Purification and characterization of ATM from human placenta - A manganese-dependent, wortmannin-sensitive serine/threonine protein kinase
Purification and characterization of ATM from human placenta - A manganese-dependent, wortmannin-sensitive serine/threonine protein kinase
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DOI：
10.1074/jbc.275.11.7803
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发表时间：
2000-03-17
影响因子：
4.8
通讯作者：
Lees-Miller, SP
中科院分区：
文献类型：
--
作者：
Chan, DW;Son, SC;Lees-Miller, SP
ATM在人类遗传疾病共济失调毛细血管扩张症中发生突变，其特征在于共济失调、免疫缺陷和癌症易感性。缺乏ATM的细胞表现出对电离辐射响应的p53的延迟上调。p53的丝氨酸15在体内响应于电离辐射而磷酸化，并且ATM的抗体免疫沉淀蛋白激酶活性，其在锰的存在下使p53的丝氨酸15磷酸化。ATM的免疫沉淀物也以锰依赖性方式磷酸化PHAS-I。在这里，我们已经纯化ATM从人类细胞使用九个色谱步骤。高度纯化的ATM磷酸化PHAS-I，RPA的32-kDa亚基，p53的丝氨酸15和Chk 2在体外。Chk 2的ATM磷酸化位点主要位于氨基端57个氨基酸。在每种情况下，磷酸化都严格依赖于锰。ATM蛋白激酶活性被渥曼青霉素抑制，IC 50约为100 nM。RPA的磷酸化，而不是p53，Chk 2，或PHAS-I，被DNA刺激。相关的蛋白质，DNA依赖性蛋白激酶催化亚基，也磷酸化PHAS-I，RPA，和Chk 2在锰的存在下，这表明锰的需求是这类酶的特征。
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 IC50 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.