DNA damage induces phosphorylation of the amino terminus of p53
DNA damage induces phosphorylation of the amino terminus of p53
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DOI：
10.1101/gad.11.24.3471
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发表时间：
1997-12-15
影响因子：
10.5
通讯作者：
Kastan, MB
中科院分区：
文献类型：
--
作者：
Siliciano, JD;Canman, CE;Kastan, MB
提供的数据表明，DNA损伤导致P53蛋白的特定翻译后修饰。利用体内标记的P53胰酶磷酸肽、重组截短的P53蛋白和合成的P53胰酶多肽的二维肽图谱，鉴定出一种独特的P53磷酸肽。该多肽代表P53的前24个氨基酸，含有三个磷酸化丝氨酸残基。一种特异性的P53磷酸肽抗体确定丝氨酸-15是P53中的两种丝氨酸之一，在多种细胞类型的电离辐射(IR)或紫外线(UV)照射引起DNA损伤后，丝氨酸-15被磷酸化。根据电泳迁移率改变分析，IR诱导的P53的磷酸化并不影响P53与DNA结合或解离的动力学。然而，DNA损伤诱导的P53磷酸化与下游P53靶基因的转录增强相关。在IR后6小时内，AT细胞中可检测到低水平的磷酸丝氨酸-15、P53，而正常人的淋巴母细胞在1小时内表现出这种修饰。相比之下，正常和AT细胞经紫外线照射后，P53在丝氨酸-15上的磷酸化程度相似。我们的结果表明，P53是在DNA损伤后被磷酸化的，这种从头开始的磷酸化可能参与了随后的P53的诱导和激活，尽管ATM影响了IR后P53的磷酸化动力学，但它并不是丝氨酸-15上P53磷酸化所必需的。
Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.