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Inhibition of Transcription by B Cell Leukemia 3 (Bcl-3) Protein Requires Interaction with Nuclear Factor κB (NF-κB) p50*

  • Patricia E. Collins
    Affiliations
    Department of Biochemistry, University College Cork, Cork, Ireland
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  • Patrick A. Kiely
    Affiliations
    Department of Life Sciences and the Materials and Surface Science Institute, University of Limerick, Limerick, Ireland
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  • Ruaidhrí J. Carmody
    Correspondence
    To whom correspondence should be addressed: Institute of Infection, Immunity, and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
    Affiliations
    Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary and Life Sciences, University of Glasgow G12 8TA, Scotland, Glasgow, United Kingdom
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  • Author Footnotes
    * This work was supported by Science Foundation Ireland Grant 08/IN.1/B1843.
    2 The abbreviations used are:RHDRel homology domainMEFmouse embryonic fibroblastCREBcAMP response element-binding proteinIRFinterferon regulatory factorPPARγperoxisome proliferator-activated receptorγIKKIκB kinase.
Open AccessPublished:January 23, 2014DOI:https://doi.org/10.1074/jbc.M114.551986
      B cell leukemia 3 (Bcl-3) is an essential negative regulator of NF-κB during Toll-like receptor and TNF receptor signaling. Bcl-3 also interacts with a number of transcriptional regulators, including homodimers of the NF-κB p50 subunit. Deletion of Bcl-3 results in increased NF-κB p50 ubiquitination and proteasomal degradation and increased inflammatory gene expression. We employed immobilized peptide array technology to define a region of p50 required for the formation of a Bcl-3·p50 homodimer immunosuppressor complex. Our data demonstrate that amino acids 359–361 and 363 of p50 are critical for interaction with Bcl-3 and essential for Bcl-3-mediated inhibition of inflammatory gene expression. Bcl-3 is unable to interact with p50 when these amino acids are mutated, rendering it incapable of inhibiting the transcriptional activity of NF-κB. Bcl-3 interaction-defective p50 is hyperubiquitinated and has a significantly reduced half-life relative to wild-type p50. Nfkb1−/− cells reconstituted with mutated p50 precursor p105 are hyperresponsive to TNFα stimulation relative to wild-type p105, as measured by inflammatory gene expression. Mutant p105 recapitulates a Bcl3−/− phenotype. This study demonstrates that interaction with p50 is necessary and sufficient for the anti-inflammatory properties of Bcl-3 and further highlights the importance of p50 homodimer stability in the control of NF-κB target gene expression.

      Introduction

      The members of the NF-κB family of transcription factors are essential regulators of the immune response and control the expression of hundreds of immunomodulatory genes. This transcription factor family is encoded by five genes, resulting in five NF subunits (p65 (RelA), c-Rel, RelB, p50, and p52) that, through the formation of hetero- and homodimers, can potentially generate 15 distinct complexes (
      • Hayden M.S.
      • Ghosh S.
      Shared principles in NF-κB signaling.
      ). All subunits of NF-κB share significant sequence homology and are characterized by the Rel homology domain (RHD)
      The abbreviations used are:
      RHD
      Rel homology domain
      MEF
      mouse embryonic fibroblast
      CREB
      cAMP response element-binding protein
      IRF
      interferon regulatory factor
      PPARγ
      peroxisome proliferator-activated receptorγ
      IKK
      IκB kinase.
      , which is essential for dimerization and DNA binding. Genetic studies in mice have demonstrated that individual NF-κB subunits carry out specific biological roles (
      • Gerondakis S.
      • Grossmann M.
      • Nakamura Y.
      • Pohl T.
      • Grumont R.
      Genetic approaches in mice to understand Rel/NF-κB and IκB function. Transgenics and knockouts.
      ), which may be due to distinct subunit specific cofactors (
      • Ghosh S.
      • Hayden M.S.
      New regulators of NF-κB in inflammation.
      ) as well as dimer-specific affinities for variable DNA binding sequences (
      • Siggers T.
      • Chang A.B.
      • Teixeira A.
      • Wong D.
      • Williams K.J.
      • Ahmed B.
      • Ragoussis J.
