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. 2006 May 22;173(4):533-44.
doi: 10.1083/jcb.200512059.

Distinct p53 Acetylation Cassettes Differentially Influence Gene-Expression Patterns and Cell Fate

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Free PMC article

Distinct p53 Acetylation Cassettes Differentially Influence Gene-Expression Patterns and Cell Fate

Chad D Knights et al. J Cell Biol. .
Free PMC article

Abstract

The activity of the p53 gene product is regulated by a plethora of posttranslational modifications. An open question is whether such posttranslational changes act redundantly or dependently upon one another. We show that a functional interference between specific acetylated and phosphorylated residues of p53 influences cell fate. Acetylation of lysine 320 (K320) prevents phosphorylation of crucial serines in the NH(2)-terminal region of p53; only allows activation of genes containing high-affinity p53 binding sites, such as p21/WAF; and promotes cell survival after DNA damage. In contrast, acetylation of K373 leads to hyperphosphorylation of p53 NH(2)-terminal residues and enhances the interaction with promoters for which p53 possesses low DNA binding affinity, such as those contained in proapoptotic genes, leading to cell death. Further, acetylation of each of these two lysine clusters differentially regulates the interaction of p53 with coactivators and corepressors and produces distinct gene-expression profiles. By analogy with the "histone code" hypothesis, we propose that the multiple biological activities of p53 are orchestrated and deciphered by different "p53 cassettes," each containing combination patterns of posttranslational modifications and protein-protein interactions.

