Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2489-2493, March 1992 Biochemistry The repeat domains of the NF-KcB precursor p105 and the protooncogene bc1-3 act as specific inhibitors of NF-cB DNA binding (transcriptional regulation/-protein interaction/) 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; and tDepartments of Pathology and Medicine, University of Chicago, Chicago, IL 60637 Communicated by Keith R. Yamamoto, November 22, 1991

ABSTRACT The inducible pleiotropic of NF-KB in vivo through binding to p65 (25). One of these NF-#cB is composed of two subunits, p50 and p65. The p5O forms is presumably identical with MAD-3 (20). We show subunit is encoded on the N-terminal half of a 105-kDa open here that the C-terminal half of p105 encodes an IKB-like reading frame and contains a rel-like domain. To date, no molecule with an affinity for p50, which has the potential to function has been described for the C-terminal portion. We bind to NF-KB as a separate molecule and inhibit DNA show here that the C-terminal half of p105, when expressed as binding. A region containing the ankyrin repeats is required a separate molecule, binds to p50 and can rapidly disrupt for this inhibition. We also show that the protooncogene protein-DNA complexes of p5O or native NF-jcB. Deletion bcl-3, which contains seven ankyrin repeats, can equally analysis of this precursor-derived inhibitor activity indicated a inhibit p50 DNA binding. These data provide experimental domain containing ankyrin-like repeats as necessary for inhi- evidence for the cellular function of bcl-3. We discuss the bition. The protooncogene bcl-3, which contains seven ankyrin functional and evolutionary implications of our findings, in repeats, can equally inhibit p50 DNA binding. These observa- the context of a general protein-interaction motif that can tions identify bd-3 as an inhibitor of NF-icB and strongly regulate rel-like factors. suggest that the ankyrin repeats in these factors are involved in protein-protein interactions with the rel-like domain of p50. MATERIALS AND METHODS Comparison with other ankyrin repeat-containing suggests that a subclass of these proteins acts as regulators of Proteins. Native NF-KB was purified from HL60, OTF-1 rel-like transcription factors. was from HeLa, and OTF-2 was from BJAB cells, as de- scribed (6, 26, 27). For p50, a BamHI-Xba I fragment The early response transcription factor NF-KB is involved in (comprising amino acids 18-502) from the p105 cDNA (6) was the regulation of a number of viral and cellular genes (for cloned into the BamHI site of pET-3b (28) and expressed as review, see refs. 1 and 2). Cloning of the p50 (3-6) and p65 described (28). Soluble bacterial extracts were passed over (7, 8) subunits of NF-KB revealed a conserved domain with Bio-Rex 70 and DNA affinity columns as described for homology to the rel protooncogene and the NF-KB purification (6). For the precursor-derived inhibitor morphogen dorsal. The rel-homologous region contains the (pdl), the p105 cDNA was cleaved with Nco I and the DNA-binding and dimerization domains as well as a con- C-terminal half (comprising amino acids 435-968) was in- served nuclear transfer signal (9). p50 is synthesized as a serted into the Nco I site of pET-3c (28). pdI was purified 105-kDa precursor protein, which must be processed in vivo from inclusion bodies, which were washed with Triton X-100 to release active p50 (3-6). In analogy to p105, p100 (10) (29). After SDS/PAGE, the band corresponding to pdI was represents a structurally closely related precursor for another excised, eluted, and renatured (6). pdI deletion constructs NF-KB-like transcription factor (unpublished data). The were obtained as follows: PCR-amplified fragments were C-terminal half of p105 contains seven repeated motifs of at generated from the p105 cDNA, cloned into the BamHI site least 33 amino acids. This motif is also found in fruitfly, of pET-3c, and introduced into Escherichia coli BL21(DE3) nematode, and or differentiation control (28). They encoded amino acids 468-968, 468-894, 468-738, proteins (11-17), in human erythrocyte ankyrin (18), in the 625-968, and 769-968 for constructs A-E, respectively, and putative human protooncogene bcl-3 (19), and in the recently were isolated from inclusion bodies. After washing with isolated human immediate-early gene MAD-3 (20). No func- Triton X-100 (29), the pellets were ruptured with urea and the tion has yet been described for the ankyrin repeat domains, proteins were renatured by dilution (29). The control sample except for the human erythrocyte ankyrin and for the p-sub- was a protein extract of cells transformed with pET vector unit ofthe transcription factor GABP (21). In human ankyrin, without insert and was processed in the same way, including a region containing 23 tandem repeats has been shown to urea solubilization of the pellet. The concentration of the confer binding to the anion-exchanger protein (22). The bacterial proteins was estimated and amounts were used as ,8-subunit of GABP contains four imperfect ankyrin-like described. Constructs for expression ofbcl-3 in were repeats, which mediate association with the a-subunit (21). obtained as follows: pET1100 contains a Nco I-Bgl II re- NF-KB and c-rel have been shown to be associated with striction fragment from the bcl-3 cDNA (amino acids 88-446) cytoplasmic proteins (23, 24). For the cytoplasmic inhibitor (19) cloned by blunt-end ligation into the BamHI site of of NF-KB, IKB, two forms have been described, both of pET3a; pET845 contains a Sau3A fragment (residues 173- which inhibit DNA binding in vitro and nuclear translocation 446) cloned into the BamHI site of pET3c. The expressed

