Relish contains both a Rel homology domain (hence the name Relish) and an IkappaB-like domain with six ankyrin repeats. Thus Relish is a dual domain protein. In this respect Relish is similar to the compound mammalian NF-kappaB precursors p100 and p105, although no obvious similarity is seen outside the two conserved domains (Dushay, 1996).

The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function in mammals of complex multidomain proteins produced by accretion of domains. With this objective, the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains has been examined. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (1) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (2) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (3) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca2+/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. However, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system (Graef, 2001).

The positions of introns in genes coding for rel domain proteins are highly conserved, with introns positioned to either side of the sequence encoding the rel domain. The exceptions to this are informative: the sequences encoding the rel domain in Relish, Dif, Dorsal, and Rel B lack an intron 5' to the coding region. If the ancestral gene contained an intron demarcating the N-terminal coding region in these genes, this intron must have been lost before the formation of Rel B, Dorsal, Dif, and Relish, because the other vertebrate genes all have retained this intron. Alternatively, if the ancestral gene lacked an intron demarcating the N-terminal coding region of the rel domain, it must have been inserted after the Relb, dif, dorsal, and relish genes had originated from the ancestral gene. By either scenario, Rel B is the closest vertebrate relative of Dorsal, Dif, and Relish. Introns could not have been randomly lost or inserted, because a number of studies have shown that their positions are highly conserved within gene families. The sequence encoding the C terminus of the rel domain is also bounded by introns for each of the proteins except Relish. Indeed, the conserved proline codon at the C terminus of all rel domains occurs within five amino acid codons of the C-terminal intronic insertion (Graef, 2001).

The most distinctive structural feature of the rel domain is the division of the dimerization and specificity domains. Remarkably, in all vertebrate rel domain-containing genes, an intron precisely separates the sequences encoding the dimerization and the DNA specificity domains within the rel domain. Again, the exceptions are informative, in that no insect gene other than Drosophila TonEBP has this intron insertion site between the recognition and dimerization domains. One possible explanation is that the ancestral gene contained an intron at this position that was lost. However, several lines of evidence bode against intron loss, particularly because there is no evidence of processing and reinsertion of the insect rel domains. A more likely scenario is that the ancestral gene gained an intron separating the sequences encoding the dimerization and specificity domains, which then allowed the rel domain to successfully recombine and disseminate in vertebrates (Graef, 2001).

In the p100, p105, and Relish proteins, a cytoplasmic retention domain is a distinct region in each protein and is characterized by the presence of ankyrin repeats. This region is processed and eventually degraded to allow translocation to the nucleus. This cis-acting cytoplasmic retention function in Relish is encoded by a single exon, which in vertebrate p105 is divided into 13 different exons and a large but as yet undetermined number of exons in the p100 gene. Cytoplasmic retention can also be provided by the cactus or IkB proteins, which have sequence similarity to p105, p100, and Relish outside the rel domain (Graef, 2001).

A rel domain related to the one found in the NFATc proteins was recently reported in the mammalian TonEBP or NFAT5. This protein is encoded by a single mammalian gene and is transcriptionally regulated by osmotic stress. A gene related to mammalian TonEBP was found in Drosophila (Misexpression Suppressor of Ras 1). This gene also has a large exon 5' to the coding sequence for the rel domain, but the protein contains neither the ankyrin repeats of the p105/Relish proteins nor the translocation domain of the NFATc family. The Drosophila protein shares some features of the human TonEBP protein outside the rel domain, including the glutamine-rich regions. The mammalian gene has been partially sequenced and found to encode a rel domain with its sequence divided by introns at sites that correspond to those present in the NFATc genes. However, outside of the rel domain, the genomic structure of TonEBP is unrelated to NFATc family members. Most definitively, TonEBP lacks the translocation exon, indicating that it is not functionally related to the NFATc proteins (Graef, 2001).

Signaling upstream of NF-kappa B

The p105 Rel protein has dual functions: it is the precursor of the p5O subunit of NF-kappaB, and it acts as an IkappaB-like inhibitor to retain other Rel subunits in the cytoplasm. The posttranslational regulation of p105 following activation of Jurkat T cells has been studied and it has been found that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-kappaB, is correlated with an increase in proteolytic processing to p5O. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, because a 68-amino acid C-terminal deletion abolishes the response to stimulation. When compounds that block Ikappa B alpha phosphorylation and degradation are tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosyl-amido-7-amino-2-heptanone block inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol do not. Thus, while regulation of the p105 IkappaB resembles that of lkappaBa, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, regulation depends on a different, redox-insensitive, signaling pathway (MacKichan, 1996).