      • Udalova I.A.
      • Smale S.T.
      • Bulyk M.L.
      Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding.
      ).
      The NF-κB p50 and p52 subunits share a number of properties that distinguish them from other NF-κB subunits. Firstly, they are both generated from the limited proteolytic processing of a precursor: p105 in the case of p50 and p100 in the case of p52. Secondly, p50 and p52 do not contain a transactivation domain and, when complexed as homodimers, they do not possess intrinsic transactivational activity (
      • Hayden M.S.
      • Ghosh S.
      Shared principles in NF-κB signaling.
      ). In this regard, p50 homodimers are generally considered as repressors of NF-κB transcription. Interestingly, p50 homodimers have also recently been identified as negative regulators of interferon regulatory factor (IRF)-mediated transcription by binding to interferon-responsive elements in interferon-inducible gene promoters, which act as an NF-κB half-site (
      • Cheng C.S.
      • Feldman K.E.
      • Lee J.
      • Verma S.
      • Huang D.B.
      • Huynh K.
      • Chang M.
      • Ponomarenko J.V.
      • Sun S.C.
      • Benedict C.A.
      • Ghosh G.
      • Hoffmann A.
      The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50.
      ). A significant number of p50 homodimers are present in the nuclei of unstimulated cells, which indicates that p50 plays a critical role in regulating the basal as well as inducible transcription of target genes (
      • Cheng C.S.
      • Feldman K.E.
      • Lee J.
      • Verma S.
      • Huang D.B.
      • Huynh K.
      • Chang M.
      • Ponomarenko J.V.
      • Sun S.C.
      • Benedict C.A.
      • Ghosh G.
      • Hoffmann A.
      The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50.
      ).
      In resting cells, NF-κB is sequestered in the cytoplasm through association with members of the IκB protein family, which include IκBα, IκBβ, IκBϵ, as well as p105 and p100. Activating stimuli trigger IκB kinase (IKK)-mediated phosphorylation of cytoplasmic IκB, leading to its ubiquitination and subsequent proteasomal degradation, thereby allowing NF-κB to translocate to the nucleus. There are also a number of atypical IκB proteins, which include Bcl-3, IκBζ, and IκBNS, which reside predominantly in the nucleus and are not degraded following activation of the IKK complex (
      • Carmody R.J.
      • Chen Y.H.
      Nuclear factor-κB. Activation and regulation during Toll-like receptor signaling.
      ). Bcl-3 is perhaps the best studied of the atypical IκB proteins. It was originally identified at the breakpoint junction t(14;19) in B cell chronic lymphocytic leukemia and is classified as an IκB family member by virtue of its seven ankyrin repeat domains that mediate selective interaction with homodimers of NF-κB p50 and p52 (
      • Palmer S.
      • Chen Y.H.
      Bcl-3, a multifaceted modulator of NF-κB-mediated gene transcription.
      ). Bcl-3 has also been reported to interact with the AP-1 transcription factors c-Jun and c-fos (
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); STAT1 (
      • Jamaluddin M.
      • Choudhary S.
      • Wang S.
      • Casola A.
      • Huda R.
      • Garofalo R.P.
      • Ray S.
      • Brasier A.R.
      Respiratory syncytial virus-inducible BCL-3 expression antagonizes the STAT/IRF and NF-κB signaling pathways by inducing histone deacetylase 1 recruitment to the interleukin-8 promoter.
      ); peroxisome proliferator-activated receptorγ (PPARγ) (
      • Yang J.
      • Williams R.S.
      • Kelly D.P.
      Bcl3 interacts cooperatively with peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α to coactivate nuclear receptors estrogen-related receptor α and PPARα.
      ); CREB-binding protein/p300 (
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); HDAC-1, -3, and -5 (
      • Viatour P.
      • Dejardin E.
      • Warnier M.
      • Lair F.
      • Claudio E.
      • Bureau F.
      • Marine J.C.
      • Merville M.P.
      • Maurer U.
      • Green D.
      • Piette J.
      • Siebenlist U.
      • Bours V.
      • Chariot A.
      GSK3-mediated BCL-3 phosphorylation modulates its degradation and its oncogenicity.
