IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells.

NF-κB dimers, inhibitor IκB proteins, and NF-κB·IκB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IκB-dependent modulation of NF-κB subcellular localization represents one of the more poorly understood processes in the NF-κB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-κB regulation exhibited by the inhibitors IκBβ and IκBα. We show that although both IκBα and IκBβ bind to NF-κB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IκBβ derives its high affinity toward NF-κB dimers by binding to both NF-κB subunit nuclear localization signals. In contrast, IκBα contacts only one NF-κB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-κB binding. We show that the presence of one free NLS in the NF-κB·IκBα complex renders it a dynamic nucleocytoplasmic complex, whereas NF-κB·IκBβ complexes are localized to the cytoplasm of resting cells.

NF-B represents a paradigm for inducible transcription factors that are regulated, in large part, through their subcellular compartmentalization (1)(2)(3). The multiple cellular activities of NF-B are controlled through interactions with members of the IB family inhibitor proteins. In resting cells, NF-B dimers reside in the cytoplasm in complex with IB proteins, which function by masking the nuclear localization signals (NLSs) 1 of NF-B. Upon cellular stimulation, the NF-B-associated IB proteins are specifically phosphorylated at two amino-terminal serines by the IB kinase complex (IKK) (4). This leads to the removal of IB by a ubiquitin-linked proteasome degradation pathway and to the migration of active NF-B into the nucleus.
Five homologous polypeptides, p50, p65, c-Rel, RelB, and p52, comprise the mammalian Rel/NF-B transcription factor family. The subunits associate in a combinatorial fashion to form transcriptionally active homo-and heterodimers. The most prevalent and well characterized species of NF-B dimer is the p50/p65 heterodimer (Fig. 1a). Rel/NF-B polypeptides are defined by the presence of the Rel homology region (RHR), a 300-amino acid segment responsible for site-specific DNA binding, subunit dimerization, nuclear translocation, and inhibitor binding (5). The regions responsible for the transcriptional activation lie outside of the RHR (6).
IB is a diverse family of transcription factor inhibitors that includes IB␣, IB␤, IB⑀ (7)(8)(9), IB␥, Bcl-3, p105, and p100 (10). Each of the IB family proteins contains six or seven imperfect copies of the 33-amino acid structural motif known as ankyrin repeats. Ankyrin repeat-containing domains, present in numerous proteins of diverse functions, are primarily protein-protein interaction modules (11). Principal among IB family proteins are the inhibitors IB␣ and IB␤ (Fig. 1b). Both proteins possess an amino-terminal signal response domain, which contains a pair of conserved serine residues for inducible phosphorylation. IB␣ and IB␤ also bear carboxyl-terminal segments rich in the amino acids proline, glutamic acid, serine, and threonine (PEST). This highly acidic PEST region represents a site for constitutive phosphorylation by protein kinase CK2 (casein kinase II) (12)(13)(14)(15).
IB␣ and IB␤ both bind preferentially to p65-and c-Relcontaining NF-B homo-and heterodimers (3,16). Despite extensive primary structural similarities, IB␣ and IB␤ exhibit significant functional differences in vivo. The most striking of these differences is that IB␤ activates NF-B persistently in a cell type-and stimulus-specific manner, whereas regulation of NF-B by IB␣ is rapid but transient (16 -18). The distinct functional roles arise due in part to the fact that active NF-B up-regulates expression of IB␣ but not IB␤. Newly synthesized IB␣ can enter the nucleus and dissociate transcriptionally competent NF-B⅐DNA complexes (19 -22). IB␤ has been shown to be a weaker inhibitor of NF-B DNA binding as compared with IB␣ (18). It is not precisely clear how an inhibitor that exhibits such weak binding affinity is capable of properly regulating NF-B activity. Finally, several recent studies have shown that both free IB␣ and NF-B⅐IB␣ complexes shuttle between the nucleus and cytoplasm (23)(24)(25)(26). Little is known about the subcellular localization status of free IB␤ and NF-B⅐IB␤ complexes.