The NF-kappaB precursor p105 has dual functions: cytoplasmic retention of attached NF-kappaB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signaling processes that are known to activate NF-kappaB p50-p65. A model has been proposed that p105 is inducibly degraded, and that its degradation liberates sequestered NF-kappaB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl-3. TNFalpha, IL-1beta or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-alpha-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaBalpha, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-alpha stimulation, but only when the IKK phospho-acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super-repressor of IKK-induced NF-kappaB transcriptional activity. Thus, the known NF-kappaB stimuli not only cause nuclear accumulation of p50-p65 heterodimers but also of Bcl-3-p50 and perhaps further transcription activator complexes which are formed upon IKK-mediated p105 degradation (Heissmeyer, 1999).

The IkappaB kinase (IKK) complex is composed of three subunits, IKKalpha, IKKbeta, and IKKgamma (NEMO). While IKKalpha and IKKbeta are highly similar catalytic subunits, both capable of IkappaB phosphorylation in vitro, IKKgamma is a regulatory subunit. Previous biochemical and genetic analyses have indicated that despite their similar structures and in vitro kinase activities, IKKalpha and IKKbeta have distinct functions. Surprisingly, disruption of the Ikkalpha locus does not abolish activation of IKK by proinflammatory stimuli and results in only a small decrease in nuclear factor (NF)-kappaB activation. The pathophysiological consequence of disruption of the Ikkbeta locus is described. IKKbeta-deficient mice die at mid-gestation from uncontrolled liver apoptosis, a phenotype that is remarkably similar to that of mice deficient in both the RelA (p65) and NF-kappaB1 (p50/p105) subunits of NF-kappaB. Accordingly, IKKbeta-deficient cells are defective in activation of IKK and NF-kappaB in response to either tumor necrosis factor alpha or interleukin 1. Thus IKKbeta, but not IKKalpha, plays the major role in IKK activation and induction of NF-kappaB activity. In the absence of IKKbeta, IKKalpha is unresponsive to IKK activators (Li, 1999).

The transcription factor NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-kappaB/p50 and NF-kappaB2/p52 . The NF-kappaB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus. The p105 precursor also acts as an NFkappaB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus. Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus. TPL-2, which is homologous to MAP-kinase-kinase kinases in its catalytic domain, forms a complex with the carboxy terminus of p105. TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats and is more than 90% identical to the human oncoprotein COT. Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50 production. This releases associated Rel subunits or p50-Rel heterodimers to generate active nuclear NF-kappaB. Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumor-necrosis factor-alpha. TPL-2 is therefore a component of a new signaling pathway that controls proteolysis of NF-kappaB1 p105 (Belich, 1999).

The role of ceramide as a second messenger in tumor necrosis factor (TNF)-mediated signal transduction has been much debated. It is supported by recent reports describing an expanding number of potential targets for this lipid, but is opposed by those describing how ceramide is not necessary for many TNF-mediated cellular events. In this paper, the effects on NFkappaB function of the cell-permeable ceramide analog, N-acetylsphingosine (C2-ceramide), are directly compared with TNF, a transcription factor whose activation is central to many TNF-mediated effects. C2-ceramide fails to drive kappaB-linked chloramphenicol acetyltransferase gene expression in either HL60 promyelocytic or Jurkat T lymphoma cells. Furthermore, it has no effect on TNF-mediated transcription of this reporter gene. However, electrophoretic mobility shift analysis following cell stimulation with this ceramide analog reveals a dose-responsive activation of NFkappaB, which is not apparent following cell treatment with the inactive dihydro form. Activated complexes from treated cells contain predominantly the p50 subunit, in contrast to complexes from TNF-treated cells, where both p50 and p65/RelA subunits are present. The specific activation of p50 homodimeric complexes by C2-ceramide, which are known to lack trans-activating activity, is strongly suggested from these data. Further investigations have shown that C2-ceramide has only a marginal effect on IkappaBalpha degradation but strongly promotes the processing of p105 to its p50 product as revealed by immunoblot analysis. The increase in p50 arising from the processing of its p105 precursor has been further established from p105/p50 ratios obtained by scanning densitometric analysis of bands from immunoblots. However, TNF stimulates both IkappaBalpha degradation and p105 processing. Furthermore, the effect of TNF on NFkappaB activation is rapid, whereas C2-ceramide requires an optimal treatment time of 1 h. Interestingly, TNF increases ceramide in cells but only after a 1-h contact time. These data therefore suggest that ceramide promotes the activation of NFkappaB complexes that lack transactivating activity by enhanced processing of p105 (Boland, 1998).

