Mechanism of Activation of eEF-2K, an Energy and Nutrient Sensor

能量和营养传感器 eEF-2K 的激活机制

• 批准号: 9289618
• 负责人: Kevin N Dalby
• 金额: $ 41.57万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9289618
• 关键词: Accounting; Address; Affinity; Alpha Cell; Alzheimer' s Disease; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biophysics; Calcium; Calcium ion; Calmodulin; Cell physiology; Cells; Complex; Cues; Disease; Disease Progression; Ensure; Equilibrium; Etiology; Eukaryotic Cell; Event; Family; Future; Goals; Homeostasis; In Vitro; Kinetics; Knock-out; Label; Learning; Length; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Memory; Mental Depression; Molecular Conformation; NMR Spectroscopy; Nature; Neurons; Nuclear Magnetic Resonance; Nutrient; Peptide Elongation Factor 2; Peptides; Phase; Phosphorylation; Phosphotransferases; Physiological; Physiological Processes; Play; Preparation; Process; Protein Biosynthesis; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Regulation; Ribosomes; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Techniques; Testing; Time; Translations; Variant; Warburg Effect; base; calmodulin-dependent protein kinase III; cancer cell; collaborative approach; cost; design; glucose uptake; member; nutrient deprivation; protein degradation; protein kinase A kinase; response; sensor; spatiotemporal

ABSTRACT Protein synthesis (translation) constitutes one of the most fundamental of all cellular processes. The balance between protein synthesis and protein degradation is critical in maintaining cellular homeostasis. Translation is one of the most energy consumptive processes in a cell, accounting for as much as 30% of the energy usage in a eukaryotic cell, making its tight regulation a necessity. Eukaryotic elongation factor 2 kinase (eEF-2K), a unique member of the α-kinase family, is a key regulator of the elongation phase of translation. eEF-2K phosphorylates and inactivates elongation factor 2 (eEF-2), leading to a reduction in global translation rates on one hand and differential translation of certain proteins on the other. The activity of eEF-2K is dependent on calmodulin, and is subject to complex regulation by calcium ions and phosphorylation . While there is accumulating evidence that the dysregulation of eEF-2K activity is involved in several disease states (e.g. Alzheimer's disease, depression, and a variety of cancers), a detailed mechanistic understanding of eEF-2K activation and regulation is lacking. Our long-term goal is to precisely define the mechanisms by which eEF-2K is activated and regulated; such information is critical to understanding its contributions to both normal cellular processes and in the etiology and progression of disease states. Toward this goal, we recently identified calmodulin-stimulated autophosphorylation on a specific residue, T348, as being the key step in the activation of eEF-2K. Our objective in the present proposal is to describe the structural and biochemical mechanisms of eEF-2K activation by calmodulin and the roles of calcium and phosphorylation at a regulatory site, S500, in modulating this process. We will achieve this goal through a multi-faceted and collaborative approach that encompasses enzymological, kinetic, solution-state nuclear magnetic resonance and cell-biological techniques. Our central hypothesis is that T348 autophosphorylation and activation of eEF-2K is dependent on calmodulin binding. Calcium ions and S500 phosphorylation play overlapping roles but utilize distinct mechanisms in regulating the calmodulin sensitivity of eEF-2K and thereby its activation. We expect our analyses will provide the basis for a thorough mechanistic understanding of how eEF-2K integrates these two disparate signals that regulate its activity. Such a detailed picture of eEF-2K regulation is critical for elucidating its contribution to a variety of fundamental cellular process and its deregulation in a variety of disease states.

摘要 蛋白质合成（翻译）是所有细胞过程中最基本的过程之一。余额 蛋白质合成和蛋白质降解之间的相互作用对维持细胞内稳态至关重要。翻译 是细胞中最耗能的过程之一，占细胞中能量使用的30%。 一个真核细胞，使其严格的调控是必要的。真核细胞延伸因子2激酶（eEF-2K）， α-激酶家族的独特成员，是翻译延伸期的关键调节因子。eEF-2K 磷酸化和失活延伸因子2（eEF-2），导致整体翻译率下降， 另一方面是某些蛋白质的差异翻译。eEF-2K的活性依赖于 钙调蛋白，并受到钙离子和磷酸化的复杂调节。虽然 越来越多的证据表明，eEF-2K活性的失调涉及几种疾病状态（例如， 阿尔茨海默病，抑郁症和各种癌症），对eEF-2K的详细机制理解 缺乏激活和调节。我们的长期目标是精确定义eEF-2K 被激活和调节;这些信息对于理解其对正常和非正常的 细胞过程以及疾病状态的病因学和进展。为了实现这一目标，我们最近发现， 钙调素刺激的特定残基T348上的自磷酸化是激活的关键步骤， eEF-2K我们在本提案中的目标是描述的结构和生化机制， 钙调素激活eEF-2K以及钙和磷酸化在调节位点S500的作用， 调节这个过程。我们将通过多方面的合作方式实现这一目标， 包括酶学、动力学、溶液状态核磁共振和细胞生物学技术。 我们的中心假设是T348自身磷酸化和eEF-2K的激活依赖于 钙调素结合钙离子和S500磷酸化发挥重叠的作用，但利用不同的 调节eEF-2K的钙调素敏感性并从而激活其的机制。我们期望我们 这些分析将为深入了解eEF-2K如何将这两种功能结合起来提供基础 调节其活动的不同信号。如此详细的eEF-2K调节图对于阐明 它对多种基本细胞过程的贡献以及它在多种疾病状态中的失调。