Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.
Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.
复制标题
蛋白激酶 PKR 突变体对痘病毒假底物 K3L 蛋白具有抗性。
DOI：
10.1073/pnas.0805524105
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发表时间：
2008
影响因子：
11.1
通讯作者：
Dever,ThomasE
中科院分区：
文献类型：
--
作者：
Seo,EunJoo;Liu,Furong;Kawagishi-Kobayashi,Makiko;Ung,TeklyL;Cao,Chune;Dar,ArvinC;Sicheri,Frank;Dever,ThomasE
作为哺乳动物细胞先天性免疫应答的一部分，双链RNA激活的蛋白激酶PKR磷酸化翻译起始因子eIF 2 α以抑制蛋白质合成，从而阻断病毒复制。痘病毒（包括牛痘病毒和天花病毒）表达PKR抑制剂，如牛痘病毒K3 L蛋白，其类似于eIF 2 α的N-末端底物靶向结构域。而高水平表达的人PKR在酵母中是有毒的，这种生长抑制被抑制的K3 L蛋白的共表达。我们使用这种酵母试验筛选对K3 L抑制有抗性的PKR突变体，并且我们鉴定了12个定位于PKR激酶结构域C-末端叶的突变。PKR突变在酵母和体外均特异性地赋予对K3 L蛋白的抗性。一致地，PKR-D486 V突变导致K3 L结合亲和力降低近15倍，但不损害eIF 2 α磷酸化。我们的结果支持在激酶结构域的C-末端叶的广泛面上鉴定eIF 2 α结合位点，并且它们表明PKR激酶结构域的细微变化可以显著影响假底物抑制，同时保持底物磷酸化完整。我们认为PKR突变对假底物与底物相互作用的这些矛盾效应反映了刚性K3 L蛋白和Ser-51磷酸化位点周围eIF 2 α的可塑性之间的差异。
As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2α to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2α. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2α phosphorylation. Our results support the identification of the eIF2α-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2α around the Ser-51 phosphorylation site.