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In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.

RelB is an unusual member of the NF-kappaB transcription factor family that acts as both a transcriptional activator as well as a repressor of NF-kappaB-dependent gene expression. Although RelB promotes gene expression when it associates with p50/NF-kappaB1 or p52/NF-kappaB2, the precise molecular mechanisms through which it represses NF-kappaB remain unclear. To examine this inhibitory function in more detail, we employed reporter gene assays and found that RelB represses at the level of RelA. Furthermore, electrophoretic mobility shift analysis revealed that in vitro translated RelB impaired the DNA binding activity of RelA and that overexpressed RelB significantly reduced tumor necrosis factor-alpha-induced RelA activity in murine embryonic fibroblasts. Intriguingly, this inhibitory effect was due to the formation of RelA.RelB heterodimers that were unable to bind to kappaB sites in vitro strongly suggesting that these newly described NF-kappaB dimers cannot bind DNA. Expression pattern analysis revealed that RelA.RelB heterodimers appeared at relatively low levels in both lymphoid and non-lymphoid cells. However, the presence of these complexes increased following stimulation with phorbolesters or lipopolysaccharide or by overexpression of constitutively active IKKbeta. Functional characterization of RelA.RelB heterodimers in NIH3T3 murine embryonic fibroblasts revealed that they are not regulated by IkappaB proteins and are located in both the cytoplasm and the nucleus. Taken together, our findings demonstrate that sequestration of RelA in transcriptionally inactive RelA.RelB complexes provides a molecular mechanism that may explain the repressive role of RelB on NF-kappaB-dependent gene expression.

RelB is an unusual member of the Rel/NF-kappaB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IkappaBs, the cytosolic NF-kappaB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IkappaBs. However, RelB degradation differs from that of IkappaBs since it is not induced by TNFalpha but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelB(DM), site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-kappaB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-kappaB activity.

We have investigated activation of nuclear factor-kappa B (NF-kappa B) in the process of primary B cell differentiation in vitro. In this system, NF-kappa B is strongly induced when B cells develop from the pre-B cell to the immature B cell stage. Unlike the typical NF-kappa B activation in response to exogenous stimuli, induction proceeds with a slow time course. NF-kappa B induction is only observed in B cells that undergo differentiation, not in Rag2-deficient cells. Nuclear DNA binding complexes predominantly comprise p50/RelA heterodimers and, to a lesser extent, c-Rel-containing dimers. The increase in NF-kappa B binding activity is accompanied by a slow and steady decrease in I kappa B beta protein levels. Interestingly, absolute RelA protein levels remain unaffected, whereas RelB and c-Rel synthesis is induced. The reason for preferential nuclear translocation of RelA complexes appears to be selective inhibition by the I kappa B beta protein. I kappa B beta can efficiently inhibit p50/RelA complexes, but has a much reduced ability to interfere with p50/c-Rel DNA binding both in vitro and in vivo. Interestingly, p50/RelB complexes are not at all targeted by I kappa B beta, and coimmunoprecipitation experiments show no evidence for an association of I kappa B beta and RelB in vivo. Consistent with these observations, I kappa B beta cotransfection can inhibit p50/RelA-mediated trans-activation, but barely affects p50/RelB mediated trans-activation.

The transcription factor NF-kappa B controls the induction of numerous cytokine promoters during the activation of T lymphocytes. Inhibition of T cell activation by the immunosuppressants cyclosporin A (CsA) and FK506 exerts a suppressive effect on the induction of these NF-kappa B-controlled cytokine promoters. We show for human Jurkat T leukemia cells, as well as human and mouse primary T lymphocytes, that this inhibitory effect is accompanied by an impaired nuclear translocation of the Rel proteins c-Rel, RelA/p65 and NF-kappa B1/p50, whereas the nuclear appearance of RelB remains unaffected. CsA does not interfere with the synthesis of Rel proteins, but prevents the inducible degradation of cytosolic NF-kappa B inhibitors I kappa B alpha and I kappa B beta upon T cell activation. CsA neither inhibits the processing of the NF-kappa B1 precursor p105 to p50, nor does it "stabilize" the C-terminal portion of p105, I kappa B gamma, which is degraded during p105 processing to mature p50. These results indicate that CsA interferes with a specific event in the signal-induced degradation of I kappa B alpha and I kappa B beta, but does not affect the processing of NF-kappa B1/p105 to p50.

A cDNA clone encoding a member of the avian interferon regulatory factor (IRF) family homologous to mammalian IRF-2 was isolated from cDNA library from poly[rI:rC]-induced chicken embryo fibroblasts (CEF). The deduced amino acid sequence shows a characteristic DNA binding domain of 124 amino acids at the amino-terminal end with 96.8% identity to human and 96% to mouse IRF-2. Identities in the C-terminal part are 77.5% and 77%, respectively. Identity to all other known members of the chicken IRF (Ch-IRF) family is distinctly lower. In C32 cells, an IRF-2 mRNA of 2.4 kb is constitutively expressed in very low amounts but is inducible by Ch-IFN in the absence or presence of cycloheximide. The Ch-IRF-2 gene is a single copy gene and was mapped by fluorescence in situ hybridization to the long arm of chromosome 4.