Proc. Nati. Acad. Sci. USAVol. 89, pp. 2489-2493, March 1992Biochemistry

The ankyrin repeat domains of the NF-KcB precursor p105 and theprotooncogene bc1-3 act as specific inhibitors of NF-cBDNA binding

(transcriptional regulation/protein-protein interaction/signal transduction)

EUNICE N. HATADA*, ALEXANDRA NIETERS*, F. GREGORY WULCZYN*, MICHAEL NAUMANN*,RALF MEYER*, GIUSEPPINA NUCIFORAt, TIMOTHY W. MCKEITHANt, AND CLAUS SCHEIDEREIT*t*Max-Planck-Institut fuer Molekulare Genetik, Otto-Warburg-Laboratorium, Ihnestrasse 73, 1000 Berlin-Dahlem, Federal Republic of Germany; andtDepartments of Pathology and Medicine, University of Chicago, Chicago, IL 60637

Communicated by Keith R. Yamamoto, November 22, 1991

ABSTRACT The inducible pleiotropic transcription factorNF-#cB is composed of two subunits, p50 and p65. The p5Osubunit is encoded on the N-terminal half of a 105-kDa openreading frame and contains a rel-like domain. To date, nofunction has been described for the C-terminal portion. Weshow here that the C-terminal half of p105, when expressed asa separate molecule, binds to p50 and can rapidly disruptprotein-DNA complexes of p5O or native NF-jcB. Deletionanalysis of this precursor-derived inhibitor activity indicated adomain containing ankyrin-like repeats as necessary for inhi-bition. The protooncogene bcl-3, which contains seven ankyrinrepeats, can equally inhibit p50 DNA binding. These observa-tions identify bd-3 as an inhibitor of NF-icB and stronglysuggest that the ankyrin repeats in these factors are involved inprotein-protein interactions with the rel-like domain of p50.Comparison with other ankyrin repeat-containing proteinssuggests that a subclass of these proteins acts as regulators ofrel-like transcription factors.

The early response transcription factor NF-KB is involved inthe regulation of a number of viral and cellular genes (forreview, see refs. 1 and 2). Cloning of the p50 (3-6) and p65(7, 8) subunits of NF-KB revealed a conserved domain withhomology to the rel protooncogene and the Drosophilamorphogen dorsal. The rel-homologous region contains theDNA-binding and dimerization domains as well as a con-served nuclear transfer signal (9). p50 is synthesized as a105-kDa precursor protein, which must be processed in vivoto release active p50 (3-6). In analogy to p105, p100 (10)represents a structurally closely related precursor for anotherNF-KB-like transcription factor (unpublished data). TheC-terminal half of p105 contains seven repeated motifs of atleast 33 amino acids. This motif is also found in fruitfly,nematode, and yeast cell cycle or differentiation controlproteins (11-17), in human erythrocyte ankyrin (18), in theputative human protooncogene bcl-3 (19), and in the recentlyisolated human immediate-early gene MAD-3 (20). No func-tion has yet been described for the ankyrin repeat domains,except for the human erythrocyte ankyrin and for the p-sub-unit ofthe transcription factor GABP (21). In human ankyrin,a region containing 23 tandem repeats has been shown toconfer binding to the anion-exchanger protein (22). The,8-subunit of GABP contains four imperfect ankyrin-likerepeats, which mediate association with the a-subunit (21).NF-KB and c-rel have been shown to be associated with

cytoplasmic proteins (23, 24). For the cytoplasmic inhibitorof NF-KB, IKB, two forms have been described, both ofwhich inhibit DNA binding in vitro and nuclear translocation

of NF-KB in vivo through binding to p65 (25). One of theseforms is presumably identical with MAD-3 (20). We showhere that the C-terminal half of p105 encodes an IKB-likemolecule with an affinity for p50, which has the potential tobind to NF-KB as a separate molecule and inhibit DNAbinding. A region containing the ankyrin repeats is requiredfor this inhibition. We also show that the protooncogenebcl-3, which contains seven ankyrin repeats, can equallyinhibit p50 DNA binding. These data provide experimentalevidence for the cellular function of bcl-3. We discuss thefunctional and evolutionary implications of our findings, inthe context of a general protein-interaction motif that canregulate rel-like factors.

