Functional Characterization of Apical Kinases in the Cellular Stress Response

细胞应激反应中顶端激酶的功能表征

• 批准号: RGPIN-2020-04242
• 负责人: Gamper, Armin
• 金额: $ 2.19万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761823
• 关键词: Functional; Characterization; Apical; Kinases; Cellular

My research program aims to understand how cells respond to different types of stress. I am particularly interested in dissecting the role of kinases that initiate stress response pathways and the underlying mechanisms. Our current focus is on the evolutionary conserved apical kinase ATM. Organisms must be able to respond to cellular stress resulting from internal and external origins to ensure their survival. Important responses include the regulation of countermeasures, cell cycle, metabolism, and - for multicellular organisms - apoptosis. Enzymatic posttranslational modifications, such as protein phosphorylation, enable rapid signal transduction and have evolved as efficient control mechanisms. Not surprisingly therefore, two apical protein kinases of stress response pathways are conserved among eukaryotes: The yeast kinases Tel1 and Mec1 share homology and functional similarity with the mammalian kinases ATM and ATR. Primarily investigated for the role these kinases have in the response to DNA damage, increasing evidence supports their role in the surveillance also of other types of insults such as oxidative, proteotoxic or mechanical stress. Having studied the role of ATM and ATR in the genotoxic stress response for many years, I propose to establish a program in the lab that uses novel approaches to expand our understanding of signal transduction pathways following cellular stress, including from different sources. We first will focus on ATM (Ataxia Telangiectasia Mutated) and in the future expand our studies to the related kinase ATR (Ataxia Telangiectasia and Rad3-related). Furthermore, based on the findings in the next five years, the program will continue studying the mechanisms of ATM regulation and the role of binding partners in determining the activation of kinases by cellular stress. To functionally characterize the role of ATM in the cellular stress response of various nature I propose: in Objective 1 to elucidate mechanisms of ATM activation by cellular stress by investigating stress-induced changes in the protein interactome by TurboID combined with cell fractionation; in Objective 2 to study the spatiotemporal dynamics of ATM localization after cellular stress by life cell imaging and static high resolution microscopy. The use of state-of-the arts techniques (including unpublished methods) and equipment will enable previously unattainable information on the regulation of ATM. Besides advancing the field, the research program will thereby also train HQP with a unique set of skills. IMPACT: Mechanistic insights into how cells respond to stress, whether arising from intrinsic or environmental factors threatening homeostasis, are fundamental to our understanding of life. Better knowledge of stress response pathways has also important implications for applied research, including the generation of induced pluripotent stem cells and tissue engineering, but also common lab techniques like tissue culture.

我的研究项目旨在了解细胞如何应对不同类型的压力。我特别感兴趣的是分析启动应激反应途径的激酶的作用和潜在的机制。我们目前的重点是进化保守的心尖蛋白激酶ATM。生物体必须能够对来自内部和外部来源的细胞压力做出反应，以确保它们的生存。重要的反应包括调控对策、细胞周期、新陈代谢，以及--对于多细胞生物体--细胞凋亡。酶促翻译后修饰，如蛋白质磷酸化，能够实现快速的信号转导，并已演变为有效的控制机制。因此，不足为奇的是，应激反应途径的两个顶端蛋白激酶在真核生物中是保守的：酵母蛋白激酶Tel1和Mec1与哺乳动物的蛋白激酶ATM和ATR具有同源性和功能相似性。最初研究这些激酶在DNA损伤反应中的作用，越来越多的证据支持它们在监测其他类型的侮辱中的作用，如氧化、蛋白毒性或机械应激。在研究了ATM和ATR在遗传毒性应激反应中的作用多年后，我建议在实验室建立一个项目，使用新的方法来扩大我们对细胞应激后的信号转导途径的理解，包括来自不同来源的信号转导。我们首先将重点放在ATM(共济失调毛细血管扩张症突变)上，并在未来将我们的研究扩展到相关的激酶ATR(共济失调毛细血管扩张症和RAD3相关)。此外，根据未来五年的研究结果，该计划将继续研究ATM调节的机制，以及结合伙伴在决定细胞应激激活激酶方面的作用。为了从功能上表征ATM在不同性质的细胞应激反应中的作用，我建议：在目标1中，通过结合细胞分离和TurboID研究应激诱导的蛋白质相互作用组的变化来阐明细胞应激激活ATM的机制；在目标2中，通过生命细胞成像和静态高分辨率显微镜来研究细胞应激后ATM定位的时空动力学。使用最先进的技术(包括未公布的方法)和设备，将使以前无法获得的关于自动取款机监管的信息成为可能。除了推动这一领域的发展，该研究计划还将因此培训HQP具有一套独特的技能。影响：对细胞如何对压力做出反应的机械论见解，无论是由威胁体内平衡的内在因素还是环境因素引起的，都是我们理解生命的基础。更好地了解应激反应途径对应用研究也有重要意义，包括诱导多能干细胞的生成和组织工程，以及组织培养等常见的实验室技术。