Bonnet M C, Weil R, Dam E, Hovanessian A G, Meurs E F
Unité de Virologie et d'Immunologie Cellulaire, URA CNRS 1930, Institut Pasteur, 75724 Paris Cedex 15, France.
Mol Cell Biol. 2000 Jul;20(13):4532-42. doi: 10.1128/MCB.20.13.4532-4542.2000.
The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-kappaB. NF-kappaB is retained in the cytoplasm through binding to its inhibitor IkappaBalpha. The critical step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. This activity releases NF-kappaB from IkappaBalpha and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-kappaB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2alpha-binding domain, can also activate NF-kappaB. This result indicated that NF-kappaB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2alpha relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR(0/0) mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKKbeta subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-kappaB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-kappaB signaling pathway without using its kinase activity.
干扰素（IFN）诱导的双链RNA激活蛋白激酶PKR通过真核起始因子2（eIF2α）α亚基的磷酸化介导蛋白质合成的抑制，并且还通过转录因子NF-κB的激活参与IFN基因的诱导。NF-κB通过与其抑制剂IκBα结合而保留在细胞质中。NF-κB激活的关键步骤是IκB激酶（IKK）复合物对IκBα的磷酸化。这种活性使NF-κB从IκBα释放并允许其转运到细胞核。在这里，我们在报告基因测定中研究了PKR激活NF-κB的能力，并且首次表明两个催化无活性的PKR突变体PKR/KR296和缺乏潜在eIF2α结合结构域的缺失突变体（PKR/Del42）也可以激活NF-κB。该结果表明PKR对NF-κB的激活不需要其激酶活性，并且它独立于PKR-eIF2α关系。在PKR(0/0)小鼠胚胎成纤维细胞中转染野生型PKR或催化无活性的PKR导致IKK复合物的激活。通过使用谷胱甘肽S-转移酶下拉试验，我们表明PKR与IKK复合物的IKKβ亚基相互作用。这种相互作用显然不需要IKK复合物的完整性，因为发现它与缺乏NF-κB必需调节剂（IKK复合物的组成成分之一）的细胞提取物发生。因此，我们的结果揭示了一种新的途径，通过该途径PKR可以在不使用其激酶活性的情况下调节NF-κB信号通路。
