A Novel NF-KB Complex Containing p65 Homodimers - Molecular and ...

MoLECULAR AND CELLULAR BIOLOGY, Dec. 1993, p. 7826-7835 0270-7306/93/127826-10$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 13, No. 12

A Novel NF-KB Complex Containing p65 Homodimers: Implications for Transcriptional Control at the Level of Subunit Dimerization PARHAM A. GANCHI,1 SHAO-CONG SUN,' WARNER C. GREENE,' AND DEAN W. BALLARD2* Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco General Hospital, San Francisco, California 94141-9100,1 and Howard Hughes Medical Institute, Vanderbilt University, 802 Light Hall, Nashville, Tennessee 37232-02952 Received 19 May 1993/Returned for modification 10 July 1993/Accepted 15 September 1993

The predominant inducible form of the NF-KB transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates KB-directed transcription, whereas p50 functions as a KB-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-cB p50-p65 heterodimer, this novel p65 homodimeric form of NF-KB is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RRPS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with IKB. These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for IKBc recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-KB/Reldirected gene expression.

Nuclear induction of the NF-KcB transcription factor complex is an important convergent step in several distinct receptor-mediated signaling pathways that potentiate the growth response of activated T lymphocytes (19, 26, 39, 62). This specific inductive event is initiated upon cellular stimulation with a variety of soluble mediators, including T-cell receptor ligands (22, 29, 33, 34), interleukin-1 (52, 63), interleukin-2 (3), tumor necrosis factor alpha (40, 52), and phorbol esters (5, 12, 62). Prior studies have revealed that the major inducible form of NF-KB corresponds to a heterodimeric complex composed of 65-kDa (p65) and 50-kDa (p50) subunits (6). Each of these DNA-binding proteins has extensive amino acid sequence homology with the N-terminal halves of the v-Rel oncoprotein, its cellular homolog c-Rel, and the Drosophila morphogen Dorsal (13, 24, 36, 42, 50, 55; reviewed in reference 25). The induced nuclear expression of this p50-p65 complex is believed to involve the phosphorylation, dissociation, and subsequent degradation of an associated cytoplasmic inhibitor now termed IKBa (4, 5, 17, 23, 27, 65). The recent finding that NF-KB also specifically activates IKBot gene expression indicates the presence of an autoregulatory feedback mechanism that ensures the rapid but transient biological action of nuclear NF-KB (17, 65). Previous investigations have firmly established that the p65 subunit of NF-KB is primarily responsible for both the *

transactivation function (8, 57, 60) and IKBct-mediated repression of the heterodimeric p65-pSO complex (6, 11, 21, 27, 50, 55, 65, 68). This DNA-binding subunit contains an N-terminal Rel homology domain of approximately 300 amino acids and a C-terminal transactivation domain that is lacking in the p50 subunit (8, 13, 24, 36, 42, 50, 55, 57, 60). Comparative functional studies with transfected genes have further suggested that p65 homodimers can induce KBdependent gene expression in the absence of p50 (8, 57, 60). In contrast, p50 homodimers (originally termed KBF1) (32, 36, 71) appear to act as KB-specific repressors in vivo (20, 34, 60), consistent with the absence of a strong activation domain within this polypeptide. Furthermore, while IKBot can trap both p65 and p50 homodimers in the cytoplasm (11, 21), this inhibitor selectively blocks the DNA binding activity of p65 only (6, 11, 21, 50, 55, 68). Recent studies indicate that dissociation of the IKcBa subunit unmasks a functional nuclear localization signal present in NF-KB (11, 21), thereby allowing its rapid translocation to the nucleus, where this inducible complex acts in concert with other constitutive transcription factors to induce the expression of select T-cell activation genes (7, 12, 19, 26, 31, 34, 39). Although conclusive biochemical evidence for inducible p5O-p65 heterodimers (6) and constitutive p5O-p5O homodimers (KBF1) (20, 32, 34, 36, 71) has been assembled, it remained unclear whether endogenous p65-p65 homodimers are in fact present in nontransfected cells. We now show that p65 homodimers exist as an inducible detergent-sensitive pool in the cytoplasm of nontransfected Jurkat human T cells