MATERIALS AND METHODSProteins. Native NF-KB was purified from HL60, OTF-1

was from HeLa, and OTF-2 was from BJAB cells, as de-scribed (6, 26, 27). For p50, a BamHI-Xba I fragment(comprising amino acids 18-502) from the p105 cDNA (6) wascloned into the BamHI site of pET-3b (28) and expressed asdescribed (28). Soluble bacterial extracts were passed overBio-Rex 70 and DNA affinity columns as described forNF-KB purification (6). For the precursor-derived inhibitor(pdl), the p105 cDNA was cleaved with Nco I and theC-terminal half (comprising amino acids 435-968) was in-serted into the Nco I site of pET-3c (28). pdI was purifiedfrom inclusion bodies, which were washed with Triton X-100(29). After SDS/PAGE, the band corresponding to pdI wasexcised, eluted, and renatured (6). pdI deletion constructswere obtained as follows: PCR-amplified fragments weregenerated from the p105 cDNA, cloned into the BamHI siteof pET-3c, and introduced into Escherichia coli BL21(DE3)(28). They encoded amino acids 468-968, 468-894, 468-738,625-968, and 769-968 for constructs A-E, respectively, andwere isolated from inclusion bodies. After washing withTriton X-100 (29), the pellets were ruptured with urea and theproteins were renatured by dilution (29). The control samplewas a protein extract of cells transformed with pET vectorwithout insert and was processed in the same way, includingurea solubilization of the pellet. The concentration of thebacterial proteins was estimated and amounts were used asdescribed. Constructs for expression of bcl-3 in bacteria wereobtained as follows: pET1100 contains a Nco I-Bgl II re-striction fragment from the bcl-3 cDNA (amino acids 88-446)(19) cloned by blunt-end ligation into the BamHI site ofpET3a; pET845 contains a Sau3A fragment (residues 173-446) cloned into the BamHI site of pET3c. The expressed

Abbreviations: NP-40, nonionic detergent Nonidet P-40; TCA, tri-chloroacetic acid; pdI, precursor-derived inhibitor.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Specificity and time course of inhibition of pdI on NF-KB DNA binding. (a) Gel retardation assay with bacterially expressed p50and purified OTF-1. Probes were either the kB site (28) (lanes 1-4) or the H2B promoter octamer site (lanes 5-8). Binding was challenged witha 50-fold molar excess of the wild-type KB (K), octamer (0), or mutant KB (M) (29) oligonucleotides or with bacterially expressed pdI. Comp.,competitor oligonucleotides. Approximately 20 ng of p50 or 10 ng of OTF-1 was used for these assays, together with 150 ng (lanes 4 and 7) or450 ng (lane 8) of pdl. Control experiments have confirmed that the inhibitory activity is trypsin sensitive (data not shown). (b) Mobility shiftassay with bacterial p50 or purified native NF-KB. Approximately 20 ng of p50, 350 ng of pdI, and 10 ng of purified NF-KB were used. (c) Timecourse of inhibition. Boxed numbers at the top indicate the time in minutes that pdI was coincubated with the preformed p50-DNA complex,for a total incubation time of 30 min. In lane 8, pdl was added immediately before loading. Lane 1, no inhibitor added. Approximately 20 ngof p50 and 150 ng of pdl were used.

proteins were partially purified by preparative SDS/PAGEfrom crude bacterial lysates as described for pdI. As acontrol, protein was isolated from gel slices in the equivalentmobility range as the bcl-3 peptides, in parallel to the bcl-3preparations, from cells containing pET3c without insert.

Mobility shift assays were performed as described (30).The nonionic detergent Nonidet P-40 (NP-40) was added tothe pSO-pdI reaction mixture in the amounts indicated; thiswas followed by addition of the radioactive probe.pdI-p5O Interaction. p50 was immobilized by incubating

200 tug of purified bacterial p50 with nitrocellulose (Schleich-er & Schuell; 2.8 cm2) at 40C for 16 hr. After blocking (5%milk powder in phosphate-buffered saline (PBS)], filters werewashed (31) and incubated with 150 ,ug of bacterial pdI orbuffer at 40C for 16 hr and then washed extensively. Boundproteins were eluted with glycine hydrochloride (0.2 M, pH2.7), trichloroacetic acid (TCA)-precipitated, and analyzedby Western blotting (31) with affinity-purified antiserum thatwas raised against a synthetic peptide (amino acids 754-768)(6). Affinity purification of the antiserum was accomplishedby allowing filter-immobilized bacterial pdI to react with theammonium sulfate-precipitated antibodies. After extensivewashing of the filters, specific antibodies were eluted with 4M MgCl2 and dialyzed against PBS.

