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

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关系是维持细胞内环境平衡的关键。翻译 是单元格中最耗能的过程之一，占 一个真核细胞，使其严格的监管是必要的。真核延长因子2激酶(EEF-2K)，a 是α-激酶家族中唯一的成员，是翻译延伸阶段的关键调节因子。EEF-2K 使延伸因子2(EEF-2)磷酸化并失活，导致全球翻译速率降低 一方面是某些蛋白质的差异翻译，另一方面是差异翻译。EEF-2K的活性依赖于 钙调蛋白，并受钙离子和磷酸化的复杂调节。趁有机会 越来越多的证据表明，EEF-2K活性的失调参与了几种疾病状态(例如， 阿尔茨海默病、抑郁症和各种癌症)，详细了解EEF-2K的机制 缺乏激活和调节。我们的长期目标是准确定义EEF-2K 是被激活和调节的；这样的信息对于理解它对正常的 细胞过程以及疾病状态的病因和进展。为了实现这一目标，我们最近确定了 钙调蛋白刺激的特定残基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细胞的结构和生化机制 钙调素激活EEF-2K及调控位点S500上钙和磷酸化的作用 调节这一过程。我们将通过多方面和协作的方法来实现这一目标 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的自动磷酸化和EEF-2K的激活依赖于 钙调素结合。钙离子和S500磷酸化作用重叠，但利用不同 调节EEF-2K钙调蛋白敏感性从而激活的机制。我们期待我们的 分析将为深入理解EEF-2K如何将这两者结合在一起提供基础 调节其活动的不同信号。如此详细的EEF-2K调控图对于阐明 它对各种基本的细胞过程的贡献，以及它在各种疾病状态下的放松调控。

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.