In this study, we have investigated the molecular mechanism of NF-B inhibition by IB␤ and compared it with IB␣. We report that, as with IB␣, IB␤ binds NF-B dimers with 1:1 stoichiometry. However, the mechanisms by which the two inhibitors interact with NF-B dimers differ significantly. IB␣ and IB␤ vary with regard to their NF-B DNA-inhibitory binding properties as well as in their abilities to mask the NF-B nuclear localization sequences. IB␤ gains significant binding energy by contacting both of the NLSs of a NF-B dimer as opposed to IB␣, which contacts only one NF-B NLS. We report that NF-B p50/p65 heterodimer⅐IB␣ complexes shuttle between the nucleus and cytoplasm by virtue of a free p50 NLS. Inactive NF-B⅐IB␤ complexes, on the other hand, reside permanently in the cell cytoplasm as both the NLSs of the NF-B dimer are masked.
Fluorescence Polarization Competition Assay-Fluorescence polarization competition assays were performed as described previously (30). Briefly, varying concentrations of IB␤ were mixed with constant amounts of NF-B pre-equilibrated with fluorescein-labeled DNA. A decrease in polarization was observed with increased IB␤ concentrations. The competition assay binding curves were analyzed for IC 50 values, defined as the concentration of IB␤ at 0.5 fractional occupancy. K I values, the dissociation constant for the NF-B/IB␤ interaction, were calculated as an average of three experiments. There was less than a 20% error between individual experiments.
Fluorescence Microscopy-All cells were grown on eight-well chamber slides (Lab Tek). Cells were transfected with a total of 0.2 g of plasmid DNA by the LipofectAMINE method (Life Technologies, Inc.). After 24 h, the cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were then permeabilized with 0.25% Nonidet P-40 in PBS for 1 min and blocked with 5 mg/ml bovine serum albumin in PBS with 0.1% Tween 20 at room temperature for 30 min. Cells were incubated with rabbit antibody alone or in a mixture as required for the experiment in PBS containing 5 mg/ml bovine serum albumin and 0.2% Nonidet P-40 at room temperature for 2 h. Cells were washed three times with PBS with 0.2% Nonidet P-40 and incubated with secondary antibody at room temperature for 1 h. Finally, cells were washed three times with PBS with 0.1% Tween 20 and covered with a drop of mounting solution (Vector).

RESULTS
Stoichiometry of NF-B⅐IB␤ Complexes-One molecule of IB␣ binds to one NF-B dimer (32). We wished to determine Amino acid numbering corresponds to murine proteins that were employed in this study. b, IB␣ (purple) and IB␤ (cyan) proteins are depicted schematically. The two letters S in the signal response domains represent the conserved serine sites of signal-dependent phosphorylation. Numbering reflects human IB␣ and murine IB␤, used in this study. the stoichiometry of the complex between NF-B and IB␤. Size exclusion chromatography on NF-B⅐IB␤ and NF-B⅐IB␣ complexes revealed that they share similar molecular masses (data not shown). This suggests that IB␤ also binds NF-B dimers with 1:1 stoichiometry. To test this hypothesis, we performed native polyacrylamide gel electrophoretic analysis of NF-B⅐IB␤ complexes (Fig. 2). Two different murine IB␤ proteins, IB␤-(54 -331) and GST-IB␤ (residues 1-359), were incubated with the RHR of NF-B p65 (residues 19 -325). The two IB␤ molecules bind NF-B dimers with identical affinities (see below) but display unique electrophoretic mobilities in complex with p65- (19 -325). When equimolar amounts of IB␤-(54 -331), GST-IB␤, and p65-(19 -325) homodimer were mixed and allowed to equilibrate, they ran as two discrete bands on the native polyacrylamide gel, the first representing the IB␤-(54 -331)⅐p65-(19 -325) homodimer complex and the second corresponding to the GST-IB␤⅐p65-(19 -325) homodimer complex. Higher order binding of IB␤ to NF-B would have resulted in the appearance of additional, intermediate mobility bands corresponding to combinations of both IB␤ molecules and p65. The absence of any such species confirms that IB␤ binds to NF-B dimers with 1:1 stoichiometry.
