Interactive Fly, Drosophila

NF-kappaB protein interactions

The inhibitory protein, IkappaBalpha (Drosophila homolog: Cactus) , sequesters the transcription factor, NF-kappaB, as an inactive complex in the cytoplasm. The structure of the IkappaBalpha ankyrin repeat domain, bound to a partially truncated NF-kappaB heterodimer (p50/ p65), has been determined by X-ray crystallography at 2.7 A resolution. It shows a stack of six IkappaBalpha ankyrin repeats facing the C-terminal domains of the NF-kappaB Rel homology regions. Contacts occur in discontinuous patches, suggesting a combinatorial quality for ankyrin repeat specificity. The first two repeats cover an alpha helically ordered segment containing the p65 nuclear localization signal. The position of the sixth ankyrin repeat shows that full-length IkappaBalpha will occlude the NF-kappaB DNA-binding cleft. The orientation of IkappaBalpha in the complex places its N- and C-terminal regions in appropriate locations for their known regulatory functions (Jacobs, 1998).

IkappaBalpha regulates the transcription factor NF-kappaB through the formation of stable IkappaBalpha/NF-kappaB complexes. Prior to induction, IkappaBalpha retains NF-kappaB in the cytoplasm until the NF-kappaB activation signal is received. After activation, NF-kappaB is removed from gene promoters through association with nuclear IkappaBalpha, restoring the preinduction state. The 2.3 A crystal structure of IkappaBalpha in complex with the NF-kappaB p50/p65 heterodimer reveals mechanisms of these inhibitory activities. The presence of IkappaBalpha allows large en bloc movement of the NF-kappaB p65 subunit amino-terminal domain. This conformational change induces allosteric inhibition of NF-kappaB DNA binding. Amino acid residues immediately preceding the nuclear localization signals of both NF-kappaB p50 and p65 subunits are tethered to the IkappaBalpha amino-terminal ankyrin repeats, impeding NF-kappaB from nuclear import machinery recognition (Huxford, 1998).

Several lines of evidence have led to the suggestion that newly synthesized IkappaB can function in the nucleus as a postinduction repressor of B-dependent gene expression.

Taken together, these results are consistent with a model in which newly synthesized IkappaB proteins can enter the nucleus, displace dimeric Rel proteins from DNA, and export Rel proteins from the nucleus to the cytoplasm. Implicit in this model is the ability of both Rel and IkappaB proteins to enter the nucleus. In contrast, this model postulates that the Rel-IkappaB complex is exported from the nucleus and is efficiently retained in the cytoplasm (Sachdev, 1998a).

The ability of the IkappaB alpha protein to sequester dimeric NF-kappaB/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-kappaB/Rel family members. IkappaB alpha can also act in the nucleus as a postinduction repressor of NF-kappaB/Rel proteins. The mechanism by which IkappaB alpha enters the nucleus is not known, as IkappaB alpha lacks a discernible classical nuclear localization sequence (NLS). Nuclear localization of IkappaB alpha is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of IkappaB alpha. A region encompassing the second ankyrin repeat of IkappaB alpha is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in IkappaB alpha suggests that cytoplasmic sequestration of the NF-kappaB/Rel-IkappaB alpha complex is a consequence of the mutual masking of the NLS within NF-kappaB/Rel proteins and the import sequence within IkappaB alpha. Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), because the ARDs from two other ARD-containing proteins, 53BP2 and GABPbeta, are also able to function as nuclear import sequences. It is proposed that the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences (Sachdev, 1998b).

The IkappaB alpha protein is able both to inhibit nuclear import of Rel/NF-kappaB proteins and to mediate the export of Rel/NF-kappaB proteins from the nucleus. The c-Rel-IkappaB alpha complex is stably retained in the cytoplasm in the presence of leptomycin B, a specific inhibitor of Crm1-mediated nuclear export. In contrast, leptomycin B treatment results in the rapid and complete relocalization of the v-Rel-IkappaB alpha complex from the cytoplasm to the nucleus. IkappaB alpha also mediates the rapid nuclear shuttling of v-Rel in an interspecies heterokaryon assay. Thus, continuous nuclear export is required for cytoplasmic retention of the v-Rel-IkappaB alpha complex. Furthermore, although IkappaB alpha is able to mask the c-Rel-derived nuclear localization sequence (NLS), IkappaB alpha is unable to mask the v-Rel-derived NLS in the context of the v-Rel-IkappaB alpha complex. Taken together, these results demonstrate that IkappaB alpha is unable to inhibit nuclear import of v-Rel. Two amino acid differences between c-Rel and v-Rel (Y286S and L302P) that link to oncogenesis have been identified: (1) the failure of IkappaB alpha to inhibit nuclear import and (2) DNA binding of a mutant c-Rel protein. These results support a model in which loss of IkappaB alpha-mediated control over c-Rel leads to oncogenic activation of c-Rel (Sachdev, 1998).

In vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their varying abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. The inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes (Tran, 1997).

The biological activity of the transcription factor NF-kappaB is differentially controlled by three IkappaB proteins: IkappaBalpha, IkappaBbeta, and IkappaBepsilon. The molecular basis for the differential inhibitory strengths of IkappaB proteins has been examined by constructing hybrid IkappaBs. The first ankyrin repeat of IkappaBalpha is responsible for its strong inhibitory effect. Swapping a putative beta-turn within the first ankyrin repeat between the strong IkappaBalpha and the weak IkappaBbeta inhibitors switches their in vivo inhibitory activity on NF-kappaB. The qualitatively distinct contacts made by this beta-turn in IkappaBalpha and IkappaBbeta with NF-kappaB determine the efficiency by which IkappaBs sequester NF-kappaB to the cytoplasm, thus explaining their distinct effects on gene activity (Simeonidis, 1999).

