REGULATION OF EUKARYOTIC PROTEIN SYNTHESIS

应激条件下哺乳动物细胞蛋白质合成的调控 条件以及特定于GCN4的翻译在 氨基酸饥饿条件下的酿酒酵母 由真核细胞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的PKR和PKR是如何 HRI和酵母蛋白激酶GCN2特异性识别和磷酸化 丝氨酸-51在eIF2的a亚基上调节翻译。我们有 建立了一种抑制酵母中PKR表达毒性的系统 通过共表达PKR的病毒抑制物。痘苗病毒K3L蛋白 是一种PKR的假底物抑制剂，能抑制PKR的毒性。 酵母。发现了一个保守的氨基酸序列基序 K3L的羧基末端和eIF2α的73-83位残基之间是 K3L对PKR的抑制至关重要。生化分析揭示了 这个主题，位于30多个残留物的地点 EIF2α的磷酸化是K3L与PKR结合的重要途径。这个 EIF2a中的相应残基是适当调节 EIF2α损伤中Gcn4的表达和该基序的突变 丝氨酸-51在体内的磷酸化。这些结果表明接触者 磷酸化部位的30多个残基对激酶很重要 对eIF2α的识别。在另一种研究激酶底物的方法中 识别我们已经鉴定出14个抗K3L的PKR等位基因 抑制力。PKR突变聚集在羧基末端的一半 基于cAMP依赖的x射线结构 蛋白激酶，被预测会改变该激酶和 底物。加州链球绦虫杆状病毒的pk2蛋白 类似于一个截短的eIF2α激动域，我们已经发现pk2 是一种eIF2α激酶抑制剂。Pk2蛋白直接相互作用。 与pkr一起，pk2被发现能保护杆状病毒免受抗病毒作用。 PKR的影响。EIF2α的底物特异性分析 蛋白激酶研究表明，PKR是一种双特异性蛋白激酶，能够 在残基-51处的丝氨酸、苏氨酸或酪氨酸上磷酸化eIF2α。一秒钟 主要研究领域涉及酵母蛋白FUN12，它 类似细菌的IF2，像eIF2一样提供的翻译因子 Met-tRNAi与核糖体相遇。在蛋白质合成的模型分析中 FUN12蛋白可替代eIF2，多聚核糖体图谱 Fun12缺失的菌株在翻译起始方面存在缺陷。这些 结果，结合在Fun12-缺失中的一些遗传观察 表明FUN12在一般或替代机制中起作用 翻译启蒙。最后，FUN12蛋白也被 在古生菌中被鉴定，我们已经鉴定出FUN12的人类同源物 这表明这一因素在人类进化过程中扮演着重要且保守的角色 细胞蛋白质合成。

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 kinases PKR and HRI and the yeast kinase GCN2 specifically recognize and phosphorylate serine-51 on the a subunit of eIF2 to regulate translation. We have established a system to suppress the toxicity of PKR expression in yeast by co-expressing viral inhibitors of PKR. The vaccinia virus K3L protein is a pseudosubstrate inhibitor of PKR, and can suppress PKR toxicity in yeast. A conserved amino acid sequence motif found near the carboxyl-terminus of K3L and between residues 73-83 in eIF2alpha is critical for K3L inhibition of PKR. Biochemical analyses have revealed that this motif, located over 30 residues from the site of phosphorylation in eIF2alpha is important for K3L binding to PKR. The corresponding residues in eIF2a are required for proper regulation of GCN4 expression and mutations in this motif in eIF2alpha impair phosphorylation of Ser-51 in vivo. These results suggest that contacts over 30 residues from site of phosphorylation are important for kinase recognition of eIF2alpha. In another approach to study kinase-substrate recognition we have identified 14 PKR alleles that are resistant to K3L inhibition. The PKR mutations cluster in the carboxyl-terminal half of the kinase domain and, based on the x-ray structure of the cAMP-dependent protein kinase, are predicted to alter contacts between the kinase and substrate. The pk2 protein from the baculovirus Autographa californica resembles a truncated eIF2alpha kinase domain and we have found that pk2 is an eIF2alpha kinase inhibitor. The pk2 protein directly interacted with PKR, and pk2 was found to protect baculovirus from the anti-viral effects of PKR. Analysis of substrate specificity of the eIF2alpha kinases revealed that PKR is a dual-specificity protein kinase capable of phosphorylating eIF2alpha on Ser, Thr, or Tyr at residue-51. A second major area of investigation involves the yeast protein FUN12 which resembles bacterial IF2, a translation factor that like eIF2 delivers Met-tRNAiMet to the ribosome. In model assays of protein synthesis the FUN12 protein could substitute for eIF2, and polyribosome profiles from fun12-deleted strains showed defects in translation initiation. These results, combined with a number of genetic observations in fun12-deletion strains, suggest that FUN12 functions in general or alternate mechanisms of translation initiation. Finally, the FUN12 protein has also been identified in archaea and we have identified a human homolog of FUN12 suggesting that this factor plays an important, and conserved, role in cellular protein synthesis.