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: $ 5.03万
• 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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=691468
• 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%的人类蛋白质在其一生中的某个时刻经历磷酸化。在人类基因组中存在的蛋白激酶中，b> 90%属于常规真核蛋白激酶（CPKs）的超家族。α -激酶家族缺乏与cpk的序列同源性，是一类有趣的非典型激酶，因其磷酸化α -螺旋底物而被发现。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，但我们将在本提案中重点关注α激酶家族。α -激酶在细胞存活、增殖和运动中起着至关重要的作用，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：TRMP7（一种参与Mg2+感知的通道激酶）和MHCK（一种作用于肌球蛋白II的thr特异性激酶），后者是我们小组确定的。我们的结构揭示了MHCK活性位点附近不可或缺的Asp残基被磷酸化，这表明磷酸盐从ATP转移到Asp，然后转移到底物，而不是cpk中ATP的直接磷酸转移。我们将使用31P-NMR和质谱分析来研究这一前所未有的两步机制。此外，我们将阐明一个新的调控特征，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活化。真核延伸因子2激酶（eEF2K）被认为是生物学上最重要的α激酶，存在多种调控因子。这并不奇怪，因为它是蛋白质合成的“主要调节器”。一个最重要的，但知之甚少的调控元件是eEF2K对钙调蛋白（CaM）激活的依赖。主要的挑战是我们最近克服的eEF2K的表达和纯化。基于这一突破，我们提出确定eEF2K-CaM复合物结构，揭示其活化的结构基础。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尚不清楚。我们计划使用与ATP结合的肽底物作为高亲和力的“双底物”来评估底物的识别和结合。基于我们发现一个保守的N/D环结合Mg2+，我们将使用膜片钳结合各种N/D突变体来剖析TRMP7的Mg2+传感的作用及其在细胞功能中的意义。综上所述，我们提出了一项综合研究计划，该计划主要基于我们自己的发现和进展。因此，我们在对未被充分表征的α -激酶做出重大贡献方面具有竞争优势，这对潜在的治疗应用很重要。