Characterization of ATM expression, localization, and associated DNA-dependent protein kinase activity
Characterization of ATM expression, localization, and associated DNA-dependent protein kinase activity
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DOI：
10.1091/mbc.9.9.2361
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发表时间：
1998-09-01
影响因子：
3.3
通讯作者：
Yen, TJ
中科院分区：
文献类型：
--
作者：
Gately, DP;Hittle, JC;Yen, TJ
共济失调毛细血管扩张症突变基因（ATM）是一个350 kDa的蛋白质，其功能在常染色体隐性遗传疾病共济失调毛细血管扩张症（AT）中有缺陷。亲和纯化的多克隆抗体用于表征ATM。ATM蛋白的稳态水平从大多数AT细胞系中检测不到HeLa、U2 OS和正常人成纤维细胞中高度表达不等。亚细胞分级显示ATM主要是与染色质和核基质相关的核蛋白。ATM蛋白水平在整个细胞周期中保持恒定，对血清没有反应。刺激.电离辐射对ATM的表达和分布均无明显影响。ATM免疫沉淀从HeLa细胞和人DNA依赖性蛋白激酶空细胞系MO59J，但不是从AT细胞，磷酸化的34 kDa亚基的复制蛋白A（RPA）复合物在一个单链和线性双链DNA依赖的方式。p34 RPA的磷酸化发生在苏氨酸和丝氨酸残基上。磷酸肽分析表明，ATM相关的蛋白激酶磷酸化p34 RPA在体内观察到类似的残基。ATM免疫复合物中观察到的DNA依赖性蛋白激酶活性，沿着ATM与染色质的结合，表明DNA损伤可以诱导ATM或稳定结合的蛋白激酶磷酸化DNA损伤反应途径中的蛋白。
Ataxia telangiectasia-mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum. stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA-dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.