      ); steroid receptor coactivator 1 (
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); TORC3 (
      • Hishiki T.
      • Ohshima T.
      • Ego T.
      • Shimotohno K.
      BCL3 acts as a negative regulator of transcription from the human T-cell leukemia virus type 1 long terminal repeat through interactions with TORC3.
      ); and the retinoic X receptor (
      • Na S.Y.
      • Choi H.S.
      • Kim J.W.
      • Na D.S.
      • Lee J.W.
      Bcl3, an IκB protein, as a novel transcription coactivator of the retinoid X receptor.
      ). Bcl-3 has also been reported to interact with the Src-related kinases Fyn (
      • Weyrich A.S.
      • Dixon D.A.
      • Pabla R.
      • Elstad M.R.
      • McIntyre T.M.
      • Prescott S.M.
      • Zimmerman G.A.
      Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets.
      ) and Lck (
      • Zhao Y.
      • Ramakrishnan A.
      • Kim K.E.
      • Rabson A.B.
      Regulation of Bcl-3 through interaction with the Lck tyrosine kinase.
      ) as well as insulin receptor substrate 3 (IRS3) (
      • Kabuta T.
      • Hakuno F.
      • Cho Y.
      • Yamanaka D.
      • Chida K.
      • Asano T.
      • Wada K.
      • Takahashi S.
      Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-κB activity.
      ) and Bag-1 (
      • Southern S.L.
      • Collard T.J.
      • Urban B.C.
      • Skeen V.R.
      • Smartt H.J.
      • Hague A.
      • Oakley F.
      • Townsend P.A.
      • Perkins N.D.
      • Paraskeva C.
      • Williams A.C.
      BAG-1 interacts with the p50-p50 homodimeric NF-κB complex. Implications for colorectal carcinogenesis.
      ). Previous data suggest that Bcl-3-mediated stabilization of p50 is required for limiting NF-κB transcriptional activity. In Bcl3−/− macrophages, p50 homodimers undergo increased ubiquitination and proteasomal degradation. Bcl3−/− cells and mice are hyperresponsive to Toll-like receptor and TNF receptor stimulation, as measured by the increased transcription of genes, including TNFα and IL-6 (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). The nature of the interaction between Bcl-3 and p50 has not been explored experimentally, and it is not clear whether Bcl-3 interaction with p50 is essential for the inhibition of cytokine expression or whether the other binding partners of Bcl-3 are also important. This information is critical for our understanding of the regulation of NF-κB activity and cytokine expression by Bcl-3 and p50 homodimers.
      Here we employed immobilized peptide array technology to identify the regions of p50 essential for interaction with Bcl-3. We identified amino acids 359–361 and 363 of p50 as critical residues required for the interaction of p50 homodimers with Bcl-3. This allowed us to generate a p50 mutant that is unable to interact with Bcl-3 but that retains normal dimerization and DNA-binding properties. p50 homodimers that are unable to interact with Bcl-3 undergo increased ubiquitination that is not inhibited by overexpression of Bcl-3. Importantly, the Bcl-3 interaction-defective p50 mutant is also defective in its ability to repress NF-κB target gene expression. Overexpression of Bcl-3 is unable to repress NF-κB-mediated gene expression in cells expressing the mutant form of p50. Our study describes the experimental characterization of the interaction between p50 and Bcl-3 and demonstrates, for the first time, that a direct interaction between Bcl-3 and p50 is required for the stability of p50 homodimers and is necessary for the anti-inflammatory function of Bcl-3.

      DISCUSSION

      Bcl-3 is an IκB protein that regulates NF-κB-dependent gene expression through interaction with NF-κB p50 and p52 homodimers (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). In addition to the NF-κB subunits, Bcl-3 interacts with a number of regulators of transcription, including c-Jun and c-fos (
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); STAT1 (
      • Jamaluddin M.
      • Choudhary S.
      • Wang S.
      • Casola A.
      • Huda R.
      • Garofalo R.P.
      • Ray S.
      • Brasier A.R.
      Respiratory syncytial virus-inducible BCL-3 expression antagonizes the STAT/IRF and NF-κB signaling pathways by inducing histone deacetylase 1 recruitment to the interleukin-8 promoter.