The RelA subunit of NF-kappaB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA associates with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behave as transdominant inhibitors of the repressive activity of wild type RelA. The 13 S E1A protein is able to interfere with the transrepressive activity of RelA. It is proposed that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor (Wissink, 1997).

Members of the Rel/NF-kappaB family of transcription factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding, dimerization, and binding to inhibitor. At the C-terminal end of the RHD, each protein has a nuclear localization signal (NLS). The crystal structures of the p50 and RelA family members show that the RHD consists of two regions: an N-terminal section that contains some of the DNA contacts and a C-terminal section that contains the remaining DNA contacts and which controls dimerization. In unstimulated cells, the homo- or heterodimeric Rel/NF-kappaB proteins are cytoplasmic by virtue of binding to an inhibitor protein (IkappaB), which somehow masks the NLS of each member of the dimer. The IkappaB proteins consist of an ankyrin-repeat-containing domain that is required for binding to dimers and N- and C-terminal domains that are dispensable for binding to most dimers. The interaction between IkappaB alpha and Rel family homodimers has been examined by mutational analysis. The dimerization regions of p50, RelA, and c-Rel are sufficient for binding to IkappaB alpha. It is shown that the NLSs of RelA and c-Rel are not required for binding to IkappaB alpha but do stabilize the interaction, while the NLS of p50 is required for binding to IkappaB alpha. Only certain residues within the p50 NLS are required for binding. In a p50-IkappaB alpha complex or a c-Rel-IkappaB alpha complex, the N terminus of IkappaB alpha either directly or indirectly masks one or both of the dimer NLSs (Latimer, 1998).

Regulation of the IkappaB alpha and IkappaB beta proteins is critical for modulating NF-kappaB-directed gene expression. Both IkappaB alpha and IkappaB beta are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, a biochemical fractionation scheme was utilized to purify a kinase activity that phosphorylates residues in the amino and carboxy termini of both IkappaB alpha and IkappaB beta. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy reveals that this kinase is the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of IkappaB alpha and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IkappaB alpha, murine severe combined immunodeficiency (SCID) cell lines that lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrates constitutive nuclear NF-kappaB DNA binding activity, which is not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IkappaB alpha that is phosphorylated by DNA-PK is a more potent inhibitor of NF-kappaB binding than nonphosphorylated IkappaB alpha. These results suggest that DNA-PK phosphorylation of IkappaB alpha increases its interaction with NF-kappaB to reduce NF-kappaB DNA binding properties (Liu, 1998).

The Groucho family includes three types of proteins. The larger proteins such as Groucho and its mammalian homologs [transducin-like enhancer of split (TLE) 1 through 3] share five domain structures. These proteins exhibit a common feature including an amino-terminal glutamine-rich region (Q domain), a glycine/proline-rich region (GP domain), a CcN domain containing a casein kinase II site and nuclear localization sequence, a serine/proline-rich region (SP domain), and COOH-terminal WD40 repeats. Three of these domains, the Q, CcN, WD40 domains, are most highly conserved. A shorter protein, the human TLE4, contains all the domains except for the amino-terminal Q domain. Shortest proteins in the Groucho family, which contain only the Q domain and the GP domain, are designated as amino-terminal enhancer of split (AES) or the Groucho-related gene (Grg). Significant homology is observed in the Q domain between AES and other Groucho proteins, except for TLE4. AES encodes a 197-amino acid protein that is homologous to the NH(2)-terminal domain of the Drosophila Groucho protein but lacks COOH-terminal WD40 repeats. Although the Drosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast two-hybrid system, a protein-protein interaction has been identified between AES and the p65 (RelA) subunit of the transcription factor nuclear factor kappaB (NF-kappaB), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed by in vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES represses p65-driven gene expression. AES also inhibits NF-kappaB-dependent gene expression induced by tumor necrosis factor alpha, interleukin-1beta, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-kappaB activation. These data indicate that AES acts as a corepressor for NF-kappaB and suggest that AES may play a pivotal role in the regulation of NF-kappaB target genes (Tetsuka, 2000).

These findings suggest that the transcriptional activity of NF-kappaB may be regulated by a balance between the counteracting effects of Groucho corepressors and coactivators, because it was previously found that NF-kappaB binds to coactivator proteins p300/CREB-binding protein. It is also speculated, although not yet proven, that the interactions of NF-kappaB with coactivators or corepressors may be modulated by signal-induced modification of these proteins. These findings suggest a putative role for vertebrate NF-kappaB in transcriptional repression. Inhibition of NF-kappaB activity by localized expression of transdominant-negative IkappaBalpha in chick embryos results in the arrest of limb growth. In this phenotype, inhibition of NF-kappaB leads to inhibition of twist expression but derepression of a vertebrate decapentaplegic homolog, the bone morphogenic protein-4. Taken together, these data indicate that the vertebrate NF-kappaB may also act as a transcriptional repressor by recruiting Groucho family corepressors, such as AES and TLEs (Tetsuka, 2000 and references therein).

In the present study, it was noted that AES and TLE1 inhibit NF-kappaB-mediated gene expression but do not actively repress NF-kappaB-driven gene expression to less than basal level (the level of gene expression without NF-kappaB and AES/TLE1). Lack of active repression may be because NF-kappaB-dependent reporter constructs, which do not contain additional repression elements, were used. In Drosophila, recruitment of Groucho is obligatory but is not sufficient for Dorsal-mediated repression. Dorsal-mediated repression requires additional repression elements in the proximity of the Dorsal binding sites and the binding of other DNA-binding repressor proteins to these elements. Dorsal-mediated repression has been shown to require the formation of a multiprotein DNA-bound complex that includes Groucho, Dorsal, and additional DNA-binding proteins, such as Cut and Dead ringer. NTF-1/Grainyhead is known to bind to the repression elements in decapentaplegic promoter, and it may play a role in decapentaplegic repression that is analogous to the role of cut and/or dead ringer in zerknüllt repression. Thus, if NF-kappaB acts as an active repressor in vertebrates, it is likely that there are additional repressor binding sites and their respective DNA-binding proteins (Tetsuka, 2000 and references therein).

