Critical Role of RelB Serine 368 for Dimerization and p100 Stabilization
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m301521200
ISSN1083-351X
AutoresHarald J. Maier, Ralf Marienfeld, Thomas Wirth, Bernd Baumann,
Tópico(s)Ferrocene Chemistry and Applications
ResumoIn mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory κB proteins. In most other cell types inhibitory κB proteins prevent nuclear translocation and activation of NFκB. 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 NFκB 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. In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory κB proteins. In most other cell types inhibitory κB proteins prevent nuclear translocation and activation of NFκB. 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 NFκB 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. NFκB represents a family of dimeric transcription factors consisting of the mammalian members p50 (NFκB1), p52 (NFκB2), RelA (p65), RelB, and c-Rel. NFκB regulates the expression of a variety of genes, many of which are involved in immune and inflammatory responses. In addition, NFκB target genes play a role in cell growth and differentiation as well as in neoplastic transformation (reviewed in Refs. 1Denk A. Wirth T. Baumann B. Cytokine Growth Factor Rev. 2000; 11: 303-320Crossref PubMed Scopus (114) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4611) Google Scholar, 3Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2451) Google Scholar, 4Karin M. Cao Y. Greten F.R. Li Z.W. Nat. Rev. Cancer. 2002; 2: 301-310Crossref PubMed Scopus (2258) Google Scholar). 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 protein-protein interaction, DNA binding, and nuclear localization. In most cell types, NFκB proteins are retained in the cytoplasm by inhibitory κB proteins, the IκBs, 1The abbreviations used are: IκBα, inhibitor κBα; IKK, IκB kinase; EMSA, electrophoretic mobility shift assay; NFκB, nuclear factor κB; NIK, NFκB-inducing kinase; PBS, phosphate-buffered saline; WT, wild type. which comprise IκBα, IκBβ, IκBγ, IκBϵ, and Bcl-3. Moreover, the precursors p100 and p105 are also known to display inhibitory functions and therefore are also classified as IκBs (5Whiteside S.T. Israel A. Semin. Cancer Biol. 1997; 8: 75-82Crossref PubMed Scopus (300) Google Scholar, 6Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4086) Google Scholar). The IκB proteins are not restricted to the cytoplasm but have been shown to retrieve NFκB heterodimers from the nucleus and escort them back to the cytoplasm (7Carlotti F. Dower S.K. Qwarnstrom E.E. J. Biol. Chem. 2000; 275: 41028-41034Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). There are different pathways for induction of NFκB. In most cell types, stimulation with a variety of substances leads to activation of the IκB kinase complex (IKK). This complex consists of the catalytic subunits IKKα and IKKβ and the regulatory IKKγ subunit (also called NEMO, NFκB essential modulator). In the classical pathway, the activated IKK, predominantly IKKβ, catalyzes the phosphorylation of IκB. Subsequently, IκB is ubiquitinated and degraded, thereby releasing the NFκB proteins. As a consequence, NFκB (in this pathway, especially the heterodimer p50-RelA) can translocate to the nucleus, bind to DNA, and activate gene transcription (8Ghosh S. Karin M. Cell. 2002; 109: S81-S96Abstract Full Text Full Text PDF PubMed Scopus (3294) Google Scholar). Recently, a new pathway for NFκB activation has been proposed, and the IKK complex mediating this alternative pathway differs from the one in the classical pathway (9Senftleben 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 (1137) Google Scholar, 10Pomerantz J.L. Baltimore D. Mol. Cell. 2002; 10: 693-695Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 11Dixit V. Mak T.W. Cell. 2002; 111: 615-619Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This pathway is dependent on new protein synthesis, involves IKKα rather than IKKβ and IKKγ (NFκB essential modulator), and the signal is not propagated via IκB, but via p100, which is processed to p52. It has also been shown that NFκB-inducing kinase (NIK) positively regulates p100 processing, but it remains unclear whether NIK exerts its effect directly upon p100 (12Xiao G. Harhaj E.W. Sun S.C. Mol. Cell. 2001; 7: 401-409Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar) or whether it does so via IKKα activation (9Senftleben 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 (1137) Google Scholar). This alternative pathway can be triggered by lymphotoxin-β receptor and B cell-activating factor receptor ligation as well as CD40L and lipopolysaccharide signaling (13Coope H.J. Atkinson P.G. Huhse B. Belich M. Janzen J. Holman M.J. Klaus G.G. Johnston L.H. Ley S.C. 