NF-kappa B: Effects of Mutation

NF-kappa B, a heterodimeric transcription factor composed of p50 and p65 subunits, can be activated in many cell types and is thought to regulate a wide variety of genes involved in immune function and development. Mice lacking the p50 subunit of NF-kappa B show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production. Mice lacking p50 are unable effectively to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae, but are more resistant to infection with murine encephalomyocarditis virus. These data support the role of NF-kappa B as a vital transcription factor for both specific and nonspecific immune responses, but do not indicate a developmental role for the factor (Sha, 1995).

The p50 subunit of NF-kappaB is produced after proteolytic processing of the p105 precursor (NF-kappaB1). Although the p105 precursor has been postulated to play a role in the regulation of the Rel/NF-kappaB activity, its physiological relevance remains unclear. To investigate this, mutant mice were generated lacking the COOH terminal half of the p105 precursor, but expressing the p50 product (p105-/-). These mutant mice display an inflammatory phenotype composed of lymphocytic infiltration in lungs and liver, and an increased susceptibility to opportunistic infections. Enlargement of multiple lymph nodes, splenomegaly due to erythrocytic extramedullary hematopoiesis, and lymphoid hyperplasia were also observed in p105-/- mice. Cytokine production in p105-/- macrophages is severely impaired, whereas proliferative responses of p105-/- B cells are increased. T cell functions are only moderately impaired in mutant mice. Loss of p105 also leads to enhanced constitutive p50 homodimer and inducible NF-kappaB activities in unstimulated and stimulated cells, respectively. Since several genes regulated by Rel/NF-kappaB are upregulated in p105-/- thymus but downregulated in p105-/- macrophages, the enhanced p50 homodimers appear to function as transcriptional activators or repressors, depending on the cell type. Thus, the p105 precursor is indispensable in the control of p50 activity, and lack of the precursor has distinct effects on different cells (Ishikawa, 1998).

Transcriptional regulation of NF-kappaB

The candidate oncoprotein BCL-3 has been shown to function as a transcriptional co-activator for homodimers of NF-kappaB p50 and p50B. When BCL-3 is ectopically expressed in pro-B cell lines, these cells exhibit a dramatic increase in nuclear kappaB motif binding activity of p50 homodimers containing BCL-3 in the complex. Co-transfection and in vitro reconstitution experiments reveal that the complex of p50 with its precursor p105 (p50-p105), which has been shown to accumulate in the cytoplasm of the pro-B cell lines, is required for induction of DNA binding of p50 homodimers by BCL-3. However, no in vivo or in vitro evidence of a BCL-3-induced increase in proteolytic processing could be found. Instead, BCL-3-mediated reorganization of NFKB1 subunits was demonstrated in vitro. Immunofluorescence staining clearly demonstrates that the transition from cytoplasmic p50-p105 to nuclear p50 homodimers is induced by BCL-3 expression. Thus BCL-3 has versatile functions: cytoplasmic activation of p50 homodimers, their nuclear translocation and modulation of the transcriptional machinery in the nucleus (Watanabe, 1997).

