INTRODUCTION

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Transcription factors of the Rel/NF-B family are present in most or all mammalian and avian cells and influence the expression of many genes (for a review, see references 1 and 19). These factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding and dimerization. Although each protein has a nuclear localization signal (NLS), the various homo- and heterodimers are cytoplasmic by virtue of an inhibitor protein whose binding masks the NLSs. Several different inhibitors (called IBs) are known, and they constitute a family of related proteins. Each IB contains five or six so-called ankyrin repeats, motifs of about 33 amino acids which are abundantly represented in the protein ankyrin. In ankyrin, these repeats apparently function in groups of about six (18) and constitute a protein-protein interaction domain. In addition, the inhibitors contain a C-terminal region which tends to be highly acidic and to resemble PEST sequences associated with rapid protein turnover (22). The IB N-terminal region contains two serine residues which, upon phosphorylation, trigger ubiquitination and proteasomal degradation of the inhibitor, thus freeing the dimer to enter the nucleus (4, 5, 7, 26-29).

While several crystal structures of Rel family members are known (6, 6a, 10, 20), none is known for the IB family. Nevertheless, studies with various mutants of the Rel family and/or IB proteins have led to some general conclusions about their interaction. First, a Rel family dimer binds a single IB molecule (8, 12, 14). Second, at least some of the contacts with IB occur in the dimerization domains of the Rel proteins. Based on the crystal structure, each monomer of p50 and RelA consists of two separate domains connected by a short hinge region. The N-terminal domain contains some (but not all) of the residues that contact DNA, while the C-terminal domain contains the remaining DNA contacts and governs dimerization. Partial deletion of the N-terminal domain of RelA did not abolish binding of IB (9). Similar results were obtained with Cactus and N-terminal deletion mutants of Dorsal (Drosophila relatives of IB and Rel proteins, respectively) (11, 25). In addition, mutation of two residues in the dimerization domain of Dorsal prevented interaction with Cactus but did not affect DNA binding or dimerization (16). These studies suggest contact between IB and the C-terminal part of the RHD. The third general conclusion is that the dimer NLSs, which were not resolved in the crystal structures but are immediately downstream of the dimerization domain in the primary structure, are somehow involved in binding to IB. While in at least some cases an intact NLS may not be required for binding to IB (11, 23), several studies have shown that altering the NLS can abolish binding (2, 9, 15, 25). Involvement of the NLS would correlate nicely with the fact that the dimer NLSs are masked in a dimer-IB complex. The final generalization is that most of the IB contacts occur within the ankyrin repeats. Mutations within the ankyrin domain disrupt binding, while deletion of the N terminus and/or the C terminus usually does not (8, 12, 13, 17, 30).

In this study, we examined in more detail the interaction between IB and Rel family homodimers. We show that (i) the dimerization domains of p50, RelA, and c-Rel are sufficient for binding to IB both in vivo and in vitro, (ii) the NLSs of RelA and c-Rel are not required for binding to IB but do stabilize the interaction, (iii) the NLS of p50 is required for binding to IB, (iv) only certain residues within the p50 NLS are required for binding, and (v) in a p50-IB complex or a c-Rel-IB complex, the N terminus of IB either directly or indirectly masks one or both of the dimer NLSs.

	MATERIALS AND METHODS

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Plasmids and mutagenesis. Human IB cDNA, a gift of Al Baldwin, was cloned into the plasmid Rc/CMV (Invitrogen). Human IB mutants from which residues 2 to 30 or 2 to 53 had been deleted were gifts of Simon Whiteside; an IB mutant from which residues 44 to 50 had been deleted was a gift of Masashi Muroi. Wild-type p50 (amino acids 1 to 399 of human p105) in Rc/CMV was a gift of Alain Israël. C-terminal truncations of c-Rel and RelA were created to eliminate their transactivation domains. The resulting c-RelC (residues 1 to 361 of human c-Rel) has been described earlier (24) and was used both in Rc/CMV and in a modified Bluescript vector containing the Rous sarcoma virus long terminal repeat (24). The plasmid RelAC was created from the plasmid pCMVINp65 (a gift of Craig Rosen) by PCR to amplify the cDNA, while the coding region after residue 337 was truncated. The codon for Ser 338 was converted to a TAA stop codon. At the same time, 5' BamHI and 3' XhoI sites were engineered up- and downstream of the coding sequence. The resulting PCR product was cleaved with BamHI and XhoI and cloned into the corresponding sites of the CMV-based expression vector pcDNA3 (Invitrogen).