RESULTSRapid Inhibition of DNA Binding of NF-cB by pdI. The

NF-KB precursor p105 cannot bind to DNA unless C-terminalparts are removed (3-6). When testing deletion mutants ofthe p105 precursor in a reticulocyte lysate system, we dis-covered a trans-inhibitory effect of a C-terminal part of p105on NF-KB DNA binding, as noted earlier (6). For a detailedanalysis of this inhibitory activity, we expressed this part ofthe precursor bacterially and purified it as described. Fig. lademonstrates that purified pdI can dissociate preformedcomplexes of bacterial p5O bound to DNA (lanes 1-4).Inhibition is specific for p5O, since binding of OTF-1 to theoctamer site (lane 7) is not affected, even at a higher con-centration of pdI (lane 8). pdl can also inhibit purified nativeNF-KB (Fig. lb, lanes 1 and 2). Clearly, pdI has a differentspecificity than the previously described inhibitors ofNF-KB,

IKBa, and IlKB3 (24), in that pdI can inhibit binding of p5O toDNA.To examine the kinetics of interaction, we performed a

time course experiment. Inhibition of preformed p50-DNAcomplexes occurs very rapidly, in <5 min (Fig. ic, lanes 1-7).In fact, even adding the inhibitor immediately before applyingthe sample to the gel reduces binding by about 70% (comparelanes 1 and 8 in Fig. ic). The rapid action of pdI, which issimilar to the fast dissociation of preformed NF-KB-DNAcomplexes by IKB (25), suggests a high affinity for p5O.

Protein-Protein Interaction Between p5O and pdI. Evidencefor a direct interaction between p50 and pdI is presented inFig. 2A. pdI was incubated with filter-immobilized bacterialp50, and bound proteins were eluted and analyzed in aWestern blot with an antiserum specific for pdI (see Mate-rials andMethods). Fig. 2A, lane 3 shows that pdI bound withhigh efficiency to immobilized p5O. In contrast, pdl was notretained significantly on filters without prebound p5O (lane 1).Lane 2 demonstrates that the antibody does not react with

A1 2 3

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rial p50 was immobilized to nitrocellulose and incubated with bac-terial pdl. The bound material was eluted and analyzed in a Westernblot with an antiserum against a synthetic peptide specific for pdL.Lane 1, eluted bacterial pdl from a control filter without immobilizedp5O to show nonspecific binding; lane 2, eluted material from filterscontaining only immobilized p50; lane 3, bacterial pdI eluted fromfilters with immobilized p50. (B) Effect of NP-40 on inhibition. p50was incubated alone (lanes 3 and 4) or with pdl (lanes 1 and 2) without(lanes 1 and 3) or with (lanes 2 and 4) 0.4%o NP-40 using the Koligonucleotide as the probe in a gel retardation assay.

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eluted traces of the immobilized p5O. This experiment sug-gests that the observed inhibition depends on an intrinsicaffinity between pdI and p5O. Further evidence for protein-protein interaction was obtained by including detergent (0.4%NP-40) in the gel retardation assay. NP-40 could release theinhibition of p5O DNA binding by pdI (Fig. 2B, lane 1 versuslane 2), whereas it did not alter the DNA binding affinity ofp5O alone (lane 3 versus lane 4). An interaction ofthe separatep5O and pdl molecules suggests that these domains might alsointeract in the intact p105 molecule.A Region in pdI Required for Inhibition Contains the

Conserved Ankyrin Repeats. To define the domain requiredfor the inhibitory activity, constructs ofdifferent lengths fromthe C-terminal part of p105 were expressed in E. coli (Fig.3a). Construct A spans amino acid residues 468-968 of p105(6). B and C start at the same position as A. Whereasconstruct B still contains all repeats, in C part of the sixthankyrin repeat and the seventh incomplete repeat are miss-ing. D does not contain the first two repeats and part of thethird, whereas E is missing all but the last incomplete ankyrinrepeat (Fig. 3a). Fig. 3A Inset shows an SDS/PAGE patternwith the bacterially expressed constructs. Their effect on

