STRUCTURAL AND FUNCTIONAL INSIGHTS INTO ALPHA-KINASES AND THEIR REGULATION

对 α 激酶及其调控的结构和功能见解

• 批准号: RGPIN-2018-04427
• 负责人: Jia, Zongchao
• 金额: $ 10.05万
• 依托单位: Queen's University
• 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=746090
• 关键词: STRUCTURAL; FUNCTIONAL; INSIGHTS; INTO; ALPHA

Protein phosphorylation represents one of the most fundamental processes in living organisms, with ~30% of all human proteins undergoing phosphorylation at some point during their lifetime. Of the protein kinases present in the human genome, >90% belong to a superfamily of conventional eukaryotic protein kinases (CPKs). The alpha-kinase family, lacking sequence homology with CPKs, is an intriguing class of atypical kinases which were discovered for their phosphorylation of alpha-helical substrates. While our long-term objective is to deepen our understanding of the function, mechanism and regulation of protein phosphorylation, we will focus on the alpha-kinase family in this proposal. The alpha-kinases play critical roles in cell survival, proliferation and motility, but they are still poorly understood. Currently, the structures of two alpha-kinases have been determined: TRMP7 (a channel kinase involved in Mg2+ sensing) and MHCK (a Thr-specific kinase which acts on myosin II), the latter of which was determined by our group. Our structure revealed that an indispensable Asp residue near the active site of MHCK is phosphorylated, suggesting that the phosphate is transferred from ATP to the Asp and then to substrate, in contrast to the direct phosphotransfer from ATP in CPKs. We will investigate this unprecedented two-step mechanism using 31P-NMR and mass spectrometry. Further, we will elucidate a novel regulatory feature, discovered in our lab, involving phospho-ligand binding to an allosteric pocket for kinase activation. For eukaryotic elongation factor 2 kinase (eEF2K), which is arguably the most biologically important alpha-kinase, multiple regulatory factors exist. This is not surprising as it is a “master regulator” of protein synthesis. One of the most important, but poorly understood, regulatory elements is eEF2K's dependence on calmodulin (CaM) for activation. The main challenge has been the expression and purification of eEF2K which we have recently conquered. Based on this breakthrough, we propose to determine an eEF2K-CaM complex structure to reveal the structural basis of its activation. Moreover, the structural basis for recognition of an alpha-helical substrate is unknown due to a lack of alpha-kinase-substrate complex structures. We plan to use a peptide substrate conjugated to ATP as a high-affinity “bisubstrate” to evaluate substrate recognition and binding. Based on our discovery that a conserved N/D loop binds Mg2+, we will dissect the role of Mg2+ sensing of TRMP7 and its implication in cellular function using patch-clamp in combination with various N/D mutants. Taken together, we have proposed a comprehensive research program which is largely based on our own discoveries and progress. As such, we have a competitive edge in making significant contributions to the under-characterized alpha-kinases which are important for potential therapeutic applications.

蛋白质磷酸化是生物体中最基本的过程之一，约30%的人类蛋白质在其生命周期的某个时间点经历磷酸化。在人类基因组中存在的蛋白激酶中，>90%属于常规真核蛋白激酶（CPK）的超家族。与CPK缺乏序列同源性的α-激酶家族是一类有趣的非典型激酶，其因其α-螺旋底物的磷酸化而被发现。虽然我们的长期目标是加深我们对蛋白质磷酸化的功能，机制和调控的理解，但我们将重点关注本提案中的α-激酶家族。α-激酶在细胞存活、增殖和运动中起着关键作用，但对它们的了解仍然很少。目前，已经确定了两种α-激酶的结构：TRMP 7（一种参与Mg 2+传感的通道激酶）和MHCK（一种作用于肌球蛋白II的Thr特异性激酶），后者是由我们小组确定的。我们的结构显示，MHCK的活性位点附近的一个不可或缺的天冬氨酸残基被磷酸化，这表明磷酸从ATP转移到天冬氨酸，然后到底物，在CPKs中从ATP的直接磷酸转移相反。我们将使用31 P-NMR和质谱来研究这种前所未有的两步机制。此外，我们将阐明一个新的监管功能，在我们的实验室发现，涉及磷酸配体结合到一个变构口袋激酶激活。对于真核细胞延伸因子2激酶（eEF 2K），其可以说是生物学上最重要的α-激酶，存在多种调节因子。这并不奇怪，因为它是蛋白质合成的“主调节器”。其中最重要但知之甚少的调节元件是eEF 2K对钙调素（CaM）的依赖性。主要的挑战是我们最近攻克的eEF 2K的表达和纯化。基于这一突破，我们建议确定eEF 2K-CaM复合物的结构，以揭示其激活的结构基础。此外，由于缺乏α-激酶-底物复合物结构，识别α-螺旋底物的结构基础是未知的。我们计划使用与ATP缀合的肽底物作为高亲和力的“双底物”来评估底物识别和结合。基于我们发现保守的N/D环结合Mg 2+，我们将使用膜片钳结合各种N/D突变体来剖析TRMP 7的Mg 2+传感作用及其在细胞功能中的意义。总之，我们提出了一个全面的研究计划，这在很大程度上是基于我们自己的发现和进展。因此，我们在对潜在治疗应用重要的特征不足的α-激酶做出重大贡献方面具有竞争优势。