Transfection. Human kidney 293 cells were seeded at 10⁶/6-cm-diameter dish. Twenty-four hours later, they were transfected by the calcium phosphate method.

Immunofluorescence. Our procedure has been described previously (3). Briefly, transfected 293 cells were seeded onto collagen-coated glass coverslips 24 h after transfection. After an additional 16 h, the cells were fixed in 2% paraformaldehyde, quenched in phosphate-buffered saline containing 50 mM glycine, rinsed, permeabilized in Tris-buffered saline (pH 7.4) containing 0.1% Nonidet P-40 (NP-40) and 0.5 M NaCl, blocked with goat serum and chicken serum albumin, and incubated with primary antibody at 1:40 or 1:80 for 1 h at room temperature. The secondary antibody was goat anti-rabbit immunoglobulin G (IgG) conjugated with fluorescein isothiocyanate (Kirkegaard and Perry Laboratories). In some cases, treatment with primary antibody was followed by incubation with biotinylated goat anti-rabbit IgG (Vector Laboratories) and then with fluorescein-conjugated streptavidin (Immunotech). After a final washing in phosphate-buffered saline, coverslips were dried and mounted on slides with Vectashield (Vector Laboratories).

Antisera. Rabbit antisera raised against synthetic peptides were employed in all experiments. Most of them have been described previously (8, 21, 24). Antisera that recognize p50 (and boundaries of the peptides in the human protein) are as follows: no. 1141 (residues 2 to 15), no. 1157 (residues 339 to 357), and no. 1613 (residues 354 to 374). Antisera that recognize RelA are no. 1207 (residues 2 to 17), no. 1774 (residues 314 to 329), and no. 1226 (residues 537 to 550). The c-Rel antiserum was no. 1136 (residues 305 to 320). Antisera that recognize IB are no. 1309 (residues 2 to 15) and no. 1258 (residues 301 to 317).

Metabolic labeling and immunoprecipitation. Twenty-four hours after transfection, cells were grown in media containing [³⁵S]methionine and [³⁵S]cysteine (each at 50 µCi/ml; 1,000 Ci/mmol; Amersham) for 1 h. For whole-cell extracts, cells were lysed and immunoprecipitated in ELB buffer (50 mM HEPES [pH 7.0], 250 mM NaCl, 5 mM EDTA, 0.1% NP-40) containing protease inhibitors. For cytoplasmic extracts, cells were lysed in HB buffer (25 mM Tris [pH 7.4], 5 mM KCl, 1 mM MgCl₂, 0.05% NP-40) and nuclei and insoluble material were removed by centrifugation at 10,000 × g for 5 min. For immunoprecipitation of the cytoplasmic extracts, NaCl and Triton X-100 were added to final concentrations of 0.1 M and 0.5%, respectively. Precipitates were collected on protein A-Sepharose (Pharmacia) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Usually 5% of the total lysate from a 6-cm-diameter dish was used per lane, giving a strong signal on X-ray film after overnight exposure.

Immunoprecipitation and immunoblotting. Transfected cells were lysed and immunoprecipitated in ELB buffer, as described above, but without prior metabolic labeling. Precipitates were subjected to SDS-PAGE, and the gel was blotted onto an Immobilon membrane (Millipore). The immunoblot was incubated first with primary antiserum at a 1:1,000 dilution and then with peroxidase-conjugated goat anti-rabbit IgG (Boehringer Mannheim) at a 1:10,000 dilution and developed by the Amersham enhanced chemiluminescence system.