By Comparison to IB␣, IB␤ Is a Weaker Inhibitor of NF-B DNA Binding-We determined the efficiency by which IB␤ functions as an inhibitor of NF-B dimer DNA binding. A solution-based competition assay was performed in which fixed amounts of fluorescein-labeled B DNA bound to the NF-B p50/p65 heterodimer or p65 homodimer were incubated with increasing concentrations of IB␤. In this assay, the IB-dependent removal of NF-B from DNA and the associated decrease in fluorescence polarization is monitored to determine the DNA binding inhibition constant at equilibrium (30).
First, experiments were performed to confirm that, like IB␣, the amino-terminal signal response domain of IB␤ does not contribute to NF-B binding affinity (data not shown). IB␤-(54 -331) inhibits the wild type DNA binding of p50-(39 -376)/p65-(19 -325) heterodimer with an apparent inhibition constant (K I ) of 150 nM ( Fig. 3a and Table I). This value is ϳ10-fold weaker than that exhibited by IB␣. We next mutated all five serines within the PEST sequence of IB␤ to phosphomimetic glutamic acids (E5-IB␤) (33). This resulted in only a slight enhancement of K I for E5-IB␤-(54 -331). Using the same fluorescence polarization competition assay, we determined the inhibition constants of these IB␤ proteins for the NF-B p65 homodimer in complex with B DNA (see Fig. 3b). IB␤-(54 -331) exhibited a 2.5-fold lower affinity for p65-(19 -325) homodimer as compared with the p50/p65 heterodimer (Table I). E5-IB␤-(54 -331) disrupts the p65 homodimer⅐DNA complex only marginally better.
IB␤ Relies More on the NF-B NLS Polypeptide(s) for Stable Complex Formation-X-ray crystal structures of the NF-B⅐IB␣ complex reveal how the segment encompassing the NLS of the NF-B p65 subunit (murine residues 291-319) is important for the stability of this complex (34,35). Specifically, IB␣ mediates numerous ionic interactions with the NF-B p65 subunit NLS residues and makes van der Waals contacts with several hydrophobic side chains carboxyl-terminal to it. This region of the NF-B RHR does not exhibit an ordered structure in the DNA-bound NF-B crystal structures (36 -38) and will be referred to as the NLS polypeptide (see Fig. 1b). To determine whether the p65 NLS polypeptide is as important for IB␤ binding, we generated a shorter version of the p65 subunit RHR that ends at residue Arg 304 . Fluorescence polarization competition assays were performed to determine the binding affinity of IB␤ for both the wild type p65-(19 -325) RHR homodimer and the truncated p65-(19 -304) (Fig. 3b).
As seen in Table II and Fig. 3b, the binding affinity of IB␤ for p65-(19 -325) is 24-fold tighter than for p65- (19 -304). In contrast, IB␣ binds to the corresponding p65 homodimers with similar affinities. In a separate study, in which direct binding of recombinant NF-B and IB proteins was assayed, we observed that IB␣ binds to p65-(19 -325) homodimer with 5-6-fold higher affinity than p65-(19 -304) (31). We currently have no explanation for the discrepancy in the values obtained from the two methods. Regardless, both assays indicate that this NLS polypeptide segment of NF-B plays a lesser role in binding to IB␣ as compared with IB␤.