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DNA complexes of p5O and native NF-KB was tested in gelretardation assays (Fig. 3b). A and B completely inhibitedbinding (lanes 2-7 and 17-22), whereas C, D, and E had noinhibitory effect (lanes 8-13 and 23-28). This equal mode ofinhibition on p5O as well as on NF-KB shows that thespecificity of the expressed constructs was identical. There-fore, the formal possibility that one of the IKB forms (25) isencoded in p105 and that differential processing of theprecursor molecule generates proteins with different speci-ficities is unlikely.The expressed construct B, although capable of complete

inhibition, could do so only at slightly higher concentrationsthan A; this effect ofB was consistently observed in all of ourassays. This result suggests that the ankyrin repeats arerequired for inhibition but that possibly C-terminal regionsmight play an additional role.The Human bcl-3 Gene Product Is an Inhibitor of pS0 DNA

Binding. The discovery that the ankyrin domain of the p105precursor is an inhibitor of p50 DNA binding encouraged usto examine the role of this domain in the human protoonco-gene bcl-3, which, among the proteins containing ankyrinrepeats, has the repeat domain most similar to p105. Bacte-rially expressed bl-3 was partially purified and tested forinhibitory activity in a gel retardation assay with p50 (Fig.4A). Two constructs were tested: pET1100, which expressesan approximately 40-kDa protein containing the completeankyrin domain of bcl-3, and pET845, which is deleted for thefirst and part of the second repeat. In Fig. 4A, lanes 2 and 3,it is evident that the 40-kDa protein from pET1100 is a potentinhibitor of p50 DNA binding. Lane 5 is a control, demon-strating that inhibition is due to the bl-3 peptide and not toa copurified bacterial contaminant. Inhibition was dependenton the integrity of the ankyrin repeat domain, since thedeletion mutant was unable to inhibit p50 binding, even at thehigher concentration (lane 4). As a control for the specificityof the inhibition, bcl-3 peptide from pET1100 was added tobinding reaction mixtures containing purified OTF-1 orOTF-2 (Fig. 4B). bcl-3 had no effect on binding of either OTFprotein at a concentration sufficient to completely inhibit ap50 shift of comparable intensity (compare lanes 5 and 6 with

A 1 2 3 4 5 6 Bi 2 3 4 5 614s.I.-M, ~~~~p5()

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FIG. 3. Delineation of the domain in pdl required for inhibition.(a) Schematic drawing of deletion mutants of pdl. Open boxesindicate the ankyrin-like repeats present in p105; the partial open boxto the left indicates the C-terminal part of the rel homology region.(Inset) SDS/PAGE of bacterially expressed mutants. (b) Effect ofthe pdl deletion constructs on DNA binding ofp5O and native NF-KBin gel-shift assays. Bacterial p50 (lanes 1-15) and native NF-KB (lanes16-30) were incubated in the binding reaction mixture alone (lanes 1and 16) or together with increasing amounts of A (lanes 2-4 and17-19), B (lanes 5-7 and 20-22), C (lanes 8, 9, 23, and 24), D (lanes10, 11, 25, and 26), E (lanes 12, 13, 27, and 28), or the control sample(lanes 14 and 15 and 29 and 30). Free DNA is not shown. Approx-imately 0.1 Ag (lanes 2, 5, 8, 10, and 12), 0.2 ug (lanes 3, 6, 9, 11, and13), or 0.36 ,ug (lanes 4 and 7) of bacterial pdl mutants was added topreformed p50-DNA complexes. For native NF-KB, amounts of themutants were as follows: approximately 0.4 Iug (lanes 17, 20, 23, 25,and 27), 0.8 ,ug (lanes 18, 21, 24, 26, and 28) and 1.6 jug (lanes 19 and22); 0.35 ,ug and 1.2 ,ug (lanes 14 and 15) or 1.5 Ag and 3.0 Ag (lanes29 and 30) of the control sample were added.