To stimulate transcriptional elongation of HIV-1 genes, the transactivator Tat recruits the positive transcription elongation factor b (P-TEFb) to the initiating RNA polymerase II (RNAPII). The activation of transcription by RelA also depends on P-TEFb. Similar to Tat, RelA activates transcription when tethered to RNA. Moreover, TNF-alpha triggers the recruitment of P-TEFb to the NF-kappaB-regulated IL-8 gene. While the formation of the transcription preinitiation complex (PIC) remains unaffected, DRB, an inhibitor of P-TEFb, prevents RNAPII from elongating on the IL-8 gene. Remarkably, DRB inhibition sensitizes cells to TNF-alpha-induced apoptosis. Thus, NF-kappaB requires P-TEFb to stimulate the elongation of transcription and P-TEFb plays an unexpected role in regulating apoptosis (Barboric, 2001).

Findings of the present study add a new dimension to how RelA finalizes its transcriptional tasks. Thus, RelA not only promotes the initiation of transcription, but, through its association with P-TEFb, it also plays an essential role in the elongation of transcription. Importantly, the transactivation domain of RelA activates transcription when tethered artificially to the RNA element in a P-TEFb-dependent manner. Thus, RelA joins the small group of activators that stimulate the elongation of plasmid transcription. They include Tat, VP16, Cyclin T1, Cdk9, and CIITA. Although not determined yet for VP16, all of these proteins associate with or are part of P-TEFb. In contrast, transcriptional activators such as Sp-1 that stimulate only the initiation of transcription are inactive in this assay. This latter group interacts with several factors that are required for PIC assembly as well as with the basal transcription factor TFIIH, whose CTD kinase activity is required for promoter clearance. Notably, it was found that chimeric proteins between Rev and the three subunits of TFIIH, Cdk7, Cyclin H, and Mat1, do not activate plasmid transcription, consistent with their having roles in promoter clearance and not elongation of transcription. Additionally, of the two kinases, only P-TEFb has the ability to confer elongating properties on early transcriptional complexes. Thus, the RNA-tethering system is useful for assessing the ability of transcription factors to stimulate P-TEFb-dependent transcriptional elongation (Barboric, 2001).

The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacts with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progress (Perkins, 1996).

Homodimers of the NF-kappaB p50 subunit are transcriptionally repressive in cells, whereas they can promote transcription in vitro, suggesting that their endogenous effects are mediated by association with other factors. Transcriptionally inactive nuclear NF-kappaB in resting cells consists of homodimers of either p65 or p50 complexed with the histone deacetylase HDAC-1. Only the p50-HDAC-1 complexes bind to DNA and suppress NF-kappaB-dependent gene expression in unstimulated cells. Appropriate stimulation causes nuclear localization of NF-kappaB complexes containing phosphorylated p65 that associates with CBP and displaces the p50-HDAC-1 complexes. Phosphorylation of p65 determines whether it associates with either CBP or HDAC-1, ensuring that only p65 entering the nucleus from cytoplasmic NF-kappaB:IkappaB complexes can activate transcription (Zhong, 2002).

The effects of HDAC-1 and CBP/p300 underscore the importance of acetylation in regulating NF-kappaB activity, although the identity of CBP/p300 targets remains to be fully determined. CBP/p300 can acetylate the four core histones, loosening chromatin and facilitating transcription. Histones associated with NF-kappaB-dependent genes are acetylated following stimulation. Alternative targets include p53, where acetylation is important for CBP-mediated p53-dependent transcription, although unlike p53 and despite many attempts, p65 acetylation by CBP/p300 has not been detected. Thus, it appears that the major effect of CBP/p300 on NF-kappaB-dependent transcription is via acetylation of histones or other proteins in the chromatin remodeling and transcriptional apparatus (Zhong, 2002).

In summary, it has been shown that p65 phosphorylation determines whether nuclear NF-kappaB associates with HDAC-1 (inactive) or CBP/p300 (active) and that p50-HDAC-1 represses NF-kappaB-dependent gene expression in resting cells. Such a regulatory mechanism ensures that only stimulus-induced NF-kappaB activates transcription, and NF-kappaB in the nucleus for any other reason is transcriptionally silent. This mechanism is unique among the inducible transcription factors, since it imposes an additional layer of control on NF-kappaB that most likely reflects the necessity of maintaining it as a true inducible transcription factor (Zhong, 2002).

The nuclear function of the heterodimeric NF-kappaB transcription factor is regulated in part through reversible acetylation of its RelA subunit. The p300 and CBP acetyltransferases play a major role in the in vivo acetylation of RelA, principally targeting lysines 218, 221 and 310 for modification. Analysis of the functional properties of hypoacetylated RelA mutants containing lysine-to-arginine substitutions at these sites and of wild-type RelA co-expressed in the presence of a dominantly interfering mutant of p300 reveals that acetylation at lysine 221 in RelA enhances DNA binding and impairs assembly with IkappaBa. Conversely, acetylation of lysine 310 is required for full transcriptional activity of RelA in the absence of effects on DNA binding and IkappaBa assembly. Together, these findings highlight how site-specific acetylation of RelA differentially regulates distinct biological activities of the NF-kappaB transcription factor complex (Chen, 2002).

In summary, these studies demonstrate that acetylation of RelA at distinct sites differentially regulates various biological functions of NF-kappaB. Acetylation of lysine 310 of RelA is required for full transactivation by the NF-kappaB complex, most likely by recruiting an unidentified cofactor. Acetylation of lysine 221 enhances RelA binding to the kappaB enhancer,while acetylation of lysine 221 alone or in combination with lysine 218 impairs the assembly of RelA with IkappaBa. Lysines 218 and 221 are highly conserved within all Rel family members, including Dorsal from Drosophila. The possibility that these evolutionarily conserved lysine residues are targets for reversible acetylation and contribute to the regulation of the biological functions of other Rel factors remains an intriguing possibility (Chen, 2002).

