Functional Analysis of the Ataxia Telangiectasia Protein

共济失调毛细血管扩张蛋白的功能分析

• 批准号: 6933052
• 负责人: Brendan D Price
• 金额: $ 30.44万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6933052
• 关键词: DNA damage; SDS polyacrylamide gel electrophoresis; ataxia telangiectasia; biological signal transduction; bleomycin; cytotoxicity; flow cytometry; immunoprecipitation; laboratory rabbit; mass spectrometry; phosphorylation; posttranslational modifications; protein kinase; protein protein interaction; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The Ataxia Telangiectasia protein kinase (ATM) is activated by the radiomimetic agent bleomycin and is required for cells to survive bleomycin-induced DNA damage. The signal transduction pathway which links the detection of bleomycin-induced DNA damage to ATM activation is poorly characterized. We have identified a novel 64 amino-acid N-terminal domain of the ATM protein which is an essential component of this signal transduction pathway. This 64 aminoacid domain of ATM is required for cells to survive bleomycin-induced DNA damage. The hypothesis to be tested is that this N-terminal domain of ATM is required for activation of ATM following exposure to bleomycin. Aim 1. Deletion analysis and site-directed mutagenesis will be used to identify the exact amino-acids which constitute this domain. ATM clones will be constructed with this N-terminal domain inactivated, and cell lines stably expressing these clones will be prepared. The cells will then be exposed to bleomycin, and the role of the N-terminal regulatory domain of ATM in regulating cell survival and ATM kinase activity will be determined. Cell lines expressing the ATM construct in which the N-terminal is inactivated will be exposed to bleomycin. The ability of this ATM construct to activate cell cycle checkpoints and key ATM targets, including chk2 and p53, will be determined. Aim 2. Proteins, which interact with the N-terminal of ATM will be identified by Mass Spectrometry. The functional role of these proteins in regulating ATM kinase activity and bleomycin sensitivity will be determined. Potential phosphorylation sites will be identified to determine how phosphorylation controls ATM activation. The identification of the exact position of this crucial N-terminal protein domain will elucidate the molecular basis by which bleomycin causes DNA damage in tumor cells and associated toxicity in normal tissue; it will contribute to the design of improved antibiotics; and it will identify new molecular targets for ATM inhibition.

描述（由申请人提供）：放射性含膜剂博来霉素激活了链移虫蛋白蛋白激酶（ATM），并且细胞需要生存于博来霉素诱导的DNA损伤。将博来霉素诱导的DNA损伤与ATM激活联系起来的信号转导途径的特征很差。我们已经确定了ATM蛋白的新型64个氨基酸N末端结构域，这是该信号转导途径的重要组成部分。细胞需要这种64个ATM的氨基酸结构域才能在博来霉素诱导的DNA损伤中生存。要测试的假设是，暴露于博来霉素后，ATM激活ATM需要的N端结构域是必需的。 AIM 1。缺失分析和定点诱变将用于识别构成该域的确切氨基酸。 ATM克隆将使用该N末端结构域灭活，并稳定地表达这些克隆的细胞系构建。然后将将细胞暴露于博来霉素，并将确定AT​​M的N末端调节结构域在调节细胞存活和ATM激酶活性中的作用。表达N端被灭活的ATM构建体的细胞系将暴露于博来霉素。将确定此ATM构建体激活细胞周期检查点和关键ATM目标（包括CHK2和P53）的能力。 AIM 2。与ATM的N末端相互作用的蛋白质将通过质谱鉴定。这些蛋白质在调节ATM激酶活性和博来霉素敏感性中的功能作用将得到确定。将确定潜在的磷酸化位点，以确定磷酸化如何控制ATM激活。鉴定该至关重要的N末端蛋白结构域的确切位置将阐明博来霉素在肿瘤细胞中引起DNA损伤并在正常组织中相关的毒性相关的分子基础。它将有助于改善抗生素的设计；它将确定AT​​M抑制的新分子靶标。