The NF-κB family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IκB 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-κB 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 NF-κB family member RelB has many properties not shared by other family members such as restricted subunit association and lack of regulation by the classical IκB 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-κB 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-κB transcription factors are formed from five family members, p50 (NF-κB1), RelA (p65), p52 (NF-κB2), c-Rel, and RelB. These proteins share an ∼300-residue long homologous region located near the N terminus. This element, referred to as the Rel homology region (RHR), 5The abbreviations used are: RHR, Rel homology region; NTD, N-terminal domain; TAD, transcriptional activation domain; IKK, IκB kinase; LZ, leucine zipper; DD, dimerization domain; GST, glutathione S-transferase; IB, immunoblotting; GFP, green fluorescent protein; HEK, human embryonic kidney; wt, wild type; MEF, mouse embryonic fibroblast; IP, immunoprecipitation; GRR, glycine-rich region; ARD, ankyrin repeat domain. 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 (1Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4610) Google Scholar). RelA and c-Rel homo- and heterodimers are tightly regulated by a class of inhibitor proteins known as IκB through the formation of stable IκB-NF-κB complexes that are unable to bind DNA. Activation of these dimers requires degradation of IκB. A large number of stimuli activate IκB degradation through phosphorylation of IκB by IκB kinase (IKK) leading to ubiquitination, 26 S proteasome recruitment, and degradation of IκB by the proteasome (3Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3293) Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4084) Google Scholar). Signaling pathways leading to NF-κB activation through degradation of classical IκB proteins (IκBα, IκBβ, and IκB∈) are classified as the canonical pathways. RelB displays characteristics that are not shared by the other NF-κB subunits: 1) Prototypical IκB proteins do not regulate RelB-containing NF-κB dimers (5Solan N.J. Miyoshi H. Carmona E.M. Bren G.D. Paya C.V. J. Biol. Chem. 2002; 277: 1405-1418Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 6Lernbecher T. Kistler B. Wirth T. EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (83) Google Scholar). 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 (7Ruben S.M. Klement J.F. Coleman T.A. Maher M. Chen C.H. Rosen C.A. Genes Dev. 1992; 6: 745-760Crossref PubMed Scopus (103) Google Scholar). 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-κB family members (8Ryseck R.P. Bull P. Takamiya M. Bours V. Siebenlist U. Dobrzanski P. Bravo R. Mol. Cell Biol. 1992; 12: 674-684Crossref PubMed Scopus (277) Google Scholar). 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 (9Huang D.B. Vu D. Ghosh G. Structure (Camb.). 2005; 13: 1365-1373Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 5) In unstimulated cells, RelB primarily associates with p100 and to a lesser extent with p50 (5Solan N.J. Miyoshi H. Carmona E.M. Bren G.D. Paya C.V. J. Biol. Chem. 2002; 277: 1405-1418Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In induced cells the predominant RelB dimer is the RelB-p52 heterodimer. This stringent specificity for both the inhibited complex (RelB-p100) and the transcriptionally active complexes (RelB-p52 and RelB-p52) is unusual in the NF-κB family. A distinct class of inducers such as LTβ, BAFF, and CD40 activate the noncanonical pathways, which result in the formation of the RelB-p52 heterodimer (10Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 11Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1136) Google Scholar, 12Dejardin E. Droin N.M. Delhase M. Haas E. Cao Y. Makris C. Li Z.W. Karin M. Ware C.F. Green D.R. Immunity. 2002; 17: 525-535Abstract Full Text Full Text PDF PubMed Scopus (777) Google Scholar, 13Claudio E. Brown K. Park S. Wang H. Siebenlist U. Nat. Immunol. 2002; 3: 958-965Crossref PubMed Scopus (577) Google Scholar, 14Coope H.J. Atkinson P.G. Huhse B. Belich M. Janzen J. Holman M.J. Klaus G.G. Johnston L.H. Ley S.C. EMBO J. 2002; 21: 5375-5385Crossref PubMed Scopus (368) Google Scholar, 15Bonizzi G. Bebien M. Otero D.C. Johnson-Vroom K.E. Cao Y. Vu D. Jegga A.G. Aronow B.J. Ghosh G. Rickert R.C. Karin M. EMBO J. 2004; 23: 4202-4210Crossref PubMed Scopus (280) Google Scholar). The key event in the noncanonical pathways is the processing of p100 into p52 by the proteasome, which requires the activation of NF-κB-inducing kinase and IKK1 (10Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 11Senftleben U. Cao Y. Xiao G. Greten F.R. Krahn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.C. Karin M. Science. 