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. An inhibitor of NF-kappaB, IBNS, has been cloned, that is rapidly expressed upon TCR-triggered but not dexamethasone- or irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to IkappaB family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of IBNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. IBNS blocks transcription from NF-kappaB reporters, alters NF-kappaB electrophoretic mobility shifts, and interacts with NF-kappaB proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of IBNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection (Fiorini, 2002).

One of the predictions of the histone code hypothesis is the existence of functional interactions between chromatin remodeling complexes, such as SWI/SNF, and histone acetylase complexes, such as GCN5. Recent studies have elucidated the temporal sequence in which these coactivators of transcription are recruited to promoters in vivo and how their enzymatic properties contribute to gene activation. The best-characterized example in mammals is provided by the human IFN-ß gene. The gene is switched on by three transcription factors (NF-kappaB, IRFs, and ATF-2/c-Jun), and an architectural protein [HMG I(Y)], all of which bind cooperatively to the nucleosome-free enhancer DNA to form an enhanceosome. The enhanceosome targets the modification and repositioning of a nucleosome that blocks the formation of a transcriptional preinitiation complex on the IFN-ß promoter. This is accomplished by the ordered recruitment of HATs, SWI/SNF, and basal transcription factors. Initially, the GCN5 HAT-containing complex is recruited, and it acetylates the nucleosome. This is followed by the recruitment of the CBP-PolII holoenzyme complex. Next, the SWI/SNF remodeling machine arrives at the promoter via bivalent interactions with CBP and the acetylated histone N tails. SWI/SNF alters the structure of the nucleosome via an unknown mechanism, thus allowing recruitment and DNA binding of TFIID to the TATA box. The DNA bending induced upon TFIID binding to the promoter causes sliding of the SWI/SNF-modified nucleosome to a new position 36 bp downstream, thus allowing the initiation of transcription. This ordered recruitment and nucleosome sliding is consistent with the view that histone acetylation sets the stage for ATP-dependent remodeling by establishing a recognition surface for the bromodomains present in SWI/SNF-like remodeling machines. Furthermore, since histone acetylation precedes the recruitment of additional complexes bearing bromodomains, such as TFIID, it is possible that this modification also regulates recruitment of TFIID to promoters (Agalioti, 2002).

One mechanism by which a cell affords protection from the transforming effects of oncogenes is via the action of the tumor suppressor, ARF, which binds to MDM2 and inhibits MDM2's E3 ligase function. Thus ARF activates p53 by inactivating Mdm2. Many oncogenes have also been shown to activate the transcription factor NF-kappaB, which can contribute toward the malignant phenotype in many ways, including an ability to antagonize p53. ARF is shown to inhibit NF-kappaB function and its antiapoptotic activity independent of Mdm2 and p53. ARF represses the transcriptional activation domain of the NF-kappaB family member RelA by inducing its association with the histone deacetylase, HDAC1. Further, it is shown that the response of NF-kappaB to the oncogene Bcr-Abl is determined by the ARF status of the cell. These results reveal an important function of ARF that can regulate the NF-kappaB response to oncogene activation (Rocha, 2003).

Repression of NF-kappaB transactivation by ARF is a result of the inducible association or RelA with histone deacetylase activity. Repression also requires threonine 505 of RelA and ATR. Furthermore, p14ARF induces phospho-threonine modification of RelA. In this report, however, it is not directly demonstrated that Thr505 itself is inducibly phosphorylated. It remains possible that other sites are also phosphorylated. It is also unlikely that threonine 505 is directly phosphorylated by ATR, since this region does not correspond to the typical consensus phosphorylation sequence for ATR/ATM, which is typically a serine or threonine followed by a glutamine residue. Nonetheless, while a number of questions remain unanswered, these observations, including the inducible association of HDAC1 with RelA through the threonine 505 region and the requirement for ATR activity for a function of ARF, define important regulatory mechanisms for both NF-kappaB and ARF. How these effects are precisely linked will require further investigation. These results are also consistent with other reports of p53-independent functions of human ARF. These include repression of E2F activity, sequestration of Hypoxia-inducible factor-1alpha in the nucleolus, inhibition of ribosomal RNA processing, and induction of antiproliferative gene expression (Rocha, 2003).

The activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial-mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. NFkappaB was identified as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition (Bachelder, 2005).

gp130-linked cytokines such as interleukin-6 (IL-6) stimulate the formation of tyrosine-phosphorylated STAT3 ( P-STAT3), which activates many genes, including the STAT3 gene itself. The resulting increase in the concentration of unphosphorylated STAT3 (U-STAT3) drives a second wave of expression of genes such as RANTES, IL6, IL8, MET, and MRAS that do not respond directly to P-STAT3. Thus, U-STAT3 sustains cytokine-dependent signaling at late times through a mechanism completely distinct from that used by P-STAT3. Many U-STAT3-responsive genes have kappaB elements that are activated by a novel transcription factor complex formed when U-STAT3 binds to unphosphorylated NFkappaB (U-NFkappaB), in competition with IkappaB. The U-STAT3/U-NFkappaB complex accumulates in the nucleus with help from the nuclear localization signal of STAT3, activating a subset of kappaB-dependent genes. Additional genes respond to U-STAT3 through an NFkappaB-independent mechanism. The role of signal-dependent increases in U-STAT3 expression in regulating gene expression is likely to be important in physiological responses to gp130-linked cytokines and growth factors that activate STAT3, and in cancers that have constitutively active P-STAT3 (Yang, 2007).