      ); PPARγ (
      • Yang J.
      • Williams R.S.
      • Kelly D.P.
      Bcl3 interacts cooperatively with peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α to coactivate nuclear receptors estrogen-related receptor α and PPARα.
      ); CREB-binding protein/p300 (
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); HDAC-1, -3, and -5 (
      • Viatour P.
      • Dejardin E.
      • Warnier M.
      • Lair F.
      • Claudio E.
      • Bureau F.
      • Marine J.C.
      • Merville M.P.
      • Maurer U.
      • Green D.
      • Piette J.
      • Siebenlist U.
      • Bours V.
      • Chariot A.
      GSK3-mediated BCL-3 phosphorylation modulates its degradation and its oncogenicity.
      ); steroid receptor coactivator 1(
      • Na S.Y.
      • Choi J.E.
      • Kim H.J.
      • Jhun B.H.
      • Lee Y.C.
      • Lee J.W.
      Bcl3, an IκB protein, stimulates activating protein-1 transactivation and cellular proliferation.
      ); TORC3 (
      • Hishiki T.
      • Ohshima T.
      • Ego T.
      • Shimotohno K.
      BCL3 acts as a negative regulator of transcription from the human T-cell leukemia virus type 1 long terminal repeat through interactions with TORC3.
      ); and retinoic X receptor (
      • Na S.Y.
      • Choi H.S.
      • Kim J.W.
      • Na D.S.
      • Lee J.W.
      Bcl3, an IκB protein, as a novel transcription coactivator of the retinoid X receptor.
      ); Fyn (
      • Weyrich A.S.
      • Dixon D.A.
      • Pabla R.
      • Elstad M.R.
      • McIntyre T.M.
      • Prescott S.M.
      • Zimmerman G.A.
      Signal-dependent translation of a regulatory protein, Bcl-3, in activated human platelets.
      ) and Lck (
      • Zhao Y.
      • Ramakrishnan A.
      • Kim K.E.
      • Rabson A.B.
      Regulation of Bcl-3 through interaction with the Lck tyrosine kinase.
      ); insulin receptor substrate 3 (IRS3) (
      • Kabuta T.
      • Hakuno F.
      • Cho Y.
      • Yamanaka D.
      • Chida K.
      • Asano T.
      • Wada K.
      • Takahashi S.
      Insulin receptor substrate-3, interacting with Bcl-3, enhances p50 NF-κB activity.
      ); and Bag-1 (
      • Southern S.L.
      • Collard T.J.
      • Urban B.C.
      • Skeen V.R.
      • Smartt H.J.
      • Hague A.
      • Oakley F.
      • Townsend P.A.
      • Perkins N.D.
      • Paraskeva C.
      • Williams A.C.
      BAG-1 interacts with the p50-p50 homodimeric NF-κB complex. Implications for colorectal carcinogenesis.
      ). The contribution of these factors to immune regulation by Bcl-3 has not been determined previously. Bcl-3 limits NF-κB-mediated transcription of proinflammatory cytokines and promote LPS tolerance, a state of hyporesponsiveness following repeated or prolonged exposure to LPS (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). Previous studies have demonstrated that Bcl-3 regulates p50 homodimer stability through the inhibition of ubiquitination and subsequent proteasomal-mediated degradation (
      • Carmody R.J.
      • Ruan Q.
      • Palmer S.
      • Hilliard B.
      • Chen Y.H.
      Negative regulation of Toll-like receptor signaling by NF-κB p50 ubiquitination blockade.
      ). However, studies employing Bcl3−/− cells and mice do not exclude the possibility that Bcl-3 may also function through p50-independent mechanisms to regulate gene expression. Here, by identifying the critical site of p50 necessary for interaction with Bcl-3, we provide the first direct evidence that Bcl-3-mediated repression of NF-κB-mediated gene expression requires interaction with p50. Furthermore, our data demonstrate that p50 that cannot interact with Bcl-3 is inherently unstable and undergoes increased ubiquitination and degradation relative to wild-type p50, even in the presence of overexpressed Bcl-3. Our data also suggest that p50-independent targets of Bcl-3 are not important in the regulation of NF-κB-dependent inflammatory gene expression.
