Zhu S, Romano P R, Wek R C
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
J Biol Chem. 1997 May 30;272(22):14434-41. doi: 10.1074/jbc.272.22.14434.
Protein kinase PKR is activated in mammalian cells during viral infection, leading to phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha) and inhibition of protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the amino terminus of PKR. Recent studies have observed that expression of mammalian PKR in yeast leads to a slow growth phenotype due to hyperphosphorylation of eIF-2alpha. In this report, we observed that while DRBD sequences are required for PKR to function in the yeast model system, these sequences are not required for in vitro phosphorylation of eIF-2alpha. To explain this apparent contradiction, we proposed that these sequences are required to target the kinase to the translation machinery. Using sucrose gradient sedimentation, we found that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences blocked kinase interaction with ribosomes. These results indicate that in addition to mediating ds-RNA control of PKR, the DRBD sequences facilitate PKR association with ribosomes. Targeting to ribosomes may enhance in vivo phosphorylation of eIF-2alpha, by providing PKR access to its substrate.
蛋白激酶PKR在病毒感染期间于哺乳动物细胞中被激活，导致真核起始因子-2（eIF-2α）的α亚基磷酸化并抑制蛋白质合成。这种抗病毒反应被认为是由病毒复制的副产物双链RNA（ds-RNA）与位于PKR氨基末端的两个双链RNA结合结构域（DRBD）结合介导的。最近的研究观察到，由于eIF-2α的过度磷酸化，哺乳动物PKR在酵母中的表达导致生长缓慢的表型。在本报告中，我们观察到虽然DRBD序列是PKR在酵母模型系统中发挥功能所必需的，但这些序列对于eIF-2α的体外磷酸化并非必需。为了解释这一明显的矛盾，我们提出这些序列是将激酶靶向翻译机制所必需的。使用蔗糖梯度沉降法，我们发现野生型PKR与核糖体相关，特别是与40 S颗粒相关。DRBD序列中的缺失或残基替换阻断了激酶与核糖体的相互作用。这些结果表明，除了介导PKR的ds-RNA控制外，DRBD序列还促进PKR与核糖体的结合。通过使PKR接近其底物，靶向核糖体可能增强eIF-2α在体内的磷酸化。
