Simon T. Whiteside

Complementation Cloning of NEMO, a Component of the IκB Kinase Complex Essential for NF-κB Activation

Abstract We have characterized a flat cellular variant of HTLV-1 Tax-transformed rat fibroblasts, 5R, which is unresponsive to all tested NF-κB activating stimuli, and we report here its genetic complementation. The recovered full-length cDNA encodes a 48 kDa protein, NEMO ( N F-κB E ssential MO dulator), which contains a putative leucine zipper motif. This protein is absent from 5R cells, is part of the high molecular weight IκB 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-κB–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-κB activity.

I kappa B epsilon, a novel member of the IκB family, controls RelA and cRel NF‐κB activity

We have isolated a human cDNA which encodes a novel IκB family member using a yeast two‐hybrid screen for proteins able to interact with the p52 subunit of the transcription factor NF‐κB. The protein is found in many cell types and its expression is up‐regulated following NF‐κB activation and during myelopoiesis. Consistent with its proposed role as an IκB molecule, IκB‐ϵ is able to inhibit NF‐κB‐directed transactivation via cytoplasmic retention of rel proteins. IκB‐ϵ translation initiates from an internal ATG codon to give rise to a protein of 45 kDa, which exists as multiple phosphorylated isoforms in resting cells. Unlike the other inhibitors, it is found almost exclusively in complexes containing RelA and/or cRel. Upon activation, IκB‐ϵ protein is degraded with slow kinetics by a proteasome‐dependent mechanism. Similarly to IκB‐α and IκB‐β, IκB‐ϵ contains multiple ankyrin repeats and two conserved serines which are necessary for signal‐induced degradation of the molecule. A unique lysine residue located N‐terminal of the serines appears to be not strictly required for degradation. Unlike IκB‐α and IκB‐β, IκB‐ϵ does not contain a C‐terminal PEST‐like sequence. IκB‐ϵ would, therefore, appear to regulate a late, transient activation of a subset of genes, regulated by RelA/cRel NF‐κB complexes, distinct from those regulated by other IκB proteins.

IκBα is a target for the mitogen‐activated 90 kDa ribosomal S6 kinase

The activity of transcription factor NFκB is regulated by its subcellular localization. In most cell types, NFκB is sequestered in the cytoplasm due to binding of the inhibitory protein IκBα. Stimulation of cells with a wide variety of agents results in degradation of IκBα, which allows translocation of NFκB to the nucleus. Degradation of IκBα is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen‐activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IκBα kinase. p90rsk1 phosphorylates IκBα at Ser32 and it physically associates with IκBα in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant‐negative mutant, IκBα degradation in response to mitogenic stimuli, e.g. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), is inhibited. Finally, NFκB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IκBα in response to mitogens.

Sequential DNA damage‐independent and ‐dependent activation of NF‐κB by UV

NF‐κB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF‐κB from IκBα and degradation of IκBα, targeting for degradation and signaling are different. Early IκBα degradation at 30 min to ∼6 h is not initiated by UV‐induced DNA damage. It does not require IκB kinase (IKK), as shown by introduction of a dominant‐negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IκBα, carrying serines at positions 32 and 36. Induced IκBα degradation requires, however, intact N‐ (positions 1–36) and C‐terminal (positions 277–287) sequences. IκB degradation and NF‐κB activation at late time points, 15–20 h after UV irradiation, is mediated through DNA damage‐induced cleavage of IL‐1α precursor, release of IL‐1α and autocrine/paracrine action of IL‐1α. Late‐induced IκBα requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the ‘alarmone’ IL‐1α may account for some of the systemic effects of sunlight exposure.

Ubiquitin-mediated Processing of NF-κB Transcriptional Activator Precursor p105 RECONSTITUTION OF A CELL-FREE SYSTEM AND IDENTIFICATION OF THE UBIQUITIN-CARRIER PROTEIN, E2, AND A NOVEL UBIQUITIN-PROTEIN LIGASE, E3, INVOLVED IN CONJUGATION

In most cases, the transcriptional factor NF-κB is a heterodimer consisting of two subunits, p50 and p65, which are encoded by two distinct genes of the Rel family. p50 is translated as a precursor of 105 kDa. The C-terminal domain of the precursor is rapidly degraded, forming the mature p50 subunit consisted of the N-terminal region of the molecule. The mechanism of generation of p50 is not known. It has been suggested that the ubiquitin-proteasome system is involved in the process; however, the specific enzymes involved and the mechanism of limited proteolysis, in which half of the molecule is spared, have been obscure. Palombella and colleagues (Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T.(1994) Cell 78, 773-785) have shown that ubiquitin is required for the processing in a cell-free system of a truncated, artificially constructed, 60-kDa precursor. They have also shown that proteasome inhibitors block the processing both in vitro and in vivo. In this study, we demonstrate reconstitution of a cell-free processing system and demonstrate directly that: (a) the ubiquitin-proteasome system is involved in processing of the intact p105 precursor, (b) conjugation of ubiquitin to the precursor is an essential intermediate step in the processing, (c) the recently discovered novel species of the ubiquitin-carrier protein, E2-F1, that is involved in the conjugation and degradation of p53, is also required for the limited processing of the p105 precursor, and (d) a novel, 320-kDa species of ubiquitin-protein ligase, is involved in the process. This novel enzyme is distinct from E6-AP, the p53-conjugating ligase, and from E3α, the “N-end rule” ligase.

Structural Motifs Involved in Ubiquitin-Mediated Processing of the NF-κB Precursor p105: Roles of the Glycine-Rich Region and a Downstream Ubiquitination Domain

ABSTRACT The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-κB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.

Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae for isolation of effectors by phenotype modulation.

NF‐κB is a ubiquitous transcription factor that contributes to the induction of many genes playing a central role in immune and inflammatory responses. The NF‐κB proteins are subject to multiple regulatory influences including post‐translational modifications such as phosphorylation and proteolytic processing. A very important component of this regulation is the control of their subcellular localization: cytoplasmic retention of NF‐κB is achieved through interaction with IκB molecules. In response to extracellular signals, these molecules undergo degradation, NF‐κB translocates to the nucleus and activates its target genes.