Figures

Figure 1.
Figure 1.
The kinetics of K320 and K373 acetylation of p53 in response to DNA damage correlate with cell cycle arrest or apoptosis. (A) Cell cycle profile of H1299 cells expressing tetracycline-inducible p53. Cells were left untreated or treated with 5 μM adozelesin or bizelesin or with 100 μM etoposide after a 24-h induction of p53 with tetracycline. Flow cytometry was performed as described previously (Avantaggiati et al., 1997). The percentage of cells displaying Sub-G1, G1, S, or G2/M DNA content is provided as an inset for each histogram. (B) H1299 cells expressing p53 were treated as indicated at the top of each panel in A. At different times after treatment, cells were harvested and cell lysates were immunoprecipitated with a polyclonal antibody recognizing acetyl-p53K320 (Acp53K320) or acetyl-p53K373 (Acp53K373) or with an antibody recognizing total p53, as indicated on the left side of each panel. The products of these reactions were then subjected to immunoblot with a mixture of monoclonal antibodies directed against the p53 NH2 terminus (DO-1) and COOH terminus (PAb421). Black lines indicate that intervening lanes have been spliced out. (C) H1299 cells expressing WT p53 were left untreated or treated with increasing concentrations of adozelesin or bizelesin (1, 50, or 500 μM), harvested after 6 h, and analyzed as in B.
Figure 2.
Figure 2.
Different effects of p53 acetylation mutants on cell growth. (A) Cell cycle profile of H1299 cells expressing p53 proteins (indicated at the top of panels). Cells were left untreated or treated with 50 μM adozelesin, 50 μM bizelesin, or 100 μM etoposide. Drugs were added 24 h after tetracycline addition, and cells were grown for an additional 24–48 h and then harvested and stained with propidium iodide (shown in all panels) and with BrdU (not depicted), and their cell cycle profile was determined. (B and C) Tetracycline-induced H1299 cells were left untreated or treated with 1 μM adozelesin for 4 h. Cells were extensively washed and incubated in drug-free media but in the presence of tetracycline for an additional 24 h. Tetracycline was then removed to shut off p53 expression, and cells were allowed to recover for 6–8 d, after which one dish was stained with Coomassie brilliant blue (B) and the remaining were used for cell counting (C). Error bars indicate SEM.
Figure 3.
Figure 3.
Interaction of cofactors with acetylated forms of p53 protein. (A) Nuclear extracts prepared from H1299 cells treated with tetracycline were immunoprecipitated with an anti-Flag antibody, eluted with Flag peptide, and run on 4–20% gradient gels. The presence of p300, PCAF, HDAC1, and mSin3 was revealed by immunoblotting with specific antibodies. (bottom) Total p53 levels contained in the anti-Flag–specific immunoprecipitation. (B) H1299 cells expressing native p53 were treated with etoposide for the indicated times. Cell extracts were immunoprecipitated with the anti-Acp53K373–specific antibody. The presence of p53, SIRT1/Sir2, or HDAC1 in these reactions was revealed via immunoblots with specific antibodies, as indicated at the side of each panel. The bottom panel represents ∼20% of the total amount of precipitated p53 protein. (C) Analysis of the interaction between truly acetylated forms of p53 and p300. A549 cells expressing WT p53 were treated for 16 h with 50 μM adozelesin and immunoprecipitated with either Acp53K320 (lane 1) or Acp53K373 (lane 2). These reactions were subjected to p53- and p300-specific immunoblots. The total levels of p300 were determined with the C-20 (Santa Cruz Biotechnology, Inc.) antibody after immunoprecipitation, whereas p53 levels were detected from total extract (lane 3).
Figure 4.
Figure 4.
Characterization of the gene-expression pattern elicited by p53 acetylation mutants. (A) Clustering of genes regulated by p53 acetylation mimics. Probes corresponding to fold change >2 were hierarchically clustered. Data from each probe are in the columns, and each experiment is shown as a row. Red and green denote increased and decreased expression levels, respectively, with the intensity reflecting the magnitude of change. (B) Validation of microarray data by semiquantitative RT-PCR. To verify oligonucleotide microarray results, semiquantitative RT-PCR was used to estimate the amount of RNA from three altered genes in WT p53–, p53Q320-, and p53Q373-expressing cells (+). To ensure that clonal variability does not contribute to the different levels of gene expression, mRNA was collected from H1299-WT, -Q320, and -Q373 cells in the absence of tetracycline (−). Densitometry was performed using Fluor Chem 3.04A software (Alpha Innotech Corp.) and normalized to the β-actin signal. Fold changes represent the difference for each mRNA observed within each individual cell line. Each band detected in the absence of tetracycline was attributed an arbitrary value of 1 and compared with the band detected in the presence of tetracycline (lanes 1, 3, and 5 were compared with lanes 2, 4, and 6, respectively). (C) In vivo interaction of p53 with its DNA binding sites derived from different promoters. Untreated H1299 or H1299 treated with tetracycline were cross-linked with formaldehyde and immunoprecipitated with a polyclonal antibody recognizing p53 (FL393). The amount of p53 proteins contained in these immunoprecipitation reactions was normalized (E), such that similar amounts of DNA-bound p53 were used for sequence-specific amplification of the promoters of p21/WAF, PIG3, BAX, and p53AIP1. (D) H1299 cells were treated with tetracycline to induce WT p53 protein and then subjected to ChIP. Acetylated p53 was precipitated