EMSA. Transfected or cotransfected cells were lysed in ELB buffer and analyzed as previously described (8). The ³²P-oligonucleotide probe contained the B binding site from the interleukin-6 promoter. Extracts and probe, with or without antisera, were mixed in electrophoretic mobility shift assay (EMSA) buffer: 10 mM HEPES (pH 7.5), 80 mM KCl, 1 mM EDTA, 1 mM EGTA, 6% glycerol, 0.05 µg of salmon sperm DNA per µl, and 0.05 µg of poly(d[IC]) per µl.

	RESULTS

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The N-terminal portion of the RHD is not required for IB binding. With the X-ray crystal structure of the p50 homodimer as a guide (10, 20), mutations were introduced in the coding sequences of the human p50, human RelA, and human c-Rel cDNAs, enabling us to delete the entire N-terminal section of the RHD. We also deleted the transactivation domains of RelA and c-Rel in order to avoid induction of endogenous IB upon transfection. The resulting constructs (RelACN and c-RelCN) consisted almost entirely of the dimerization region of the RHD (Fig. 1). These truncated proteins were then expressed in human 293 cells either alone or in combination with the human IB protein. Subcellular localization of the overexpressed proteins was assayed by indirect immunofluorescence with antisera specific for the NF-B proteins. As shown in Fig. 2A, the N-terminally truncated p50 construct (p50N) was strictly nuclear (panel a). When this p50 mutant was coexpressed with IB in excess, however, it was cytoplasmic (panel b), indicating that IB bound to the mutant and retained it in the cytoplasm. Similarly, the comparable RelA mutant protein (RelACN) was nuclear when expressed alone (panel c) but was retained in the cytoplasm when it was coexpressed with IB (panel d). The same results were obtained with c-RelCN (panels e and f). Based on these results, we conclude that there are no sites in the N-terminal domains of these NF-B proteins that are necessary for IB binding in vivo or retention of the NF-B homodimers in the cytoplasm.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Deletion mutants of p50, RelA, and c-Rel. Within each of the three sets, the wild-type protein is diagrammed on top. N- and C-terminal residue numbers in the N and CN mutants are indicated. Numbers for the NLS mutants indicate the deleted residues. A summary of results presented in the text is shown at the right.

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Rel family proteins lacking the N-terminal domain are able to bind IB. (A) 293 cells were transfected with N-terminal deletion mutants of p50, RelA, or c-Rel or cotransfected with IB in excess as indicated for each panel. Localization of the mutant proteins was assayed by immunofluorescence with antisera against p50 (no. 1613), RelA (no. 1774), or c-Rel (no. 1136). The targets of the antisera in the cotransfection experiments (b, d, and f) are underlined. (B) 293 cells were transfected, cotransfected, or mock transfected, as indicated. Whole-cell lysates were immunoprecipitated with anti-IB (no. 1309) (lanes 1, 2, and 4), preimmune serum (lane 3) or antiserum raised against the N terminus of RelA (no. 1207, RelA[N]) (lane 5), a peptide located within RelACN (no. 1774, RelA[I]) (lane 6), the C terminus of wild-type RelA (no. 1226, RelA[C]) (lane 7), or a peptide located within c-Rel CN (no. 1136, c-Rel[I]) (lanes 8 to 11). Immunoprecipitates were fractionated by SDS-PAGE, and immunoblots were analyzed with anti-p50 (no. 1157) (lanes 1 and 2) or anti-IB (no. 1309) (lanes 3 to 11). IB did not precipitate with antisera raised against the N or C terminus of wild-type RelA (lanes 5 and 7), proving that the precipitated IB in lane 6 was bound to RelACN and not to endogenous full-length RelA. Ab for IP and Western, antibody used for immunoprecipitation and immunoblotting, respectively.