Corresponding author. 7826

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and appear within the nucleus following cellular stimulation with phorbol 12-myristate 13-acetate (PMA). Furthermore, the domain of p65 that mediates homodimerization is located within a segment of the N-terminal Rel homology domain that is required for IKBa binding (21). Site-directed mutagenesis studies have identified two important amino acid residues within this Rel homology subregion that are required for p65 homodimerization but not for heterodimerization with p50. One of these determinants corresponds to the only conserved cysteine residue present within this interaction domain (Cys-216). The other represents a conserved serine residue present within a consensus recognition sequence for cyclic AMP (cAMP)-dependent protein kinase (RRPS; amino acids 273 to 276) (25, 35). Unexpectedly, mutations affecting these specific amino acids failed to prevent functional association of the resultant p65 monomers with the inhibitor IKBa, thus indicating that dimerization is not an absolute prerequisite for the assembly of p65-IKBa complexes in the cytoplasm. MATERIALS AND METHODS Expression vectors and reporter plasmids. The pCMV4based expression vectors (2) encoding human NF-KB p65 (8, 55), p50 (amino acids 1 to 461) (36), IKBa (27), and all truncation mutants of p65 have been described previously (8, 21). Site-directed mutations were introduced into wild-type p65 by using phosphorothioate chemistry (47) and specific oligonucleotide primers that introduced diagnostic restriction sites. The KB reporter plasmid containing the human immunodeficiency virus type 1 (HIV-1) KB enhancer motif and the chloramphenicol acetyltransferase (CAT) gene has been previously described (64). Cell transfections, extract preparations, and CAT assays. The monkey kidney cell line COS and human Jurkat T cells were maintained in Iscove's and RPMI media, respectively, supplemented with 10% fetal calf serum, 2 mM L-glutamine, and antibiotics. Effector and reporter plasmids were introduced into both cell lines by using DEAE-dextran (30). COS cells were metabolically radiolabeled with both [35S]methionine and [35S]cysteine, and whole-cell extracts were prepared under previously described detergent lysis conditions (21). Jurkat nuclear and cytoplasmic extracts were prepared before or after stimulation with PMA (50 ng/ml; 2.5 h) by the method of Schreiber et al. (61). CAT assays were performed with whole-cell extracts from Jurkat cell transfectants as described previously (15, 49). DNA binding studies. A functional palindromic variant of the interleukin-2 receptor alpha KB enhancer sequence (5'CAACGGCAGQQAA 2JCCCCTCTCCer-3') (KB-pd) (9) was used as a probe (-10 fmol, 200,000 cpm) in DNA binding reaction mixtures (20 p,l) that contained protein extract (10 to 20 p,g) and 0.2 p,g of double-stranded poly(dIdC) in 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid) (pH 7.9)-5% glycerol-1 mM EDTA-0.5 mg of bovine serum albumin per ml-1% Nonidet P-40-5 mM dithiothreitol (7, 21). Deoxycholate (0.5%) (5) or specific polyclonal antisera (1 pl) were added as indicated. Nucleoprotein complexes were resolved on native 5% polyacrylamide gels and visualized by autoradiography (12). For UV cross-linking analyses (7, 44), polyacrylamide gels containing the fractionated nucleoprotein complexes were irradiated at 300 nm for 20 min. Gel slices corresponding to specific nucleoprotein complexes were excised, boiled in the presence of 4% sodium dodecyl sulfate (SDS)-5% ,-mercaptoethanol-8 M urea, and subjected to SDS-polyacrylamide gel

