Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells
Biochemical characterization of the ataxia-telangiectasia mutated (ATM) protein from human cells
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DOI：
10.1016/j.dnarep.2004.03.041
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发表时间：
2004-07-02
期刊：
影响因子：
3.8
通讯作者：
Lees-Miller, SP
中科院分区：
文献类型：
--
作者：
Goodarzi, AA;Lees-Miller, SP
共济失调-毛细血管扩张症突变（ATM）是一种丝氨酸/苏氨酸蛋白激酶，在控制细胞对电离辐射和其他DNA损伤剂的反应中起核心作用。ATM是一种3056个氨基酸的多肽，以低丰度存在于人细胞核中。在这里，我们描述了从HeLa细胞的核级分的ATM的纯化和表征。制备微克量的高度稳定的激酶活性ATM。纯化的ATM在丝氨酸1981上被磷酸化，并且对多种已知的ATM底物（包括p53和Bloom综合征解旋酶BLM）具有活性。ATM的蛋白激酶活性可被渥曼青霉素、咖啡因和LY 294002选择性抑制，并可被带电荷的生物聚合物（包括单链M13 DNA（ssDNA）、剪切的双链小牛胸腺DNA、硫酸肝素和聚ADP-核糖（PAR））刺激，这表明带电荷的结构可能有助于ATM活性的调节。然而，化学抑制细胞中聚ADP-核糖的形成对电离辐射激活ATM依赖性通路没有影响。使用凝胶过滤色谱法，我们还表明，纯化的ATM，以及ATM的粗核提取物从未经辐照和辐照的细胞洗脱估计约600 kDa的天然分子量。此外，丝氨酸1981的去磷酸化不影响辐照提取物中ATM的表观分子量。我们的研究结果表明，丝氨酸1981磷酸化单独可能不直接调节ATM的亚基组成。(C)2004 Elsevier B. V.保留所有权利。
Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to ionizing radiation and other DNA-damaging agents. ATM is a 3056 amino acid polypeptide that is present in low abundance in the nucleus of human cells. Here, we describe the purification and characterization of ATM from the nuclear fraction of HeLa cells. Microgram quantities of highly stable, kinase-active ATM were prepared. purified ATM was phosphorylated on serine 1981 and was active towards a variety of known ATM substrates, including p53 and the Bloom Syndrome helicase, BLM. The protein kinase activity of ATM was selectively inhibited by wortmannin, caffeine and LY294002 and was stimulated by charged biological polymers, including single-stranded M 13 DNA (ssDNA), sheared double-stranded calf thymus DNA, heparin sulfate and poly ADP-ribose (PAR), raising the possibility that charged structures may contribute to regulation of ATM activity. However, chemical inhibition of the formation of poly ADP-ribose in cells had no effect on the activation of ATM-dependent pathways by ionizing radiation. Using gel filtration chromatography, we also show that purified ATM, as well as ATM in crude nuclear extracts from unirradiated and irradiated cells elutes with an estimated native molecular weight of approximately 600 kDa. Moreover, dephosphorylation of serine 1981 did not affect the apparent molecular weight of ATM in irradiated extracts. Our results suggest that phosphorylation of serine 1981 alone may not directly regulate the subunit composition of ATM. (C) 2004 Elsevier B.V. All rights reserved.