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The exact mechanisms of this constitutive activity remain unclear; the alternative pathway just mentioned may be involved. Whereas predominantly p50-RelA heterodimers are responsible for inducible NFκB activity, RelB- and partially c-Rel-containing complexes are involved in this constitutive nuclear activity (19Lernbecher T. Müller U. Wirth T. Nature. 1993; 365: 767-770Crossref PubMed Scopus (211) Google Scholar, 20Dobrzanski P. Ryseck R.-P. Bravo R. EMBO J. 1994; 13: 4608-4616Crossref PubMed Scopus (106) Google Scholar). The finding that RelB expression is enhanced in tissues with constitutive activity and the observation that RelB-containing complexes are less susceptible to inhibition by IκBα further underline the important role of RelB in this context (21Lernbecher T. Kistler B. Wirth T. EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (83) Google Scholar). However, it is not clear how RelB is protected from inhibition in mature B cells; one possibility could be some kind of post-translational modification that only occurs in lymphoid tissue. RelB itself cannot form homodimers, nor does it apparently dimerize with RelA or c-Rel; however, it can form heterodimers with p50 and p52 (20Dobrzanski P. Ryseck R.-P. Bravo R. EMBO J. 1994; 13: 4608-4616Crossref PubMed Scopus (106) Google Scholar, 22Ryseck 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). These heterodimers can induce transcription because of the carboxyl- and amino-terminal transactivation domains of RelB, both of which are required for full transactivation (23Dobrzanski P. Ryseck R.-P. Bravo R. Mol. Cell Biol. 1993; 13: 1572-1582Crossref PubMed Scopus (72) Google Scholar). The NFκB/Rel protein family does not only exist in mammalians but is also found in Drosophila, where the three known family members are called Dorsal, Dif, and Relish. Dorsal, a Rel homologue, is retained in the cytoplasm by the IκB protein Cactus, which can be phosphorylated and degraded in a signal-dependent manner. This process finally results in Dorsal release and nuclear translocation (24Belvin M.P. Jin Y. Anderson K.V. Genes Dev. 1995; 9: 783-793Crossref PubMed Scopus (151) Google Scholar, 25Drier E.A. Steward R. Semin. Cancer Biol. 1997; 8: 83-92Crossref PubMed Scopus (47) Google Scholar). In the early embryo, however, Dorsal itself is phosphorylated and subsequently translocates to the nucleus, an event that is crucial for establishing a ventral to dorsal nuclear gradient and thus determining polarity of the embryo (26Roth S. Stein D. Nusslein-Volhard C. Cell. 1989; 59: 1189-1202Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 27Drier E.A. Huang L.H. Steward R. Genes Dev. 1999; 13: 556-568Crossref PubMed Scopus (80) Google Scholar). The site of this phosphorylation event, serine 317, is evolutionarily conserved in all NFκB/Rel proteins, suggesting an important role also in the mammalian system (27Drier E.A. Huang L.H. Steward R. Genes Dev. 1999; 13: 556-568Crossref PubMed Scopus (80) Google Scholar). When we compared the amino acid sequence in the neighborhood of this serine, it became clear that the highest conservation is observed in the RelB protein. While phosphorylation of serine 317 regulates constitutive nuclear import of Dorsal, the constitutive presence of RelB in lymphoid tissue apparently also depends on a so-far unknown post-translational modification of RelB in mature B cells (21Lernbecher T. Kistler B. Wirth T. EMBO J. 1994; 13: 4060-4069Crossref PubMed Scopus (83) Google Scholar). Thus, serine 368 might be involved in the tissue-specific constitutive nuclear translocation of RelB. In this study, we therefore addressed the question whether nuclear targeting of RelB in mature B cells is regulated by phosphorylation at serine 368, the conserved residue of serine 317 in Dorsal. To examine this hypothesis, we generated serine to alanine and serine to glutamic acid mutations at position 