Metabolic stress responses and eIF2 kinase GCN2

描述(申请人提供)：真核生物起始的磷酸化 因子-2(EIF2)是调节蛋白质合成的重要机制。 对不同细胞压力的反应。本提案侧重于其中一项 EIF2激酶，GCN2，在氨基酸限制过程中调节翻译。在……里面 酵母，在氨基酸饥饿过程中积累的不带电荷的tRNA结合 与组氨酰-tRNA合成酶(HisRS)同源的GCN2序列，诱导 EIF2激酶活性。EIF2的磷酸化水平升高刺激 相关基因转录调控因子GCN4的翻译表达 在不同的代谢途径中。除了氨基酸饥饿，Gcn4 翻译是对葡萄糖限制的反应，TOR抑制是由 雷帕霉素和接触高浓度的氯化钠。我们将描述 GCN2的激活机制及GCN4翻译整合的研究 这些应激途径。GCN2的调控也涉及C-末端序列 被提出用来调节多种功能，包括核糖体靶向 和二聚化。我们将在以下内容中说明这些功能的重要性 GCN2的调控。有趣的是，我们发现GCN2的C末端可以 功能性地替换相关eIF2激酶的调控结构域，即PKRI 这个所谓的PKR嵌合体系统将被利用来识别不同的 寡聚和靶向结构域并选择突变来研究 这些功能在天然蛋白质中的重要性。考虑到饥饿对 氨基酸刺激哺乳动物细胞eIF2磷酸化，我们寻找 并在小鼠体内鉴定出GCN2同源物。像它的酵母菌一样，哺乳动物 GCN2含有FlisRS相关序列，与激酶催化作用并列 域。我们将使用GCN2(+/+)和(-/-)小鼠胚胎成纤维细胞来研究 GCN2I的调节和功能此外，酵母和哺乳动物系统 将用于解决泄漏扫描机制是否对 酵母中GCN4的翻译也在eIF2磷酸化的反应中发挥作用 哺乳动物。最后，将使用微阵列杂交试验来筛选 在应对营养压力时诱导的基因并评估 GCN2活性的贡献。为了追寻这些根本问题，我们 提出四个具体目标：1)描述C-末端的作用 在应激反应中激活GCN2；2)表征GCN2的作用 酵母中多种应激途径中的GCN2蛋白激酶；3)特性 GCN2调控哺乳动物基因特异性翻译的机制；以及4) EIF2作用下基因表达变化的特征 GCN2的磷酸化。

DESCRIPTION (provided by applicant): Phosphorylation of eukaryotic initiation factor -2 (eIF2) is an important mechanism regulating protein synthesis in response to different cellular stresses. This proposal focuses on one of these eIF2 kinases, GCN2, that regulates translation during amino acid limitation. In yeast, uncharged tRNA that accumulates during amino acid starvation combines with GCN2 sequences homologous to histidyl-tRNA synthetases (HisRS), inducing eIF2 kinase activity. Elevated phosphorylation of eIF2 stimulates the translational expression of GCN4, a transcriptional regulator of genes involved in different metabolic pathways. In addition to amino acid starvation, GCN4 translation is induced in response to glucose limitation, TOR Inhibition by rapamycin and exposure to elevated NaC1 concentrations. We will characterize the mechanisms activating GCN2 and study the integration of GCN4 translation in these stress pathways. Regulation of GCN2 also involves C-terminal sequences that are proposed to mediate multiple functions, including ribosome targeting and dimerization. We will address the importance of each of these functions in the regulation of GCN2. Interestingly, we found that the C-terminus of GCN2 can functionally replace the regulatory domain of the related eIF2 kinase, PKRI This so-called PKR chimera system will be exploited to identify diverse oligomerization and targeting domains and select for mutations to study the importance of these functions in the native protein. Given that starvation for amino acids stimulates eIF2 phosphorylation in mammalian cells, we searched for and identified a GCN2 homologue in mice. Like its yeast counterpart, mammalian GCN2 contains FlisRS-related sequences juxtaposed to the kinase catalytic domain. We will use GCN2 (+/+) and (-/-) mouse embryo fibroblast cells to study the regulation and function of GCN2I Furthermore, yeast and mammalian systems will be used to address whether the mechanism of leaky scanning important for GCN4 translation in yeast also function in response to eIF2 phosphorylation in mammals. Finally, will use the microarray hybridization assay to screen for genes that are induced in response to nutritional stress and assess the contribution of GCN2 activity. To pursue these fundamental questions, we propose four specific aims: 1) Characterization of the role of the C-terminus in activation of GCN2 in response to stress; 2) Characterization of the role of GCN2 protein kinase in multiple stress pathways in yeast; 3) Characterization of mechanisms regulating mammalian gene-specific translation by GCN2; and 4) Characterization of changes in gene expression in response to eIF2 phosphorylation by GCN2.