HISRS-DOMAIN AND REGULATING GCN2 PROTEIN KINASE

拟议研究的目标是通过以下方式了解这些机制 哪些真核细胞调节它们的代谢途径以响应 生长条件的变化。模型体系中的酵母菌 Cerevisiae，氨基酸饥饿诱导GCN2蛋白激酶 使真核细胞翻译起始的α-亚基磷酸化 因子2(EIF-2)。该因子的磷酸化刺激 氨基酸转录激活因子GCN4的翻译 生物合成基因。此前，有研究表明， GCN2的C末端区域与GCN2的整个序列同源 酿酒酵母和人的组氨酰-tRNA合成酶(HisRS)。vt.给出 氨酰-tRNA合成酶结合不带电荷的tRNA作为底物 为了区分带电荷和不带电荷的tRNA，有人建议 GCN2中的HisRS相关结构域监控未带电tRNA的浓度 在细胞内并激活相邻的蛋白激酶部分 当未带电的tRNA积累时的饥饿状态。一个主要的 这项建议的目标是在净化系统中测试 不带电荷的tRNA水平调节着eIF-2-α的GCN2磷酸化。 为了检验这一模型，提出了以下调查路线。 首先，GCN2在体内监测哪些氨基酸。众所周知， Gcn4翻译被刺激以响应饥饿 至少十种不同的氨基酸。每个项目是否需要GCN2 限制？其次，不同物种之间的相互作用 不带电荷的tRNA和GCN2蛋白激酶将通过体外测定 结合分析和显示与GCN2形成复合体的tRNA将被检测 进一步确定它们是否在动力学上增强了磷酸化 EIF-2-α被GCN2激活。最终提议的实验使用的是基因工具 该系统可用于分离和鉴定GCN2的突变 底物专一性改变。 更好地理解GCN2的调节和底物亲和力 将提供有关哺乳动物应激反应的有用信息 细胞。已知与GCN2相关的eIF-2-α激酶抑制 病毒感染后的蛋白质合成(双链RNA 导致网织红细胞中DAI激酶的激活和血红素的剥夺 (激活HCR蛋白激酶)。

The goal of the proposed research is to understand the mechanisms by which eukaryotic cells regulate their metabolic pathways in response to changes in growth conditions. In the model system Saccharomyces cerevisiae, starvation for amino acids induces the GCN2 protein kinase to phosphorylate the alpha-subunit of eukaryotic translation initiation factor-2 (eIF-2). Phosphorylation of this factor stimulates the translation of GCN4, a transcriptional activator of amino acid biosynthetic genes. Previously, it was shown that the sequence of the C-terminal region of GCN2 is homologous to the entire sequence of histidyl-tRNA synthetase (HisRS) from S. cerevisiae and humans. Given that aminoacyl-tRNA synthetases bind uncharged tRNA as a substrate and distinguish between charged and uncharged tRNA, it was proposed that the HisRS-related domain in GCN2 monitors the concentration of uncharged tRNA in the cell and activates the adjacent protein kinase moiety under starvation conditions when uncharged tRNA accumulates. A primary objective of this proposal is to test in a purified system whether the levels of uncharged tRNA regulate GCN2 phosphorylation of eIF-2-alpha. To test this model the following line of investigation is proposed. First, which amino acids are monitored in vivo by GCN2. It is known that GCN4 translation is stimulated in response to starvation for any one of at least ten different amino acids. Is GCN2 required for each limitation? Secondly, the interaction between different species of uncharged tRNA and GCN2 protein kinase will be measured by in vitro binding assays and the tRNAs shown to complex with GCN2 will be examined further to determine if they kinetically enhance the phosphorylation of eIF-2-alpha by GCN2. The final proposed experiments use genetic tools available in this system to isolate and characterize mutations in GCN2 with altered substrate specificity. A better understanding of the regulation and substrate affinity of GCN2 will provide useful information about stress responses in mammalian cells. It is known that eIF-2-alpha kinases related to GCN2 inhibit protein synthesis in response to viral infection (double-stranded RNA leads to activation of DAI kinase) and heme deprivation in reticulocytes (activates HCR protein kinase).