2001; 293: 1495-1499Crossref PubMed Scopus (1136) Google Scholar, 16Xiao G. Fong A. Sun S.C. J. Biol. Chem. 2004; 279: 30099-30105Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Fong A. Sun S.C. J. Biol. Chem. 2002; 277: 22111-22114Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Secondary lymphoid organ development and maintenance are defective in mice deficient in RelB, p52, LTβ, IKK1, or NF-κB-inducing kinase, suggesting a close functional connection between components of the non-canonical pathway (18Weih F. Carrasco D. Durham S.K. Barton D.S. Rizzo C.A. Ryseck R.P. Lira S.A. Bravo R. Cell. 1995; 80: 331-340Abstract Full Text PDF PubMed Scopus (710) Google Scholar, 19Miyawaki S. Nakamura Y. Suzuka H. Koba M. Yasumizu R. Ikehara S. Shibata Y. Eur. J. Immunol. 1994; 24: 429-434Crossref PubMed Scopus (300) Google Scholar, 20Ishikawa H. Carrasco D. Claudio E. Ryseck R.P. Bravo R. J. Exp. Med. 1997; 186: 999-1014Crossref PubMed Scopus (159) Google Scholar, 21Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Crossref PubMed Scopus (368) Google Scholar, 22Fu Y.X. Chaplin D.D. Annu. Rev. Immunol. 1999; 17: 399-433Crossref PubMed Scopus (566) Google Scholar, 23Shinkura R. Kitada K. Matsuda F. Tashiro K. Ikuta K. Suzuki M. Kogishi K. Serikawa T. Honjo T. Nat. Genet. 1999; 22: 74-77Crossref PubMed Scopus (381) Google Scholar). The present study aims to investigate the structural and functional relationships between RelB and p100/p52. Understanding the reliance of RelB on specific NF-κB and IκB subunits may shed light into the mechanism of p100 processing and the formation of RelB-p52 heterodimer. In this report, we show that the stability of RelB protein requires the presence of p100/p52 and p105/p50. However, RelB uses two distinct modes to interact with p100 and p105. RelB binds to p105 through the N-terminal p50 region of p105, whereas contacts between p100 and RelB are more extensive. Protein-protein contact between RelB and p52 also involves the N-terminal domains, which is not known to occur in non-RelB NF-κB dimers. The intimate association between p100/p52 and RelB explains why RelB preferentially forms a complex with p100. This association may imply their reliance for each other for their biological function. Escherichia coli Expression and Protein Purification—Human p100 CTD(344–899), p100 CTDΔGRR(406–899), p100 and p100 into with an N-terminal into with a N-terminal and a and into from with an N-terminal and a into used for and into used to form the RelB-p52 complex used for and RelB the as RelB DD, which (9Huang D.B. Vu D. Ghosh G. Structure (Camb.). 2005; 13: 1365-1373Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). proteins using by of the used in this study by DNA of the of protein and binding partner in a and and for of of The for The by with The with and proteins by and by or and RelB and as The heterodimer formed by a of to p52 in a and with a protein of The heterodimer formed by by using the the complex by to RelB and the heterodimer and p52 homodimer A from to The and the S by as S. T. Ghosh G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of p100 with of RelB-p52 heterodimer with DNA in and DNA p100 and a of p100 by and into which by fluorescent protein and a or C-terminal RelB by and into to C-terminal proteins in embryonic cells with with and The cells in and and the using The when to that of DNA RelB and and all from and from and p52 a from Cell of the into the and as S. H. V. E. C.A. Ware C.F. Ghosh G. A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). and and cells in with and The by cells by with and inhibitor of protein from the by by to using specific with of with of or of by the in the in the presence of of protein complex and by by cells of wild and by in and and for The with by the of and by in the by and specific for RelB by a of the p100 into a by transcription in the presence of a The with by with and the by The products and by The the of protein as a of the p52 and p52 as D.B. Wang Vu D. Ghosh G. J. Mol. Biol. PubMed Google using a DNA the human and as the p100 RelB in the between RelB and p100 in we the of RelB in wild and mouse embryonic RelB is in cells the association between RelB and p100 in cells We using that the level of RelB in cells is not reduced when with cells p50 and p52 do not an role in the transcription of RelB. The level of RelB is in cells, which that RelB is induced by NF-κB However, the p100 protein level is reduced in cells, suggesting that the p100 in regulated by and The of p100/p52 protein level to be the for reduced of RelB. in RelB protein with p100 by RelB in In these cells, of both RelB and p100 are that in cells and these that in to other modes of RelB is also regulated the level of protein the proteasome is involved in the regulation of RelB protein we used the proteasome inhibitor and the presence of the inhibitor RelB protein and cells with for a in the RelB protein level as with cells The lack of of RelB protein level in the inhibitor cells that the proteasome role in RelB factors may also be p52 RelB by the RelB-p52 cells, RelB associates with p52, the processed of p100. This that the stabilization of RelB in induced cells also be by p52. investigate and p52 stabilizes RelB, we used a the RelB protein or with or RelB with in the of degradation products when RelB is with p52 or p100 as with when RelB is In p52 as a stable protein of the presence or absence of RelB and We also the stability of RelB in RelB is also an