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electrophoresis (SDS-PAGE) (7). Alternatively, radiolabeled adducts were passively eluted from crushed gel slices in 50 mM Tris (pH 7.5)-140 mM NaCl-1% deoxycholate-1% Triton X-100-0.1% SDS-0.1 mg of bovine serum albumin per ml-2.5% glycerol-1 mM dithiothreitol at 4°C for 15 to 20 h and subjected to immunoprecipitation analysis as described below. Immunoprecipitation and immunoblot analyses. Expression of transfected wild-type and mutated p65 cDNAs was monitored by using specific antipeptide antibodies raised against the unique N- and C-terminal sequences of p65 (amino acids 1 to 21 and 535 to 551, respectively). Briefly, 100 [lI of 35S-labeled COS cell extract was sequentially precleared, incubated with the appropriate p65-specific antiserum, washed extensively in lysis buffer, and analyzed on an SDS-10% polyacrylamide gel (21). Alternatively, COS cell extracts were fractionated on SDS-10% polyacrylamide gels, transferred to nitrocellulose filters, and probed with an antibody recognizing the C terminus of IKBBa (amino acids 289 to 317). Immunoreactive species were detected by an enhanced chemiluminescence assay (21) as specified by the manufacturer (Amersham, Inc.). RESULTS Activation of human Jurkat T cells induces nuclear expression of p65 homodimers. As described above, it has remained unclear whether endogenous p65 homodimeric complexes actually exist in nontransfected cells as a physiologically relevant transcription factor species. To directly address this possibility, optimum parameters for the detection of p65 homodimer DNA binding were first determined by using whole-cell extracts from COS cells transfected with pCMV4p65, a recombinant cDNA expression vector that directs the high-level synthesis of human p65 (8). Consistent with previous studies using either renatured or recombinant p65 protein (9, 38), we observed that both the concentration of the nonspecific competitor poly(dI-dC) added and the precise sequence of the radiolabeled KB enhancer probe significantly influenced the detection of p65 homodimers. Cytoplasmic extracts from human Jurkat cells were tested by using these p65 DNA binding conditions in combination with deoxycholate, a detergent known to dissociate IKBBa from the NF-KB p65-p5O heterodimer in vitro (5). As shown in Fig. 1A, a discrete nucleoprotein complex was detected at low concentrations of poly(dI-dC) (10 ng/p,l; lane 4); this complex comigrated with COS-derived p65 homodimers (lane 2) but exhibited a mobility that was significantly slower than that of the prototypical p65-p50 heterodimeric complex (lane 1). Similarly, small amounts of a complex with an indistinguishable electrophoretic mobility were detected in nuclear extracts prepared from these cells following stimulation with PMA (Fig. 1A, lane 6), an agent known to induce the rapid nuclear translocation of NF-KB (5, 12, 62). Two complementary experimental approaches were employed to assess whether this slowly migrating nucleoprotein complex in fact corresponded to authentic p65 homodimers. First, as shown in Fig. 1B, DNA binding reaction mixtures containing these various complexes were reacted with either anti-p65- or anti-p50-specific antibodies prior to electrophoresis under native gel retardation conditions. In accord with its indistinguishable mobility relative to COS-derived p65 (Fig. 1B, lanes 4 to 6), the slowly migrating complex detected in either cytoplasmic or nuclear extracts from Jurkat cells were "supershifted" in the presence of anti-p65 antiserum (lanes 8 and 11) but not with either preimmune

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FIG. 1. Endogenous p65 homodimers exist as an inducible detergent-sensitive pool in Jurkat T cells. (A) Cytoplasmic (CYTO) and nuclear (NUC) extracts from the indicated cell lines were incubated with a radiolabeled KB enhancer probe and resolved on a native 5% polyacrylamide gel. The reaction mixture for the DNA binding reaction analyzed in lane 4 was supplemented with 0.5% deoxycholate prior to electrophoresis. NUC-PMA refers to nuclear extracts prepared from Jurkat cells stimulated with 50 ng of PMA per ml for 2.5 h prior to analysis. The arrows identify nucleoprotein complexes containing p65 homodimers. (B) DNA binding reaction mixtures containing extracts described for panel A were supplemented with preimmune (PI), anti-p65 (oap65), or anti-pSO (ap5O) antiserum prior to electrophoresis. Supershifted species are identified by arrows. (C) DNA binding reaction mixtures containing nuclear extracts from PMA-stimulated (45 min) Jurkat cells were supplemented with preimmune (PI) or antipeptide antisera raised against the indicated Rel-related proteins prior to gel shift analysis. (D) Gel slices containing p65 homodimers were irradiated with UV light in situ and excised, and radiolabeled adducts were either resolved directly by SDS-PAGE (lanes 1 to 3) or eluted and subjected to immunoprecipitation analysis using a p65-specific antiserum (lanes 5 to 7). A gel slice containing p65-pSO heterodimers from PMA-stimulated Jurkat nuclear extracts was analyzed in lane 4. UV-cross-linked p65-DNA and p5O-DNA adducts are as indicated.