by using antibodies directed against Acp53K320 or Acp53K373/K382, whereas total p53 was precipitated with the polyclonal p53 antibody. Promoter binding was assessed as described in C. Input represents ∼2% of the total DNA content used for precipitation. (E) Equalization of p53 proteins used for the ChIP assays in C. p53 levels in each cell line were predetermined after DNA–protein cross-linking by direct immunoblot and before the immunoprecipitation reaction (not depicted). The amounts of cell extract were then adjusted based on this preliminary assessment, so that equivalent amounts of each acetylation mutant were subjected to immunoprecipitation and subsequent PCR amplification. The two panels show typical levels of p53 proteins after this equalization, derived from two different experiments. (F) H1299 cells were left untreated (time 0) or were treated with tetracycline for 12 and 24 h. The type of p53-expressing cell line is indicated at the top of the panels. Cell lysates were prepared and subjected to immunoblots with antibodies recognizing p53, p21/WAF, Bax, and actin.
Figure 5.
Figure 5.
Acetyl-mimic Q320 alters the nuclear-cytoplasmic shuttling of p53. (A and B) Tetracycline-induced H1299 cells were grown on glass coverslips, and 24 h later cells in A and B were stained with an anti-p53 polyclonal antibody (red) and with DAPI (blue). (C) Tetracycline-induced cells expressing p53Q320 and p53DM were grown in the presence of leptomycin B and subjected to indirect immunofluorescence.
Figure 6.
Figure 6.
Acetylation of K320 increases the cytoplasmic localization of p53. (A) Tetracycline-induced WT p53–expressing H1299 cells were left untreated or were treated for 16 h with 50 μM adozelesin and then stained with a polyclonal antibody recognizing 320-acetylated p53 (Ac-320; red), a monoclonal antibody recognizing total p53 (green) and DAPI. (B) WT p53–H1299 cells were transfected with a PCAF or a p300 expression vector for 24 h and then stained with DAPI and with antibodies recognizing p300, p53, or PCAF, as indicated at the top of each panel.
Figure 7.
Figure 7.
Phosphorylation profile of p53 acetylation mutant proteins. (A) Cell extracts derived from tetracycline-induced H1299 cells expressing p53, p53Q320, or p53Q373 were subjected immunoblot with antibodies recognizing total or S15-phosphorylated p53. p53 levels were equalized among the different cell lines (bottom), so that similar amounts of each p53 acetylation mutant was probed with anti-phosphorylation–specific antibodies. S46 or S392 phosphorylation was detected via immunoprecipitation with a p53 polyclonal antibody, followed by immunoblot with anti-p53 antibodies directed against phospho-S46 or -S392. Fold induction for each phosphorylation site was determined by densitometry by using Fluor Chem 3.04A software, whereby cells expressing native p53 were attributed an arbitrary value of 1 and signals were normalized for total p53 levels. (B) WT p53, p53Q320, and p53Q373 were purified from baculovirus-infected cells and immunoprecipitated (IP) with different antibodies, each recognizing a different epitope, at the concentrations indicated at the top of each panel. The products of these immunoprecipitations were subjected to immunoblot (IB). The combination of antibodies used is indicated at the left side of each panel. (C) p53 mutants purified from baculovirus-infected cells were visualized via Coomassie blue staining (1) or by direct immunoblot with the DO-1 antibody (2).
Figure 8.
Figure 8.
p53 cassettes, created by combinations of posttranslational modifications and protein–protein interactions, control cell fate. By analogy with the “histone code” hypothesis elaborated by Strahl and Allis (2000) and based on data shown in this study, we propose that specific combinations of posttranslational modifications generate distinct p53 cassettes that direct p53 toward precise cellular functions. These p53 cassettes, in addition to directly influencing key biochemical properties of p53, such as its DNA binding affinity, also interact specifically with effector proteins that participate in deciphering and eliciting a particular response. We envision that these combination cassettes may exist for many p53 posttranslational modifications and establish a modular link between multiple upstream regulators of p53 and downstream events.
Figure 9.
Figure 9.
Conservation of K373 but not K320 between human and D. melanogaster p53. Human and D. melanogaster p53 were aligned using LALIGN from GeneStream (http://www.eng.uiowa.edu/∼tscheetz/sequence-analysis/examples/LALIGN/lalign-guess.html). Identical residues are shaded in gray, whereas conserved residues are colored in blue. K320 and K373 are indicated on human p53.

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References

    1. Appella, E., and C.W. Anderson. 2001. Post-translational modifications and activation of p53 by genotoxic stresses. Eur. J. Biochem. 268:2764–2772. - PubMed
    1. Avantaggiati, M.L., V. Ogryzko, K. Gardner, A. Giordano, A.S. Levine, and K. Kelly. 1997. Recruitment of p300/CBP in p53-dependent signal pathways. Cell. 89:1175–1184. - PubMed
    1. Bates, S., E.S. Hickman, and K.H. Vousden. 1999. Reversal of p53-induced cell-cycle arrest. Mol. Carcinog. 24:7–14. - PubMed
    1. Bode, A.M., and Z. Dong. 2004. Post-translational modification of p53 in tumorigenesis. Nat. Rev. Cancer. 4:793–805. - PubMed
    1. Brodsky, M.H., W. Nordstrom, G. Tsang, E. Kwan, G.M. Rubin, and J.M. Abrams. 2000. Drosophila p53 binds a damage response element at the reaper locus. Cell. 101:103–113. - PubMed

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