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The Ankyrin Repeat Domains of the NF-Kcb Precursor P105 and The Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2489-2493, March 1992 Biochemistry The ankyrin 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-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; and tDepartments of Pathology and Medicine, University of Chicago, Chicago, IL 60637 Communicated by Keith R. Yamamoto, November 22, 1991 ABSTRACT The inducible pleiotropic transcription factor 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 proteins 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 Drosophila 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 yeast cell cycle 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 bacteria 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. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2489 Downloaded by guest on October 3, 2021 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.
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