Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity
Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity
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DOI：
10.1128/mcb.25.13.5389-5395.2005
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发表时间：
2005-07-01
影响因子：
5.3
通讯作者：
Xu, Y
中科院分区：
文献类型：
--
作者：
Feng, LJ;Lin, TX;Xu, Y
抑癌基因p53的翻译后修饰在调节其稳定性和活性方面起着重要作用。p53 C末端的6个赖氨酸残基可以通过多种机制进行后修饰，包括乙酰化、泛素化、neddylation、甲基化和sumo化。先前的细胞系转染研究表明，这些赖氨酸残基的泛素化是p53的泛素依赖性降解所必需的。此外，生物化学和细胞系研究表明，p53在C末端的乙酰化可能稳定p53并激活其转录活性。为了研究这些C-末端修饰在调节p53稳定性和活性中的生理功能结果，我们在小鼠胚胎干（ES）细胞中将6个赖氨酸残基（K6 R）处的错义突变（赖氨酸至精氨酸）引入内源性p53基因中。K6 R突变阻止了这些位点的所有翻译后修饰，但保留了p53的结构。与以前的研究结论相反，在K6 R ES细胞，小鼠胚胎成纤维细胞和胸腺细胞中的p53稳定性分析表明，在K611细胞DNA损伤之前和之后，p53的稳定性正常，这表明这些赖氨酸残基的泛素化不是有效的p53降解所必需的。然而，p53依赖的基因表达受损的K6 R ES细胞和胸腺细胞在启动子特异性的方式DNA损伤后，表明在C末端的翻译后修饰的净结果是激活p53的转录活性后DNA损伤。
Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K611 cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.