The publication costs of this article were defrayed in part by page charge Abbreviations: NP-40, nonionic detergent Nonidet P-40; TCA, tri- payment. This article must therefore be hereby marked "advertisement" chloroacetic acid; pdI, precursor-derived inhibitor. in accordance with 18 U.S.C. ยง1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

2489 2490 Biochemistry: Hatada et al. Proc. Natl. Acad. Sci. USA 89 (1992)

I I *N M 1 -) (K(. -f " .. .; 11 ()[ i! F\ NM )d

II ",j.',

Ni 1! ba lie' I);)(I S .- I

A. MM,,PdlwfAL

FIG. 1. Specificity and time course of inhibition of pdI on NF-KB DNA binding. (a) Gel retardation assay with bacterially expressed p50 and 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 with a 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) or 450 ng (lane 8) of pdl. Control experiments have confirmed that the inhibitory activity is trypsin sensitive (data not shown). (b) Mobility shift assay 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) Time course 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 ng of p50 and 150 ng of pdl were used. proteins were partially purified by preparative SDS/PAGE IKBa, and IlKB3 (24), in that pdI can inhibit binding of p5O to from crude bacterial lysates as described for pdI. As a DNA. control, protein was isolated from gel slices in the equivalent To examine the kinetics of interaction, we performed a mobility range as the bcl-3 peptides, in parallel to the bcl-3 time course experiment. Inhibition of preformed p50-DNA preparations, from cells containing pET3c without insert. complexes occurs very rapidly, in <5 min (Fig. ic, lanes 1-7). Mobility shift assays were performed as described (30). In fact, even adding the inhibitor immediately before applying The nonionic detergent Nonidet P-40 (NP-40) was added to the sample to the gel reduces binding by about 70% (compare the pSO-pdI reaction mixture in the amounts indicated; this lanes 1 and 8 in Fig. ic). The rapid action of pdI, which is was followed by addition of the radioactive probe. similar to the fast dissociation of preformed NF-KB-DNA pdI-p5O Interaction. p50 was immobilized by incubating complexes by IKB (25), suggests a high affinity for p5O. 200 tug of purified bacterial p50 with nitrocellulose (Schleich- Protein-Protein Interaction Between p5O and pdI. Evidence er & Schuell; 2.8 cm2) at 40C for 16 hr. After blocking (5% for a direct interaction between p50 and pdI is presented in milk powder in phosphate-buffered saline (PBS)], filters were Fig. 2A. pdI was incubated with filter-immobilized bacterial washed (31) and incubated with 150 ,ug of bacterial pdI or p50, and bound proteins were eluted and analyzed in a buffer at 40C for 16 hr and then washed extensively. Bound Western blot with an antiserum specific for pdI (see Mate- proteins were eluted with glycine hydrochloride (0.2 M, pH rials andMethods). Fig. 2A, lane 3 shows that pdI bound with 2.7), trichloroacetic acid (TCA)-precipitated, and analyzed high efficiency to immobilized p5O. In contrast, pdl was not by Western blotting (31) with affinity-purified antiserum that retained significantly on filters without prebound p5O (lane 1). was raised against a synthetic peptide (amino acids 754-768) Lane 2 demonstrates that the does not react with (6). Affinity purification of the antiserum was accomplished by allowing filter-immobilized bacterial pdI to react with the A B 1 2 3 4 ammonium sulfate-precipitated . After extensive 1 2 3 washing of the filters, specific antibodies were eluted with 4 M MgCl2 and dialyzed against PBS. X, P50 RESULTS ''pU - pdl Rapid Inhibition of DNA Binding of NF-cB by pdI. The NF-KB precursor p105 cannot bind to DNA unless C-terminal parts are removed (3-6). When testing deletion mutants of the p105 precursor in a reticulocyte lysate system, we dis- 'WVS covered a trans-inhibitory effect of a C-terminal part of p105 on NF-KB DNA binding, as noted earlier (6). For a detailed FIG. 2. (A) Western blot analysis of pdl-p50 interaction. Bacte- analysis of this inhibitory activity, we expressed this part of rial p50 was immobilized to nitrocellulose and incubated with bac- the precursor bacterially and purified it as described. Fig. la terial pdl. The bound material was eluted and analyzed in a Western demonstrates that purified pdI can dissociate preformed blot with an antiserum against a synthetic peptide specific for pdL. to DNA Lane 1, eluted bacterial pdl from a control filter without immobilized complexes of bacterial p5O bound (lanes 1-4). p5O to