The NLS of p50, but not that of RelA or c-Rel, is required for IB interaction. Having shown that the C-terminal portions of the RHDs of p50, RelA, and c-Rel are sufficient for binding to IB, we looked for critical residues within that region. Several groups have reported that the NLSs of Rel family proteins are required for IB binding (2, 9, 15, 25), while others have found that they are not (11, 23). To investigate this problem, oligonucleotide-directed mutagenesis was used to delete the NLSs of p50, RelA, and c-Rel. These mutant proteins were then assayed in vivo and in vitro for their subcellular localizations and abilities to bind IB.

View larger version (73K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   View larger version (37K): [in this window] [in a new window]   FIG. 3.   IB binds to RelA- and c-Rel mutants lacking an NLS but not to NLS p50. (A) 293 cells were transfected or cotransfected as indicated, and localization of the expressed proteins was assayed by immunofluorescence. For cotransfected cells, the targets of the antisera are underlined. Nonunderlined partners were present in excess in panels d, e, i, j, and m. (B) 293 cells were transfected, cotransfected, or mock transfected, as indicated. Twenty-four hours later, the cells were incubated in medium containing 35S-amino acids and whole-cell lysates were immunoprecipitated with anti-p50 (no. 1141) (lanes 1 and 3), anti-IB (no. 1309) (lanes 2 and 4), or anti-c-Rel (no. 1136) (lanes 9 to 13). Precipitates were analyzed by SDS-PAGE. For lanes 5 to 8, samples were first precipitated with anti-RelA (no. 1207) and then boiled and reprecipitated with anti-RelA (no. 1207) (lanes 5 and 7) or anti-IB (no. 1309) (lanes 6 and 8). Ab for IP, antibody used for immunoprecipitation. (C) 293 cells were transfected, as indicated, with plasmids encoding IB, full-length RelA, or full-length RelANLS or were mock transfected (lane 3). Twenty-four hours later, the cells were cultured in medium containing 35S-amino acids and cytoplasmic extracts were immunoprecipitated with anti-RelA (no. 1226). Precipitates were analyzed by SDS-PAGE.

IB must mask both NLSs of a dimer. We sought to determine whether a dimer needs both of its NLSs for nuclear entry or whether one is sufficient. To answer this question, we cotransfected RelACNLS with excess p50 so that RelACNLS would be found predominantly in heterodimers with p50. The localization of the RelA mutant was determined by immunofluorescence with anti-RelA. Despite its lack of an NLS, the RelA mutant was found in the nucleus (Fig. 3A, panel j), indicating that a single NLS is sufficient for nuclear entry of a dimer. We also observed nuclear localization of a p50 dimer in which only one monomer had an NLS (data not shown). Thus, if a dimer is to be retained in the cytoplasm, IB binding must mask both NLSs.

Deletion of the c-Rel or RelA NLS weakens interaction of the dimer with IB. Although the NLS of c-Rel or RelA is not required for binding to IB, its presence does stabilize the binding. We demonstrated this several ways. First, we asked whether IB could inhibit the DNA-binding activity of these NLS-deficient mutants in vitro. Extracts of 293 cells expressing RelACNLS alone or RelACNLS together with excess IB were tested for DNA-binding activity by EMSA. Whereas IB completely inhibited DNA binding by RelAC (Fig. 4A, lane 2), IB inhibited RelACNLS only partially (lane 4). The same results were obtained with c-RelC and c-RelCNLS (lanes 6 and 8). (Supershift analysis with anti-IB revealed no IB in the RelACNLS-DNA complexes or the c-RelCNLS-DNA complexes [data not shown].) Therefore, while the NLSs of these proteins are not essential for IB binding, they appear to play an important role in IB-mediated inhibition of DNA-binding activity, suggesting that they stabilize the NFB-IB complex.

