Extensive post-translational modification of active and inactivated forms of endogenous p53.

TitleExtensive post-translational modification of active and inactivated forms of endogenous p53.
Publication TypeJournal Article
Year of Publication2014
AuthorsDeHart, CJ, Chahal, JS, Flint, SJ, Perlman, DH
JournalMol Cell Proteomics
Volume13
Issue1
Pagination1-17
Date Published2014 Jan
ISSN1535-9484
KeywordsAcetylation, Adenovirus E1B Proteins, Animals, Cercopithecus aethiops, COS Cells, DNA-Binding Proteins, Fibroblasts, Humans, Methylation, Phosphorylation, Protein Processing, Post-Translational, Proteolysis, Tumor Suppressor Protein p53, Ubiquitin-Protein Ligases
Abstract

<p>The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues-for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated.</p>

DOI10.1074/mcp.M113.030254
Alternate JournalMol. Cell Proteomics
PubMed ID24056736
PubMed Central IDPMC3879606
Grant ListR01 AI058172 / AI / NIAID NIH HHS / United States
R01AI1058172 / AI / NIAID NIH HHS / United States
R56 AI091785 / AI / NIAID NIH HHS / United States
R56A111091785 / / PHS HHS / United States