During human cytomegalovirus (HCMV) infection, the promoters for the classical NF-kappaB subunits (p65 and p105/p50) are transactivated. The viral immediate-early (IE) proteins (IE1-72, IE2-55, and IE2-86) are involved in this upregulation. However, these viral factors alone, can not account for the entirety of the increased levels of transcription. Because one of the hallmarks of HCMV infection is the induction of cellular transcription factors, it is hypothesized that one or more of these induced factors is also critical to the regulation of NF-kappaB during infection. Sp1 was one such factor that might be involved because p65 promoter activity is upregulated by Sp1 and both of the NF-kappaB subunit promoters are GC rich and contain Sp1 binding sites. Therefore, to detail the role that Sp1 plays in the regulation of NF-kappaB during infection, Sp1 levels were examined for changes during infection. HCMV infection results in increased Sp1 mRNA expression, protein levels, and DNA binding activity. Because both promoters are transactivated by Sp1, it was reasoned that the upregulation of Sp1 plays a role in p65 and p105/p50 promoter activity during infection. To address the specific role of Sp1 in p65 and p105/p50 promoter transactivation by HCMV, both promoters were mutated. These results demonstrate that the Sp1-specific DNA binding sites are involved in the virus-mediated transactivation. To further dissect the role of HCMV in the Sp1-mediated induction of NF-kappaB, the role that the viral IE genes play in Sp1 regulation was examined. The IE gene products (IE1-72, IE2-55, and IE2-86) cooperate with Sp1 to increase promoter transactivation and physically interacted with Sp1. In addition, the IE2-86 product increases Sp1 DNA binding by possibly freeing up inactive Sp1. These data support the hypothesis that Sp1 is involved in the upregulation of NF-kappaB during HCMV infection through the Sp1 binding sites in the p65 and p105/p50 promoters and additionally demonstrate a potential viral mechanism that might be responsible for the upregulation of Sp1 activity (Yurochko, 1997).

The gene encoding NFKB1 is autoregulated, responding to NF-kappa B/Rel activation through NF-kappa B binding sites in its promoter, which also contains putative sites for Ets proteins. One of the Ets sites, which is referred to as EBS4, is located next to an NF-kappa B/Rel binding site, kB3, which is absolutely required for activity of the promoter in Jurkat T cells in response to activation by phorbol 12-myristate 13-acetate (PMA), PMA/ionomycin, or the Tax protein from human T cell leukemia virus type I. EBS4 is required for the full response of the nfkb1 promoter to PMA or PMA/ionomycin in Jurkat cells. EBS4 is bound by Ets-1, Elf-1, and other species. Overexpression of Ets-1 augments the response to PMA/ionomycin and this is reduced by mutation of EBS4. Elf-1 has less effect in conjunction with PMA/ionomycin, but by itself activates the promoter 12-fold. This activation is only partly affected by mutation of EBS4. A mutant promoter that binds Ets-1, but not Elf-1, at the EBS4 site responds to PMA/ionomycin as efficiently as the wild-type. Ets proteins may be responsible for fine-tuning the activity of the nfkb1 gene in a cell-type-specific manner (Lambert, 1997).

Translation of NF-kappa B and formation of p50/p52

The NFkappaB1 gene encodes two functionally distinct proteins termed p50 and p105. p50 corresponds to the N terminus of p105 and with p65 (RelA) forms the prototypical NF-kappaB transcription factor complex. In contrast, p105 functions as a Rel-specific inhibitor (IKB) and has been proposed to be the precursor of p50. p50 is generated by a unique cotranslational processing event involving the 26S proteasome, whereas cotranslational folding of sequences near the C terminus of p50 abrogates proteasome processing and leads to p105 production. These results indicate that p105 is not the precursor of p50 and reveal a novel mechanism of gene regulation that ensures the balanced production and independent function of the p50 and p105 proteins (Lin, 1998).

Processing of the p105 precursor to form the active subunit p50 of the NF-kappaB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Following IkappaB kinase (IkappaK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918-934) serves as a recognition motif for the SCF(beta)(-TrCP) ubiquitin ligase. Expression of IkappaKbeta dramatically increases processing of wild-type p105, but not of p105-Delta918-934. Dominant-negative beta-TrCP inhibits IkappaK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Delta918-934 associate physically, following phosphorylation. In vitro, SCF(beta)(-TrCP) specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IkappaBs, beta-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Delta918-934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs (Orian, 2000).

Generation of the NF-kappaB p50 transcription factor is mediated by the proteasome. p50 is generated during translation of the NFKB1 gene and this cotranslational processing allows the production of both p50 and p105 from a single mRNA. The Rel homology domain in p50 undergoes cotranslational dimerization and this interaction is required for efficient production of p50. This coupling of dimerization and proteasome processing during translation uniquely generates p50-p105 heterodimers. Accordingly, after the primary cotranslational event, additional posttranslational steps regulate p50 homodimer formation and the intracellular ratio of p50 and p105. This cellular strategy places p50 under the control of the p105 inhibitor early in its biogenesis, thereby regulating the pool of p50 homodimers within the cell (Lin, 2000).

