Functional Analysis of the Ataxia Telangiectasia Protein

描述（由申请方提供）：共济失调毛细血管扩张蛋白激酶（ATM）被拟放射性药物博来霉素激活，是细胞在博来霉素诱导的DNA损伤中存活所必需的。将博莱霉素诱导的DNA损伤检测与ATM活化联系起来的信号转导途径的特征很差。我们已经确定了一个新的64个氨基酸的N-末端结构域的ATM蛋白，这是一个重要组成部分，这一信号转导途径。ATM的这个64个氨基酸结构域是细胞在博来霉素诱导的DNA损伤中存活所必需的。待检验的假设是，暴露于博来霉素后ATM的活化需要ATM的该N-末端结构域。目标1。缺失分析和定点诱变将用于鉴定构成该结构域的确切氨基酸。将用该N-末端结构域失活构建ATM克隆，并制备稳定表达这些克隆的细胞系。然后将细胞暴露于博来霉素，并确定ATM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。将表达N-末端失活的ATM构建体的细胞系暴露于博来霉素。将确定该ATM构建体激活细胞周期检查点和关键ATM靶点（包括chk 2和p53）的能力。目标二。将通过质谱法鉴定与ATM的N-末端相互作用的蛋白质。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被确定。将鉴定潜在的磷酸化位点以确定磷酸化如何控制ATM活化。这一关键的N-末端蛋白结构域的确切位置的鉴定将阐明博来霉素在肿瘤细胞中引起DNA损伤和在正常组织中引起相关毒性的分子基础;它将有助于改进抗生素的设计;并且它将鉴定ATM抑制的新分子靶点。

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.