HISRS DOMAIN AND REGULATING GCN2 PROTEIN KINASE

许多生物体中调节蛋白质合成的重要机制 真核细胞起始因子-2(eIF-2)的磷酸化。许多 已发现不同的应激信号可诱导eIF-2 磷酸化，包括病毒感染、氨基酸或血清 剥夺、缺铁、钙动员与脑 缺血症。此外，对eIF-2的不当调控 磷酸化被发现会导致恶性转化。这个 这项提议的目标有两个。第一个目标集中在 酵母中eIF-2激酶GCN2的调控。GCN2感官氨基酸 在酵母中饥饿并通过磷酸化转导这一信号。 EIF-2，以刺激转录激活因子Gcn4的翻译 参与氨基酸生物合成的基因。GCN2同源序列 与组氨酰-tRNA合成酶(HisRSs)通过以下途径刺激激酶活性 与氨基酸过程中积累的未带电的tRNA相互作用 饿死了。我们将讨论核糖体靶向和 自磷酸化与HisRS相关结构域的特异性 GCN2激酶的激活。这项提议的第二个目标是 了解蛋白质启动减少的分子基础 缺乏氨基酸的哺乳动物细胞中的合成。给定 GCN2在低等真核生物翻译调控中的重要作用 我们搜索并鉴定了一个与酵母同源的哺乳动物cdna。 GCN2。这种新的哺乳动物激酶既含有激酶又含有与HisRS相关的 结构域，在多种组织中表达，包括肝脏和 通过Northern印迹分析判断为脑组织。我们建议 GCN2激酶对eIF-2的磷酸化降低翻译起始 在饥饿的哺乳动物细胞中。为了实现上述两个目标，我们将 解决四个具体目标。1)鉴定核糖体结合部位 酵母GCN2激酶刺激所需的翻译调控。2) 表征磷酸化和HisRS相关结构域的作用 通过不带电荷的tRNA水平激活酵母GCN2激酶。3) 研究哺乳动物GCN2在翻译控制中的作用 酵母菌作为模型系统。4)研究GCN2蛋白在细胞周期中的作用 在缺乏氨基酸的哺乳动物细胞中eIF-2的磷酸化。 这些研究将进一步加深我们对以下机制的理解： TRNA和核糖体相互作用通过以下途径促进eIF-2的磷酸化 GCN2激酶。此外，这些研究将确定 新的哺乳动物GCN2蛋白在蛋白质合成中的调控作用 缺乏营养的哺乳动物细胞。

An important mechanism regulating protein synthesis in many organisms in phosphorylation of eukaryotic initiation factor-2 (eIF-2). Many different stress signals have been identified that elicit eIF-2 phosphorylation, including viral infection, amino acid or serum deprivation, iron deficiency, calcium mobilization and cerebral ischemia. In addition, inappropriate regulation of eIF-2 phosphorylation was found to lead to malignant transformation. The goals of this proposal are twofold. The first goal focuses on the regulation of an eIF-2 kinase from yeast, GCN2. GCN2 senses amino acid starvation in yeast and transduces this signal, via phosphorylation of eIF-2, to stimulate translation of GCN4, a transcriptional activator of genes involved in amino acid biosynthesis. Sequences in GCN2 homologous with histidyl-tRNA synthetases (HisRSs) stimulate kinase activity by interacting with uncharged tRNAs that accumulate during amino acid starvation. We will address the roles of ribosome targeting and autophosphorylation and the specificity of the HisRS-related domain in the activation of GCN2 kinase. The second goal of this proposal is to understand the molecular basis for reduced initiation of protein synthesis in mammalian cells starving for amino acids. Given the important role of GCN2 in translational control in lower eurkaryotes, we searched for and identified a mammalian cDNA with homology to yeast GCN2. This new mammalian kinase contains both kinase and HisRS-related domains and is expressed in a variety of tissues, including liver and brain, as judged by Northern blot analysis. We propose that phosphorylation of eIF-2 by GCN2 kinase reduces translation initiation in starving mammalian cells. To achieve the two stated goals, we will address four specific aims. 1) Characterize the ribosomal binding site required for yeast GCN2 kinase stimulation of translation control. 2) Characterize the roles of phosphorylation and the HisRS-related domain in the activation of yeast GCN2 kinase by uncharged tRNA levels. 3) Characterize the role of mammalian GCN2 in translation control using yeast as a model system. 4) Characterize the role of GCN2 kinase in the phosphorylation of eIF-2 in mammalian cells starving for amino acids. These studies will further our understanding to the mechanisms by which tRNA and ribosome interaction facilitate phosphorylation of eIF-2 by GCN2 kinase. Additionally, these studies will determine the role of the new mammalian GCN2 kinase in the control of protein synthesis in nutrient-deprived mammalian cells.