368 of RelB and analyzed whether these mutations had an impact on nuclear translocation of RelB in mammalian cells. We report here that serine 368 is not involved in a phosphorylation-dependent mechanism regulating the subcellular localization of RelB but is essential for RelB dimerization. In addition, our results also demonstrate that RelB dimerization with p100 protects p100 from processing to p52. Site-directed Mutagenesis—For mutagenesis of the RelB MexNeo and pCDNA3 RelA plasmids, a kit from Stratagene (QuikChange XL site-directed mutagenesis kit) was used according to the manufacturer's protocol. The plasmids were denatured, and the mutagenic primers were annealed. After extension of the oligonucleotide primers, non-mutated parental DNA was removed by treatment with DpnI endonuclease. After transformation into competent bacteria, the received constructs were sequenced to confirm the point mutations and the integrity of the remaining protein. Mutagenic primers used were RelB S368A, sense (GATGGG GTGTGCGCCGAGCCGCTGCC), antisense (GGCAGCGGCTCGGCGCACACCCCATC); RelB S368E, sense (GATGGGGTGTGCGAGGAGCCGCTGCC), antisense, (GGCAGCGGCTCCTCGCACACCCCATC); RelA S281A, sense (CCGACCGGGAGCTCGCTGAGCCCATGGAATTCC), antisense (GGAATTCCATGGGCTCAGCGAGCTCCCGGTCGG); RelA S281E, sense (CCGACCGGGAGCTCGAGGAGCCCATGGAATTCC), antisense (GGAATTCCATGGGCTCCTCGAGCTCCCGGTCGG). Cell Culture—S107, Jurkat T, and NIH 3T3 cells were grown in Dulbecco's modified Eagle medium (Invitrogen) containing 10% heat-inactivated fetal calf serum (PAN Systems, Aidenbach, Germany), 100 units/ml penicillin, 100 μg/ml streptomycin, supplemented with 50 μm β-mercaptoethanol (final concentration). Stable and Transient Transfection—For generation of stable transfectants, S107 cells were electroporated with 40 μg of wild type or mutated RelB plasmids. Cells were transfected in 300 μl of culture medium by electroporation with a Bio-Rad gene pulser at 975 μF and 250 V and immediately resuspended in 20 ml of medium (28Baumann B. Weber C.K. Troppmair J. Whiteside S. Israel A. Rapp U.R. Wirth T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4615-4620Crossref PubMed Scopus (154) Google Scholar). Cell clones with genomically integrated vectors were selected on medium containing 2 mg/ml G418 by limited dilution. For transient transfections of stable S107 clones, cells were transfected with 20 μg of a NFκB-dependent luciferase reporter (3× κB.luc, containing three copies of the κB motif immediately upstream of the β-globin TATA box) and 30 ng of a Renilla luciferase reporter under the control of the ubiquitin promoter (29Baumann B. Bohnenstengel F. Siegmund D. Wajant H. Weber C. Herr I. Debatin K.M. Proksch P. Wirth T. J. Biol. Chem. 2002; 16: 16Google Scholar). For transient cotransfections of stable S107 clones, cells were transfected with 20 μg of the respective plasmid (empty vector, p50 and p52 expression vector) together with 4 μg of the NFκB-dependent luciferase reporter and 30 ng of the ubiquitin-dependent Renilla luciferase reporter. For transient cotransfections of S107 cells and Jurkat T cells, cells were transfected with 20 μg of TexMex (empty vector) or 20 μg of the respective plasmid (RelB Mex-Neo, RelB S368A MexNeo, RelB S368E MexNeo), together with 4 μg of the NFκB-dependent luciferase reporter and 30 ng of the ubiquitin-dependent Renilla luciferase reporter. NIH 3T3 cells were transiently transfected with 20 μg of pCDNA3 (empty vector) or 20 μg of the respective plasmid (RelA, RelA S281A, RelA S281E), together with 4 μg of the NFκB-dependent luciferase reporter and 30 ng of the ubiquitin-dependent Renilla luciferase reporter. NIH 3T3 cells (107 cells/sample) were electroporated in 200 μlof PBS using a Bio-Rad gene pulser at 250 μF and 450 V (cuvette, 4 mm), immediately put on ice (5 min), and after 5 min of incubation at room temperature finally resuspended in 20 ml of medium. All cells were harvested after 18-20 h, and luciferase activity was measured. Renilla luciferase activity was measured to normalize for differences in transfection efficiency. Cell Extract Preparation—Whole cell extracts were prepared by freeze-thaw lysis as follows. Cells were washed twice in PBS and resuspended in three packed cell volumes of buffer C (30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). The samples were subjected to three cycles of freezing in liquid nitrogen