PPAPAPAPAS SGQHHDFLSD LFSDDYGGKN CKKPABYGYV SLGRLGAARG ALHPGCFAPL NPPPPPPPPP AELKAEPGFE PADCKR EAGAPGGAGMA 201 AGFPYALRAY LGYQAVPSGS SGSLSTSSSS SPPGTPSPADARAPPTACYA 251 GAGPAPSQVK SKAKKTVDKH SDEYKIRRER NNIAVRKSRD KAKMRNLETQ

The NF-cB-pSO polypeptide, a member of the Rel family of transcription factors, was produced as a fusion protein containing amino-terminal peptide additions that facilitate purification and detection with a monoclonal antibody and specific radiolabeling by phosphorylation in vitro. The 32P-labeled NF-#cB-p5O fusion polypeptide was used as the probe in Western blotting experiments and in screenings of a bacteriophage expression library to isolate cDNAs encoding interacting protein domains. As expected, cDNAs encoding proteins of the Rel family were identified. Surprisingly, the 32P-labeled NF-uB protein also specifically bound to proteins encoded by cDNAs for the human NF-1L6 transcription factor. The NF-#cB-p5O and NF-IL6 proteins directly interact, and the Rel homology domain and leucine-zipper motif, respectively, are important for this interaction. Since induction ofthe NF-ucB and NF-EL6 factors are important events in immune and acute-phase responses, this interaction could permit coregulation of genes. The NF-KB complex was characterized as a critical transcription factor in B-lymphoid cell development (1, 2). Further analysis revealed that it is important in controlling the expression of many inducible genes and that several viruses, including the simian virus 40, cytomegalovirus, and human immunodeficiency viruses, contain at least one copy of an NF-KB binding site in their enhancers (reviewed in ref. 3). Biochemical purification and subsequent molecular cloning experiments demonstrated that the NF-KB transcription factor is a complex oftwo proteins, NF-KB-p50 and NF-KB-p65, which are members of a larger protein family. Analysis of the protein encoded by the human NF-KB-pSO cDNA revealed that it is initially synthesized as a 105-kDa polypeptide, which contains several ankyrin repeat domains in the carboxyl-terminal portion (4). Posttranslational processing generates the mature DNA-binding NF-KB-p50 form by removal ofthe segment containing the ankyrin repeats (5). The processed protein contains an "'300-amino acid region (the Rel domain) with sequence similarity to proteins encoded by the rel oncogene and the dorsal gene ofDrosophila (reviewed in ref. 6). The Rel domain ofNF-KB-p50 is required for DNA binding and for the ability to form homodimers and heterodimers with other Rel family proteins (4, 7). Studies of the c-Rel protein and the retrovirus-transduced form v-Rel indicated that these proteins associate with other cellular proteins (8-10). It is not apparent whether all of the cellular proteins that associate with these Rel proteins are themselves members of the Rel protein family. To study the interactions between NF-KB-p50 and other cellular proteins, aform ofNF-KB-p5O was generated that has a unique antigenic determinant for purification and detection and a separate element that enables labeling in vitro by phosphorylation with [y32P]ATP. When 32P-labeled NF-KBp50 was used as a molecular probe in Western blots and in Agtll expression library screenings, interactions with other members of the Rel protein family were observed. In addition, these analyses indicated that the NF-KB-p50 protein also associates with other cellular proteins that are not members of the Rel family, including the NF-IL6 protein, a transcription factor ofthe basic domain, leucine-zipper class. MATERIALS AND METHODS Bacterial Expression. The NF-KB-p5O cDNA was obtained by the polymerase chain reaction using human Jurkat T-cell cDNA and oligonucleotide primers based on the NF-KB-p50 sequence (4). The construction of the T7 polymerase expression vector (referred to as the FLAG-HMK vector) has been described (11). The 343-amino acid NF-KB-Spe protein was generated by cleaving and religating the NF-KB cDNA at a unique Spe I site. The human c-rel cDNA (12) and the NF-IL6 cDNA fiagments in phages B and C were subcloned to the FLAGHMK expression vector to produce the c-Rel, NF-IL6-B, and NF-IL6-C proteins. Recombinant proteins were expressed in the BL21 pLysS bacterial strain after isopropyl j-Dthiogalactoside induction (13). Purification and Labeling of Proteins. NF-KB and NF-IL6 proteins were purified by conventional chromatography or low pH elution from M2 anti-FLAG antibody resin (provided by William Brizzard, IBI-Kodak, New Haven, CT). Proteins were labeled in vitro with ['y-32P]ATP (NEG-002Z; DuPont/ NEN) by using protein kinase (P-2645; Sigma) as described (11). Western Blotting and Agtll Library Screening. Eukaryotic RIPA extracts (14) and nuclear extracts (15) were prepared, electrophoresed, and transferred to nitrocellulose. Transferred proteins were denatured in 6 M guanidine hydrochloride at 40C, renatured, and then incubated with 32P-labeled protein for 4 hr at 40C in buffer of 1% Nonidet P-40, 50 mM Hepes (pH 7.5), 5 mM DTT, 1 mM EDTA, 1 mM PMSF, and 5 mM MgCl2, with the NaCl concentration indicated in the figure legends. A Agtll library constructed from poly(A)+ RNA from -interferon-treated HeLa cells (Stratagene) was plated and screened (16, 17) with 32P-labeled NF-KB-p50 protein as described (11). Transcription and Translation in Vitro. The NF-KB-p50 and NF-IL6-C cDNA inserts were subcloned into the Bluescript vector (Stratagene). To generate a shorter form of the NFKB-p50 protein, the DNA template was digested at the Spe I site (18). The NF-IL6 cDNA was linearized at a Sac I site to produce a form of the NF-IL6-C protein (NF-IL6-Sac) that terminates at amino acid 306 (19). Linearized DNAs were Abbreviations: DTT, dithiothreitol; HMK, heart muscle kinase; IL, interleukin; PMSF, phenylmethylsulfonyl fluoride. tPresent address: Division ofImmunology, Department ofMedicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215. 8145 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Nadl. Acad. Sci. USA 89 (1992) transcribed in the presence of m7G(5')ppp(5')G cap (Pharmacia) using T3 or T7 polymerase (Stratagene). RNA was translated in the presence of [35S]methionine (NEG-009A; DuPont/NEN) using rabbit reticulocyte lysate (Promega). Immunoprecipitatlon. For immunoprecipitation analysis, 35S-labeled proteins were combined with control bacterial extract (BL21) or with extracts containing NF-KB-p50 or NF-IL6-C proteins in an immunoprecipitation buffer ofphosphate-buffered saline with 10% glycerol, 0.1 mM EDTA, 2.5 mM MgCl2, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF, and trypsin inhibitor at 0.02 mg/ml. After addition of M2 anti-FLAG antibody (IBI-Kodak) and protein G beads (Pharmacia), immune complexes were precipitated by centrifugation. Immunoprecipitated proteins were electrophoresed on 12% polyacrylamide/SDS gels and then treated with EN3HANCE solution (DuPont/NEN) for autoradiography.