(lanes 7 and 10) or anti-p5O (lanes 9 and 12) antiserum. Furthermore, as shown in Fig. 1C, this novel p65-associated complex failed to react with a panel of monospecific antipeptide antisera raised against other known members of the NF-KB-Rel family of transcription factors, including p52 (14, 48, 59), c-Rel (18), and RelB (56, 58). Second, when these putative cytoplasmic and nuclear p65 complexes were cross-linked in situ with UV light to a 32P-labeled KB enhancer probe and the specific complexes were isolated, a single photoreactive adduct of -72 kDa was detected following SDS-PAGE (Fig. 1D, lanes 2 and 3). This adduct comigrated with authentic p65-DNA adducts derived from transfected COS cells (lane 1), which migrate as a slightly larger species relative to native p65 because of the presence of the cross-linked oligonucleotide probe (9). In contrast, when the more rapidly migrating p5O-p65 com-

plexes were cross-linked, both p50 and p65 species were identified (Fig. 1D, lane 4). Consistent with these results, a single 32P-radiolabeled adduct of the expected size was specifically immunoprecipitated with p65-specific antiserum (lanes 6 and 7), thus confirming the presence of p65 epitopes. Taken together, these findings strongly suggest that inactive but inducible p65 homodimers exist in the cytoplasm of nonstimulated human T cells. Sequences mediating p65 homodimer formation are contained within the IKBa binding domain. In order to delineate the intrinsic sequences that mediate p65 homodimerization, a series of C-terminal deletions was introduced into the full-length human p65 cDNA. Monkey COS cells were cotransfected with recombinant expression vectors containing these truncated cDNAs together with a full-length wildtype p65 construct and metabolically radiolabeled with both

VOL. 13, 1993

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together with full-length p65. Radiolabeled whole-cell extracts were prepared from these cells and subjected to coimmunoprecipitation analyses with an antiserum specific for the unique N terminus of p65 (amino acids 1 to 21). As shown in Fig. 2B, truncated mutants of p65 (lane 1) lacking up to 200 N-terminal amino acids were readily coimmunoprecipitated with the full-length protein (lanes 2 to 5), suggesting that the proximal two-thirds of the Rel homology domain are dispensable for the homodimerization function of p65. However, removal of an additional 50 amino acids containing a highly conserved cysteine residue (Cys-216; see below) completely disrupted homodimer formation (lane 6).

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[35SJcysteine and [35S]methionine, and then whole-cell exwere immunoprecipitated with an antipeptide antiserum raised against the C terminus of p65 (amino acids 535 to 551). As shown in Fig. 2A, this antibody reacted specifically with a radiolabeled 65- to 72-kDa polypeptide present in COS cells transfected with the full-length p65 expression tracts

vector (lane 2) but not in COS cells transfected with the parental pCMV4 expression vector lacking a cDNA insert (lane 1). When cDNAs encoding the C-terminal truncation mutants of p65 were cotransfected with wild-type p65, many

of the smaller radiolabeled proteins lacking the antibody epitope were coimmunoprecipitated, indicating the formation of stable homodimeric complexes between the fulllength and truncated p65 proteins. Specifically, progressive deletions that removed the p65 transactivation domain (amino acids 450-551; Fig. 2A, lane 3) (8, 57, 60), the minileucine zipper (amino acids 436 to 450; lane 4) (57), and the nuclear localization signal (amino acids 301 to 304; lane 5) (11, 21) did not disrupt homodimerization. However, further deletion to amino acid 270, which ablates a consensus recognition site for protein kinase A (amino acids 272 to 276; RRPS) (35) completely disrupted this function (Fig. 2A, lane 6). Immunoblot analyses of all extracts used in these experiments confirmed that the vectors encoding these truncated mutants expressed comparable amounts of the relevant protein species, including the p65 (1-270) construct (data not shown). These results localize the C-terminal limits of the p65 homodimerization domain to a segment between amino acids 270 and 291. As shown in Fig. 3A, this boundary is binding but distinct from the C-terminal boundary for is coincident with that required for p65 DNA binding, a function that appears to be strictly dependent on dimer

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formation (21). In reciprocal studies, cDNAs encoding a series of N-terminal deletion mutants of p65 were expressed in COS cells

