In vivo Analysis of ATM-Regulated Pathways

描述(由申请人提供)：本申请的主要目的是：(I)功能性地表征将ATM(共济失调-毛细血管扩张-突变)连接到环状AMP反应元件结合蛋白(CREB)转录因子的信号转导通路；以及(Ii)鉴定和功能性地表征新型ATM底物。ATM是一种DNA损伤激活的蛋白激酶，在遗传不稳定和神经退行性综合征(共济失调-毛细血管扩张)中发生突变，而CREB是一种神经保护性转录因子，调节细胞的生长、代谢和生存。我们已经定义了一种新的CREB调控模式，在这种模式下，ATM和酪蛋白激酶1和2(CK1/CK2)协同磷酸化被称为RAX结构域(共同调节的ATM和酪蛋白激酶位点)的5个簇位置上的CREB，以响应遗传毒性压力。ATM和CK1/CK2对CREB的磷酸化抑制了CREB与其共激活因子CREB结合蛋白(CBP)之间的相互作用，提示ATM途径可能抑制了CREB对DNA损伤的转录功能。ATM和CREB之间的联系耐人寻味，因为这两个因素都具有神经保护功能。在这项提议中，我们将检验这一假设，即ATM通过分别抑制和刺激未受干扰和DNA损伤的细胞中的Rax结构域磷酸化，在CREB调节中发挥双重作用。这项工作的一个重要目标是定义在没有DNA损伤的情况下控制CREB Rax结构域磷酸化的上游信号，并阐明其修饰在完整细胞中的生化结果。此外，我们将使用从CREB磷酸化范例中收集的信息来发现和功能表征由ATM和CK1/CK2协调调节的蛋白质底物，以响应DNA损伤。这些研究将对ATM功能和CREB调控的机制产生基本的见解，并可能改变目前对ATM信号对DNA损伤的反应的观点。该项目的目标是了解由ATM基因突变引起的神经变性/癌症易感性综合征--共济失调-毛细血管扩张症(A-T)的分子基础。ATM是细胞对DNA损伤反应的关键调节因子，本申请中提出的工作将表征ATM的一个特别重要的下游靶标，称为CREB(循环AMP反应元件结合蛋白)，它是基因表达的重要调节因子。我们特别感兴趣的是研究CREB的放松是否有助于A-T相关表型的表现，包括癌症和神经元死亡。

DESCRIPTION (provided by applicant): The major objectives of this application are to: (i) functionally characterize a signal transduction pathway linking ATM (ataxia-telangiectasia-mutated) to the cyclic AMP response element-binding protein (CREB) transcription factor; and (ii) identify and functionally characterize novel ATM substrates. ATM is a DNA damage-activated protein kinase that is mutated in the genetic instability and neurodegeneration syndrome, ataxia-telangiectasia, whereas CREB is a neuroprotective transcription factor that regulates cell growth, metabolism, and survival. We have defined a new mode of CREB regulation whereby ATM and casein kinases 1 and 2 (CK1/CK2) collaboratively phosphorylate CREB on five clustered sites termed the RAX domain (co-Regulated ATM and Casein Kinase Sites) in response to genotoxic stress. Phosphorylation of CREB by ATM and CK1/CK2 inhibits the interaction between CREB and its coactivator, CREB-binding protein (CBP) suggesting that the ATM pathway may repress CREB transcriptional functions in response to DNA damage. The linkage between ATM and CREB is intriguing given the neuroprotective functions of both factors. In this proposal we will test the hypothesis that ATM plays a dual role in CREB regulation through suppression and stimulation of RAX domain phosphorylation in unperturbed and DNA-damaged cells, respectively. An important goal of the work is to define the upstream signals controlling CREB RAX domain phosphorylation in the absence of DNA damage and to elucidate the biochemical outcomes of its modification in intact cells. In addition, we will use information gleaned from the CREB phosphorylation paradigm to discover and functionally characterize protein substrates that are coordinately regulated by ATM and CK1/CK2 in response to DNA damage. These studies should yield fundamental insights into the mechanisms of ATM function and CREB regulation, and may alter current views of ATM signaling in response to DNA damage. The goal of this project is to understand the molecular basis for the neurodegeneration/cancer susceptibility syndrome, ataxia-telangiectasia (A-T), which is caused by mutations in the ATM gene. ATM is a critical regulator of cellular responses to DNA damage and the work proposed in this application will characterize a particularly important downstream target of ATM, termed CREB (cyclic AMP response element-binding protein), which is an important regulator of gene expression. We are specifically interested in examining whether deregulation of CREB contributes to the manifestation of A-T-related phenotypes, including cancer, and neuron demise.