View larger version (47K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   FIG. 4.   Deletion of the c-Rel or RelA NLS weakens binding to IB. (A) 293 cells were transfected or cotransfected as indicated. Whole-cell lysates were analyzed by EMSA (top panel). Arrows point at the DNA-binding complexes. Lysates were also analyzed by immunoblotting with antisera raised against RelA (no. 1207) or c-Rel (no. 1136) (middle panel) or against IB (no. 1309) (lower panel). Arrows indicate detected proteins. (B) Human 293 cells were transfected with the indicated plasmid DNAs (lanes 1 to 5) or were mock transfected (lane 6). Whole-cell lysates were immunoprecipitated with anti-IB, and precipitates were collected on protein A-Sepharose. The washed precipitates were resuspended in 50 µl of EMSA buffer which included a 32P-labeled oligonucleotide containing the B site from the interleukin-6 promoter. After incubation (with agitation) at 25°C for 20 min, the samples were centrifuged at 13,000 × g for 1 min and the supernatants were analyzed by EMSA (top panel). The two lower panels are immunoblots of the whole-cell lysates probed with anti-c-Rel (upper) or anti-IB (lower), demonstrating that each of the plasmids was indeed expressed. Some degradation products are evident in the c-Rel panel, presumably the results of freezing and thawing of the lysate. IP, immunoprecipitate. (C) Human 293 cells were cotransfected with c-RelC and IB or c-RelCNLS and IB. Twenty-four hours later, the cells were cultured in medium containing 35S-amino acids for 1 h. Whole-cell extracts were incubated overnight at 4°C with nonradioactive whole-cell extracts from mock-transfected cells (lanes 1 and 4) or cells transfected with c-RelC (lanes 2 and 5) or were incubated without extract (lanes 3 and 6). The ratio of nonradioactive to radioactive extract was about 5:1. Following incubation, the samples were immunoprecipitated with anti-IB, and the precipitates were analyzed by SDS-PAGE.

Binding of IB to NLS mutants of p50. To map more precisely the p50 residues required for IB interaction, we created a set of substitution mutants within the p50 NLS. The mutants were first tested in vivo for subcellular localization in the absence of IB. Their behavior is summarized in Table 1. As expected, simultaneous substitution of all four positively charged residues of the NLS (RKRQK  AAAQA) resulted in a protein that was cytoplasmic when it was expressed in 293 cells. However, the effects of individual mutations varied greatly. Mutation of the first (R362), fourth (Q365), or fifth (K366) residue to alanine had little or no effect on localization of the protein. In contrast, mutation of either K363 or R364 to alanine had a drastic effect and resulted in a cytoplasmic protein. Since K363 (unlike R364) is conserved at the same relative position in the NLSs of all Rel family proteins, we wondered if even a conservative change would be tolerated. To answer this question, we mutated K363 to arginine. Even this comparatively minor alteration gave rise to a protein that was predominantly cytoplasmic. Thus, it appears that K363 and R364 are necessary for NLS function.

View larger version (46K): [in this window] [in a new window]   FIG. 5.   Binding of p50 NLS mutants to IB. 293 cells were transfected with IB alone (a) or with IB plus an excess of p50 NLS mutant. The cells were examined by immunofluorescence with anti-IB (no. 1309) or anti-p50 (no. 1141). The sequences of the mutant NLSs are shown above their respective panels. The targets of the antisera in the cotransfection experiments are underlined.