unstable protein that to products. In the presence of p100 or p52, the of RelB are These to the that unstable RelB be stabilized by both p100 and p52 We a of in to the between RelB and p52. In to other NF-κB RelB is to from an E. coli because The is that the stability of RelB is E. coli to the regions of RelB RelB is with p52 RHR, a stable RelB-p52 heterodimer is formed that be to using an and to a heterodimer between RelB and p52 not of the RelB of p52 a The RelB does not bind to the and in the through of the a of the complex be which to degradation This that the of p52 is for the stability of the RelB-p52 heterodimer and that the of p52 a role in the RelB-p52 heterodimer. to the RelB-p52 we also that the formation of the heterodimer the of p50 not In both the heterodimer and the RelA heterodimer be formed with S. T. Ghosh G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the of p52 and p50 for the stability of RelB to be unique to RelB. the stabilization role of p52 for RelB, we have p52 in In these cells, RelB protein level is significantly the RelB protein level in cells deficient in p100/p52 and These that RelB be stabilized by p52 and p50 and that RelB has a unique interaction with p52 and has that the processing of p100 into p52 in the on DNA. is possible that the stabilization of RelB by p52 may p52, which is in DNA this we have stable cells the p52 These two are the NF-κB family and have to contact DNA in all in RelA and p50 significantly their DNA binding both in and in cells Ghosh D. F. Mol. Cell Biol. 13: PubMed Google Scholar). the is defective in DNA binding show that this DNA of p52 is of RelB in cells these that both p100 and p52 RelB. to RelB RelB interaction specificity for both p100 and p105. We to p105 and RelB also show that p105 interacts with RelB to a extent as RelA these that RelB interacts with both the p100/p52 and p105/p50. These also an for the stabilization of RelB protein in the absence of p100/p52. has that unlike p100, p105 unable to RelB in the cytoplasm (5Solan N.J. Miyoshi H. Carmona E.M. Bren G.D. Paya C.V. J. Biol. Chem. 2002; 277: 1405-1418Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We that and structural p105 and p100 might bind RelB we both p100 and p105 as proteins and in the of p105 does not bind the binds the p50 homodimer complex and In the of p100 binds both RelB-p52 heterodimer and p52 that the RelB-p105 complex is unique RelB the of p50 to interact with the of p105. The modes of complex formation may explain in the of the RelB-p100 and RelB-p105 The RelB-p100 and RelB-p52 of p100 and the mechanism of specific complex formation between RelB and p100, we the role of each of the functional and structural domains of p100. p100 be into five regions on the known domain functional the NTD, DD, and the flexible also known as which the glycine-rich region ankyrin repeat domain and the domain in and that the of p52 interacts with RelB and These are with that the p52 a role in the RelB-p52 heterodimer. However, be that this interaction is and the of this contact is in the of the complex contacts to the complex. this interaction to be specific to the RelB-p52 complex because such interaction is in the complex not we p52 and RelA into RelA does not interact with the of p52 interaction between the of p100 with the RelB-p52 heterodimer (5Solan N.J. Miyoshi H. Carmona E.M. Bren G.D. Paya C.V. J. Biol. Chem. 2002; 277: 1405-1418Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). in show that the of p100 interacts with RelB and RelB and The association has by using of cells and RelB or RelB and A in interaction between RelB and the of p100 as with RelB implies that the of RelB may be involved in complex formation with p100. We in the regions the of the of p100 are involved in complex formation with the of p100 to of the binding we a more DNA binding to We used p100 p100 and p100 to their to DNA binding of the RelB-p52 heterodimer in a show that the of the and C-terminal regions reduced the activity of the of p100, suggesting that in the of p100 the is involved in the RelB-p52 The with to each of the RelB structural domains are involved in the RelB-p100 complex RelB be into NTD, DD, and the The of RelB a long the N terminus. for p100, we have the binding by two using proteins in and of proteins in that the RelB associates with p100 but not with p52 or The presence of a of RelB to the by and the role of this region in p100 using proteins revealed a in the binding interaction of the RelB-p52 heterodimer for p100 and We the role of the domain using a DNA binding This revealed that the presence of the N-terminal for the to the DNA binding of the heterodimer of RelB to p100 or near the of the role of the RelB in p100 show that p100 interacts with the of RelB. the C-terminal which the domain the of induced are not for stable interaction with RelB In of the N to the of p100 binding in binding The of RelB is involved in complex formation with both p100 and the and However, the RelB binds more to p100 p100 is possible that the in the This is by binding between the of RelB and p100 the of p100 to the of the ARD, binding to RelB and We that