Chan D W, Son S C, Block W, Ye R, Khanna K K, Wold M S, Douglas P, Goodarzi A A, Pelley J, Taya Y, Lavin M F, Lees-Miller S P
Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
J Biol Chem. 2000 Mar 17;275(11):7803-10. doi: 10.1074/jbc.275.11.7803.
ATM is mutated in the human genetic disorder ataxia telangiectasia, which is characterized by ataxia, immune defects, and cancer predisposition. Cells that lack ATM exhibit delayed up-regulation of p53 in response to ionizing radiation. Serine 15 of p53 is phosphorylated in vivo in response to ionizing radiation, and antibodies to ATM immunoprecipitate a protein kinase activity that, in the presence of manganese, phosphorylates p53 at serine 15. Immunoprecipitates of ATM also phosphorylate PHAS-I in a manganese-dependent manner. Here we have purified ATM from human cells using nine chromatographic steps. Highly purified ATM phosphorylated PHAS-I, the 32-kDa subunit of RPA, serine 15 of p53, and Chk2 in vitro. The majority of the ATM phosphorylation sites in Chk2 were located in the amino-terminal 57 amino acids. In each case, phosphorylation was strictly dependent on manganese. ATM protein kinase activity was inhibited by wortmannin with an IC(50) of approximately 100 nM. Phosphorylation of RPA, but not p53, Chk2, or PHAS-I, was stimulated by DNA. The related protein, DNA-dependent protein kinase catalytic subunit, also phosphorylated PHAS-I, RPA, and Chk2 in the presence of manganese, suggesting that the requirement for manganese is a characteristic of this class of enzyme.
共济失调毛细血管扩张症是一种人类遗传性疾病，其特征为共济失调、免疫缺陷和癌症易感性，该疾病中ATM发生了突变。缺乏ATM的细胞在受到电离辐射后，p53的上调出现延迟。p53的丝氨酸15在体内会因电离辐射而发生磷酸化，并且针对ATM的抗体可免疫沉淀一种蛋白激酶活性，在有锰存在的情况下，该活性会使p53的丝氨酸15发生磷酸化。ATM的免疫沉淀物也以锰依赖的方式使PHAS-I发生磷酸化。在这里，我们通过九个色谱步骤从人细胞中纯化了ATM。高度纯化的ATM在体外可使PHAS-I、RPA的32 kDa亚基、p53的丝氨酸15以及Chk2发生磷酸化。Chk2中大多数ATM磷酸化位点位于氨基末端的57个氨基酸中。在每种情况下，磷酸化都严格依赖于锰。渥曼青霉素对ATM蛋白激酶活性具有抑制作用，其IC(50)约为100 nM。DNA可刺激RPA的磷酸化，但不刺激p53、Chk2或PHAS-I的磷酸化。相关蛋白DNA依赖性蛋白激酶催化亚基在有锰存在的情况下也可使PHAS-I、RPA和Chk2发生磷酸化，这表明对锰的需求是这类酶的一个特征。