View this table: [in this window] [in a new window]   TABLE 2.   Binding of NLS mutants of p50 to IBa

The N-terminal region of IB masks the NLS of p50. The results presented above suggest that one or more contacts between the p50 NLS and IB are crucial for establishing a stable protein-protein complex. A clue to the location of these contacts in IB is provided by the behavior of certain IB mutants which are missing some or all of the N-terminal domain upstream of the ankyrin repeats. For example, when we cotransfected p50 with an IB mutant from which residues 4 to 61 had been deleted (IB4-61), the immunofluorescence assay revealed that the p50 was predominantly nuclear, even when the mutant IB was supplied in substantial excess (Fig. 6, panel b). This result was surprising, since it has been known for some time that N-terminal deletion mutants of IB are able to bind to p50 homodimers in vitro (13, 17). In agreement with these previous experiments, we were able to coprecipitate either p50 or c-RelC with IB4-61 from lysates of cotransfected cells (Fig. 7). We also showed by immunoblotting that failure to retain p50 in the cytoplasm could not be attributed to low levels of expression of IB N-terminal deletion mutants. Per microgram of plasmid DNA, the expression of the IB mutants was comparable to that of wild-type IB when either was cotransfected with p50 or c-RelC DNA (data not shown). The immunofluorescence result suggested, therefore, that the mutant IB was bound to p50 in the transfected cell but that one or both p50 NLSs were unmasked, leading to nuclear localization of the p50-IB complex.

View larger version (61K): [in this window] [in a new window]   FIG. 6.   The IB N terminus masks the NLS of p50 and c-Rel. 293 cells were transfected or cotransfected, as indicated, and analyzed by immunofluorescence with anti-p50 (no. 1141), anti-c-Rel (no. 1136), or anti-IB (no. 1258 or no. 1309). The targets of the antisera in the cotransfection experiments are underlined. Nonunderlined partners were present in excess in all experiments. IB61 is IB4-61, IB30 is IB2-30, IB53 is IB2-53, IB (43 to 47) is IB with a substitution mutation in residues 43 to 47 (EQMVK  QAAAA), IB39-43 is IB with residues 39 to 43 deleted, IB (48 to 52) is IB with a substitution mutation in residues 48 to 52 (ELQEI  QAAQA), and p50 NLS has RKAQN instead of the wild-type NLS RKRQK.

View larger version (38K): [in this window] [in a new window]   FIG. 7.   IB4-61 coprecipitates with c-RelC and with p50. 293 cells were transfected as indicated. Twenty-four hours later, cells were cultured in medium containing 35S-amino acids for 1 h, and cytoplasmic extracts were immunoprecipitated with the indicated antisera. Precipitates were analyzed by SDS-PAGE. The mobility of wild-type IB (lane 1) demonstrates that c-RelC and p50 coprecipitated with IB4-61 and not with endogenous wild-type IB.

	DISCUSSION

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In this study, we have concentrated on potential interactions between the NLSs of a Rel/NF-B dimer and IB. The binding of IB (or the other IBs) to a dimer somehow results in the masking of both NLSs, with the consequence that the complex is cytoplasmic. Does this masking involve direct contact between IB and the NLSs? Several previous studies reported that the NLS is required for IB binding (2, 9, 15, 25), strongly suggesting direct contact. More recently, however, it has been shown that neither the binding of IB to the avian v-Rel and c-Rel proteins nor the binding of Cactus to Dorsal requires the NLS (11, 23). In the present study, we show that neither human RelA nor c-Rel homodimers require NLSs to bind IB. The difference between earlier and more recent studies is the type of NLS mutation that was tested. Whereas most of the studies suggesting an NLS requirement were with substitution mutants, the experiments showing no requirement for the NLS were with deletion mutants. It now seems likely that the results with the substitution mutants should be interpreted not as a requirement for the NLS but, rather, as the prevention of binding due to the substituted residues. Thus, there is no direct evidence for contact between the NLSs of RelA, c-Rel or v-Rel (or Dorsal), and IB (or Cactus), although there is some very strong indirect evidence. First, the drastic effect of the NLS substitution mutations in blocking the binding of IB argues at least for their proximity. Second, deletion of the chicken c-Rel NLS rendered association with IB dependent on the presence of the PEST domain, suggesting that loss of the NLS had significantly weakened binding (23). Third, we showed that in the absence of an NLS in RelA or c-Rel, IB has a significantly reduced ability to inhibit DNA binding. Finally, we showed that complexes of IB with NLS deletion mutants of RelA or c-Rel were significantly less stable than complexes of IB with wild-type RelA or c-Rel. Therefore, it seems reasonable to conclude that IB contacts one or both of the RelA or c-Rel NLSs, although this contact is not required for binding.