the of RelB directly interacts with the processing region of p100 the and RelA does not show binding interaction with p100, suggesting that RelB interacts with p100 in a unique In these that all known functional of RelB in the complex formation with of p100 NF-κB dimers are stable with the of RelB. is not the and domains that RelB the of RelB to be the for its lack of proteins are by degradation of which of the protein degradation is the We show that the proteasome is responsible for RelB However, is possible that other are involved in RelB RelB is from degradation by p100, its processed p52 and might also be other specific proteins, such as which has to interact with RelB through its C.F. E. Li P. G. F. Mol. 21: PubMed Scopus Google Scholar). The of flexible regions in the complex a mechanism by which RelB degradation by stabilization of proteins through association with their partners is a mechanism by Cell inhibitor and have to degradation are or to partner proteins J. M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. P. Y. C. Mol. Cell. 2005; 17: Full Text Full Text PDF PubMed Scopus Google Scholar, G. J. L. C. Y. U. S. A. 2002; PubMed Scopus Google Scholar). of protein stabilization also be from the In cells, is primarily by RelA and c-Rel and in their absence protein are significantly reduced A. A. D. Science. 2002; PubMed Scopus Google Scholar). the of The structure of the RelB homodimer revealed a lack of in these domains, which in explains why the might not be to fold and forms a domain swapped homodimer in the crystal (9Huang D.B. Vu D. Ghosh G. Structure (Camb.). 2005; 13: 1365-1373Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also in the RelB x-ray crystal structure D.B. Wang Vu D. Ghosh G. J. Mol. Biol. PubMed Google Scholar). These are with that the of RelB its degradation in cells R. F. A. E. M. 2001; PubMed Scopus Google Scholar). these structural and that the of RelB is to degradation because of its is that the lack of stability of RelB to interact with its partners more which not be possible RelB a more stable the presence of RelB in the absence of its partners result in between RelB and other proteins that be to the all the partner proteins of RelB, p100 to be the in its to RelB from does by a stable complex through binding between RelB and p100. from binding a complex of interaction between RelB and p100, different structural domains and flexible regions of both proteins in the complex The unique complex between RelB and p100 also that RelB may a role in the processing of p100 into p52. The interaction between the of RelB and the of processing of p100 that RelB may p100 the RelB-p100 complex to be an complex. This interaction is unique to the RelB-p100 complex because the interaction is not in the complex. The complex of RelB-p100 also an role for RelB in p100 with this that RelB p100 the specific domain arrangement in the RelB-p100 complex might as a to bind other NF-κB and IκB proteins. This large complex in cells specific NF-κB dimers through degradation of p100. that has is the and heterodimers through the degradation of p100 S. H. V. E. C.A. Ware C.F. Ghosh G. A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). RelB also interacts in a stable with p52. However, the RelB-p52 heterodimer is not to be as stable as the RelB-p100 complex because the and of RelB are in these are these RelB domains stabilized in the RelB-p52 do RelB from is that these RelB heterodimers in vivo as dimers. to the and with DNA and are involved in transcriptional these transcriptionally active dimers in the interacts with DNA and the interacts with other proteins. The unique between the p52 and RelB a This interaction the domains of both subunits in a a that is with DNA However, in the of non-RelB NF-κB the two in which DNA are two by which the of RelB-p52 heterodimer to bind is possible that the p52 binds and to the RelB primarily through contacts and between the dimers and DNA the to the is also possible that an active such as phosphorylation of of these proteins may the of the p52 from the RelB this is to that p52 has to be by G. Fong A. Sun S.C. J. Biol. Chem. 2004; 279: 30099-30105Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Although we do not these in p52 might a or role in the binding interaction with RelB, such a p105 and p50 also RelB through stable to p52. We that the binding is in both the RelB-p105 and the contacts are through the of p50 and RelB. complex formation between RelB and p50 explains RelB that are of p100. is in mice deficient in but not in mice deficient in The heterodimer may as a of key and such activity of RelB does not p100. In show that RelB is an unstable protein in vivo and requires specific partners for its However, the lack of RelB stability is not because many proteins are stabilized in a The unique complex between RelB and p100/p52 and p50 explain RelB specificity p100, p52, and p50 in are to the involved and the mechanism RelB and the role stable between RelB and p100 have on the processing of p100. We and for the and the for in the structural and We for the
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