Mechanisms of ATM activation

描述(申请人提供)：ATM蛋白激酶是细胞对染色体DNA双链断裂反应的主要调节器。这种类型的DNA损伤发生在DNA复制过程中，由于代谢中间体的损害，以及暴露于电离辐射和拟辐射化合物之后。作为对DNA损伤的反应，ATM使许多细胞底物磷酸化，其中几种是已知的人类肿瘤抑制因子。这些底物的磷酸化启动细胞周期停滞、细胞凋亡和DNA修复。在共济失调-毛细血管扩张症(A-T)患者中，ATM的丢失会导致基因组的不稳定性增加，双链断裂诱导的细胞周期检查点完全丧失，癌症发生率显著增加。A-T患者对氧化应激和DNA双链断裂的反应也严重降低，慢性氧化应激被证明是这些患者神经变性的原因之一。哺乳动物细胞中的ATM蛋白以非活性同源二聚体的形式存在，并被DNA双链断裂激活，与DNA修复复合体Mre11/Rad50/Nbs1(MRN)结合。在以前的工作中，我们已经用重组纯化的蛋白重构了ATM的激活过程，并证明了MRN可以作为ATM的双链断裂传感器。在目前的提案中，我们使用这个系统来更详细地描述ATM激活的机制，并研究ATM调节的新途径。具体目标1阐述了ATM通过氧化损伤激活的分子基础，并试图确定这一过程中涉及的ATM结构域和残基。对氧化激活缺陷的特定突变体的分析将被用来研究这一途径在人类细胞中的功能。目的研究Nbs1在双链断裂激活ATM中的作用机制，并通过鉴定MRN-ATM蛋白相互作用和ATM同源二聚基序，更详细地阐述MRN与ATM之间的功能关系。目的3研究在人类细胞中调节ATM激活和底物磷酸化的其他蛋白质的作用，并研究ATM乙酰化和自动磷酸化的功能效应。该项目的总体目标是用生物化学方法破译控制人类细胞ATM活动的多个层次的调控，以了解这种重要的蛋白质是如何如此迅速地、特别是对DNA双链断裂和氧化应激做出反应的。与公共健康相关：ATM蛋白激酶被DNA损伤激活，从而启动细胞周期停滞、细胞程序性死亡和DNA修复。这些反应对于防止人类的致癌转化是必不可少的，ATM的缺失已被证明促进了肿瘤的发生。更多地了解ATM激活的机制对于我们理解针对基因组不稳定性和肿瘤进展的主要细胞防御是至关重要的。

DESCRIPTION (provided by applicant): The ATM protein kinase is a master regulator of the cellular response to chromosomal DNA double-strand breaks. This type of DNA damage occurs during DNA replication, as a result of damage from metabolic intermediates, and after exposure to ionizing radiation and radiomimetic compounds. In response to DNA damage, ATM phosphorylates many cellular substrates, several of which are known tumor suppressors in humans. Phosphorylation of these substrates initiates cell cycle arrest, apoptosis, and DNA repair. Loss of ATM, as seen in patients with Ataxia-Telangiectasia (A-T), results in increased genomic instability, a complete loss of double-strand break-induced cell cycle checkpoints, and a significant increase in cancer frequency. A-T patients also suffer from severely reduced responses to oxidative stress as well as to DNA double-strand breaks, and chronic oxidative stress has been shown to contribute to neurodegeneration seen in these patients. The ATM protein in mammalian cells exists as an inactive homodimer and becomes activated by DNA double-strand breaks through association with the DNA repair complex Mre11/Rad50/Nbs1 (MRN). In previous work we have reconstituted the ATM activation process with recombinant purified proteins and showed that MRN acts as a double-strand break sensor for ATM. In the current proposal we use this system to characterize the mechanisms of ATM activation in greater detail and investigate novel pathways of ATM regulation. Specific Aim 1 addresses the molecular basis of ATM activation through oxidative damage and seeks to identify ATM domains and residues involved in this process. Analysis of specific mutants deficient in oxidative activation will be used to investigate the functions of this pathway in human cells. Aim 2 investigates the mechanistic role of Nbs1 in ATM activation by double-strand breaks and addresses the functional relationship between MRN and ATM in greater detail through the identification of MRN-ATM protein-protein interactions and ATM homodimerization motifs. Aim 3 characterizes the roles of other proteins that are known to regulate ATM activation and substrate phosphorylation in human cells, and investigates the functional effects of ATM acetylation and autophosphorylation. The overall goal of the project is to biochemically decipher the many layers of regulation that govern ATM activity in human cells in order to understand how this important protein responds so rapidly and specifically to DNA double-strand breaks and oxidative stress. PUBLIC HEALTH RELEVANCE: The ATM protein kinase is activated by DNA damage to initiate cell cycle arrest, programmed cell death, and DNA repair. These responses are essential for preventing oncogenic transformation in humans, and loss of ATM has been shown to promote tumorigenesis. Greater understanding of the mechanisms of ATM activation is essential for our understanding of the primary cellular defense against genomic instability and tumor progression.