FIG. 4. bcl-3 inhibits p50 DNA binding in vitro. (A) A gelretardation assay was performed using 5 ng of p50 per reaction.Reaction mixtures contained p50 alone (lane 1) with 15 ng (lane 2) or150 ng (lane 3) of bc1-3 protein from pET1100, with 150 ng of bc1-3protein from pET845 (lane 4), with control protein (lane 5), or with30 ng of pdl (lane 6). (B) Gel retardation assay using the H2Bpromoter octamer site (lanes 1-4) or the KB site (lanes 5 and 6).Preformed complexes were challenged either with 25 ng of bcl-3peptide from pET1100 or with an equal volume of control protein.Lanes 1 and 2, 8 ng of purified OTF-1 together with control (lane 1)or bcl-3 (lane 2); lanes 3 and 4, 8 ng of OTF-2 plus control protein(lane 3) or bc1-3 (lane 4); lanes 5 and 6, 5 ng of bacterial p50 pluscontrol protein (lane 5) or bcl-3 (lane 6).

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FIG. 5. Alignment of the con-served ankyrin repeat domain inp105 (6), bcl-3 (19), and MAD-3/IKB (20). p105 is represented in thefirst line, bl-3 is in the secondline, and MAD-3 is in the thirdline. The sequences are showncontinuously, each block of threelines corresponding to one repeatunit. Generally conserved resi-dues are lightly boxed and con-served p105/bcl-3/MAD-3-spe-cific residues are boxed withheavy lines. Amino acids in italicsindicate the acidic region, which isabsent in bc-3. Numbers at theleft are the positions in the proteinsequences; numbers on top arepositions in the repeats. Consen-sus sequences (lower panel) arefor erythrocyte ankyrin (ANK)(18) and for p105, bcl-3, and MAD-3/IKB (CON). qi, Hydrophobic; 4,

hydrophilic.

lanes 1 and 2 or lanes 3 and 4). These results confirm that thistype of ankyrin repeat domain is a protein interaction motifspecific for the rel family of transcription factors and stronglysuggest that bcl-3 is a cellular regulator of these factors.The Ankyrin Repeats in p105, bcl-3, and MAD-3/IcB Share

a High Homology. Haskill et al. (20) reported the cloning ofMAD-3/1KB, which specifically inhibits NF-KB DNA bindingdependent on the p65 subunit and which encodes six ankyrinrepeats. An alignment ofthe repeat regions in p105, bcl-3, andMAD-3/IKB is shown in Fig. 5. The most prominent differ-ence among the three aligned sequences is the lack of theseventh repeat in MAD-3. p105 and MAD-3 contain, after thesixth repeat, a stretch rich in acidic amino acids. The indi-vidual repeats show highest homology in the order theyappear (e.g., the second repeat in p105 is most similar to thesecond repeat in bcl-3 and MAD-3, etc.). The repeats have aminimal length of 33 amino acids, but, unlike erythrocyteankyrin, they are not phased regularly. The consensus se-quence in p105, MAD-3, and bcl-3 differs from that of theerythrocyte ankyrin at several positions (Fig. 5) and is clearlydistinct from that of GABP repeats (ref. 21; not shown).There are conservations of additional amino acids among thethree proteins in the positions interspersed among the gen-erally conserved residues (Fig. 5, bold boxes), including apotential protein kinase C substrate sequence in the sixthrepeat of p105 and MAD-3 (amino acid residues 728-733 and259-264, respectively) (Fig. 5). These conservations ofaminoacids outside of the generally conserved residues were notobserved in the erythrocyte ankyrin or in GABPP, whichpossess different functions. Furthermore, the conservationsare also absent from the other ankyrin-containing proteins(notch, lin-12, glp-1, etc.; data not shown). Possibly some ofthese conserved amino acids could account for the specificityof the three proteins for the rel domain in p65 and p50.

DISCUSSIONWe have demonstrated that the C-terminal half of p105, whenexpressed as a separate molecule (pdl), can inhibit DNA

