STRUCTURAL AND FUNCTIONAL INSIGHTS INTO ALPHA-KINASES AND THEIR REGULATION

蛋白质磷酸化是生物体中最基本的过程之一，大约30%的人类蛋白质在其一生中的某个时间点经历了磷酸化。在人类基因组中存在的蛋白激酶中，90%属于传统的真核蛋白激酶(CPK)超家族。α-激酶家族与CPK缺乏序列同源性，是一类有趣的非典型激酶，被发现是因为它们对α-螺旋底物的磷酸化。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，但在这项提案中，我们将重点放在阿尔法激酶家族上。α-激酶在细胞的存活、增殖和运动中起着关键作用，但人们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(参与镁离子感应的通道激酶)和MHCK(作用于肌球蛋白II的Thr特异性激酶)，后者是本课题组确定的。我们的结构表明，MHCK活性中心附近不可或缺的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CPKs中直接从ATP转移磷。我们将使用31P-核磁共振和质谱学来研究这一前所未有的两步反应机理。此外，我们将阐明在我们的实验室中发现的一个新的调节功能，涉及到磷酸配体与变构口袋结合以激活激酶。对于真核细胞延伸因子2激酶(EEF2K)来说，它是最具生物学意义的α-激酶，存在多种调节因素。这并不令人惊讶，因为它是蛋白质合成的“主要调节器”。最重要但知之甚少的调控元件之一是eEF2K依赖钙调蛋白(CaM)进行激活。主要的挑战是我们最近攻克的eEF2K的表达和纯化。基于这一突破，我们建议确定eEF2K-CaM的络合物结构，以揭示其激活的结构基础。此外，由于缺乏α-激酶-底物的复杂结构，识别α-螺旋底物的结构基础尚不清楚。我们计划使用与三磷酸腺苷结合的多肽底物作为高亲和力的双底物来评估底物的识别和结合。基于我们的发现，保守的N/D环与镁离子结合，我们将结合各种N/D突变体，分析TRMP7对镁离子的感知作用及其在细胞功能中的意义。综上所述，我们提出了一个在很大程度上基于我们自己的发现和进展的全面研究计划。因此，我们在对未充分表征的α-激酶做出重大贡献方面具有竞争优势，这些α-激酶对潜在的治疗应用非常重要。

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.