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NF-kappa B/Rel family members are physically associated phosphoproteins.

ABSTRACT: We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.

SUBMITTER: Li CC

PROVIDER: S-EPMC1137355 | biostudies-other | 1994 Oct

REPOSITORIES: biostudies-other

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Cell transformation by v-Rel reveals distinct roles of AP-1 family members in Rel/NF-kappaB oncogenesis.

Project description:Cell transformation by the v-rel oncogene is mediated by the aberrant expression of genes that are normally tightly regulated by other Rel/NF-kappaB family members. Although a number of genes inappropriately activated or suppressed by v-Rel have been identified, their contributions to the v-Rel transformation process have been poorly characterized. Here, we examine the role of individual AP-1 proteins in v-Rel-mediated transformation. v-Rel-transformed cells exhibit elevated RNA and protein expression of c-Fos, c-Jun and ATF2 and sustained repression of Fra-2. c-Fos and c-Jun are essential in both the initiation and maintenance of v-Rel-mediated transformation, whereas Fra-2 is dispensable. By employing a c-Jun dimerization mutant, we further identified Fos/Jun heterodimers as major contributors to the v-Rel transformation process. The inability of c-Rel to induce the expression of c-Fos and c-Jun contributes to its weaker oncogenic potential relative to v-Rel. Our studies also demonstrate that v-Rel may induce AP-1 members by directly upregulating gene expression (c-fos and ATF2) and by activating pathways that stimulate AP-1 activity. Although elevated expression of ATF2 is also required for v-Rel-mediated transformation, its ectopic overexpression is inhibitory. Investigating the mode of ATF2 regulation revealed a positive feedback mechanism whereby ATF2 induces p38 MAPK phosphorylation to further induce its own activity. In addition, these studies identified Ha-Ras as an effector of v-Rel-mediated transformation and reveal a novel role for ATF2 in the inhibition of the Ras-Raf-MEK-ERK signaling pathway. Overall, these studies reveal distinct and complex roles of AP-1 proteins in Rel/NF-kappaB oncogenesis.

| S-EPMC2932816 | biostudies-literature

TRAF family proteins link PKR with NF-kappa B activation.

Project description:The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.

| S-EPMC400457 | biostudies-literature

The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).

Project description:The transcription factors of the Rel/NF-kappaB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-kappaB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-kappaB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-kappaB raised the issue of whether NF-kappaB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-kappaB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-x(L). While Bcl-x(L) was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-kappaB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-kappaB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-kappaB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-kappaB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-x(L) and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-kappaB activity.

| S-EPMC85484 | biostudies-literature

Characterization of three alternatively spliced isoforms of the Rel/NF-kappa B transcription factor Relish from the mosquito Aedes aegypti.

Project description:The Rel/NF-kappa B transcription factor Relish performs a central role in the acute-phase response to microbial challenge by activating immune antibacterial peptides. We cloned and molecularly characterized the gene homologous to Drosophila Relish from the mosquito Aedes aegypti. Unlike Drosophila Relish, Aedes Relish has three alternatively spliced transcripts encoding different proteins. First, the predominant Aedes Relish transcript of 3.9 kb contains both the Rel-homology domains and the inhibitor kappa B (I kappa B)-like domain, which is similar to Drosophila Relish and to the mammalian p105 and p100 Rel/NF-kappa B transcription factors. Second, Aedes Relish transcript contains Rel-homology domains identical to those of the major transcript but it completely lacks the I kappa B-like domain-coding region, which has been replaced by a unique 3'-untranslated region sequence. In the third transcript, a deletion replaces most of the N-terminal sequence and Rel-homology domains; however, the I kappa B-like domain is intact. All three Aedes Relish transcripts were induced by bacterial injection but not by blood feeding. In vitro-translated protein from the Rel-only construct specifically binds to the kappa B motif from Drosophila cecropin A1 and Aedes defensin genes. PCR and Southern blot hybridization analyses show that these three transcripts originated from the same large inducible mRNA encoded by a single Relish gene.

| S-EPMC126610 | biostudies-literature

Bifunctional Activity of members of the Arabidopsis NF-YA Transcription Factor Family

Project description:Five members of the Arabidopsis thaliana NF-YA gene family are strongly induced by several stress conditions via transcriptional and miR169-related posttranscriptional mechanisms. These transcription factors participate in gene regulation via two different mechanisms, one depending on binding to the CCAAT-box in the promoter of regulated genes and the other, independent of the CCAAT-box, in which NF-YA prevents the interaction of the NF-YB/YC heterodimer with transcription factors.

2012-04-25 | GSE36092 | GEO

Bifunctional Activity of members of the Arabidopsis NF-YA Transcription Factor Family

2012-04-24 | E-GEOD-36092 | biostudies-arrayexpress

NF-?B1, NF-?B2 and c-Rel differentially regulate susceptibility to colitis-associated adenoma development in C57BL/6 mice.