These bidirectional deletion mapping studies thus established that the maximal boundaries of the p65 homodimerization domain lie between amino acids 201 and 291, a segment positioned within Rel homology sequences that also mediates the IKcBa and DNA binding functions of this protein (Fig. 3A) (11, 21, 50, 55). Similar results were obtained when these truncation mutants were cotransfected with p50, indicating that the homo- and heterodimerization domains of p65 are either coincident or significantly overlapping (data not shown). Two distinct classes of point mutations within the Rel homology domain of p65 segregate its homo- and heterodimerization functions. Biochemical studies with purified p65 suggest that this NF-KcB subunit engages its cognate enhancer as a homodimer, presumably reflecting a requirement for the apposition of certain key amino acids from each monomer to form a functional DNA binding domain. In this regard, NF-KB DNA binding activity in vitro is potently inhibited by sufhydryl and oxidizing agents (37, 43, 66, 67). Recently, Toledana et al. (66) have shown that cysteine 62 of human NF-KB p50 is critically involved in mediating these redox effects. Inspection of the predicted p65 primary sequence (55) revealed a total of nine cysteine residues that are exclusively clustered in the Rel homology/DNA binding domain of this protein. Six of these cysteine residues are highly conserved among all other known Rel-related proteins (25, 55). On the basis of these functional and structural considerations, each of the six highly conserved p65 cysteines was replaced with serine, and the resultant mutants were assessed for changes in p65 DNA binding activity and homodimer formation. Whole-cell extracts from COS cells expressing each of these full-length p65 mutants in the presence or absence of IKBa were first analyzed for DNA binding activity in gel retardation assays using a 32P-labeled KB enhancer probe. As shown in Fig. 3B, in vivo-synthesized p65 supported the formation of a distinct nucleoprotein complex (lane 3) which was completely blocked by coexpression with IKBa (lane 4). This wild-type pattern of IKBa-regulated DNA binding activity was fully recapitulated by cysteine mutants altered at amino acid positions 95 (lanes 7 and 8), 160 (lanes 11 and 12), and 197 (lanes 13 and 14). Consistent with results reported for v-Rel (37), substitution of serine for conserved cysteine 38 produced modestly enhanced p65 DNA binding activity (lanes S and 6). In contrast, point mutations introduced into p65 at residues 120 lane 9) and 216 lane 15) signifi(C-120-- S cantly attenuated the DNA binding activity of p65, although Western blot analyses indicated comparable levels of protein expression (data not shown). Of note, five of these six highly conserved cysteine residues are positioned outside the homodimerization domain as localized by deletion mapping. Only cysteine 216 is present within this critical subregion. Thus, one plausible mechanistic explanation for these DNA

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FIG. 3. A single highly conserved cysteine located within the homodimerization domain is essential for p65 DNA binding and homodimer formation. (A) Primary domain structure of p65 with the maximal amino acid endpoints for the DNA binding (amino acids 21 to 291), IKB binding (amino acids 151 to 312), and dimerization (amino acids 201 to 291) domains. The N-terminal Rel homology domain, which contains two highly conserved tetrapeptide motifs, a functional nuclear localization signal (NLS) (KRKR; amino acids 301 to 304) and a consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (RRPS, amino acids 273 to 276), is shown in conjunction with the C-terminal sequences encoding the transcriptional activation domain and the mini-leucine zipper motif (MLZ). The single highly conserved cysteine residue contained within the dimerization domain is also indicated (C216). (B) Extracts from COS cell transfectants expressing unmodified pCMV4, wild-type (WT) p65, or the indicated cysteine mutants in the absence (-) or presence (+) of IKBa were incubated with a radiolabeled KB enhancer probe and analyzed on a native 5% polyacrylamide gel. The arrow indicates nucleoprotein complexes containing homodimers of p65. (C) Full-length (amino acids 1 to 551; FL) and truncated (amino acids 1 to 312) forms of wild-type (WT) p65 and the C120S and C216S mutants were coexpressed in COS cells. Extracts were prepared and subjected to immunoprecipitation analysis with an antiserum specific for the C terminus of p65.

