Critical Role of RelB Serine 368 for Dimerization and p100 Stabilization

Abstract

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory B proteins. In most other cell types inhibitory B proteins prevent nuclear translocation and activation of NFB. 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 NFB 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.NFB represents a family of dimeric transcription factors consisting of the mammalian members p50 (NFB1), p52 (NFB2), RelA (p65), RelB, and c-Rel. NFB regulates the expression of a variety of genes, many of which are involved in immune and inflammatory responses. In addition, NFB target genes play a role in cell growth and differentiation as well as in neoplastic transformation (reviewed in Refs. 1-4). Whereas the three Rel proteins are directly synthesized as mature proteins, p50 and p52 are generated by proteolytical processing from their p105 and p100 precursors, respectively. A characteristic of all family members is the Rel homology domain, which comprises about 300 amino acids and is responsible for proteinprotein interaction, DNA binding, and nuclear localization. In most cell types, NFB proteins are retained in the cytoplasm by inhibitory B proteins, the IBs, 1 which comprise IB␣, IB␤, IB␥, IB⑀, and Bcl-3. Moreover, the precursors p100 and p105 are also known to display inhibitory functions and therefore are also classified as IBs (5, 6). The IB proteins are not restricted to the cytoplasm but have been shown to retrieve NFB heterodimers from the nucleus and escort them back to the cytoplasm (7). There are different pathways for induction of NFB. In most cell types, stimulation with a variety of substances leads to activation of the IB kinase complex (IKK). This complex consists of the catalytic subunits IKK␣ and IKK␤ and the regulatory IKK␥ subunit (also called NEMO, NFB essential modulator). In the classical pathway, the activated IKK, predominantly IKK␤, catalyzes the phosphorylation of IB. Subsequently, IB is ubiquitinated and degraded, thereby releasing the...

Role for Protein Kinase Cθ (PKCθ) in TCR/CD28-mediated Signaling through the Canonical but Not the Non-canonical Pathway for NF-κB Activation

“…The generation of p52-containing nuclear NF-B complexes results in long term NF-B activation that is required in developmental events such as the generation and maintenance of lymphoid architecture. However, the stimulation of p100 generation by TCR/CD28 signaling may have special consequences for T cell activation signaling and survival because the constellation of genes bound and activated by p52-containing hetero- (20,29) or homodimers (30) could be different from those genes that are activated by the p50/RelA heterodimers that are active early on in the response to TCR/ CD28 signaling. One potential gene target of p52 is the antiapoptotic gene Bcl-2, the transcription of which can be stimulated by p50 or p52 homodimers in combination with Bcl-3 (30).…”

Post-translational modifications regulating the activity and function of the nuclear factor kappa B pathway

“…RelB is phosphorylated at Ser-368, the equivalent of Ser-281 in RelA, and this regulates its dimerization with p100 (Maier et al, 2003). Similarly, as discussed above, p50 is phosphorylated at the RelA Ser-276 equivalent, that is, Ser-337 (Hou et al, 2003;Guan et al, 2005).…”

RelA repression of RelB activity induces selective gene activation downstream of TNF receptors

“…Surprisingly, the increased RelB protein level in the nucleus does not lead to an increased DNA-binding activity, suggesting a nuclear control of RelB in these cells. p100 and phosphorylation of RelB on serine 368 seem to contribute to this negative control (19)(20)(21), but additional mechanisms clearly account for the nuclear control of RelB DNAbinding activity in these cells.…”