STRUCTURE/FUNCTION OF EFF-2 KINASE

我们最近发现了一类新的蛋白激酶， 在结构上和进化上与 真核生物蛋白激酶超家族 的原型成员 这一类是Ca 2 +/钙调蛋白依赖性延伸因子-2 激酶（eEF-2激酶），一种普遍存在于 各种真核生物 eEF-2激酶的主要功能 是磷酸化并激活eEF-2，从而调节 蛋白质合成的延伸阶段。 初步证据 表明eEF-2激酶具有新型催化结构域， 并利用了底物识别的新机制。 本建议的目的是确定和描述 eEF-2激酶的功能结构域，研究其作用机制。 底物识别，并揭示其特定的生理 角色。 将进行人eEF-2激酶的体外诱变， 鉴定和表征催化和钙调蛋白结合 域. 我们还将研究底物的作用机理 通过eEF-2激酶识别。 定向肽文库将 筛选以鉴定eEF识别的共有序列- 2激酶。 接下来，我们将测试阿尔法- 在磷酸化位点肽的螺旋构象 的底物是被eEF-2激酶识别所必需的。 肽模拟具有不同程度的磷酸化位点， α-螺旋度将被合成并测试它们的能力 进行磷酸化。 为了鉴定eEF-2激酶的其他潜在底物， 方法将被使用：固相磷酸化表达 筛选方法和酵母双杂交系统。 为了揭示eEF-2激酶的确切生理功能， 将使用秀丽隐杆线虫。 我们将分析影响 eEF-2激酶基因失活对表型和总体 使用三种不同方法的发展模式：Tc 1 诱变、缺失诱变和质粒拯救 转基因和反义方法。 这项工作将揭示的结构组织和功能， 一类新的蛋白激酶，这将提供新的， 关于蛋白质作用机制的重要信息 磷酸化

We have recently identified a new class of protein kinases, which are structurally and evolutionarily unrelated to members of the eukaryotic protein kinase superfamily. The prototype member of this class is Ca2+/calmodulin-dependent elongation factor-2 kinase (eEF-2 kinase), a ubiquitous protein kinase present in various eukaryotic organisms. The major function of eEF-2 kinase is to phosphorylate and inactivate eEF-2, and thus, regulate the elongation phase of protein synthesis. Preliminary evidence suggests that eEF-2 kinase has a novel type of catalytic domain, and utilizes a novel mechanism of substrate recognition. The aim of this proposal is to identify and characterize the functional domains of eEF-2 kinase, to study its mechanism of substrate recognition, and to reveal its specific physiological role(s). In vitro mutagenesis of human eEF-2 kinase will be performed to identify and characterize the catalytic and calmodulin-binding domains. We will also study the mechanism of substrate recognition by eEF-2 kinase. An oriented peptide library will be screened to identify the consensus sequence recognized by eEF- 2 kinase. Next, we will test the hypothesis that an alpha- helical conformation of the peptide at the phosphorylation site of the substrate is required for recognition by eEF-2 kinase. Peptides mimicking phosphorylation sites with varying degrees of alpha-helicity will be synthesized and tested for their ability to undergo phosphorylation. To identify other potential substrates for eEF-2 kinase, two approaches will be used: a solid-phase phosphorylation expression screening method and the yeast two-hybrid system. To reveal the exact physiological function of eEF-2 kinase, Caenorhabditis elegans will be used. We will analyze the effect of eEF-2 kinase gene inactivation on phenotype and overall developmental pattern using three different approaches: Tc1 mutagenesis, deletion mutagenesis with rescue by plasmid transgenics, and an antisense approach. This work will reveal the structural organization and function of a new class of protein kinases, which will provide new and important information about the mechanism of protein phosphorylation.