Extensive post-translational modification of active and inactivated forms of endogenous p53 - PubMed (original) (raw)

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

Caroline J DeHart et al. Mol Cell Proteomics. 2014 Jan.

Abstract

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.

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Figures

Fig. 1.

Fig. 1.

Properties of p53 isolated from normal human fibroblasts. A, HFFs were infected with 100 pfu/cell AdEasy E1 (WT) or AdEasyE1Δ2347 (ΔE1B) for the periods indicated, or mock infected (M). Whole cell extracts were prepared and the proteins listed were examined via immunoblotting as described under “Experimental Procedures.” B, p53 present in HFFs infected with AdEasyE1Δ2347 for 44 h was examined using the antibodies indicated at the top. C, Whole cell extracts were prepared from cells infected with AdEasyE1Δ2347 (ΔE1B) or exposed to 125 μ

m

etoposide (E) for 44 h, and the quantity of p53 present in the increasing extract volumes indicated at the top was compared via immunoblotting.

Fig. 2.

Fig. 2.

Analysis of DNA binding by p53. The relative concentrations of p53 present in whole cell extracts of HFFs infected with 100 pfu/cell AdEasyE1(Δ)2347 (ΔE1B p53) or exposed to 125 μ

m

etoposide (E p53) for 44 h were determined via immunoblotting and quantification of signals, with β-actin as an internal control. The binding of equal quantities of the two forms of p53 to a consensus DNA binding site was then examined as a function of p53 concentration, as described under “Experimental Procedures.” All panels show the means and standard deviations of triplicate technical replicates. The results of independent experimental replicates are shown in panels A and C, and the initial portion of the binding curve shown in panel A is expanded in panel B.

Fig. 3.

Fig. 3.

Examples of immunoaffinity purification of p53. HFFs were infected with 100 p.f.u./cell AdEasyE1Δ2347 for 44 h, and following lysate preparation, p53 was purified via immunoaffinity chromatography. A, lysates were prepared in buffer containing 1% (v/v) Triton X-100, and p53 was eluted in 200 m

m

TEA. The relative concentrations of p53, β-actin, and IgG heavy chain (IgG) in the lysate (L), flow-through (F), wash fractions (W1–W3), and eluates (E1 and E2) were determined via immunoblotting. B, lysates were prepared in buffer containing 1% (v/v) N-laurylsarcosine, and p53 was eluted from the immunoaffinity matrix via heat treatment. p53 and IgG heavy chain were visualized as described in panel A.

Fig. 4.

Fig. 4.

Representative MS/MS spectra of ΔE1B p53. Peptides prepared from p53 isolates were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown are representative examples of tandem mass spectra displaying their PTM-bearing peptide fragment ion assignments. Prominent fragment ions are labeled with their empirical m/z values and b- and y-ion designations or annotations indicating neutral losses from precursor species. The peptide sequences are displayed above the spectra, with all of the fragment ions matched in the spectra indicated by flags. Modified residues are color-coded by PTM in the following manner: phosphorylation, red; acetylation, green; ubiquitinylation, purple; monomethylation, navy; dimethylation, cerulean; trimethylation, turquoise; oxidation, tan; and carbamidomethylation, orange.

Fig. 5.

Fig. 5.

Representative MS/MS spectra of Ε p53. Peptides prepared from p53 isolated from HFFs treated with etoposide were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown are representative examples of tandem mass spectra displaying their PTM-bearing peptide fragment ion assignments, labeled as described in the legend to Fig. 4.

Fig. 6.

Fig. 6.

Post-translational modification of p53 isolated from AdEasyE1Δ2347-infected HFFs. The modifications reproducibly identified across three independent biological replicate samples of ΔE1B p53 are summarized, classified according to whether they have been previously described (A) or are novel (B). AD, activation domain; DBD, DNA-binding domain; NLS, nuclear localization signal; TD, tetramerization domain; BD, basic domain.

Fig. 7.

Fig. 7.

Representative MS/MS spectra of COS-1 p53. Peptides prepared from p53 isolated from COS-1 cells were subjected to reversed-phase nano-LC-MS and MS/MS on a UPLC-Orbitrap Velos platform as described under “Experimental Procedures.” Shown are representative examples of tandem mass spectra displaying their PTM-bearing peptide fragment ion assignments, labeled as described in the legend to Fig. 4.

Fig. 8.

Fig. 8.

Spectral count profiles of modified Ser, Thr, and Arg residues. Peptides from equivalent populations of ΔE1B p53 and E p53 isolated in parallel were subjected to nano-UPLC-MS and MS/MS analyses on the Orbitrap Velos platform. Modification profiles were generated from technical triplicate LC-MS runs for each sample by means of spectral counting–based quantification of matched PTMs per residue, as described under “Experimental Procedures.” Shown are the modification profiles for (A) phosphorylation of Ser and Thr residues and (B) mono-, di-, and trimethylation of Arg residues present on both p53 populations.

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