Project description:NF-?B signalling is an important factor in the development of inflammation-associated cancers. Mouse models of Helicobacter-induced gastric cancer and colitis-associated colorectal cancer have demonstrated that classical NF-?B signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF-?B proteins, including NF-?B1/p50, NF-?B2/p52, and c-Rel, differentially regulate the development of gastric pre-neoplasia. To investigate the effect of NF-?B subunit loss on colitis-associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1(-/-), Nfkb2(-/-), and c-Rel(-/-) mice. Animals lacking the c-Rel subunit were more susceptible to colitis-associated cancer than wild-type mice, developing 3.5 times more colonic polyps per animal than wild-type mice. Nfkb2(-/-) mice were resistant to colitis-associated cancer, developing fewer polyps per colon than wild-type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2(-/-) mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c-Rel(-/-) mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild-type counterparts. These observations demonstrate different functions of specific NF-?B subunits in this model of colitis-associated carcinogenesis. NF-?B2/p52 is necessary for the development of colitis, whilst c-Rel-mediated signalling regulates colonic epithelial cell turnover following DNA damage.

| S-EPMC4737252 | biostudies-literature

Viral targeting of the interferon-{beta}-inducing Traf family member-associated NF-{kappa}B activator (TANK)-binding kinase-1.

Project description:Expression of the antiviral cytokines IFN-alpha/beta is among the most potent innate defenses of higher vertebrates to virus infections, which is controlled by the inducible transcription factor IFN regulatory factor (IRF)3. Borna disease virus (BDV) establishes persistent noncytolytic infections in animals and tissue culture cells, indicating that it can circumvent this antiviral reaction by an unexplained activity. In this study, we identify the BDV P protein as microbial gene product that associates with and inhibits the principal regulatory kinase of IRF3, Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK-1). We demonstrate that the P protein counteracts TBK-1-dependent IFN-beta expression in cells and, hence, the establishment of an antiviral state. Furthermore, our data show that the BDV P protein itself is phosphorylated by TBK-1, suggesting that P functions as a viral decoy substrate that prevents activation of cellular target proteins of TBK-1. Thus, our findings provide evidence for a previously undescribed mechanism by which a viral protein interferes with the induction of the antiviral IFN cascade.

| S-EPMC1224622 | biostudies-literature

CAPERalpha is a novel Rel-TAD-interacting factor that inhibits lymphocyte transformation by the potent Rel/NF-kappaB oncoprotein v-Rel.

Project description:The Rel/NF-kappaB transcription factors are constitutively activated in many human cancers. The Rel proteins in this family are implicated in leukemia/lymphomagenesis, but the mechanism is not completely understood. Previous studies showed that the transcription activation domains (TADs) of the viral oncoprotein v-Rel and its cellular Rel/NF-kappaB homologues c-Rel and RelA are key determinants of their different transforming activities in primary lymphocytes. Substitution of a Rel TAD for that of RelA conferred a strong transforming phenotype upon RelA, which otherwise failed to transform cells. To gain insights into protein interactions that influence cell transformation by the Rel TADs, we identified factors that interact with the TAD of v-Rel, the most oncogenic member of the Rel/NF-kappaB family. We report that the coactivator for transcription factors AP-1 and estrogen receptors, CAPERalpha, interacts with the v-Rel TAD and potently synergizes v-Rel-mediated transactivation. Importantly, coexpression of CAPERalpha markedly reduced and delayed v-Rel's transforming activity in primary lymphocytes, whereas a dominant-negative mutant enhanced the kinetics of v-Rel-mediated transformation. Furthermore, small interfering RNA-mediated knockdown of CAPERalpha in v-Rel-transformed lymphocytes significantly enhanced colony formation in soft agar. Since the potency of Rel-mediated transactivation is an important determinant of lymphocyte transformation, as is Rel's ability to induce transcriptional repression, these data suggest that CAPERalpha's interaction with the Rel TAD could modulate Rel/NF-kappaB's transforming activity by facilitating expression or dampening repression of specific gene subsets important for oncogenesis. Overall, this study identifies CAPERalpha as a new transcriptional coregulator for v-Rel and reveals an important role in modulating Rel's oncogenic activity.

| S-EPMC2573166 | biostudies-literature

NF-κB Rel subunit exchange on a physiological timescale.

Project description:The Rel proteins of the NF-κB complex comprise one of the most investigated transcription factor families, forming a variety of hetero- or homodimers. Nevertheless, very little is known about the fundamental kinetics of NF-κB complex assembly, or the inter-conversion potential of dimerised Rel subunits. Here, we examined an unexplored aspect of NF-κB dynamics, focusing on the dissociation and reassociation of the canonical p50 and p65 Rel subunits and their ability to form new hetero- or homodimers. We employed a soluble expression system to enable the facile production of NF-κB Rel subunits, and verified these proteins display canonical NF-κB nucleic acid binding properties. Using a combination of biophysical techniques, we demonstrated that, at physiological temperatures, homodimeric Rel complexes routinely exchange subunits with a half-life of less than 10 min. In contrast, we found a dramatic preference for the formation of the p50/p65 heterodimer, which demonstrated a kinetic stability of at least an order of magnitude greater than either homodimer. These results suggest that specific DNA targets of either the p50 or p65 homodimers can only be targeted when these subunits are expressed exclusively, or with the intervention of additional post-translational modifications. Together, this work implies a new model of how cells can modulate NF-κB activity by fine-tuning the relative proportions of the p50 and p65 proteins, as well as their time of expression. This work thus provides a new quantitative interpretation of Rel dimer distribution in the cell, particularly for those who are developing mathematical models of NF-κB activity.

| S-EPMC8376415 | biostudies-literature

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