show nonspecific binding; lane 2, eluted material from filters Inhibition is specific for p5O, since binding of OTF-1 to the containing only immobilized p50; lane 3, bacterial pdI eluted from octamer site (lane 7) is not affected, even at a higher con- filters with immobilized p50. (B) Effect of NP-40 on inhibition. p50 centration of pdI (lane 8). pdl can also inhibit purified native was incubated alone (lanes 3 and 4) or with pdl (lanes 1 and 2) without NF-KB (Fig. lb, lanes 1 and 2). Clearly, pdI has a different (lanes 1 and 3) or with (lanes 2 and 4) 0.4%o NP-40 using the K specificity than the previously described inhibitors ofNF-KB, oligonucleotide as the probe in a gel retardation assay. Biochemistry: Hatada et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2491 eluted traces of the immobilized p5O. This experiment sug- DNA complexes of p5O and native NF-KB was tested in gel gests that the observed inhibition depends on an intrinsic retardation assays (Fig. 3b). A and B completely inhibited affinity between pdI and p5O. Further evidence for protein- binding (lanes 2-7 and 17-22), whereas C, D, and E had no protein interaction was obtained by including detergent (0.4% inhibitory effect (lanes 8-13 and 23-28). This equal mode of NP-40) in the gel retardation assay. NP-40 could release the inhibition on p5O as well as on NF-KB shows that the inhibition of p5O DNA binding by pdI (Fig. 2B, lane 1 versus specificity of the expressed constructs was identical. There- lane 2), whereas it did not alter the DNA binding affinity of fore, the formal possibility that one of the IKB forms (25) is p5O alone (lane 3 versus lane 4). An interaction ofthe separate encoded in p105 and that differential processing of the p5O and pdl molecules suggests that these domains might also precursor molecule generates proteins with different speci- interact in the intact p105 molecule. ficities is unlikely. A Region in pdI Required for Inhibition Contains the The expressed construct B, although capable of complete Conserved Ankyrin Repeats. To define the domain required inhibition, could do so only at slightly higher concentrations for the inhibitory activity, constructs ofdifferent lengths from than A; this effect ofB was consistently observed in all of our the C-terminal part of p105 were expressed in E. coli (Fig. assays. This result suggests that the ankyrin repeats are 3a). Construct A spans residues 468-968 of p105 required for inhibition but that possibly C-terminal regions (6). B and C start at the same position as A. Whereas might play an additional role. construct B still contains all repeats, in C part of the sixth The Human bcl-3 Gene Product Is an Inhibitor of pS0 DNA ankyrin repeat and the seventh incomplete repeat are miss- Binding. The discovery that the ankyrin domain of the p105 ing. D does not contain the first two repeats and part of the precursor is an inhibitor of p50 DNA binding encouraged us third, whereas E is missing all but the last incomplete ankyrin to examine the role of this domain in the human protoonco- gene bcl-3, which, among the proteins containing ankyrin repeat (Fig. 3a). Fig. 3A Inset shows an SDS/PAGE pattern repeats, has the repeat domain most similar to p105. Bacte- with the bacterially expressed constructs. Their effect on rially expressed bl-3 was partially purified and tested for a p105 inhibitory activity in a gel retardation assay with p50 (Fig. 4A). Two constructs were tested: pET1100, which expresses an approximately 40-kDa protein containing the complete A ankyrin domain ofbcl-3, and pET845, which is deleted for the first and part of the second repeat. In Fig. 4A, lanes 2 and 3, B it is evident that the 40-kDa protein from pET1100 is a potent C inhibitor of p50 DNA binding. Lane 5 is a control, demon- A B C D E ki;: D strating that inhibition is due to the bl-3 peptide and not to a copurified bacterial contaminant. Inhibition was dependent ,~-ww - 71 E on the integrity of the ankyrin repeat domain, since the -_ - 44 deletion mutant was unable to inhibit p50 binding, even at the - 25 higher concentration (lane 4). As a control for the specificity - 18 of the inhibition, bcl-3 peptide from pET1100 was added to binding reaction mixtures containing purified OTF-1 or OTF-2 (Fig. 4B). bcl-3 had no effect on binding of either OTF I 1 1 i) 6 ( 9 1 l 1 t3 5 7 0 l12 1"41 protein at a concentration sufficient to completely inhibit a p50 shift of comparable intensity (compare lanes 5 and 6 with A 1 2 3 4 5 6 Bi 2 3 4 5 6 Uiim 4s.I.-M,1 ~~~~p5()

