Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53
Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53
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DOI：
10.1074/mcp.m113.030254
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发表时间：
2014-01-01
影响因子：
7
通讯作者：
Perlman, David H.
中科院分区：
文献类型：
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作者：
DeHart, Caroline J.;Chahal, Jasdave S.;Perlman, David H.
P53肿瘤抑制蛋白在感染5型腺病毒的正常人成纤维细胞中积累到非常高的浓度，这种突变体不能直接组装病毒E1B 55-kDa蛋白所含的E3泛素连接酶，该酶以p53为靶点进行降解。尽管核内P53的浓度很高，但在这些感染的细胞中并没有诱导P53的转录程序。我们利用这个系统来检测转录惰性的内源性人p53以及对依托泊苷处理正常的人成纤维细胞而激活的p53上存在的部分翻译后修饰(PTM)。通过免疫亲和层析和SDS-PAGE从全细胞裂解产物中纯化出这些形式的p53，并在高分辨率精确质量MS平台上进行纳米超高效LC-MS和MS/MS分析(数据可从ProteomeXchange，PXD000464获得)。我们发现了出人意料的大量PTM，包括Ser和Thr残基的磷酸化，Arg残基的甲基化，以及Lys残基的乙酰化、泛素化和甲基化。例如，从感染细胞中分离出约150个以前未描述的P53修饰。这些修饰分布在两种形式的内源性人类P53蛋白的所有功能结构域，以及从COS-1细胞分离的同源P53群体的所有功能结构域。尽管在活性上存在差异，包括从依托泊苷处理的细胞中分离的P53显示出更强的体外序列特异性DNA结合活性，但在两种人P53上的PTM的位置、性质或相对频率上几乎没有观察到差异。事实上，我们已经确定的PTM的丰富与PTM对P53的复杂程度的组合调控比先前预期的要一致得多。
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 residuesfor 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.