Functional Characterization of the ATM Gene Product

描述（由申请人提供）：了解细胞对DNA损伤反应的分子控制对癌症发展和癌症治疗都有重要意义。我们的细胞需要应对的最关键的DNA损伤类型是磷酸二酯主干的断裂。ATM蛋白激酶是调节细胞对DNA断裂反应的中心信号分子。在此资助的前一个资助期，在阐明参与ATM激酶激活的机制方面取得了重大进展。此外，我们能够识别ATM酶的特定蛋白靶点，并破译这些磷酸化事件的功能意义。在这个应用中，我们建立在这些成功的基础上，并提出将进一步阐明参与细胞对DNA断裂和其他类型DNA损伤反应的分子机制的实验。我们发现ATM以同型二聚体的形式存在于细胞中，并在DNA损伤后被丝氨酸1981的分子间自磷酸化激活，导致二聚体解离。我们最近在ATM蛋白中发现了一种额外的丝氨酸，它在DNA损伤时被磷酸化。我们提出了实验来探索这种新的翻译后修饰的功能意义。特别是，我们期望这种磷酸化事件有助于调节ATM激酶初始激活后的细胞活性。此外，研究人员还提出了一些实验来探索ATM与染色体蛋白相互作用的本质，以便我们更好地了解ATM如何与染色质相关联，并感知高阶染色质结构的改变而被激活。ATM激活机制的阐明也使我们能够在没有可检测的DNA损伤的情况下激活酶。这导致了原理验证实验，证明在辐照前激活ATM-p53通路会导致暴露于全身辐照的小鼠的辐射防护。初步数据还显示，激活ATM-p53通路可以在多种小鼠模型中预防癌症的发展，包括由电离辐射、化学致癌物或激活的癌基因引起的癌症。我们建议通过实验进一步探讨ATM活化的辐射防护和癌症预防作用的分子机制。

DESCRIPTION (provided by applicant): Understanding the molecular control of cellular responses to DNA damage has significance for both cancer development and cancer therapies. Among the most critical types of DNA damage with which our cells need to cope are breaks in the phosphodiester backbone. The ATM protein kinase is a central signaling molecule in modulating cellular responses to DNA breakage. In the previous funding period of this grant, significant progress was made in elucidating the mechanisms involved in the activation of the ATM kinase. In addition, we were able to identify specific protein targets of the ATM enzyme and to decipher the functional significance of these phosphorylation events. In this application, we build upon these successes and propose experiments that will further elucidate molecular mechanisms involved in cellular responses to DNA breakage and other types of DNA damage. We found that ATM exists in cells as a homodimer and is activated after DNA damage by an intermolecular autophosphorylation on serine 1981 that causes dissociation of the dimer. We recently identified an additional serine in the ATM protein that becomes phosphorylated in response to DNA damage. Experiments are proposed to explore the functional significance of this new post-translational modification. In particular, we expect that this phosphorylation event contribute to modulating the cellular activities of the ATM kinase after its initial activation. In addition, experiments are proposed that will explore the nature of ATM interactions with chromosomal proteins so that we can better understand how ATM associates with chromatin and senses alterations in higher order chromatin structures to become activated. The elucidation of the ATM activation mechanism also led to our ability to activate the enzyme in the absence of detectable DNA damage. This led to proof-of-principle experiments demonstrating that activation of the ATM-p53 pathway prior to irradiation leads to radioprotection of mice exposed to total body irradiation. Preliminary data is also presented demonstrating that activation of the ATM-p53 pathway can prevent cancer development in multiple mouse models, including cancers caused by ionizing irradiation, chemical carcinogens, or activated oncogenes. Experiments are proposed to further explore the molecular mechanisms involved in both the radioprotective effects and the cancer preventative effects of ATM activation.