nfkb2 encodes two members of the NF-kappa B/Rel family of proteins: p52 and p100. The p100 polypeptide has been proposed to serve as a precursor of p52, which corresponds to the N-terminal half of p100. While p52 functions as a Rel transcription factor, the larger p100 protein acts as a cytoplasmic inhibitor of select NF-kappa B/Rel transcription factor complexes. Because of their distinct functions, the biochemical basis for the production of these two nfkb2-derived gene products has been studied. Like the p50 product of the nfkb1 gene, p52 is principally generated in a cotranslational manner involving proteolytic processing by the proteasome. The generation of p52 is dependent on a glycine-rich region (GRR) located upstream of the p52 C-terminus, and repositioning of this GRR alters the location of proteasome processing. In most cells, small amounts of p52 are produced relative to the levels of p100, unlike the usually balanced production of nfkb1-derived p50 and p105. Using p100/p105 chimeras containing different segments of the nfkb1 and nfkb2 genes, it has been found that diminished p52 processing is a property conferred by peptide sequences located downstream of the GRR, flanking the site of p52 processing (Heusch, 1999).

Nuclear factor kappaB1 (NF-kappaB) is a heterodimeric complex that regulates transcription of many genes involved in immune and inflammatory responses. Its 50-kDa subunit (p50) is generated by the ubiquitin-proteasome pathway from a 105-kDa precursor (p105). This proteolytic process has been reconstituted in HeLa cell extracts and the responsible enzymes have been purified. Ubiquitination of p105 requires E1, and either of two types of E2s: E2-25K (for which p105 is the first proven substrate) or a member of the UBCH5 (UBC4) family. It also requires a new E3 of 50 kDa, which has been termed E3kappaB. This set of enzymes differs from the E2s and E3 reported by others to catalyze p105 ubiquitination in reticulocytes. The ubiquitinating enzymes purified in this study, together with 26S proteasomes, allow formation of p50. Thus, the 26S proteasome provides all the proteolytic activities necessary for p105 processing. Interestingly, in the reconstituted system, as observed in cells, the C-terminally truncated form of p105, p97, is processed into p50 more efficiently than normal p105, even when both species are ubiquitinated to a similar extent. Therefore, some additional mechanism involving the C-terminal region of p105 influences the proteolytic processing of the ubiquitinated precursor (Coux, 1998).

Domain structure of NF-kappaB and processing

Transcription factor NF-kappaB is generally considered to be a heterodimer with two subunits, p50 and p65. The p50 subunit has been suggested to be generated from its precursor, p105, via the ubiquitin-proteasome pathway. During processing, the C-terminal portion of p105 is rapidly degraded whereas the N-terminal portion (p50) is left intact. A 23-amino-acid, glycine-rich region (GRR) in p105 functions as a processing signal for the generation of p50. A GRR-dependent endoproteolytic cleavage downstream of the GRR releases p50 from p105, and this cleavage does not require any specific downstream sequences. p50 can be generated from chimeric precursor p105N-GRR-IkappaBalpha, while the C-terminal portion (IkappaBalpha) can also be recovered, suggesting that p105 processing includes two steps: a GRR-dependent endoproteolytic cleavage and the subsequent degradation of the C-terminal portion. The GRR can direct a similar processing event when it is inserted into a protein unrelated to the NF-kappaB family and it is therefore an independent signal for processing (Lin, 1996).

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-kappaB, 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. 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, 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 (Orian, 1999).

Negative regulation of toll-like receptor signaling by NF-kappaB p50 ubiquitination blockade

Toll-like receptors (TLRs) trigger the production of inflammatory cytokines and shape adaptive and innate immunity to pathogens. This study reports the identification of B cell leukemia (Bcl)-3 as an essential negative regulator of TLR signaling. Bcl-3 is a nuclear member of the inhibitor of NF-kappaB (IkappaB) family, that interacts exclusively with the transcriptionally inactive homodimers of p50 and p52, two members of the NF-kappaB family. Bcl3 deficiency in mice disrupts the microarchitecture of lymphoid organs but does not affect the development of lymphoid or myeloid cells By blocking ubiquitination of p50, a member of the NFkappa-B family, Bcl-3 stabilizes a p50 complex that inhibits gene transcription. As a consequence, Bcl-3-deficient mice and cells are hypersensitive to TLR activation and unable to control responses to lipopolysaccharides. Thus, p50 ubiquitination blockade by Bcl-3 limits the strength of TLR responses and maintains innate immune homeostasis. These findings indicate that the p50 ubiquitination pathway can be selectively targeted to control deleterious inflammatory diseases (Carmody, 2007).