17 29 16(S 1t8I l 293)2121 i22; 23 211 2)526 2-)7 238 '30 a_ 0 fth

I WWcVWWi N' k13

FIG. 3. Delineation of the domain in pdl required for inhibition. (a) Schematic drawing of deletion mutants of pdl. Open boxes indicate the ankyrin-like repeats present in p105; the partial open box to the left indicates the C-terminal part of the rel homology region. (Inset) SDS/PAGE of bacterially expressed mutants. (b) Effect of the pdl deletion constructs on DNA binding ofp5O and native NF-KB in gel-shift assays. Bacterial p50 (lanes 1-15) and native NF-KB (lanes FIG. 4. bcl-3 inhibits p50 DNA binding in vitro. (A) A gel 16-30) were incubated in the binding reaction mixture alone (lanes 1 retardation assay was performed using 5 ng of p50 per reaction. and 16) or together with increasing amounts of A (lanes 2-4 and Reaction mixtures contained p50 alone (lane 1) with 15 ng (lane 2) or 17-19), B (lanes 5-7 and 20-22), C (lanes 8, 9, 23, and 24), D (lanes 150 ng (lane 3) of bc1-3 protein from pET1100, with 150 ng of bc1-3 10, 11, 25, and 26), E (lanes 12, 13, 27, and 28), or the control sample protein from pET845 (lane 4), with control protein (lane 5), or with (lanes 14 and 15 and 29 and 30). Free DNA is not shown. Approx- 30 ng of pdl (lane 6). (B) Gel retardation assay using the H2B imately 0.1 Ag (lanes 2, 5, 8, 10, and 12), 0.2 ug (lanes 3, 6, 9, 11, and promoter octamer site (lanes 1-4) or the KB site (lanes 5 and 6). 13), or 0.36 ,ug (lanes 4 and 7) of bacterial pdl mutants was added to Preformed complexes were challenged either with 25 ng of bcl-3 preformed p50-DNA complexes. For native NF-KB, amounts of the peptide from pET1100 or with an equal volume of control protein. mutants were as follows: approximately 0.4 Iug (lanes 17, 20, 23, 25, Lanes 1 and 2, 8 ng of purified OTF-1 together with control (lane 1) and 27), 0.8 ,ug (lanes 18, 21, 24, 26, and 28) and 1.6 jug (lanes 19 and or bcl-3 (lane 2); lanes 3 and 4, 8 ng of OTF-2 plus control protein 22); 0.35 ,ug and 1.2 ,ug (lanes 14 and 15) or 1.5 Ag and 3.0 Ag (lanes (lane 3) or bc1-3 (lane 4); lanes 5 and 6, 5 ng of bacterial p50 plus 29 and 30) of the control sample were added. control protein (lane 5) or bcl-3 (lane 6). 2492 Biochemistry: Hatada et al. Proc. Natl. Acad. Sci. USA 89 (1992)

