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具有重要意义。我们的细胞需要应对的最关键的DNA损伤类型之一是磷酸二酯骨架的断裂。ATM蛋白激酶是调节细胞对DNA断裂反应的中心信号分子。在这笔赠款的前一个资助期，在阐明ATM激酶激活所涉及的机制方面取得了重大进展。此外，我们能够识别ATM酶的特定蛋白质靶标，并破译这些磷酸化事件的功能意义。在这一应用中，我们将在这些成功的基础上，提出进一步阐明细胞对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以同源二聚体的形式存在于细胞中，在DNA损伤后，通过1981年丝氨酸上的分子间自动磷酸化而激活，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，它会在DNA损伤时被磷酸化。通过实验来探索这一新的翻译后修饰的功能意义。特别是，我们预计这种磷酸化事件有助于调节ATM激酶在最初激活后的细胞活动。此外，还提出了探索ATM与染色体蛋白质相互作用的本质的实验，以便我们能够更好地理解ATM如何与染色质相关联，并感觉到高阶染色质结构的变化而被激活。ATM激活机制的阐明也使我们能够在没有检测到DNA损伤的情况下激活酶。这导致了原理验证实验表明，在照射前激活ATM-P53通路会导致暴露在全身照射下的小鼠具有辐射防护作用。初步数据还表明，ATM-P53通路的激活可以防止多种小鼠模型的癌症发展，包括由电离辐射、化学致癌物或激活的癌基因引起的癌症。建议进行实验，以进一步探索ATM激活的辐射防护作用和癌症预防作用所涉及的分子机制。

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.