Stabilization of RelB Requires Multidomain Interactions with p100/p52

Abstract

The NF-B family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IB proteins. We show that the protein level of RelB is significantly reduced in the absence of p100 and reduced even more when both p100 and p105 are absent. RelB stabilizes itself by directly interacting with p100, p105, and their processed products. However, RelB forms complexes with its partners using different interaction modes. Although the C-terminal ankyrin repeat domain of p105 is not involved in the RelB-p105 complex formation, all domains and flexible regions of each protein are engaged in the RelB-p100 complex. In several respects the RelB-p52 and RelB-p100 complexes are unique in the NF-B family. The N-terminal domain of p100/p52 interacts with RelB but not RelA. The transcriptional activation domain of RelB, but not RelA, directly interacts with the processing region of p100. These unique protein-protein contacts explain why RelB prefers p52 as its dimeric partner for transcriptional activity and is retained in the cytoplasm as an inhibited complex by p100. This association-mediated stabilization of RelB implies a possible role for RelB in the processing of p100 into p52.The dimeric NF-B transcription factors are formed from five family members, p50 (NF-B1), RelA (p65), p52 (NF-B2), c-Rel, and RelB. These proteins share an ϳ300-residue long homologous region located near the N terminus. This element, referred to as the Rel homology region (RHR), 5 is responsible for DNA binding, dimerization, inhibitor binding, and nuclear localization. p50 and p52 are the processed products of precursor proteins, p105 and p100, respectively (1, 2). RelA and c-Rel homo-and heterodimers are tightly regulated by a class of inhibitor proteins known as IB through the formation of stable IB-NF-B complexes that are unable to bind DNA. Activation of these dimers requires degradation of IB. A large number of stimuli activate IB degradation through phosphorylation of IB by IB kinase (IKK) leading to ubiquitination, 26 S proteasome recruitment, and degradation of IB by the proteasome (3, 4). Signaling pathways leading to NF-B activation through degradation of classical IB proteins (IB␣, IB␤, and IB⑀) are classified as the canonical pathways.RelB displays characteristics that are not shared by the other NF-B subunits: 1) Prototypical IB proteins do not regulate RelB-containing NF-B dimers (5, 6). 2) The RelB homodimer does not have DNA binding activity, suggesting that unlike other members, RelB may not form a stable detectable homodimer in vivo (7). 3) RelB has a N-terminal extension, known as the leucine zipper (LZ) domain because of the presence of a leucine-rich heptad repeat that is not present in other NF-B family members (8). 4) The x-ray crystal structure of the RelB dimerization domain (DD) revealed an intertwined domain swapped arrangement of the two monomers, suggesting that this RelB domain fold might be unstable and is stabilized by domain intertwining (9...

A single NFκB system for both canonical and non-canonical signaling

“…RelB associates with p100 through multiple protein-protein contacts. This multidomain interaction results in protein co-stabilization, as nfκb2 −/− cells showed reduced level of RelB protein and relb −/− cells have decreased amounts of p100 protein [69,70]. Counterintuitively, this interaction also inhibits p100 processing and RelB:p52 formation, suggesting that RelB:p52 dimer formation may occur after p100 processing, though it may involve alternate, transient interactions with p100.…”

The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100

“…However, RelB must enable some processing of p100 to p52. This challenging dual tasking of RelB is solved elegantly: RelB uses its unique ability to form the p100-centric kappaBsome within which other NF-kB subunits may interact with p100 (32)(33)(34). Our study suggests that the generation of p52 occurs within the context of such a multiprotein complex.…”

p100/IκBδ sequesters and inhibits NF-κB through kappaBsome formation

“…Our work on p105:IκBγ revealed that p105 forms a dimer sequestering two NF-κB monomers although the precise mode of how p105 assembles with two NF-κB monomers is unknown. Interestingly, the C-terminal domain (CTD) of p105/IκBγ fails to bind RelB, although p50, the processed product of p105, interacts with RelB to form the p50: RelB heterodimer (26). We also showed that, like p105, p100 forms a large complex of unknown stoichiometry with NF-κB.…”

“…RelB was subcloned into pEGFP-N1 vector (Clontech), and RelA was subcloned into pCMV HA vector. Transfection and fractionation by size-exclusion chromatography were done as previously described (26).…”