binding results is that the C216S mutant is defective in its ability to homodimerize, a prerequisite for DNA binding, whereas the C120S mutant lacks an essential determinant(s) required for appropriate contacts with the KB enhancer. To address this possibility, coimmunoprecipitation studies were performed with radiolabeled extracts from COS cells expressing full-length (amino acids 1 to 551) and C-terminally truncated (amino acids 1 to 312) forms of these mutant p65 proteins. As shown in Fig. 3C, variants lacking the C terminus of p65 were immunoprecipitated with antiserum reactive with a C-terminal epitope when coexpressed with either wild-type p65 (lane 1) or the C120S mutant (lane 2), suggesting that each of these proteins is capable of forming stable homodimers. However, full-length and truncated (amino acids 1 to 312) forms of p65 containing the C216S mutation failed to coimmunoprecipitate, thus confirming the inability of this cysteine mutant to homodimerize. As only dimers of Rel proteins appear to be capable of binding to DNA, these results likely explain the attenuated DNA

binding activity of the C216S mutant observed in gel shift experiments (Fig. 3B, lane 15). In addition to cysteine 216, the p65 homodimerization domain also contains a consensus recognition site for cAMPdependent protein kinase (RRPS; amino acids 273 to 276) (35). Although its physiological function remains unclear, this site is conserved in almost all known Rel polypeptides and is adjacent to a basic tetrapeptide motif that appears to serve as a common nuclear localization signal for this transcription factor family (25) (Fig. 3A). In this regard, we (69) and others (45) have previously shown that altering serine to alanine within the RRPS motif of the related v-Rel oncoprotein has no effect on its DNA binding and heterodimerization functions. To assess whether this site is a requisite determinant for p65 DNA binding and/or homodimer formation, serine 276 was substituted with four distinct amino acids (RRPS--RRPA, RRPG, RRPD, and RRPE). Notably, two of these amino acid replacements, RRPD and RRPE, introduce negatively charged residues at

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VOL. 13, 1993

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,0 \ a,p WPO °.1W FIG. 4. Alteration of the serine residue withini the conserved consensus RRPS motif of p65 disrupts DNA b inding and homodimerization. (A) Extracts from COS transfecitants containing wild-type (WT) p65 or the indicated RRPS mutants Nwere assayed for DNA binding activity in the absence (-) or presenc(e (+) of IKBa by using a radiolabeled KB enhancer probe. Nucleopr(otein complexes containing p65 are indicated. (B) Full-length (FL RRPA, and (amino acids 1 to 312) forms of wild-type (WT) p RRPD were coexpressed in COS cells. Extracts ifrom 35S-labeled cells were analyzed by immunoprecipitation wit h an antiserum

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the phosphate acceptor position, thus potenti;ally mimicking the local charge distribution associated withi phosphorylation. As shown in Fig. 4A, DNA binding assays revealed that none of these p65 mutants formed stable com;)lexes with the radiolabeled KB enhancer probe (lanes 3 and 5 and data not A

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shown). In addition, cotransfection experiments performed with cDNAs encoding full-length (amino acids 1 to 551) and truncated (amino acids 1 to 312) forms of each of these mutants demonstrated a loss of homodimerization (Fig. 4B, lanes 2 and 3, and data not shown). As all of these mutants were stably expressed as assessed by independent immunoblotting studies (data not shown), these RRPS mutations define a second distinct class of homodimerization-defective variants of p65 that fall within the dimerization domain as defined by deletion mapping studies. In complementary experiments designed to assess the abilities of these various mutants to form heterodimers with p5O, extracts from COS cells coexpressing each mutant and p5O were subjected to coimmunoprecipitation analyses with p65-specific antiserum. As shown in Fig. 5A, p5O and a smaller N-terminal degradation product of p50 were coimmunoprecipitated with wild-type p65 (lane 1). Surprisingly, both the C216S mutant (lane 2) and the two RRPS mutants (lanes 3 and 4), which failed to homodimerize efficiently, retained the ability to interact with p5O. Thus, the C216S and RRPS p65 mutations segregate the homo- and heterodimerization functions of this transcription factor. In order to determine whether these homodimerization mutants could be functionally rescued by heterodimerization with p5O, two in vivo experiments were performed. First, Jurkat human T cells were cotransfected with an HIV-1 KB CAT reporter plasmid (KB-TATA-CAT) in the presence or absence of expression vectors encoding p5O and each p65 mutant. As shown in Fig. SB, cotransfection of wild-type p65 and KB-TATA-CAT resulted in potent activation of KBdirected transcription (-180 fold) compared with that observed with either the unmodified parental expression vector (pCMV4) or p5O alone. As expected, the C216S mutant and

