Mechanisms of ATM activation

描述（由申请人提供）：ATM蛋白激酶是细胞对染色体DNA双链断裂反应的主要调节剂。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伤，以及暴露于电离辐射和拟辐射化合物之后。在对DNA损伤的反应中，ATM使许多细胞底物磷酸化，其中一些是人类已知的肿瘤抑制因子。这些底物的磷酸化启动细胞周期阻滞、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的缺失导致基因组不稳定性增加，双链断裂诱导的细胞周期检查点的完全缺失，以及癌症频率的显著增加。A-T患者对氧化应激和DNA双链断裂的反应也严重降低，慢性氧化应激已被证明有助于这些患者的神经退行性变。在哺乳动物细胞中，ATM蛋白以非活性同二聚体的形式存在，并通过DNA双链断裂与DNA修复复合体Mre11/Rad50/Nbs1 （MRN）的结合而激活。在之前的工作中，我们用重组纯化蛋白重构了ATM的激活过程，并表明MRN作为ATM的双链断裂传感器。在目前的建议中，我们使用该系统来更详细地表征ATM激活的机制，并研究ATM调节的新途径。Specific Aim 1研究了通过氧化损伤激活ATM的分子基础，并试图识别参与这一过程的ATM结构域和残基。对缺乏氧化激活的特定突变体的分析将用于研究这一途径在人类细胞中的功能。目的2研究Nbs1在ATM双链断裂激活中的机制作用，并通过鉴定MRN-ATM蛋白-蛋白相互作用和ATM同源二聚化基序更详细地探讨MRN和ATM之间的功能关系。目的3描述了已知在人类细胞中调节ATM活化和底物磷酸化的其他蛋白质的作用，并研究了ATM乙酰化和自磷酸化的功能作用。该项目的总体目标是通过生物化学方法破译控制人体细胞中ATM活动的多层调控机制，以了解这种重要的蛋白质是如何对DNA双链断裂和氧化应激做出如此迅速和特异性的反应的。公共卫生相关性：ATM蛋白激酶被DNA损伤激活，启动细胞周期阻滞、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，并且已经证明ATM的丢失会促进肿瘤的发生。更好地了解ATM活化的机制对于我们理解基因组不稳定性和肿瘤进展的主要细胞防御至关重要。

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.