These results establish that Bcl-3 promotes p50 homodimer occupancy of target gene promoters by inhibiting the ubiquitination and subsequent degradation of DNA-bound p50 homodimers. It is proposed that this state of Bcl-3-p50 homodimer-mediated promoter hyporesponsiveness is the molecular basis of TLR tolerance. Neither Bcl-3 nor p50 alone is sufficient to maintain the tolerant state of gene promoters. In the absence of Bcl-3-p50 complex, the loading of NF-kappaB subunits on target promoters and the subsequent dimer exchange, critical for appropriate gene expression, are disrupted, leading to aberrant expression of inflammatory cytokines. Thus, TLR tolerance and suppression are dependent on the coordinated action of both the inhibitor p50 and its stabilizer, Bcl-3. These findings provide important insights into the molecular mechanisms of TLR signaling and suggest that deleterious inflammatory responses can be effectively controlled by targeting the NF-kappaB p50 ubiquitination pathway (Carmody, 2007).

Subcellular distribution of NF-kappa B

Transcription factors of the NF-kappa B/Rel family are retained in the cytoplasm as inactive complexes through association with I kappa B inhibitory proteins. Several NF-kappa B activators induce the proteolysis of I kappa B proteins, which results in the nuclear translocation and DNA binding of NF-kappa B complexes. A novel mechanism is reported of NF-kappa B regulation mediated by p105 (NF-kappa B1) precursor of p50 directly at the nuclear level. In Epstein-Barr virus-immortalized B cells, p105 is found in the nucleus, where it is complexed with p65. In concomitance with NF-kappa B activation, mitomycin C induces the processing of p105 to p50 in the nucleus, while it does not affect the steady-state protein levels of I kappa B alpha and p105 in the cytoplasm. In contrast, phorbol 12-myristate 13-acetate induces a significant proteolysis of both I kappa B alpha and p105 in the cytoplasm, while it does not affect the protein level of p105 in the nucleus. These results suggest that in Epstein-Barr virus-positive B cell lines, the nuclear processing of p105 can contribute to NF-kappa B activation in response to specific signaling molecules, such as DNA-damaging agents (Baldassarre, 1995).

Defective processing of NF-kappa B

The multisubunit proteasome complex is the principal mediator of nonlysosomal protein degradation. The proteasome subunit varies minimally between cells with the exception of LMP2, LMP7, and LMP10 subunits in rodent and human cells. LMP2 and LMP7 subunits are encoded by the human lymphocyte antigen region, and they optimize proteolytic mediated antigen presentation. The proteasome is also important for the function of transcription factor NF-kappaB. It is required for NF-kappaB subunits p50 and p52 generation and catalyzes degradation of phosphorylated IkappaBalpha. These proteasome-mediated reactions have now been shown to be defective in T2 cells, a human lymphocyte cell line that lacks both LMP2 and LMP7. Although T2 cells contain normal expression of p100 and p105, the abundance of p50 and p52 is greatly reduced. TNF-alpha induces normal phosphorylation of IkappaBalpha but fails to induce degradation of phosphorylated IkappaBalpha. Both DNA binding assays and luciferase assays reveal that TNF-alpha-induced NF-kappaB activation is defective in T2 cells. Unlike parental cells, T2 cells are susceptible to TNF-alpha-induced apoptosis. These data indicate that human leukocyte antigen-linked proteasome subunits are essential for NF-kappaB activation and protection of cells from TNF-alpha-induced apoptosis (Hyashi, 2000).

The p50 subunit of NF-kappa B is generated by proteolytic processing of a 105-kDa precursor (p105) in yeast and mammalian cells. Yeast mutants in the ubiquitin-proteasome pathway inhibit or abolish p105 processing. Specifically, p105 processing is inhibited by a mutation in a 20 S proteasome subunit (pre1-1), by mutations in the ATPases located in the 19 S regulatory complexes of the proteasome (yta1, yta2/sug1, yta5, cim5), and by a mutation in a proteasome-associated isopeptidase (doa4). A ubiquitinated intermediate of the p105 processing reaction accumulates in some of these mutants, strongly suggesting that ubiquitination is required for processing. However, none of the ubiquitin conjugating enzyme mutants tested (ubc1, -2, -3, -4/5, -6/7, -8, -9, -10, -11) had an effect on p105 processing, suggesting that more than one of these enzymes is sufficient for p105 processing. Interestingly, a mutant 'N-end rule' ligase does not adversely affect p105 processing, showing that the N-end rule pathway is not involved in degrading the C-terminal region of p105. Unexpectedly, it was found that a glycine-rich region of p105, which is required for p105 processing in mammalian cells, is not required for processing in yeast. Thus, p105 processing in both yeast and mammalian cells requires the ubiquitin-proteasome pathway, but the mechanisms of processing, while similar, are not identical (Sears, 1998).