IkappaB independent regulation of NF-kappaB

Phosphatidylinositol 3-kinase (PI3K: see Drosophila Pi3K92E) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. IL-1 is found to stimulate interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression has no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, it was found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB (Sizemore, 1999).

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 is 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. p50 or p52 homodimers generated by processing of p105 or p100 are the known targets for the IkappaB homolog Bcl-3. Bcl-3 is a unique IkappaB member, since it is most abundant in the nucleus and is not degraded upon activation of NF-kappaB-stimulating pathways. Bcl-3 can have different effects on p52 or p50 binding to DNA, depending on its phosphorylation status, concentration or interaction with nuclear co-factors. The interaction of Bcl-3 with p50 or p52 homodimers can result in the dissociation of these homodimers from DNA. Since neither p50 nor p52 contain transactivation domains, it has been proposed that Bcl-3 thus may antagonize p50-mediated inhibition. Alternatively, Bcl-3 can form ternary complexes with p50 or p52 homodimers bound to DNA and act as a transcription activator. Transcription activation requires the presence of N- and C-terminal proline- and serine-rich domains. The activation potential of Bcl-3-p50 complexes can be stimulated further by interaction of Bcl-3 with the histone acetylase Tip60 (Heissmeyer, 1999 and references).

TNF, IL-1 or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-beta-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaB, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta (see Drosophila Ird5) physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-beta 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 that are formed upon IKK-mediated p105 degradation (Heissmeyer, 1999).

The data reported here provide evidence that the p105 precursor and small IkappaBs are equivalent targets of the IKK complex. This implies that various agents and pathways known to activate IkappaB kinases result in release of NF-kappaB/Rel not only from small IkappaBs, but also from p105. Thus, depending on the cell type-specific abundance of the different inhibitor complexes, transcriptional activators other than classical p50-p65, such as Bcl-3-p50, will be activated rapidly by the same signaling pathways. This strongly suggests the involvement of inducible Bcl-3-p50 complexes, and not only p50-p65, in diverse NF-kappaB signaling-controlled processes such as apoptosis, cellular proliferation and the immune response. Specifically, it is tempting to speculate that cytokine-induced Bcl-3-p50, like p50-p65, is involved in regulating genes that protect cells from apoptosis (Heissmeyer, 1999).

NF-kappaB activity is induced by cytokines, stress, and pathogens. IKK1 and IKK2 are critical IkappaB kinases in NF-kappaB activation. Mice lacking IKK1 and IKK2 died at E12. Additional defect in neurulation associated with enhanced apoptosis in the neuroepithelium is observed. MEF cells from IKK1^-/-/IKK2^-/- embryos do not respond to NF-kappaB inducers. Upon crossing with kappaB-lacZ transgenic mice, double-deficient embryos also lose lacZ transgene expression in vascular endothelial cells during development. These data suggest that IKK1 and IKK2 are essential for NF-kappaB activation in vivo and have an important role in protecting neurons against excessive apoptosis during development (Li, 2000).

Transcription factors within a family usually share the ability to recognize similar or identical consensus sites. For example, the five mammalian NF-kappaB/Rel proteins generate more than 12 dimers recognizing 9-11 nucleotide kappaB sites. Each dimer selectively regulates a few target promoters; however, several genes are redundantly induced by more than one dimer. Whether this property simply generates redundancy in target gene activation or underlies more complex regulatory mechanisms is an open issue. During dendritic cell maturation, rapidly activated dimers (e.g., p50/RelA) bound to a subset of target promoters are gradually replaced by slowly activated dimers (e.g., p52/RelB). Since the dimers have different transcriptional activity at each promoter, the dimer exchange allows fine tuning of the response over time. Further, due to the insensitivity of p52/RelB to the NF-kappaB inhibitors, the IkappaBs, dimer exchange contributes to sustained activation of selected NF-kappaB targets in spite of the resynthesis of IkappaBalpha (Saccani, 2003).

Glucocorticoid receptor (GR)-mediated transrepression of the transcription factors AP-1 and NF-kappaB, responsible for most of the anti-inflammatory effects of glucocorticoids, is initiated by the tethering of GR to the promoters of target genes. This tethering is mediated by a nuclear isoform of the focal adhesion LIM domain protein Trip6. Trip6 functions as a coactivator for both AP-1 and NF-kappaB. As shown by chromatin immunoprecipitation, Trip6 is recruited to the promoters of target genes together with AP-1 or NF-kappaB. In the presence of glucocorticoids, GR joins the Trip6 complex. Reducing the level of Trip6 by RNA interference or abolishing its interaction with GR by dominant-negative mutation eliminates transrepression. It is proposed that GR tethering to the target promoter through Trip6 forms the basis of transrepression, and that Trip6 exerts its nuclear functions by acting as a molecular platform, enabling target promoters to integrate activating or repressing signals (Kassel, 2004).

The role of chromatin structure in the regulation of transcription of NF-kappaB dependent genes: Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome

NF-kappaB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. This study used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, it was found that NF-κB is able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-kappaB dependent genes (Lone, 2013).