The situation is quite different for p50, for which the NLS is required for binding to IB. We showed that either the deletion or the substitution of the p50 NLS resulted in the complete loss of interaction both in vitro and in vivo. The mutation of individual NLS residues revealed that K363 is required for binding, together with at least one other basic residue. Since arginine can substitute for K363, a charge-dependent interaction is suggested.

Which region of IB does the p50 NLS contact? We report here a new function for the N-terminal region of IB: it is required for masking one or both of the NLSs in a p50 or a c-Rel homodimer. The N terminus cannot be the sole IB contact for both NLSs because (i) the p50 NLS is required for binding to IB and (ii) p50 can bind IB in the absence of the latter's N-terminal region. However, various alternative models are possible. For example, the IB N-terminal region might contact one of the NLSs of a dimer (a dispensable contact, since the complex is stable in the absence of the IB N terminus), while a different IB region masks the second NLS (a required contact, since the p50 NLS is necessary for binding). This model predicts that a p50 dimer with only one NLS would be able to bind IB, which is consistent with the results of our current studies. Alternatively, the IB N-terminal region might not contact either of the NLSs but might sterically mask one or both of them by virtue of its contact with some other region of the dimer.

In accordance with previous mutational studies (9, 11, 16, 25) and with the crystal structure of p50 and RelA (6, 6a, 10, 20), we found that IB interacts predominantly (or perhaps even exclusively) with the dimerization region of the RHD of p50, c-Rel, and RelA. In our experiments, this interaction was stable not only in vivo but also in vitro under conditions of moderate salt concentration and nonionic detergent. Of course, we cannot rule out the possibility that contacts are also normally made with the N-terminal region of the RHD and that these contacts might be crucial for IB's ability to inhibit DNA binding by RelA- and c-Rel-containing dimers. However, several considerations should be taken into account when such hypothetical contacts are postulated. First, the ankyrin domain seems to be an unlikely source of such contacts, simply because binding is so sensitive to alteration in the ankyrin repeats (i.e., if the ankyrins cannot be altered without the loss of binding, it is unlikely that a target of the ankyrins could be altered or removed without the loss of binding). The N terminus of IB also now seems unlikely to contact the N-terminal region of the RHD. We demonstrate here that the N terminus of IB is somehow involved with the dimer NLS(s), which is suggested (but not proved) by the p50 crystal structure to be at some distance from the N-terminal domains of the monomers. Is the C-terminal PEST region of IB a possible source of contacts with the N-terminal region of the RHD? We know that the PEST region binds somewhere, because it is required for the inhibition of DNA binding by RelA- and c-Rel-containing dimers and because the IBPEST mutant does not bind stably to c-Rel homodimers in vitro (8). We suggested previously that this crucial contact with c-Rel might occur in the N-terminal portion of the RHD (8). If this were the case, however, deletion of the c-Rel N-terminal region would abrogate binding to IB in vitro just as deletion of the IB PEST region abolished binding to wild-type c-Rel. The results presented here, however, show that IB binds quite stably in vitro to c-Rel lacking its entire N-terminal region. Thus, it seems likely that the major contact with the PEST region occurs in the c-Rel dimerization region. In summary, all contacts between the dimer and IB may be confined to the dimerization domain.

Finally, one must be mindful of the significant difference between IB's binding to p50 and its binding to c-Rel or RelA. IB can inhibit DNA binding by c-Rel and RelA but cannot inhibit p50. The structural basis for this functional difference is unknown but the difference suggests that discoveries regarding interaction of the IB with c-Rel or RelA will not always apply to interaction with p50 and vice versa.