Lakin N D, Hann B C, Jackson S P
Wellcome Trust/Cancer Research Campaign Institute of Cancer and Developmental Biology, Department of Zoology, Cambridge University, UK.
Oncogene. 1999 Jul 8;18(27):3989-95. doi: 10.1038/sj.onc.1202973.
Levels of the tumour suppressor protein p53 are increased in response to a variety of DNA damaging agents. DNA damage-induced phosphorylation of p53 occurs at serine-15 in vivo. Phosphorylation of p53 at serine-15 leads to a stabilization of the polypeptide by inhibiting its interaction with Mdm2, a protein that targets p53 for ubiquitin-dependent degradation. However, the mechanisms by which DNA damage is signalled to p53 remain unclear. Here, we report the identification of a novel DNA-activated protein kinase that phosphorylates p53 on serine-15. Fractionation of HeLa nuclear extracts and biochemical analyses indicate that this kinase is distinct from the DNA-dependent protein kinase (DNA-PK) and corresponds to the human cell cycle checkpoint protein ATR. Immunoprecipitation studies of recombinant ATR reveal that catalytic activity of this polypeptide is required for DNA-stimulated phosphorylation of p53 on serine-15. These data suggest that ATR may function upstream of p53 in a signal transduction cascade initiated upon DNA damage and provide a biochemical assay system for ATR activity.
肿瘤抑制蛋白p53的水平会因多种DNA损伤剂而升高。在体内，DNA损伤诱导的p53磷酸化发生在丝氨酸15位点。p53在丝氨酸15位点的磷酸化通过抑制其与Mdm2的相互作用而导致该多肽的稳定，Mdm2是一种将p53靶向泛素依赖性降解的蛋白质。然而，DNA损伤向p53发出信号的机制仍不清楚。在此，我们报告鉴定出一种新型的DNA激活蛋白激酶，它能使p53在丝氨酸15位点磷酸化。对HeLa细胞核提取物进行分级分离和生化分析表明，这种激酶不同于DNA依赖性蛋白激酶（DNA-PK），对应于人细胞周期检查点蛋白ATR。对重组ATR的免疫沉淀研究表明，该多肽的催化活性是DNA刺激p53在丝氨酸15位点磷酸化所必需的。这些数据表明，ATR可能在DNA损伤引发的信号转导级联反应中在p53的上游发挥作用，并为ATR活性提供了一种生化检测系统。
