to indicate that Lys-382 acetylation is suppressed in the K305A mutant, but enhanced in the K305R mutant. It remains to be examined whether this is the primary mechanism responsible for the altered transcriptional activity of these mutants. It is also intriguing to examine whether the modification of other sites may also be affected by Lys-305 acetylation.


    FOOTNOTES
 
* This work was supported by National Science Council Grant NSC90-2321-B002-003 and the Institute of Biological Chemistry, Academia Sinica, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Inst. of Molecular Medicine, National Taiwan University, Taipei, Taiwan. Tel.: 886-2-2312-3456 (ext. 2982); Fax: 886-2-2321-0977; E-mail: slee{at}ccms.ntu.edu.tw.

1 The abbreviations used are: HAT, histone acetyltransferase; CBP, cAMP response element-binding protein-binding protein; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SIT, selected ion tracing; CAT, chloramphenicol acetyltransferase; Ab, antibody; CID, collision-induced dissociation; HMG, high mobility group. Back

2 Y.-G. Tsay, unpublished data. Back

3 Available at www.lf2.cuni.cz/projects/germline_mut_p53.htm. Back


    ACKNOWLEDGMENTS
 
We thank Drs. Ming-Yang Lai, Li-Jung Juan, and Young-Sun Lin for the plasmids. We also thank Li-Ping Tu and Jui Chao for excellent technical assistance and Ming-Yue Lee for editing the manuscript.



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