Gale M, Tan S L, Wambach M, Katze M G
Department of Microbiology, School of Medicine, University of Washington, Seattle 98195, USA.
Mol Cell Biol. 1996 Aug;16(8):4172-81. doi: 10.1128/MCB.16.8.4172.
Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.
双链RNA激活蛋白激酶（PKR）的表达由干扰素诱导，PKR活性在建立干扰素诱导的抗病毒和抗增殖状态中起关键作用。PKR直接受与特异性抑制剂P58IPK（四肽重复序列（TPR）家族的一种细胞蛋白）以及痘苗病毒相应基因产物K3L的物理结合调控。P58IPK和K3L抑制PKR的激活和活性。为了研究P58IPK和K3L介导的PKR抑制机制，我们结合了体外和体内结合试验来确定这些蛋白的相互作用区域。PKR与P58IPK相互作用的位点被定位到一个52个氨基酸的片段（第244至296位氨基酸），该片段跨越蛋白激酶催化结构域的ATP结合区域。与PKR的相互作用不需要P58IPK的C末端DNA-J同源区域，但依赖于真核起始因子2-α同源区域的存在，该区域位于第六个P58IPK TPR基序内的34个氨基酸处。与其他TPR蛋白一致，P58IPK在体内形成多聚体：N末端的166个氨基酸对于复合物形成既必要又充分。一项平行的体内分析用于定位PKR的K3L结合区域，结果显示，与P58IPK一样，K3L仅与PKR蛋白激酶催化结构域相互作用。然而，相比之下，PKR的K3L结合区域位于第367至551位氨基酸之间，表明每种抑制剂在独特的、不重叠的结构域中与PKR结合。综合这些数据表明，P58IPK和K3L可能通过不同机制介导PKR抑制。最后，我们将提出一个PKR抑制模型，其中P58IPK或P58IPK复合物结合PKR并干扰核苷酸结合和自身调节，而PKR-K3L复合物的形成则干扰活性位点功能和/或底物结合。