The nonobese diabetic (NOD) mouse is an animal model of human type I diabetes with a strong genetic component that maps to the major histocompatibility complex (MHC) of the genome. A specific proteasome defect has been identified in NOD lymphocytes that results from the lack of the LMP2 subunit. The pronounced proteasome defect results in defective production and activation of the transcription factor NF-kappaB, which plays an important role in immune and inflammatory responses as well as in preventing apoptosis induced by tumor necrosis factor alpha. The defect in proteasome function in NOD mouse splenocytes is evident from impaired NF-kappaB subunit p50 and p52 generation by proteolytic processing and impaired degradation of the NF-kappaB-inhibitory protein IkappaBalpha. An obligatory role of MHC-linked proteasome subunits in transcription factor processing and activation has been established in a spontaneous-disease model and mutant cells, similarly lacking the MHC-encoded subunit. These data suggest that NOD proteasome dysfunction is due to a tissue- and developmental-stage-specific defect in expression of the MHC-linked Lmp2 gene, resulting in altered transcription factor NF-kappaB activity, and that this defect contributes to pathogenesis in NOD mice. These observations are consistent with the diverse symptomatology of type I diabetes and demonstrate clear sex-, tissue-, and age-specific differences in the expression of this error that parallel the initiation and disease course of insulin-dependent (type I) diabetes mellitus (Hayashi, 1999).

Protein interactions of NF-kappa B

NF-kappaB family of transcription factors plays a pivotal role in regulation of immune and inflammatory responses. NF-kappaB is known to function by binding to the B enhancer and directly activating target gene transcription. Another function of NF-kappaB has been demonstrated, in which the NFkappab1 gene product p105 regulates MAP kinase signaling triggered by the bacterial component lipopolysaccharide. p105 exerts this signaling function by controlling the stability and function of an upstream kinase, Tpl2. In macrophages, Tpl2 forms a stable and inactive complex with p105, and activation of Tpl2 involves its dissociation from p105 and subsequent degradation. Thus, p105 functions as a physiological partner and inhibitor of Tpl2, which provides an example of how a transcription factor component regulates upstream signaling events (Waterfield, 2003).

Transcriptional targets of NF-kappa B

The transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing several consensus binding sites for the ubiquitous transcription factor NF-kappa B. To verify whether MCMV, like the human CMV, can activate NF-kappa B-dependent transcription, murine embryo fibroblasts cells were transfected with a construct containing three copies of the NF-kappa B element in front of the homologous minimal MCMV IE1-3 promoter. Upon MCMV infection the reporter gene activity is transactivated to about three-fold above the basal level. The specificity of this transactivation was demonstrated by the lack of any significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with a NF-kappa B probe reveal that MCMV infection activates DNA binding proteins showing NF-kappa B characteristics. The DNA-binding activity remains elevated during the course of infection and is associated with an increase in the steady-state mRNA levels for the NF-kappa B subunit p105/p50. Since the promoter of the p105/p50 gene is transactivated by MCMV infection during the period in which the IE proteins are expressed, the role of the two major IE transcriptional regulatory proteins was examined. In cotransfection experiments, the IE1 protein transactivates the p105/p50 promoter, whereas the IE3 is ineffective in increasing the transcription of the reporter gene. Taken as a whole, these results demonstrate that MCMV, like its human counterpart, regulates the cellular NF-kappa B activity needed for the initial induction of the IE genes and the progression of the viral replicative cycle (Gribaudo, 1996).

NF-kappa B and development

The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses have revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrate that DNA-binding complexes containing p50 and RelA are abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice is highest during pregnancy, decreases to near undetectable levels during lactation, and is elevated during involution. This highly regulated pattern of activity is consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha (Brantley, 2000).

Relish : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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