and subsequent thawing on ice. After centrifugation, the supernatant was used as whole cell extract. For preparing cytoplasmic and nuclear extracts, cells were washed twice in PBS and incubated for 15 min in five packed cell volumes of buffer A (30Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). Swollen cells were then aspirated 20 times with a 26-gauge needle. Nuclei were pelleted and washed twice with buffer A. Nuclear proteins were extracted for one hour in two packed cell volumes of buffer C; after centrifugation, the supernatant was used as nuclear extract. The supernatant from the first nuclear spin was supplemented with 10% glycerol and centrifuged; the supernatant was used as cytoplasmic extract. Western Blotting—50-100 μg of protein extract were separated by SDS-PAGE and electrophoretically blotted to a polyvinylidene difluoride membrane. Membranes were incubated with specific antibodies and developed by enhanced chemoluminescence (ECL Western blotting detection kit; Amersham Biosciences). Specific antibodies were purchased from Santa Cruz Biotechnology (anti-RelA sc-372, anti-RelB sc-226, anti-p50/p105 sc-114X, anti-IκBα sc-371, anti-Bcl-3 sc-185, anti-IKK α/β sc-7607) and Upstate Biotechnology (anti-p52/p100; catalogue no. 06-413). Electrophoretic Mobility Shift Assay (EMSA)—Protein extracts (5 μg) were incubated for 30 min at room temperature with 3 μg of poly(dI/dC), 10 μg of bovine serum albumin in buffer containing 50 mm NaCl, 1 mm dithiothreitol, 10 mm Tris-HCl, 1 mm EDTA, 5% glycerol, and radiolabeled double-stranded oligonucleotides containing an Ig-κ enhancer consensus NFκB site or a Sp-1-specific site (5′-attcgatcggggcggggcgagc-3′). The DNA-protein complexes formed were then separated from free oligonucleotides on a native 4% polyacrylamide gel. For supershift experiments, 2.5 μg of protein extract were preincubated for 30 min with specific antibody before being treated as described before. Cell Labeling—Cells were washed twice with PBS, once with methionine/cysteine-free medium supplemented with dialyzed, heat-inactivated fetal calf serum (10%), and incubated in this medium for 15 min at 37 °C. The cells were resuspended in the same medium (4 × 106 cells/ml) containing [35S]Met-Cys-Promix (100 μCi/ml; Amersham Biosciences) and incubated for 2 h at 37 °C (pulse). Thereafter, cells were washed once with prewarmed PBS, twice with complete medium, resuspended in an excess of this medium, and incubated again at 37 °C. At specific chase time points (0, 2, 4, 8, and 12 h) cells (8 × 106 cells/time point) were harvested and cell extracts prepared as described under "Immunoprecipitation." Immunoprecipitation—Cells were washed twice with cold PBS and resuspended in lysis buffer (containing 25 mm Tris-HCl, pH 8.0., 150 mm NaCl, 2 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10% glycerol, and 1% Nonidet P-40). Following incubation on ice for 15 min, lysates were clarified by microcentrifugation for 15 min. To avoid unspecific binding, cell lysates containing 500 μg of protein were then precleared with protein A-agarose for one hour at 4 °C on a rocking platform. After that, the specific antibody (cited under "Western Blotting") together with protein A-agarose was added to the supernatant and incubated for four hours at 4 °C. The beads were washed twice with lysis buffer and once with PBS at 4 °C prior to elution by boiling in Laemmli loading buffer and resolution by SDS-PAGE. Gels were then subjected to Western blotting. In the case of the pulse-chase studies, the gels were fixed (30 min, 40% methanol and 10% acetic acid), washed twice in H2O, and treated with activation buffer (1 m sodiumsalicylate and 3% glycerol) for 30 min. Finally gels were dried and exposed to film or PhosphorImager analysis (Amersham Biosciences). Mutagenesis of RelB—The prominent role of serine 317 in phosphorylation-dependent nuclear translocation of Dorsal prompted us to mutate the homologous serine 368 of RelB. Interestingly, RelB shows a higher conservation in the amino acids surrounding Serine 368 than do the other NFκB/Rel proteins (Fig. 1). To abolish the potential phosphorylation site, serine 368 was mutated to alanine (RelB S368A). We also tried to mimic the effect of a potential serine phosphorylation by introducing a glutamic acid residue (RelB S368E). Each construct was completely sequenced to confirm the point mutation in a wild type context. The murine S107 plasmacytoma cell line has previously been shown to be an appropriate model system for investigations of inducible and constitutive NFκB activity. The S107 cell line displays a specific defect in inducible NFκB activity and, more important in this context, completely lacks RelB expression and constitutive NFκB activity (31Baumann B. Kistler B. Kirillov A. Bergman Y. Wirth T. J. Biol. Chem. 1998; 273: 11448-11455Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 32Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nature Genet. 