binding of native NF-KB as well as of p5O via a directprotein-protein interaction that is sensitive to detergent.Even preformed p5O-DNA complexes were inhibited rapidlyby pdI. Furthermore, we have evidence that pdI can alsointeract with p65 (E.N.H. and C.S., unpublished data).Deletion analysis indicated that a conserved ankyrin repeatdomain was required for this activity. This repeat domain hasa significant homology to a similar domain in the putativeprotooncogene bcl-3 (19). We predicted that bcl-3 would alsobe able to inhibit p5O DNA binding and confirmed thisprediction experimentally. Thus, the C-terminal half of p105as a separate molecule, bcl-3, and MAD-3/IKB share func-tional and structural similarities, with the most strikingdifference being that IKB cannot bind to p5O, whereas pdI andbcl-3 can. Given the similarity between the domains in pdI,bcl-3, and MAD-3/IKB, we propose that this type of ankyrin-like repeat domain confers specific interactions with parts ofthe conserved rel domain in p5O and p65. Since p105, bcl-3,and MAD-3/IKB share no significant homology outside oftherepeats, except for an acidic region after the sixth repeat inplO5 and MAD-3, we propose that the repeats per se aremediating the interaction, with possible contribution of shortimmediately adjacent or interspersed sequences. We furtherpropose that other cytoplasmic inhibitors of the rel class oftranscription factors may have a similar repeat domain andthat they may act in inhibiting DNA binding in vitro andnuclear translocation in vivo. Very recently, Thompson et al.(21) have shown that the p9-subunit of the transcription factorGABP contains ankyrin-like repeats, which are necessary forassociation with the a-subunit. The consensus sequence ofthe ankyrin repeat domain in GABP/B differs from that of therel inhibitors described in the legend to Fig. 5 in severalpositions, indicating that this domain in GABPB belongs to adifferent class of ankyrin repeats, in agreement with itsdistinct mode of action. In the same way, the repeats in thethree rel inhibitors in Fig. 5 are more similar to each otherthan to those in the other ankyrin-repeat-containing proteins(data not shown), which would indicate that they form astructural and functional class of their own. The ankyrin

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repeat structure might provide a general structural scaffoldfor protein-protein interaction, where specificity is mediatedby residues outside ofthe widely conserved ones (e.g., by thespecifically conserved amino acids in p105, bcl-3, andMAD-3; Fig. 5).The product(s) of the C-terminal part of the p105 precursor

after processing events in the cell has yet to be analyzedfunctionally. Our immunological data reveal a putative prod-uct of about 40 kDa, which is recognized by antisera raisedagainst bacterial pdI and p105; however, this protein isapparently present in much lower abundance than either p105or p50 (data not shown). This result suggests that the ankyrinrepeats of p105 function predominantly in cis. The mecha-nism of action of the repeat domain in cis or in trans might besimilar. Signal-induced phosphorylation of IKB might lead toa conformational change, releasing the rel-like transcriptionfactor (as demonstrated in vitro in ref. 32), which migrates tothe nucleus, and leading to a spontaneous degradation of IKBdue to unshielded protease-sensitive regions. In an analogousfashion, signal-induced phosphorylation might induce a con-formational change in p105, unmasking the pdl domain,which is then rapidly processed by proteases to release p50.The function of the C-terminal domain in p105, as of IKB forNF-KB, would then be to prevent the translocation of therel-like protein to the nucleus, accomplished probably bymasking the conserved nuclear transfer signal; this sameinteraction would also be responsible for inhibition ofDNAbinding in vitro. Evolutionarily, p105 might have arisen by acombination of a rel-like gene and a member of the lKB-likegenes or, conversely, p105 could have been the predecessorfor the separated genes.The discovery that the ankyrin domain of bl-3 mediates

inhibition of p50 DNA binding in vitro provides the first clueto the cellular function of this putative protooncogene. Ourresults strongly suggest that bl-3 is an IKB form with a novelspecificity, since previously characterized 1KB forms areunable to inhibit binding of p50 homodimers (25). We shouldpoint out that our results are not sufficient to prove that p50is the cellular target for bcl-3; characterization of the speci-ficity of bl-3 for the various rel-like proteins requires furtherinvestigation.Our findings provide a first step toward understanding the

function of ankyrin repeats in the regulation of rel proteins.Future studies will be necessary to analyze in detail theinteraction of the repeat domain with subregions of theconserved rel-like domain and to determine whether pdIbinding interferes with dimerization. Apart from mechanisticaspects, it will be important to characterize the maturation ofp105, which seems to be one of the main cytoplasmic storageforms of NF-KB.We thank Dr. H.-P. Hohmann for discussion, Dr. A. Bindereif for

critically reading the manuscript, Dr. Alain Israel for the gift of p105antiserum, and S. Franke for purified OTF-1 and OTF-2. This workwas supported by a grant from the Deutsche Forschungsgemein-schaft to C.S.

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