We also determined the binding affinities of IB␤-(54 -331) for both the wild type NF-B p50-(39 -376)/p65-(19 -325) heterodimer and the truncated heterodimer p50-(39 -363)/p65- (19 -304). As summarized in Table II, the binding affinity of the truncated heterodimer is nearly 10-fold weaker as compared with the wild type p50/p65 heterodimer. In contrast, IB␣ binds both the long and short NF-B heterodimers with nearly equal affinities (Table II). Again, analysis of IB␣⅐NF-B complex formation by direct binding assay showed a 4-fold higher affinity for the longer p50/p65 heterodimer as compared with p50-(39 -363)/p65-(19 -304) (31). In all, these experiments indicate that the NLS polypeptide segment of NF-B p65 homodimers and p50/p65 heterodimers makes greater contributions to overall binding affinity in the case of IB␤ but not IB␣.
In addition to fluorescence polarization experiments, the sta-bility of these complexes was analyzed by qualitative analytical size exclusion chromatography. A 5-fold molar excess of IB␤ was incubated with either the truncated p65-(19 -304) or p65-(19 -325) homodimer, and the complexes were separated on a

IB␤ Is a Resident Cytoplasmic NF-B Inhibitor
size exclusion column (data not shown). The NF-B p65-(19 -304)⅐IB␤ complex exhibits one broad peak, suggestive of an unstable complex. The p65-(19 -325) homo-dimer⅐IB␤ complex, on the other hand, displays one sharp peak corresponding to the NF-B⅐IB␤ complex followed by a second peak that contains free IB␤. These experiments lend support to the hypothesis that the NLS polypeptides of NF-B subunits play a greater role in binding to IB␤ than they do with IB␣. IB␤ Interacts Strongly with NF-B-The fact that IB␤ inhibits the DNA binding of NF-B more weakly than does IB␣ may not necessarily indicate that interactions between IB␤ and NF-B dimers are of low affinity. To test the affinity of direct physical association between IB␤ and NF-B dimers, we employed an electrophoretic gel mobility shift competition assay (31). In this assay, 32 P-radiolabeled IB␣ in complex with NF-B p50/p65 heterodimer was competed out by increasing amounts of unlabeled IB␤ (Fig. 4a). We performed similar experiments using IB␣ as the unlabeled competitor. Each experiment was performed at least four times, and the concentrations of unlabeled IB␤ and IB␣ required to dissociate half of the radiolabeled IB␣ were calculated.
It is clear from these experiments that IB␣ is a stronger competitor than IB␤, suggesting that IB␣ binds more tightly to the NF-B p50/p65 heterodimer. However, the difference in affinity of IB␣ and IB␤ toward NF-B is only on the order of 2-3-fold compared with a much larger disparity observed in the abilities of these inhibitors to disrupt NF-B DNA binding. Furthermore, the pattern of competitive replacement of radiolabeled IB␣ differs between the unlabeled IB␣ and IB␤. IB␤ begins to interfere with NF-B⅐IB␣ binding at a lower concentration and exhibits a more gradual competitive binding profile (Fig. 4a, compare lanes 7 and 13). Similar experiments have been performed with the p65 homodimer (Fig. 4b). Again we observe that IB␤ binds p65 homodimer with only 4-fold lower affinity than IB␣.
IB␤ Masks Both NF-B NLSs-A model that accounts for both the increased dependence of IB␤ on the NF-B NLS polypeptides and its weaker ability to inhibit NF-B DNA binding is that, in contrast to IB␣, IB␤ forms high affinity complexes with NF-B by contacting both NLSs. To test this hypothesis, we performed controlled proteolysis experiments and observed the protection patterns of NF-B subunit NLSs. E5-IB␤-(54 -331) was complexed with a p65 subunit construct containing only the protease-resistant dimerization domain with the p65 NLS polypeptide attached (amino acids 191-321). After a 1-h incubation with the nonspecific protease thermolysin, most of this p65 remains intact (Fig. 5a, lanes 3-5). Only at longer time points does the NF-B p65-(191-321) homodimer begin to exhibit partial digestion. The resulting doublet, corresponding to one intact p65-(191-321) and one with the NLS polypeptide removed, suggests that both p65 NLS polypeptides contact IB␤, albeit asymmetrically. We have analyzed the digested product of the complex by mass spectrometry and observe that the lower band of the doublet corresponds to p65 amino acids 191-311. By comparison, both NLS polypeptides of free p65-(191-321) homodimer are proteolyzed to the stable dimerization domain fragment within minutes of thermolysin treatment (Fig. 5a, lanes 6 -8). Interestingly, when the p65-(19 -321)⅐IB␣ complex is treated with thermolysin, approxi-mately half of the p65 is proteolyzed to the dimerization domain fragment within minutes (Fig. 5b, lanes 2-8). The resulting doublet remains stable indefinitely. This suggests that, unlike IB␤, IB␣ is capable of protecting only one p65 NLS polypeptide from proteolytic cleavage.
