Radiation Activation of the ATM and p53 proteins

共济失调毛细血管扩张症基因产物ATM蛋白激酶是细胞在电离辐射（IR）诱导的DNA损伤中存活所必需的。IR诱导的DNA损伤与ATM激活之间的信号转导通路的特征不明显。我们已经确定了一个新的114个氨基酸的N-末端结构域的ATM，这是一个重要组成部分，这一信号转导途径。ATM的氨基酸1-114是细胞在IR诱导的DNA损伤中存活以及p53与ATM结合所必需的。待检验的假设是ATM的N-末端是ATM结合和磷酸化关键效应蛋白所必需的，包括p53、chk 2、Brca 1和NBS。在具体目标1中，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。ATM与此N-末端结构域失活将在共济失调毛细血管扩张细胞中表达。将这些细胞暴露于IR，并确定ATM的N-末端调节结构域在调节细胞存活、ATM激酶活性和与p53的关联中的作用。在具体目标2中，我们将确定ATM的N-末端在暴露于IR后调节细胞存活的机制。将表达N-末端失活的ATM构建体的细胞系暴露于IR，并确定ATM与关键ATM效应物（包括Brca 1、p53、chk 2和NBS蛋白）缔合和磷酸化的能力。ATM的N-末端将在用人端粒酶永生化的人乳腺上皮细胞中表达为ATM的显性负性抑制剂。这些细胞将用于检查未受病毒或癌基因转化损害的细胞中的ATM功能。确定这个关键的N-末端蛋白结构域的确切位置将阐明ATM激活DNA损伤诱导的信号转导的分子基础，并将确定ATM抑制的新分子靶点。

The Ataxia Telangiectasia gene product, the ATM protein kinase, is required for cells to survive Ionizing Radiation (IR)-induced DNA damage. The signal transduction pathway which links IR-induced DNA damage to ATM activation is poorly characterized. We have identified a novel 114 amino-acid N-Terminal domain of ATM which is an essential component of this signal transduction pathway. Amino-acids 1-114 of ATM are required for cells to survive IR-induced DNA damage and for p53 binding to ATM. The hypothesis to be tested is that the N-terminal of ATM is required for ATM to bind to and phosphorylate key effector proteins, including p53, chk2, Brca1 and NBS. In specific aim 1, deletion analysis and site-directed mutagenesis will be used to identify the exact amino-acids which constitute this domain. ATM with this N-terminal domain inactivated will be expressed in Ataxia Telangiectasia cells. These cells will be exposed to IR, and the role of the N-Terminal regulatory domain of ATM in regulating cell survival, ATM kinase activity and association with p53 will be determined. In specific aim 2, we will determine the mechanism by which the N-Terminal of ATM regulates cell survival following exposure to IR. Cell lines expressing the ATM construct in which the N-terminal is inactivated will be exposed to IR and the ability of ATM to associate with and phosphorylate key ATM effectors, including the Brca1, p53, chk2 and NBS proteins, will be determined. The N- terminal of ATM will be expressed as a dominant negative inhibitor of ATM in human breast epithelial cells immortalized with human Telomerase. These cells will be used to examine ATM function in cells which are not compromised by viral or oncogene transformation. The identification of the exact position of this crucial N-terminal protein domain will elucidate the molecular basis by which ATM activates DNA damage induced signal-transduction and it will identify new molecular targets for ATM inhibition.