Most studies of gene induction by inflammatory stimuli have focused on transcription factors that recognize specific DNA sequences and the cytoplasmic events that regulate the activation of these transcription factors. However, transcriptional activation of eukaryotic genes is also influenced by chromatin structure. Studies on the alterations in the chromatin structure required for productive NF-κB binding are essential for understanding the control of expression of inflammatory genes. However, the available data on this important topic are scarce and contradictory. This study used a combination of EMSA, hydroxyl radical (^.OH) and UV laser footprinting to analyze how NF-κB binds to nucleosomes and the effect of histone H1 on the binding. The data provide definitive evidence that NF-κB is able to bind specifically to its cognate sequence when inserted at the end of the nucleosome, but not when it was inserted in vicinity to the nucleosome dyad. The accessibility to the ends of the nucleosome could be explained by the weaker histone-DNA interactions at these sites and their spontaneous unwrapping. At the center (the dyad) of the nucleosome, where the histone-DNA interactions are very strong, NF-κB is unable to specifically bind its cognate site. By contrast, several studies in the past have reported that some transcription factors, including NF-κB, were able to invade the nucleosome and to bind to nucleosome-embedded recognition sequences even when located in the center of nucleosomal DNA. However, these studies were carried out at low nucleosome concentrations at which sub-nucleosomal (hexameric and tetrameric) particles tend to appear. In order to understand whether the nucleosomes per se act as barriers for transcription factor binding, it is imperative to have homogenous population of nucleosomes devoid of any sub-nucleosomal entities. These sub-nucleosomal entities are formed by the loss of one or both H2A-H2B dimers and hence contain disorganized nucleosomal DNA which most likely would permit the specific binding of transcription factors. The results demonstrate that eviction of H2A/2B dimers is required for the binding of NF-κB. This can be achieved by the binding of factors that can disrupt the nucleosomes either directly by themselves indirectly through the recruitments of other nucleosome disrupting activity (Lone, 2013).

It has also been reported that the remodeling of 156 bp nucleosome core particles by SWI-SNF leads to complete and specific binding of NF-κB to its binding sites buried inside the nucleosome. However, this study observed only partial binding of NF-κB at the nucleosomal dyad when the nucleosomes are repositioned by the ATP dependent remodeler RSC. This discrepancy could be attributed to partial histone eviction under the very high concentration of the SWI-SNF that was used to remodel the nucleosomes and/or a presumable instability of the non-canonical (loss of the dyad axis) slid core particles (Lone, 2013).

Unexpectedly, in contrast to the partial accessibility to dimeric restriction enzymes at the dyad and efficient base excision repair (BER) initiatio remosomes did not show specific binding of NF-κB. Thus, in line with the available structural information , the specific binding of NF-κB requires much higher perturbations in histone-DNA interactions and unpeeling of its cognate sequence from the histone surface allowing it to 'embrace' DNA and to productively bind to it. The experimental results further demonstrate that such specific and productive binding could be efficiently achieved when the H2A–H2B dimer is removed from the nucleosome or when the histone octamer is repositioned in a way that the binding site nears the edge (Lone, 2013).

The compaction of chromatin by the linker histone in general has a global and repressive impact on transcription. Linker histone H1 brings the two helices close to each other and leads to the formation of a so-called 'stem' structure (Syed, 2010). Binding of H1 to DNA at the termini of nucleosomes inhibit spontaneous wrapping and unwrapping of DNA and hence would prevent the binding of transcription factors. Another possibility in which H1 could affect transcription is by occupying the binding sequences of those transcription factors whose binding sites are located in the linker region. This suggests that TF will have to compete with H1 to bind to their cognate sites. Several studies have provided the evidence that in certain cases linker histone can be directly displaced by transcription factor. In agreement with these studies, this study found that the presence of histone H1 does not prevent the specific binding of NF-κB when their binding regions overlap. In fact, NF-κB binding completely displaces histone H1 from the nucleosomes. It was also observed that H1 cannot displace the specifically bound NF-κB. In vivo, this competition might be even more in favor of NF-κB as H1 is quite mobile and dynamic (Lone, 2013).

The in vitro data sheds light on the in vivo requirements for the alterations in chromatin structure necessary for the productive binding of NF-κB. These include either a removal of H2A-H2B dimers from the nucleosome and/or chromatin remodeler induced mobilization of the histone octamer. The H2A-H2B dimers are more easily displaced from the histone core than H3 and H4 and they are extensively exchanged in vivo. Moreover, in mammalian cells the nucleosomes in the vicinity of the TSS contain the histone variant H2A.Z. A tentative hypothesis is that specific chaperones, recognizing variant H2A.Z nucleosomes, could be involved in the removal of H2A.Z-H2B variant dimer, thus allowing binding of the NF-κB transcription factors to any site of the nucleosomal DNA (Lone, 2013).

Signaling upstream of NF-kappa B

The two members of the atypical protein kinase C (aPKC) subfamily of isozymes (zetaPKC and lambda/iotaPKC) are involved in the control of NF-kappaB through IKKbeta activation. The previously described aPKC-binding protein, p62, selectively interacts with RIP but not with TRAF2 in vitro and in vivo. p62 bridges the aPKCs to RIP, whereas the aPKCs link IKKbeta to p62. In this way, a signaling cascade of interactions is established from the TNF-R1 involving TRADD/RIP/p62/aPKCs/IKKbeta. These observations define a novel pathway for the activation of NF-kappaB involving the aPKCs and p62. Consistent with this model, the expression of a dominant-negative mutant lambda/iotaPKC impairs RIP-stimulated NF-kappaB activation. In addition, the expression of either an N-terminal aPKC-binding domain of p62, or its C-terminal RIP-binding region are sufficient to block NF-kappaB activation. Furthermore, transfection of an antisense construct of p62 severely abrogates NF-kappaB activation. Together, these results demonstrate that the interaction of p62 with RIP serves to link the atypical PKCs to the activation of NF-kappaB by the TNFalpha signaling pathway (Sanz, 1999).

Glycogen synthase kinase-3 (GSK-3)-alpha and -beta are closely related protein-serine kinases, which act as inhibitory components of Wnt signaling during embryonic development and cell proliferation in adult tissues. Disruption of the murine GSK-3beta gene results in embryonic lethality caused by severe liver degeneration during mid-gestation, a phenotype consistent with excessive tumor necrosis factor (TNF) toxicity, as observed in mice lacking genes involved in the activation of the transcription factor NF-kappaB. GSK-3beta-deficient embryos are rescued by inhibition of TNF using an anti-TNF-alpha antibody. Fibroblasts from GSK-3beta-deficient embryos are hypersensitive to TNF-alpha and show reduced NF-kappaB function. Lithium treatment (which inhibits GSK-3) sensitizes wild-type fibroblasts to TNF and inhibits transactivation of NF-kappaB. The early steps leading to NF-kappaB activation (degradation of I-kappaB and translocation of NF-kappaB to the nucleus) are unaffected by the loss of GSK-3beta, indicating that NF-kappaB is regulated by GSK-3beta at the level of the transcriptional complex. Thus, GSK-3beta facilitates NF-kappaB function (Hoeflich, 2000).