IB␤ Is a Cytoplasmic Inhibitor of NF-B, whereas IB␣ Is Nucleocytoplasmic-Having established that IB␤ masks both NF-B subunit NLSs, we investigated the in vivo consequences of differential NF-B interactions by IB␣ and IB␤. First, we determined the subcellular distribution of endogenous IB␤ in p65 Ϫ/Ϫ MEF cells (Fig. 6a). We observe that IB␤ is a predominantly cytoplasmic protein, while IB␣ is present both in the cytoplasm and nucleus, although a larger fraction is present in the cytoplasm. While the vast majority of IB proteins exist as complexes with NF-B, it is possible that at least a fraction of IB␣ and IB␤ are present as free proteins in p65 Ϫ/Ϫ MEF cells. Compartmental distribution of IB␣ and IB␤ was also determined in cells treated with the nuclear export inhibitor leptomycin B (LMB) (39 -42). The subcellular distribution of IB␤ does not change significantly upon the addition of LMB. This result indicates that endogenous IB␤ is cytoplasmic irrespective of its status as a free protein or in complex with NF-B dimers such as the p50/c-Rel heterodimer or c-Rel homodimer in p65 Ϫ/Ϫ MEF cells. On the other hand, the addition of LMB results in nuclear staining of IB␣. This result suggests that the entire population of IB␣, both free and in complex with NF-B, participates in dynamic shuttling between the nuclear and cytoplasmic cellular compartments. Because it is unclear in these studies whether the IB proteins are free or in complex with NF-B dimers, we further tested for IB compartmentalization in the presence of excess NF-B binding partner. MEF p65 Ϫ/Ϫ cells were transfected with p65 cDNA to test whether nuclear localization of IB␤ and IB␣ is influenced by the presence of excess p65 homodimer (Fig. 6b). Immunofluorescence staining of p65-transfected cells with anti-IB␣ antibody indicated that IB␣ localizes exclusively to the cytoplasm. It appears that overexpressed p65 localizes mostly to the nucleus; we do, however, observe some cytoplasmic staining of p65. Treatment with LMB, however, resulted in nuclear IB␣ staining. Immunofluorescence staining of p65 revealed that p65 co-localizes with IB␣. This result is consistent with reports published previously (23)(24)(25). In contrast, IB␤ exhibits a unique pattern of subcellular localization in p65-transfected cells (Fig. 6c). We observe that, in p65transfected p65 Ϫ/Ϫ MEF cells, IB␤ remains primarily cytoplasmic in both the LMB-treated and -untreated cells. This result is even clearer when both IB␤ and p65 are co-transfected in HeLa cells (Fig. 6d). The NF-B p65 homodimer⅐IB␤ complex is, therefore, a resident cytoplasmic complex.