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FIG. 5. Homodimerization-defective p65 variants are functionally rescued by coexpression with p50. (A) Radiolabeled extracts from COS cells expressing p50 and the indicated p65 variants were immunoprecipitated with p65-specific antiserum and fractionated by SDS-PAGE. The positions of p65 and p50 are as shown. The asterisk denotes an N-terminal degradation product of p50. (B) Human Jurkat T cells were cotransfected with expression vectors encoding the indicated polypeptides and a reporter CAT plasmid containing the HIV-1 KB enhancer motif (KB-TATA-CAT). CAT activity is presented as fold induction relative to basal levels measured with pCMV4 lacking a cDNA insert. (C) Whole-cell extracts from COS cells expressing the indicated p65 variants in the absence or presence (+p50) of p50 were resolved by SDS-PAGE and transferred to nitrocellulose. The induction of endogenous IKBa (IKB,) expression was assessed by Western blot analysis with an IKBa-specific antiserum, using an enhanced chemiluminescense detection system.

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both RRPS mutants (RRPA and RRPD) were minimally active when transfected alone. However, when these p65 mutants were expressed in the presence of the p50 subunit of NF-KB, each mediated significant transcriptional activation (200- to 300-fold). To extend these initial findings with exogenous CAT reporter plasmids to a more physiologically relevant setting, changes in endogenous IKBa protein expression were monitored by immunoblotting with extracts from COS cells expressing each of these p65 variants. We have previously demonstrated that when transiently introduced into these cells, wild-type p65 homodimers potently induce endogenous IKBot mRNA and protein expression (65). As shown in Fig. 5C, endogenous IKBa was readily detected in extracts from COS cells expressing wild-type p65 (lane 2) but not in those from pCMV4-transfected cells (lane 1). Consistent with their impaired DNA binding activity, neither the C216S mutant (lane 3) nor the RRPS mutants (lanes 4 and 5) alone significantly induced endogenous IcBa expression. However, all three of these p65 constructs were fully functional when expressed in the presence of p50 (lanes 8 to 10). Monomeric p65 mutants form stable complexes with IKBa in vivo. Prior studies have demonstrated that amino acids 151 to 312 of the p65 Rel homology domain confer its IKB binding function and contain a functional nuclear localization signal that is efficiently masked upon binding by IKBa (11, 21). The dimerization domain of p65 is contained entirely within these sequences required for IKBa binding (Fig. 3A). These findings raised the possibility that mutations introduced within the dimerization domain of p65 may also affect its ability to bind IKBBa. To test this hypothesis, COS cells were cotransfected with expression vectors encoding the wild-type IKBBa protein together with the cysteine 216 and RRPS p65 mutants and immunoprecipitated with p65-specific antiserum (Fig. 6). As expected, IKBBa was readily detected in immunoprecipitates from cells cotransfected with wild-type p65 (lane 2). Similarly, as shown in lanes 3 to 5, IKBxa was also efficiently coimmunoprecipitated with each of these homodimerization-defective mutants. These results suggest that the assembly of p65-IKBa complexes in vivo is not obligately dependent upon prior dimerization of p65. DISCUSSION The prototypical form of NF-KB is an inducible heterodimeric pSO-p65 complex that can activate transcription from a variety of cellular genes and viruses, notably HIV (25, 39, 46). This pSO-p65 complex is sequestered within the cytoplasmic compartment of resting T cells because of its association with various inhibitors, including IKBao (4, 5, 11, 21, 27, 41, 54). Upon cellular stimulation, IKBa is phosphorylated and rapidly degraded, thereby permitting nuclear translocation of the heterodimeric NF-KB complex (4, 5, 17, 23, 65). Although earlier studies suggested that only the p50 subunit of NF-KB possessed intrinsic DNA binding activity (6), subsequent investigations have shown that the p65 subunit of NF-KB is also capable of binding DNA (8, 9, 50, 55). Furthermore, p65 contains a potent transcriptional activation domain that is largely responsible for the functional effects attributed to the heterodimeric NF-KB complex (8, 57, 60). It is also now well recognized that p50 homodimers (previously termed KBF1) (36, 71) may be constitutively expressed in the nuclei of intact human T lymphocytes (20, 34). These p50 homodimers function as specific repressors of KB-directed transcription, presumably because of the ab-

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