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4^-8^- thymocytes and is down-regulated across the CD4^-8^- to CD4⁺8⁺ transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics show an increased number of thymocytes, particularly late CD4^-8^- cells, a failure to down-regulate CD25 in post-CD4^-8^- subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas develop with high penetrance. Tumors sustain characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3 (Bellavia, 2000).

The role that zetaPKC plays in NF-kappaB activation has been addressed using mice in which this kinase has been inactivated by homologous recombination. These mice, although grossly normal, show phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-ß receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected that requires the previous degradation of IkappaB. Although in zetaPKC^-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited (Leitges, 2001).

The activation of NF-kappaB by TNFalpha provides a potent antiapoptotic signal that prevents TNFalpha-induced cell death. The inhibition of NF-kappaB signaling in the pathways of the different knockout mice sensitizes cells to the proapoptotic actions of TNFalpha. In this regard, there are intriguing differences in the phenotypes of the GSK-3ß, T2K, and zetaPKC mutant mice. Whereas both the GSK-3ß- and T2K-deficient mice die of liver apoptosis, only the EFs from the GSK-3ß-deficient mice undergo apoptosis in response to TNFalpha, whereas the T2K^-/- EFs do not. The zetaPKC^-/- EFs are sensitive to the proapoptotic actions of TNFalpha, like the GSK-3ß EFs, but the livers of the zetaPKC mutant mice do not show signs of apoptosis. It is worth mentioning that not all the mice with a knockout of genes shown to be essential for NF-kappaB activation display liver cell death. Thus, for example, whereas mice deficient in RelA (p65), IKKß, and Nemo/IKKgamma (see Drosophila Kenny), in addition to the already discussed T2K, or GSK-3ß, die around day 14 of gestation due to severe liver degeneration and apoptosis, other mutant mice, such as the TNF-R1, TRAF2, TRAF6, RIP, MyD88, and IRAK, do not. It may be that the former proteins control the expression of NF-kappaB-dependent antiapoptotic genes that are essential for survival of liver cells, whereas the latter do not. In the case of the zetaPKC^-/- mice, a possible explanation for the lack of liver cell death may rely on the fact that this tissue expresses little or no zetaPKC and, therefore, other kinases may have substituted for zetaPKC for this function. Upon injection of LPS, the activation of NF-kappaB in lungs is severely inhibited in the zetaPKC^-/- mice but not in the livers of these animals (Leitges, 2001).

The phenotype of the zetaPKC^-/- mice is complex and consistent with the notion that this kinase participates in distinct NF-kappaB signaling pathways. Not surprisingly, the zetaPKC-deficient mice have at least some of the features characteristic of mutant mice for the TNF-R1 and LT-ßR. For example, the zetaPKC^-/- mice have a significantly reduced number of Peyers patches (PP), which is characteristic of the mutant mice for the TNF-R1. However, the PP of the zetaPKC-deficient mice display an impaired segregation of B and T cells, which has not been reported in the PP of the TNF-R1 animals. However, the PP of both mutant mice are similar in that they have decreased follicular dendritic cell networks. The overall structure of the spleen is normal in the zetaPKC^-/- mice, similar to what has been reported for the TNF-R1 mutant mice. In addition, the zetaPKC^-/- mice have detectable lymph nodes, like the TNF-R1 mutant mice and unlike the LT-ßR-deficient mice, which display a complete lack of lymph nodes. However, the zetaPKC^-/- lymph nodes are smaller and have a lower content of B cells. Therefore, overall, the zetaPKC^-/- phenotype, although less penetrant, has characteristics of both the TNF-R1 and LT-ßR mutant phenotypes, but is more similar to the former. There is an additional important observation in the zetaPKC^-/- mice, which is the apparent delay in the maturation of B cells. A similar phenotype has been described for mutants in the BCR signaling cascade. Studies are in progress to determine if zetaPKC could play a nonredundant role in this pathway that could explain, at least in part, this phenotype. Taken together, the data suggest that, rather than a fundamental defect in TNF-R1 or LT-ßR signaling in the spleen, the zetaPKC deficiency causes a developmental delay in the delivery of these signals during the first few weeks of life. Thus, it may be that the zetaPKC requirement to transduce these signals in lymphoid tissues is developmentally regulated, and as the animal matures, other, perhaps redundant, signaling pathways can compensate. This study highlights the role that zetaPKC plays in the transduction of various receptor signals to NF-kappaB; illustrates the complexity of zetaPKC regulation in multiple tissue and cell types, and demonstrates that this kinase plays an essential role in NF-kappaB activation at two different levels: IKK stimulation and NF-kappaB transcriptional activity (Leitges, 2001).

It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. This study shows that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. Both S-nitrosocysteine and cytokine-activated NOS2 inhibits NF-kappaB in human respiratory cells or murine macrophages. This inhibition is reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity does not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro results in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect is reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 is detected in cells, and the level is modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation (Marshall, 2001).