The p50 Subunit NLS Is Responsible for Nuclear Translocation of the NF-B p50/p65 Heterodimer⅐IB␣ Complex-In an effort to determine whether NF-B p50/p65 heterodimer⅐IB complexes behave similarly to p65 homodimer⅐IB complexes, p65 Ϫ/Ϫ MEF cells were co-transfected with both p65 and p50 cDNAs. Immunofluorescence staining revealed that the NF-B p50/p65⅐IB␣ complex is cytoplasmic (Fig. 7a, left panels). We observe staining of overexpressed p50 in both the cytoplasm and nucleus. Nuclear p50 is likely to be due to excess p50 homodimer, which is not retained in the cytoplasm by IB proteins (23). However, similar to complexes between p65 homodimer and IB␣, LMB treatment resulted in nuclear complex staining. Similar experiments performed with the NF-B p50/p65 heterodimer⅐IB␤ complex revealed that it is primarily cytoplasmic and that LMB treatment of cells does not alter this localization profile (Fig. 7b, left panels).
We next tested whether nuclear translocation of the NF-B p50/p65 heterodimer⅐IB␣ complex is dependent on the free p50 NLS as suggested by the x-ray crystal structures (34,35) and protease sensitivity studies of the NF-B p65 homodimer⅐IB␣ complex presented in this study. p65 Ϫ/Ϫ MEF cells were transfected with p65 and an excess of NLS-deficient p50 (residues 1-352). We observe that the NF-B p50-(1-352)/ p65 heterodimer⅐IB␣ complex localizes to the cytoplasm in both LMB-treated and -untreated cells (Fig. 7a, right panels). As expected, the analogous NF-B p50-(1-352)/p65⅐IB␤ complex displays the same cytoplasmic staining pattern as the wild type NF-B⅐IB␤ complex (Fig. 7b, right panels). These results confirm that the free p50 subunit NLS of the NF-B p50/p65 heterodimer⅐IB␣ complex is a necessary component for dynamic nucleocytoplasmic shuttling of the complex. Further-FIG. 6. IB␤ is a cytoplasmic inhibitor of NF-B, whereas IB␣ is nucleocytoplasmic. a, the subcellular distribution of IB␣ (left panels) and IB␤ (right panels) in resting p65 Ϫ/Ϫ MEF cells in the presence (above) and absence (below) of nuclear export receptor inhibitor LMB. Note that IB␣ localizes to the nucleus only upon LMB treatment. b, localization of IB␣ in p65 Ϫ/Ϫ MEF cells transfected with HA-tagged p65. Immunofluorescence staining is directed toward IB proteins (left panels, green) and HA (right panels, red). c, IB␤ localization (left panels, red) in p65 Ϫ/Ϫ MEF cells transfected with HA-p65 (right panels, green). Note that IB␤ remains primarily cytoplasmic in both the presence and absence of LMB. d, co-transfection and immunofluorescence detection of IB␤ (left panels, red) and p65 (right panels, green) in HeLa cells. more, our studies suggest that in contrast to IB␣, IB␤ retains NF-B dimers in the cytoplasm by masking both the nuclear localization signals of NF-B.

IB␤ Binds Strongly to NF-B Dimers but Inhibits NF-
B⅐DNA Complexes Weakly-It is generally believed that IB␣ is a stronger inhibitor of NF-B dimers than IB␤. This conclusion is based principally on their relative abilities to inhibit NF-B⅐DNA complexes (18). Inhibition of NF-B⅐DNA complexes may not, however, directly correlate with the ability of these inhibitors to associate with NF-B dimers. We have shown previously by direct binding assay that IB␣ exhibits high binding affinity toward NF-B dimers with dissociation constants in the low nanomolar range (31). It is clear, however, that in most cell types both IB␣ and IB␤ are present and capable of forming stable complexes with NF-B p50/p65 in roughly similar ratios (16). This suggests that IB␤ must also be capable of high affinity NF-B binding.