1 10 20 30 .537 Q ~~~~~Nax I LiEMZ R_Dit e3VV 2 0 E~~LIsDDIXL 1I vi*D D I I 121 T~a~j3D~isII3Zvj~LIVlLpuwoj7G 68 Q OL XDJXDXP AXaKA VXIRQVI DL1L 576 DjX YIX X BQ! Lv1LIA I I 1116D LLJRas 158 DITI xXLR Q ePLLA X 3 DL a v x LT LA aa t IL 105 X 7 Q Q ePi L L a I lN I Ena~aya 0 609 JLw41 8 L AXA 2 D a I ;K t xx A a L 191 . ~J3D20 TAAXLaC 8 TCUIRaLLD i[x 0GTILIDL 138 lIR a J! L LX a C Q C L A 8 V!T Q9C TIf eXI L3I L FIG. 5. Alignment of the con- i served ankyrin repeat domain in 645 ~IDr1SgIii .WI AITX XIHDP8 C L IdALAalA p105 (6), bcl-3 (19), and MAD-3/ 228 IkIIaII VWJa'AO L ima NIJC*Y1QL1LLIQEGX L A IKB (20). p105 is represented in the 177 v a C L Aa 1 SOY I 2 L L|0lA rt* fq]T first line, bl-3 is in the second 679 QfJ a gToALN LIA V D I DA aWLK 2 D A v line, and MAD-3 is in the third 262 actsis~~~~~rAxsx vav N 8 ;QtlQ Q xiat 11 line. The sequences are shown 211 IA*ADL[EQLOby Q8 kLL Cl aDS continuously, each block of three 713 D tDSSTP L I A >^a LJALwKabetsO lines corresponding to one repeat 295 fl A Q T aYRa LIL3Na Ru QQJLR T L R unit. Generally conserved resi- 244 1!1s~Y~A~tQ~LQSyw Q 2 Q X dues are lightly boxed and con- served p105/bcl-3/MAD-3-spe- 749 N LL D D SW EN AG F[P cific residues are boxed with 282 DR heavy lines. Amino acids in italics LPESEDMESYDTZSZFTEFT indicate the acidic region, which is absent in bc-3. Numbers at the 766 L 1x13jKIYrA D D L L a P D left are the positions in the protein 328 L K[C D NV[AR R R I IxL 2J1Ia RIAla Q P D P __ sequences; numbers on top are positions in the repeats. Consen- ANK X - - - -(- tPL3EtAA- - G X - - - V- - L L - - a A- - sus sequences (lower panel) are D for erythrocyte ankyrin (ANK) (18) and forp105, bcl-3, and MAD-