1996; 13: 435-441Crossref PubMed Scopus (209) Google Scholar). The other NFκB/Rel proteins are expressed but retained in the cytoplasm. Transfection of this cell line with a RelB expression vector results in the appearance of nuclear p50-RelB complexes and restores κB-dependent transcriptional activity. In contrast, transfection with a RelA expression vector does not have these consequences (32Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nature Genet. 1996; 13: 435-441Crossref PubMed Scopus (209) Google Scholar). To test whether the mutations affect RelB function, we first performed transient transfection assays. S107 cells were transfected with wild type RelB (termed wt), RelB S368A (termed A), and RelB S368E (termed E), respectively, together with a κB-dependent luciferase reporter to determine κB-dependent transcriptional activity. The parental S107 cell line shows only weak constitutive κB-dependent transcriptional activity (Fig. 2A). This activity is strongly increased upon wild type RelB transfection. Cotransfection of RelB bearing a Ser to Ala mutation (RelB S368A) showed a significantly reduced activity. Surprisingly, the transcriptional activity of transfected RelB S368E was even lower than that of RelB S368A. From these results we concluded that introduction of the phosphomimetic mutation did not result in the proposed constitutive nuclear translocation, because we would have expected an enhanced transcriptional activity in this case. To analyze whether these results were because of a S107 cell-specific effect, we performed similar transient transfection experiments with Jurkat T cells (Fig. 2B). Consistent with the data obtained in S107 cells, transcriptional activation is significantly lower in Jurkat T cells transfected with RelB S368A as compared with the wild type RelB transfected cells. Again, the cells transfected with RelB S368E showed the lowest activation. The results of the S368A mutation would be consistent with the initial hypothesis, whereas the behavior of the S368E mutation does not fit to the proposed model of constitutive nuclear translocation. Subcellular Localization of RelB Mutants Is Not Altered in S107 Cells—To explain the defects displayed by RelB bearing the Ser to Ala and Ser to Glu mutations, several possibilities could be considered. The mutations could affect nuclear translocation of RelB. Alternatively, they could impair DNA binding and/or transactivation capabilities. Finally, the mutations could also alter RelB dimerization, which is a prerequisite for DNA binding and transactivation. To address these questions, we generated S107 cell clones constitutively expressing the different RelB variants, which we designated S107 wt, A, and E clones, for RelB wild type, RelB S368A, and RelB S368E mutations, respectively. We first tested several of the S107 wt, A, and E clones for RelB expression in whole cell extracts to select clones with comparable RelB expression levels (Fig. 3 and data not shown). Several such clones were retrieved, and they behaved similarly; only the results of one representative clone for each construct are shown in subsequent analyses. To examine subcellular localization, we prepared nuclear and cytoplasmic extracts of S107 wt, A, and E clones and monitored RelB distribution by Western blot assays (Fig. 3). Interestingly, the subcellular localization of RelB is not affected either by the A or by the E mutation; both RelB and the mutants are almost equally distributed between the cytoplasmic and the nuclear compartment. The mutations also do not interfere with protein stability. The quality of the nuclear and cytoplasmic fractions was controlled by the detection of the cytoplasmic proteins RelA and IKKα/β (Fig. 3, lower panels). We conclude that despite the strong evolutionary