p53核输入功能在鼠双微体2调节的p53蛋白降解和泛素化中的作用

目的研究p53核输入功能在鼠双微体2(murine double minute2,MDM2)调节的p53降解与泛素化中的作用。方法由直接点突变的方法构建质粒,将决定p53-GFP核位置信号(NLS)的两个部分5个基本氨基酸(赖氨酸和精氨酸)单一替换为丙氨酸,构建了p53-NLS突变型053KRKKK-GFP,并且经DNA序列确定。用DNA克隆技术将p53KRKKK与pEGF-Nuc融合,构建了p53KRKKK-NLS-GFP,然后分别转染U2OS细胞,在荧光显微镜下直接观察活细胞的蛋白表达部位;同时用间接免疫荧光染色、Western blot和泛素化分析的方法,观察p53KRKKK与MDM2野生型或MDM2-NLS突变型共转染U2OS细胞时的蛋白表达部位、降解和泛素化现象。结果p53KRKKK-GFP蛋白表达完全在细胞浆,并且不能被野生型和NLS突变型的MDM2降解,但是可以被泛素化。p53KRKKK-NLS-GFP可以使p53蛋白表达重新回到细胞核,此时能被MDM2野生型和NLS突变型降解和泛素化。p53野生型和NLS突变型均可被MDM2野生型和NLS突变型泛素化,当p53KRKKK和MDM2-NLS同时表达在细胞浆时,p53蛋白出现了更高效率的泛素化,但不能被降解。结论p53核输入对由MDM2调节的p53降解是必须的;MDM2调节的p53泛素化可以出现在细胞浆,而且效率更高。

Nuclear import of p53 in relation to MDM2-mediated degradation and ubiquitination

Objective To study the function of p53 nuclear import in murine double minute 2(MDM2) mediated ubiquitination and degradation. Methods Plasmid containing mutant p53 GFP was constructed by site directed mutagenesis by which 5 amino scid residues in the nuclear localization signal (NLS) were replaced by alanine to produce mutant p53KRKKK GFP. After being fused with pEGF Nuc(NLS containing SV40)to produce p53KRKKK NLS GFP,it was transfected into U20S cells. Localization, degradation and ubiquitination of p53 and MDM2 proteins were assessed by fluorescent staining, Western blot and ubiquitination analysis in MDM2 or MDM2 NLS co transfected U20S cells. Results p53KRKKK GFP was located in cytoplasm, and was not degraded by either MDM2 or MDM2 NLS mutation, but could be ubiquitinated; p53KRKKK NLS GFP could be brought back to nucleus by SV 40 NLS, so could be both degraded and ubiquitinated by either MDM2 or MDM2 NLS; Wild type p53 and mutant NLS could be ubiquitinated by either wild type MDM2 or mutant NLS. Ubiquitination happened to be even more efficient in cytoplasm when p53KRKKK and MDM2 NLS co localization, but not degraded. Conclusion Nuclear import is required for p53 degradation mediated by MDM2, but not for ubiquitination. p53 can be efficiently ubiquitinated in cytoplasm.