Nerve growth factor (NGF) binding to both p75 and TrkA neurotrophin receptors activates the transcription factor nuclear factor kappaB (NF-kappaB). The atypical protein kinase C-interacting protein, p62, that binds TRAF6, selectively interacts with TrkA but not p75. In contrast, TRAF6 interacts with p75 but not TrkA. The formation is demonstrated of a TRAF6-p62 complex that serves as a bridge linking both p75 and TrkA signaling. Of functional relevance, transfection of antisense p62-enhanced p75-mediated cell death and diminished NGF-induced differentiation occur through a mechanism involving inhibition of IKK activity. These findings reveal a new function for p62 as a common platform for communication of both p75-TRAF6 and TrkA signals. Moreover, p62 serves as a scaffold for activation of the NF-kappaB pathway, which mediates NGF survival and differentiation responses (Wooten, 2001).

Mice deficient in the B cell adaptor for phosphoinositide 3-kinase (BCAP: Drosophila homolog - Stumps) have reduced numbers of mature B lymphocytes, which show defects in cell survival and proliferation. The NF-kappa B (Rel) pathway is impaired in BCAP-deficient mature B cells, and NF-kappa B target genes, indispensable for cell survival and division, are not induced in response to B cell receptor (BCR) stimulation. Among the NF-kappa B (Rel) family, expression of c-Rel is specifically reduced in BCAP-deficient B cells. Retrovirus-mediated reintroduction of c-Rel restores the pool size of immunoglobulin (Ig)M(lo)IgD(hi) mature B cells in the spleen as well as proliferative responses to BCR stimulation. These results indicate BCAP is essential in the maintenance of mature B cells through functional coupling with c-Rel (Yamazaki, 2004).

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-kappaB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE - glycation is an uncontrolled, non-enzymatic reaction of sugars with proteins) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, was investigated in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produce activation of NF-kappaB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increase the activity of IKKbeta, HMGB1 exposure results in activation of both IKKalpha and IKKbeta. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-kappaB-dependent transcription by HMGB1. Transfections with dominant negative constructs have demonstrated that TLR 2 and TLR 4 are both involved in HMGB1-induced activation of NF-kappaB. In contrast, RAGE plays only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS (Park, 2004)

The ARF tumour suppressor (p14ARF in humans, p19ARF in mice) is a central component of the cellular defence against oncogene activation. The expression of ARF, which shares a genetic locus with the p16INK4a tumour suppressor, is regulated by the action of transcription factors such as members of the E2F family. ARF can bind to and inhibit the Hdm2 protein (Mdm2 in mice), which functions as an inhibitor and E3 ubiquitin ligase for the p53 transcription factor. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor-alpha induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor (Rocha, 2005).

Bruton's tyrosine kinase (see Btk family kinase at 29A) has been shown to participate in the induction of NFkappaB-dependent gene expression by the lipopolysaccharide (LPS) receptor Toll-like receptor-4 (TLR4). The mechanism whereby Btk participates in this response has been examined. Treatment of a murine monocytic cell line with LFM-A13, a specific Btk inhibitor, blocks LPS-induced NFkappaB-dependent reporter gene expression but not IkappaB alpha degradation. Transient transfection of HEK293 cells with Btk has no effect on NFkappaB-dependent reporter gene expression but strongly promotes transactivation of a reporter gene by a p65-Gal4 fusion protein. IkappaB alpha degradation activated by LPS is intact in macrophages from X-linked immunodeficiency (Xid) mice, which contain inactive Btk. Transfection of cells with a dominant negative form of Btk (BtkK430R) inhibits LPS-driven p65 mediated transactivation. Additionally LFM-A13 impairs phosphorylation of serine 536 on p65 induced by LPS in HEK293-TLR4 cells, and in Xid macrophages this response is impaired. This study therefore reveals a novel function for Btk. It is required for the signaling pathway activated by TLR4 that culminates in phosphorylation of p65 on serine 536 promoting transactivation by NFkappaB (Doyle, 2005).

G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-kappaB activation, the molecular mechanism of GPCR-induced NF-kappaB activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, this study showed that CARMA3 is required for GPCR-induced NF-kappaB activation. Mechanistically, it was found that CARMA3 deficiency impairs GPCR-induced IkappaB kinase (IKK) activation, although it does not affect GPCR-induced IKKα/β phosphorylation, indicating that inducible phosphorylation of IKKα/β alone is not sufficient to induce its kinase activity. It was also found that CARMA3 is physically associated with NEMO/IKKγ, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, it was found TRAF6 deficiency also abrogates GPCR-induced NF-kappaB activation. Together, these results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-kappaB activation (Grabiner, 2007).

NF-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) that activates NF-κB, is essential for NF-κB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor α (TNFα)-induced NF-κB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKα and IKKβ, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-κB activation. In contrast, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFα receptor 1 (TNFR1). This study shows that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFα-induced NF-κB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFα-induced NF-κB activity. These results reveal a physiologic role for optineurin in dampening TNFα signaling, and this role might provide an explanation for its association with glaucoma (Zhu, 2007).

While NF-kappaB is considered to play key roles in the development and progression of many cancers, the mechanisms whereby this transcription factor is activated in cancer are poorly understood. A key oncoprotein in a variety of cancers is the serine- threonine kinase Akt, which can be activated by mutations in PI3K, by loss of expression/activity of PTEN, or through signaling induced by growth factors and their receptors. A key effector of Akt-induced signaling is the regulatory protein mTOR (mammalian target of rapamycin). This study shows that mTOR downstream from Akt controls NF-kappaB activity in PTEN-null/inactive prostate cancer cells via interaction with and stimulation of IKK. The mTOR-associated protein Raptor is required for the ability of Akt to induce NF-kappaB activity. Correspondingly, the mTOR inhibitor rapamycin is shown to suppress IKK activity in PTEN-deficient prostate cancer cells through a mechanism that may involve dissociation of Raptor from mTOR. The results provide insight into the effects of Akt/mTOR-dependent signaling on gene expression and into the therapeutic action of rapamycin (Dan, 2008).

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. This study reports that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency (Rahighi, 2009).

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