conservation, serine 368 of RelB is not involved in the regulation of nuclear translocation of the protein, unlike the serine 317 residue in Dorsal. The same results were obtained by immunofluorescence (data not shown). RelB Mutants Are Impaired in DNA Binding—To recapitulate the findings of the transient transfection experiments, S107 wt, A, and E clones were transfected in parallel with a κB-dependent firefly luciferase reporter (3× κB.luc) and the same reporter lacking the κB motifs. As expected, κB-dependent transcription is strongly activated in the wt clone, whereas this is not the case for the A and E clones (Fig. 4A). We had noted previously that the activity of wild type RelB can be augmented by cotransfection of a p50 expression vector (data not shown); we therefore asked whether this is also the case for the mutated forms of RelB. Upon cotransfection of wt and A clones with a p50 expression vector, there is a strong increase in NFκB-dependent transcription, with the wt and A cells reaching almost the same levels of activity (Fig. 4B). Thus, the defect in the A clone can be compensated to a large extent by transient overexpression of p50. Interestingly, the defect in the E clone cannot be reverted in that way. As compared with p50, cotransfection of p52 reveals a less efficient stimulation of both S107 wt and A cells, with the values for the E clone again remaining at a very low level (Fig. 4B). From control Western blots we conclude that we do not succeed in expressing p52 as efficiently as we do for p50 (data not shown); this could be responsible for the difference in co-stimulation of RelB-dependent transcription by p52. We next addressed the question whether reduced DNA binding activity is responsible for the low activity in the A and E clones. Using EMSA with a κB-specific probe, we observed severe defects of the mutated RelB proteins. The κB binding activity is strongly reduced in the A clone and almost abolished in the E clone (Fig. 5). We detected four κB-specific complexes (a, b, c, and d), which represent complexes of different subunit composition. Previous experiments (32Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergman Y. Nature Genet. 1996; 13: 435-441Crossref PubMed Scopus (209) Google Scholar) suggest that complex d is composed of p50 homodimers that are found in comparable amounts in whole cell and nuclear extracts of all clones. In the cytoplasmic fraction this complex is absent, indicating that there is little or no contamination with nuclear proteins. Complex c, representing the main part of DNA binding activity, is most probably composed of RelB-containing dimers. This complex is highly enriched in nuclear extracts and strongly reduced in the A and E clones. Complexes a and b were also decreased in the A and E clones. They are found at similar levels in the cytoplasmic and nuclear extracts. As a quality control, we performed an EMSA with the same extracts, using a SP-1-specific probe. To further characterize the composition of the four different DNA binding complexes (a, b, c, and d) detected in Fig. 5, we performed supershift assays with specific antibodies. In these assays we used nuclear extracts from A, E, and wt clones and antibodies directed against different NFκB/Rel proteins (Fig. 6). Of the four DNA binding complexes, complex a is shifted to some extent with a RelA-specific antibody, whereas the other complexes do not react strongly with this antibody. In the presence of a RelB-specific antibody, complexes a, b, and c are almost completely supershifted, indicating that they all contain RelB. These three complexes are also affected to a different extent in the presence of p50/p105- and p52/p100-specific antibodies. Complex a is almost completely reduced by a p50/p105-specific antibody and less affected by a p52/p100-specific antibody, whereas complex b is completely supershifted by a p52/p100-specific antibody and to a lesser extent by a p50/p105-specific antibody. Complex c is strongly supershifted by a p50/p105-specific antibody but also affected by a p52/p100-specific antibody, suggesting that this DNA binding activity consists of mainly p50-RelB but also of p52-RelB heterodimers. Complex d has previously been shown to consist of p50 homodimers (32Kirillov A. Kistler B. Mostoslavsky R. Cedar H. Wirth T. Bergma
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