We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-kappaB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO (NF-kappaB Essential MOdulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IkappaB kinase complex, and is required for its formation. In vitro, NEMO can homodimerize and directly interacts with IKK-2. The NEMO cDNA was also able to complement another NF-kappaB-unresponsive cell line, 1.3E2, in which the protein is also absent, allowing us to demonstrate that this factor is required not only for Tax but also for LPS, PMA, and IL-1 stimulation of NF-kappaB activity.

The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-B. NF-B is retained in the cytoplasm through binding to its inhibitor IB␣. The critical step in NF-B activation is the phosphorylation of IB␣ by the IB kinase (IKK) complex. This activity releases NF-B from IB␣ and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-B 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 eIF2␣-binding domain, can also activate NF-B. This result indicated that NF-B activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2␣ 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 IKK␤ 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-B essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-B signaling pathway without using its kinase activity.

The transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and serves as a pivotal mediator of inflammatory response. In the first part of this review, we discuss the NF-κB inducers, signaling pathways, and regulators involved in immune homeostasis as well as detail the importance of post-translational regulation by ubiquitination in NF-κB function. We also indicate the stages of central and peripheral tolerance where NF-κB plays a fundamental role. With respect to central tolerance, we detail how NF-κB regulates medullary thymic epithelial cell (mTEC) development, homeostasis, and function. Moreover, we elaborate on its role in the migration of double-positive (DP) thymocytes from the thymic cortex to the medulla. With respect to peripheral tolerance, we outline how NF-κB contributes to the inactivation and destruction of autoreactive T and B lymphocytes as well as the differentiation of CD4+-T cell subsets that are implicated in immune tolerance. In the latter half of the review, we describe the contribution of NF-κB to the pathogenesis of autoimmunity and autoinflammation. The recent discovery of mutations involving components of the pathway has both deepened our understanding of autoimmune disease and informed new therapeutic approaches to treat these illnesses.