p53 K370位乙酰化调控紫外线诱导的p53活化及细胞凋亡反应

Acetylation at K370 is critical for mediating p53 transactivation and apoptotic response under UVB exposure

目的探讨p53 K370位乙酰化修饰反应在紫外线B(UVB)诱导p53活化及细胞凋亡反应中的作用。方法体外培养野生型和p53基因缺陷型小鼠胚胎成纤维细胞(wt-MEFs和p53-/-MEFs)。以UVB为刺激源,双荧光素酶报告基因法分析wt-MEFs细胞中p53的转录诱导活化情况;Western印迹检测UVB诱导p53在K370位发生乙酰化修饰反应及p53总蛋白表达情况;构建p53点突变体K370R并在p53-/-MEFs细胞中比较UVB诱导wt-p53和p53 K370R的转录活化情况及野生型和突变体蛋白介导细胞凋亡反应的差异。结果 UVB刺激wt-MEFs细胞后p53转录激活活性呈剂量和时间依赖性明显上调;同样条件下UVB能够有效诱导p53在K370位发生乙酰化修饰反应;p53 K370位突变后并不影响UVB刺激作用下p53的蛋白质稳定性,但其转录激活活性显著下降;同时K370位突变后能够有效抑制UVB诱导的细胞凋亡反应。结论 p53 K370位乙酰化修饰反应在UVB诱导的p53活化及细胞凋亡反应中具有重要作用。

Objective To explore the role of p53 acetylation at Lys370 in mediating p53 transactivation and its proapoptotic effect under UVB exposure.Methods Wild type（wt） and p53-/-murine embryonic fibroblasts（MEFs） were exposed to UVB.Luciferase assay was used to detect the transactivation of p53.The accumulation and acetylation of p53 at Lys370 in the UVB response were tested by Western-blot assay.p53-/-MEFs were transfected with the plasmids containing wild-type p53（wt p53） or p53 with a Lys370Arg mutation（K370R） generated by mutagenic PCR and exposed to UVB,then the transactivation of p53 was detected by the luciferase assay,and the apoptosis of transfected cells was determined by a flow cytometric assay.Results Both dose-and time-dependent experiments indicated a significant increase of p53 transactivity under UVB exposure,along with the strong induction of p53 acetylation at Lys370 under the same conditions.Abrogation of p53 acetylation at Lys370 by mutagenesis did not affect p53 protein stability but remarkably decreased UVB-induced transactivation of p53 and the apoptotic response.Conclusion p53 Acetylation at Lys370 plays an important role in the transactivation of p53 and the induced apoptotic response under UVB exposure.