- CON - - -3 -- C e L XtA ------v - - L L- - GA- - 3/IKB (CON). qi, Hydrophobic; 4, D is a hydrophilic. lanes 1 and 2 or lanes 3 and 4). These results confirm that this binding of native NF-KB as well as of p5O via a direct type of ankyrin repeat domain is a protein interaction motif protein-protein interaction that is sensitive to detergent. specific for the rel family oftranscription factors and strongly Even preformed p5O-DNA complexes were inhibited rapidly suggest that bcl-3 is a cellular regulator of these factors. by pdI. Furthermore, we have evidence that pdI can also The Ankyrin Repeats in p105, bcl-3, and MAD-3/IcB Share interact with p65 (E.N.H. and C.S., unpublished data). a High Homology. Haskill et al. (20) reported the cloning of Deletion analysis indicated that a conserved ankyrin repeat MAD-3/1KB, which specifically inhibits NF-KB DNA binding domain was required for this activity. This repeat domain has dependent on the p65 subunit and which encodes six ankyrin a significant homology to a similar domain in the putative repeats. An alignment ofthe repeat regions in p105, bcl-3, and protooncogene bcl-3 (19). We predicted that bcl-3 would also MAD-3/IKB is shown in Fig. 5. The most prominent differ- be able to inhibit p5O DNA binding and confirmed this ence among the three aligned sequences is the lack of the prediction experimentally. Thus, the C-terminal half of p105 seventh repeat in MAD-3. p105 and MAD-3 contain, after the as a separate molecule, bcl-3, and MAD-3/IKB share func- sixth repeat, a stretch rich in acidic amino acids. The indi- tional and structural similarities, with the most striking vidual repeats show highest homology in the order they difference being that IKB cannot bind to p5O, whereas pdI and appear (e.g., the second repeat in p105 is most similar to the bcl-3 can. Given the similarity between the domains in pdI, second repeat in bcl-3 and MAD-3, etc.). The repeats have a bcl-3, and MAD-3/IKB, we propose that this type ofankyrin- minimal length of 33 amino acids, but, unlike erythrocyte like repeat domain confers specific interactions with parts of ankyrin, they are not phased regularly. The consensus se- the conserved rel domain in p5O and p65. Since p105, bcl-3, quence in p105, MAD-3, and bcl-3 differs from that of the and MAD-3/IKB share no significant homology outside ofthe erythrocyte ankyrin at several positions (Fig. 5) and is clearly repeats, except for an acidic region after the sixth repeat in distinct from that of GABP repeats (ref. 21; not shown). plO5 and MAD-3, we propose that the repeats per se are There are conservations of additional amino acids among the mediating the interaction, with possible contribution of short three proteins in the positions interspersed among the gen- immediately adjacent or interspersed sequences. We further erally conserved residues (Fig. 5, bold boxes), including a propose that other cytoplasmic inhibitors of the rel class of potential protein kinase C substrate sequence in the sixth transcription factors may have a similar repeat domain and repeat of p105 and MAD-3 (amino acid residues 728-733 and that they may act in inhibiting DNA binding in vitro and 259-264, respectively) (Fig. 5). These conservations ofamino nuclear translocation in vivo. Very recently, Thompson et al. acids outside of the generally conserved residues were not (21) have shown that the p9-subunit ofthe transcription factor observed in the erythrocyte ankyrin or in GABPP, which GABP contains ankyrin-like repeats, which are necessary for possess different functions. Furthermore, the conservations association with the a-subunit. The consensus sequence of are also absent from the other ankyrin-containing proteins the ankyrin repeat domain in GABP/B differs from that of the (notch, lin-12, glp-1, etc.; data not shown). Possibly some of rel inhibitors described in the legend to Fig. 5 in several these conserved amino acids could account for the specificity positions, indicating that this domain in GABPB belongs to a of the three proteins for the rel domain in p65 and p50. different class of ankyrin repeats, in agreement with its distinct mode of action. In the same way, the repeats in the three rel inhibitors in Fig. 5 are more similar to each other DISCUSSION than to those in the other ankyrin-repeat-containing proteins We have demonstrated that the C-terminal halfofp105, when (data not shown), which would indicate that they form a expressed as a separate molecule (pdl), can inhibit DNA structural and functional class of their own. The ankyrin Biochemistry: Hatada et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2493 repeat structure might provide a general structural scaffold 2. Baeuerle, P. A. (1991) Biochim. Biophys. Acta 1088, 171-182. for protein-protein interaction, where specificity is mediated 3. Bours, V., Villabos, J., Burd, P. R., Kelly, K. & Siebenlist, U. by residues outside ofthe widely conserved ones (e.g., by the (1990) Nature (London) 348, 76-80. 4. Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, specifically conserved amino acids in p105, bcl-3, and G. P. & Baltimore, D. (1990) Cell 62, 1019-1029. MAD-3; Fig. 5). 