      In this study, we employed peptide array techniques to identify residues of p50 necessary for interaction with Bcl-3. This approach, which uses immobilized peptides representing the p50 protein sequence, has distinct advantages over deletional mutagenic approaches. Perhaps chief among these is the avoidance of potential artifacts on the formation of multisubunit complexes, which may result from the deletion of significant portions of a protein. For example, the RHD of p50 is essential for dimerization as well as DNA binding. Even partial deletion of the RHD may have consequences on the formation of a Bcl-3·p50 complex. In this study, the immobilized peptide array approach allowed us to identify a series of overlapping peptides with a strong affinity for purified recombinant Bcl-3 corresponding to amino acids 337–378 at the C-terminal region of p50. The serial substitution of amino acids 355–372 with alanine as immobilized peptide arrays further increased the resolution of the site and identified Arg-359, Lys-360, Arg-361 and Lys-363 as essential residues for interaction with Bcl-3. Subsequently, the mutation of Arg-359, Lys-360, and Arg-361 to alanine in full-length p50 blocked the interaction with purified, recombinant Bcl-3. These data validate peptide array technology as a valuable tool in identifying sites of protein-protein interaction with amino acid level resolution.
      The region of p50 containing Arg-359, Lys-360, and Arg-361 is C-terminal to previously identified ankyrin repeat domain interaction sites determined from the crystal structure of a p65/p50 heterodimer complexed with IκBα. Unfortunately, amino acids 359–361 are not represented on the crystal structures of p50-containing complexes, and, therefore, no structural data are available. However, a hydrophobicity plot reveals that these amino acids lie in a region of low hydrophobicity and, thus, are expected to be available for interaction with Bcl-3. Importantly, mutation of these amino acids does not alter the hetero- or homodimer formation properties of p50 or interfere with DNA binding of p50. This allows us to rule out the loss of repressor function of p50RKR homodimers because of a lack of dimerization or DNA binding. Moreover, despite being located in a region reported previously to be important for nuclear localization (
      • Latimer M.
      • Ernst M.K.
      • Dunn L.L.
      • Drutskaya M.
      • Rice N.R.
      The N-terminal domain of IκB α masks the nuclear localization signal(s) of p50 and c-Rel homodimers.
      ), the mutation of Arg-359, Lys-360, and Arg-361 to alanine had no effect on the nuclear localization of p50, as monitored by subcellular fractionation analysis and DNA binding assays. Our data suggest that additional sequences of p50 are important in its nuclear translocation.
      The mutation of Arg-359, Lys-360, and Arg-361 in p50 (p50RKR) functionally recapitulates the phenotype of Bcl3−/− cells described previously. Thus, p50RKR undergoes increased ubiquitination corresponding to a reduced half-life, and cells expressing p50RKR display increased NF-κB transcriptional activity relative to wild-type p50. Critically, Bcl-3 is unable to rescue the increased NF-κB activity in p50RKR-expressing cells. NF-κB transactivation is not inhibited by overexpression of Bcl-3 in cells expressing p50RKR, and Bcl-3 is unable to inhibit p50RKR ubiquitination. The importance of p50 in regulating transcriptional programs during inflammation has been highlighted by two recent studies of Toll-like receptor- (
      • Yan Q.
      • Carmody R.J.
      • Qu Z.
      • Ruan Q.
      • Jager J.
      • Mullican S.E.
      • Lazar M.A.
      • Chen Y.H.
      Nuclear factor-κB binding motifs specify Toll-like receptor-induced gene repression through an inducible repressosome.
      ) and interferon-induced responses (
      • Cheng C.S.
      • Feldman K.E.
      • Lee J.
      • Verma S.
      • Huang D.B.
      • Huynh K.
      • Chang M.
      • Ponomarenko J.V.
      • Sun S.C.
      • Benedict C.A.
      • Ghosh G.
      • Hoffmann A.
      The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50.
      ). Our study further highlights the importance of Bcl-3 and p50 interaction in the regulation of the inflammatory response independently of the interaction of Bcl-3 with other transcriptional regulators.

      Acknowledgment

      We thank Karen Keeshan for critical evaluation of the manuscript.

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