HISRS DOMAIN AND REGULATING GCN2 PROTEIN KINASE

在许多生物体中调节蛋白质合成的重要机制 在真核起始因子-2（eIF-2）的磷酸化中。 许多 不同的应激信号已经被鉴定为诱导eIF-2 磷酸化，包括病毒感染、氨基酸或血清 缺铁、缺铁、钙动员和脑 缺血 此外，eIF-2的不适当调节 发现磷酸化导致恶性转化。 的 这项建议有两个目标。 第一个目标是 来自酵母的eIF-2激酶GCN 2的调节。 GCN 2传感氨基酸 饥饿在酵母和转导这一信号，通过磷酸化 eIF-2，刺激GCN 4的翻译，GCN 4是一种转录激活因子， 参与氨基酸生物合成的基因。 GCN 2同源序列 与组氨酰-tRNA合成酶（HisRS）刺激激酶活性， 与氨基酸合成过程中积累的不带电荷的转运RNA相互作用， 饥饿 我们将讨论核糖体靶向的作用， 自磷酸化和HisRS相关结构域的特异性， GCN 2激酶的激活。 该提案的第二个目标是 了解蛋白质起始减少的分子基础 在哺乳动物细胞中合成氨基酸。 鉴于 GCN 2在低等真核生物翻译调控中重要作用， 我们寻找并鉴定了与酵母同源的哺乳动物cDNA GCN 2。 这种新的哺乳动物激酶含有激酶和HisRS相关的 结构域，并在多种组织中表达，包括肝脏和 脑，如通过北方印迹分析判断的。我们建议 通过GCN 2激酶磷酸化eIF-2减少翻译起始 在饥饿的哺乳动物细胞中。 为了实现这两个目标，我们将 实现四个具体目标。 1)鉴定核糖体结合位点 所需的酵母GCN 2激酶刺激的翻译控制。 （二） 表征磷酸化和HisRS相关结构域的作用 在不带电的tRNA水平激活酵母GCN 2激酶。 第三章 使用以下方法表征哺乳动物GCN 2在翻译控制中的作用： 酵母作为模型系统。 4)表征GCN 2激酶在细胞凋亡中的作用。 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这些机制的理解， tRNA和核糖体的相互作用促进eIF-2的磷酸化， GCN 2激酶。此外，这些研究将确定 新的哺乳动物GCN 2激酶在蛋白质合成控制中的作用 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.