5. Kieran, M., Blank, V., Logeat, F., Vanderkerckhove, J., The product(s) ofthe C-terminal part ofthe p105 precursor Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., after processing events in the cell has yet to be analyzed Baeuerle, P. A. & Israel, A. (1990) Cell 62, 1007-1018. functionally. Our immunological data reveal a putative prod- 6. Meyer, R., Hatada, E. N., Hohmann, H.-P., Haiker, M., uct of about 40 kDa, which is recognized by antisera raised Bartsch, C., Rdthlisberger, U., Lahm, H. W., Schlaeger, E. J., against bacterial pdI and p105; however, this protein is Van Loon, A. P. G. M. & Scheidereit, C. (1991) Proc. Natl. apparently present in much lower abundance than either p105 Acad. Sci. USA 88, 966-970. 7. Nolan, G. P., Ghosh, S., Liou, H.-C., Tempst, P. & Baltimore, or p50 (data not shown). This result suggests that the ankyrin D. (1991) Cell 64, 961-969. repeats of p105 function predominantly in cis. The mecha- 8. Ruben, S., Dillon, P. J., Schreck, R., Henkel, T., Chen, C.-H., nism of action ofthe repeat domain in cis or in trans might be Maher, M., Baeuerle, P. A. & Rosen, C. (1991) Science 251, similar. Signal-induced phosphorylation of IKB might lead to 1490-1493. a conformational change, releasing the rel-like transcription 9. Logeat, F., Israel, N., Ten, R., Blank, V., Le Bail, O., factor (as demonstrated in vitro in ref. 32), which migrates to Kourilsky, P. & Israel, A. (1991) EMBO J. 10, 1827-1832. the nucleus, and leading to a spontaneous degradation of IKB 10. Schmid, R. M., Perkins, N. D., Duckett, C. S., Andrews, due to unshielded protease-sensitive regions. In an analogous P. C. & Nabel, G. J. (1991) Nature (London) 352, 733-736. fashion, signal-induced phosphorylation might induce a con- 11. Kidd, S., Kelley, M. R. & Young, M. W. (1986) Mol. Cell. Biol. 6, 3094-3108. formational change in p105, unmasking the pdl domain, 12. Yochem, J., Weston, K. & Greenwald, I. (1988) Nature (Lon- which is then rapidly processed by proteases to release p50. don) 335, 547-550. The function of the C-terminal domain in p105, as of IKB for 13. Yochem, J. & Greenwald, I. (1989) Cell 58, 553-563. NF-KB, would then be to prevent the translocation of the 14. Spence, A. M., Coulson, A. & Hodgkin, J. (1990) Cell 60, rel-like protein to the nucleus, accomplished probably by 981-990. masking the conserved nuclear transfer signal; this same 15. Aves, S. J., Durkacz, B., Carr, A. & Nurse, P. (1985) EMBO interaction would also be responsible for inhibition of DNA J. 4, 457-463. binding in vitro. Evolutionarily, p105 might have arisen by a 16. Andrews, B. J. & Herskowitz, I. (1989) Nature (London) 342, combination of a rel-like gene and a member of the lKB-like 830-833. 17. Breeden, L. & Nasmyth, K. (1987) Nature (London) 329, genes or, conversely, p105 could have been the predecessor 651-654. for the separated genes. 18. Lux, S. E., John, K. M. & Bennet, V. (1990) Nature (London) The discovery that the ankyrin domain of bl-3 mediates 344, 36-42. inhibition of p50 DNA binding in vitro provides the first clue 19. Ohno, H., Takimoto, G. & McKeithan, T. W. (1990) Cell 60, to the cellular function of this putative protooncogene. Our 991-997. results strongly suggest that bl-3 is an IKB form with a novel 20. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yu- specificity, since previously characterized 1KB forms are rochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P. unable to inhibit binding of p50 homodimers (25). We should & Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289. out that our results are not sufficient to prove that p50 21. Thompson, C. C., Brown, T. A. & McKnight, S. L. (1991) point Science 253, 762-766. is the cellular target for bcl-3; characterization of the speci- 22. Davis, L. H. & Bennett, V. (1990) J. Biol. Chem. 265, 10589- ficity of bl-3 for the various rel-like proteins requires further 105%. investigation. 23. Kochel, T., Mushinski, J. F. & Rice, N. R. (1991) Oncogene 6, Our findings provide a first step toward understanding the 615-626. function of ankyrin repeats in the regulation of rel proteins. 24. Govind, S. & Steward, R. (1991) Trends Genet. 7, 119-125. Future studies will be necessary to analyze in detail the 25. Zabel, U. & Baeuerle, P. A. (1990) Cell 61, 255-265. interaction of the repeat domain with subregions of the 26. Murphy, S., Pierani, A., Scheidereit, C., Melli, M. & Roeder, conserved rel-like domain and to determine whether pdI R. G. (1989) Cell 59, 1071-1080. interferes with from mechanistic 27. Scheidereit, C., Heguy, A. & Roeder, R. G. (1987) Cell 51, binding dimerization. Apart 783-793. aspects, it will be important to characterize the maturation of 28. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, p105, which seems to be one of the main cytoplasmic storage J. W. (1990) Methods Enzymol. 185, 60-89. forms of NF-KB. 29. Marston, F. A. 0. (1987) DNA Cloning:A Practical Approach, We thank Dr. H.-P. Hohmann for discussion, Dr. A. Bindereif for ed. Glover, D. M. (IRL, Oxford), Vol. 3. critically reading the manuscript, Dr. Alain Israel for the gift of p105 30. Kawakami, K., Scheidereit, C. & Roeder, R. G. 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