REGULATION OF EUKARYOTE PROTEIN SYNTHESIS

真核生物蛋白质合成的调控

• 批准号: 2575666
• 负责人: T E DEVER
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2575666
• 关键词: Baculoviridae; eukaryote; gene induction /repression; gene mutation; genetic regulation; genetic strain; oxidative stress; phosphorylation; protein biosynthesis; protein kinase; protein tyrosine kinase; translation factor; vaccinia virus; yeasts

The regulation of protein synthesis in mammalian cells under stress conditions as well as the regulation of GCN4-specific translation in the yeast Saccharomyces cerevisiae under amino acid starvation conditions is mediated by phosphorylation of the eukaryotic translation initiation factor (eIF)-2. We have been studying how the mammalian PKR and HRI kinases and the yeast GCN2 kinase specifically recognize and phosphorylate serine-51 on the a subunit of eIF2 to regulate translation. One approach has been to study the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. We have established a system to suppress the toxicity of PKR expression in yeast by co-expressing K3L. Mutational analyses have revealed that residues near the carboxyl-terminus of K3L, that are conserved in eIF2a (residues 73-83), are critical for K3L activity. The corresponding residues in eIF2a are required for proper regulation of GCN4 expression suggesting that contacts over 20 residues from site of phosphorylation are important for kinase recognition of eIF2alpha. The suppression of PKR toxicity in yeast has also been used to identify a novel eIF2alpha kinase inhibitor from the baculovirus Autographa californica. The pk2 protein of baculovirus resembles a truncated eIF2 kinase domain and we have found that when expressed in yeast pk2 can inhibit both the PKR and GCN2 kinases. Similarly, the corresponding domain of GCN2 also acts as a dominant-negative inhibitor. In an analysis of substrate specificity of the eIF2 kinases it was found that all three eIF2 kinases readily phosphorylated threonine in place of eIF2alpha serine-51, and PKR was able to phosphorylate tyrosine at this position as well. In addition, PKR was found to autophosphorylate on tyrosine. These results identify PKR as a dual-specificity protein kinase. Finally, we have initiated a project examining a novel yeast protein, FUN12, which resembles a bacterial translation factor that performs the same role as eIF2.

在应激下哺乳动物细胞中蛋白质合成的调节 条件以及对GCN4特异性翻译的调节 在氨基酸饥饿条件下酿酒酵母的酵母糖疗法是 通过真核翻译起始的磷酸化介导 因子（EIF）-2。 我们一直在研究哺乳动物PKR和HRI 激酶和酵母GCN2激酶专门识别和 在EIF2的A亚基上磷酸化丝氨酸51以调节翻译。 一种方法是研究谷菌病毒K3L蛋白，A PKR的假基底抑制剂。 我们已经建立了一个系统 通过共表达K3L抑制PKR表达在酵母中的毒性。 突变分析表明，附近的残基 K3L的羧基末端，在EIF2A中保守（残基73-83）， 对于K3L活性至关重要。 EIF2A中的相应残基是 适当调节GCN4表达所必需的，这表明 从磷酸化部位的20多个残基的接触对于 激酶对EIF2Alpha的识别。 抑制PKR毒性 酵母也已用于识别新型的EIF2Alpha激酶抑制剂 来自加利福尼亚州的杆状病毒Autographa。 PK2蛋白 杆状病毒类似于截短的EIF2激酶结构域，我们发现 在酵母菌中表达时，PK2可以抑制PKR和GCN2 激酶。 同样，GCN2的相应域也充当 显性阴性抑制剂。 在分析底物特异性 EIF2激酶发现所有三个EIF2激酶很容易 磷酸化的苏氨酸代替EIF2alpha丝氨酸-51，PKR为 能够在该位置磷酸化酪氨酸。 此外， 发现PKR在酪氨酸上自磷酸化。 这些结果确定 PKR作为双特异性蛋白激酶。 最后，我们发起了 研究一种新型酵母蛋白Fun12的项目，类似于一个 与EIF2相同的作用的细菌翻译因子。