Metabolic stress responses and eIF2 kinase GCN2

描述（由申请人提供）：真核起始的磷酸化 因子-2（eIF 2）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 eIF 2激酶，GCN 2，在氨基酸限制期间调节翻译。在 酵母，在氨基酸饥饿期间积累的不带电荷的tRNA结合 具有与组氨酰-tRNA合成酶（HisRS）同源的GCN 2序列，诱导 eIF 2激酶活性。eIF 2的磷酸化水平升高， GCN 4的翻译表达，GCN 4是参与基因的转录调节因子， 不同的代谢途径。除了氨基酸饥饿，GCN 4 翻译是响应于葡萄糖限制而诱导的， 雷帕霉素和暴露于升高的NaCl浓度。我们将描述 GCN 2的激活机制，并研究GCN 4翻译的整合， 这些压力路径。GCN 2的调节还涉及C末端序列 它们被提议介导多种功能，包括核糖体靶向， 和二聚化。我们将讨论这些功能中每一个的重要性， GCN 2的调节。有趣的是，我们发现GCN 2的C-末端可以 在功能上取代相关eIF 2激酶PKRI的调节结构域 这种所谓的PKR嵌合体系统将被用来识别不同的 寡聚化和靶向结构域，并选择突变来研究 这些功能在天然蛋白中的重要性。鉴于饥饿， 在哺乳动物细胞中，氨基酸刺激eIF 2磷酸化，我们寻找 并在小鼠中鉴定了GCN 2同源物。与酵母菌类似，哺乳动物 GCN 2含有与激酶催化酶并置的FlisRS相关序列。 域我们将使用GCN 2（+/+）和（-/-）小鼠胚胎成纤维细胞进行研究 GCN 2 I调节和功能此外，酵母和哺乳动物系统 将被用来解决泄漏扫描的机制是否对 酵母中的GCN 4翻译也响应于eIF 2磷酸化而起作用。 哺乳动物最后，将使用微阵列杂交分析来筛选 基因是诱导响应营养压力，并评估 GCN 2活性的贡献。为了解决这些基本问题，我们 提出了四个具体目标：1）C-末端的作用的表征 在响应于应激的GCN 2的活化中; 2）表征 酵母中多种应激途径中的GCN 2蛋白激酶; 3）表征 通过GCN 2调节哺乳动物基因特异性翻译的机制;以及4） 响应eIF 2的基因表达变化的表征 通过GCN 2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.