Functional Characterization of Apical Kinases in the Cellular Stress Response

我的研究项目旨在了解细胞如何应对不同类型的压力。我特别感兴趣的是剖析启动应激反应途径和潜在机制的激酶的作用。我们目前的重点是进化保守的顶端激酶ATM。生物体必须能够对来自内部和外部的细胞压力作出反应，以确保其生存。重要的反应包括调控对策、细胞周期、代谢和多细胞生物的凋亡。酶的翻译后修饰，如蛋白质磷酸化，可以实现快速的信号转导，并已发展成为有效的控制机制。因此，在真核生物中，应激反应途径的两种顶端蛋白激酶是保守的：酵母激酶Tel1和Mec1与哺乳动物激酶ATM和ATR具有同源性和功能相似性。主要研究这些激酶在DNA损伤反应中的作用，越来越多的证据支持它们在监视其他类型的损伤（如氧化，蛋白质毒性或机械应力）中的作用。在研究了ATM和ATR在基因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，使用新颖的方法来扩展我们对细胞应激后信号转导途径的理解，包括来自不同来源的信号转导途径。我们首先将重点放在ATM （Ataxia telangiecasia Mutated）上，未来将扩大我们对相关激酶ATR （Ataxia telangiecasia and rad3相关）的研究。此外，基于这些发现，该项目将在未来五年内继续研究ATM调节机制和结合伙伴在决定细胞应激激活激酶中的作用。为了从功能上表征ATM在各种性质的细胞应激反应中的作用，我提出：在目标1中，通过TurboID结合细胞分离研究应激诱导的蛋白质相互作用组的变化，阐明ATM在细胞应激下激活的机制；目的2利用生命细胞成像和静态高分辨率显微镜研究细胞应激后ATM定位的时空动态。使用最先进的技术（包括未发表的方法）和设备将使以前无法获得的关于ATM管制的信息成为可能。除了推动该领域的发展，该研究项目还将为HQP提供一套独特的技能。影响：关于细胞如何应对压力的机制见解，无论是来自威胁体内平衡的内在因素还是环境因素，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和组织工程的产生，以及组织培养等常见实验室技术。

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.