In this study, we determined that, as with IB␣, IB␤ binds NF-B dimers at a 1:1 molar ratio. We determined the magnitude of NF-B DNA binding inhibition by IB␤ and observed that IB␤ inhibits DNA binding by both the NF-B p50/p65 heterodimer and p65 homodimer with ϳ30-fold lower efficiency than does IB␣. In contrast, both IB␣ and IB␤ compete away radiolabeled IB␣ from NF-B with only marginal differences. The estimated variation in NF-B⅐IB␤ and NF-B⅐IB␣ complex binding affinity is 2-4-fold. These results suggest that, although IB␣ and IB␤ differ in their abilities to inhibit NF-B DNA binding, both inhibitors are capable of forming high affinity complexes with NF-B dimers. The PEST Sequences of IB␣ and IB␤ Play Different Roles-Three-dimensional x-ray structures of the NF-B⅐IB␣ complex revealed a mechanism by which IB␣ blocks the DNA binding of NF-B (34,35). The PEST sequence and the sixth ankyrin repeat of IB␣ interact with the regions of NF-B that are involved in DNA recognition and binding. The removal of this carboxyl-terminal PEST region renders IB␣ incapable of inhibiting NF-B⅐DNA complexes (30,32). We reported previously that the binding constants of NF-B⅐IB␣ complexes measured using direct binding assay and DNA binding competition assays produce roughly similar results (30,31). We concluded from these experiments that both the ankyrin repeat domain and the PEST sequence of IB␣ are critical in forming stable complexes with NF-B p50/p65 heterodimer and the p65 homodimer.
In this report, we show that the two activities of binding to NF-B and inhibition of NF-B DNA binding are not directly linked in the context of recombinant IB␤. It has been shown previously that phosphorylation at serine 313 and serine 315 of human IB␤ is essential for efficient inhibition of DNA binding by NF-B family homodimers p65 and c-Rel (12,18,33). We have converted five carboxyl-terminal serine residues, including those corresponding to human positions 313 and 315, to glutamic acid, however, and this E5-IB␤ behaves similarly to murine IB␤ with regard to NF-B binding and inhibition of DNA binding. It is, therefore, possible that glutamic acid substitution does not adequately mimic phosphorylation in this case and that the phosphorylated IB␤ functions differently than does the E5-IB␤.
An explanation for the apparent discrepancy between the ability of IB␤ to bind tightly to NF-B dimers and its relative inability to disrupt NF-B DNA binding comes from previous observations that wild type and PEST-deleted IB␤ are capable of forming ternary complexes with NF-B and DNA (17,18). These experiments lend support to the idea that the PEST sequence of IB␤ is not a critical component for stable complex formation with NF-B dimers. They also indicate that in the presence of both NF-B and DNA, IB␤ participates in two somewhat independent binding equilibria; one is for formation of the NF-B⅐IB␤ complex, and the other involves the ternary complex of NF-B⅐DNA⅐IB␤. Depending on the reaction conditions, reactant concentrations, and, possibly, the post-translational modification status of the factors involved, the active fraction of IB␤ can shift from one equilibrium to the other. As a result, the presence and status of target DNA fail to provide an accurate measure of IB␤ binding affinity toward NF-B dimers.
The NLSs of NF-B Dimers Interact Differently with IB Proteins-Within the context of the p65 homodimer, we have demonstrated that the NF-B NLS polypeptide contributes a 24-fold enhancement in binding affinity for IB␤. By contrast, this same NLS polypeptide segment contributes only 5-6-fold enhancement toward IB␣ binding affinity. This greater dependence by IB␤ on the p65 NLS polypeptides results from the involvement of both NLSs in NF-B binding to IB␤. We have shown that both p65 homodimer NLSs are protected from proteolytic digestion in the NF-B p65 homodimer⅐IB␤ complex. In contrast, IB␣ protects only one p65 homodimer NLS. As was first revealed by the NF-B p50/p65 heterodimer⅐IB␣ crystal structure, IB␣ directly contacts only the p65 subunit NLS (34,35). Interestingly, our results also indicate that IB␤ does not identically protect both p65 NLSs. One NLS is clearly better protected. This is not surprising, since the symmetric p65 homodimer and pseudosymmetric p50/p65 heterodimer are contacted asymmetrically by one IB molecule. It is likely that, in complex with IB␤, the fully protected NLS of the p65 homodimer and p50/p65 heterodimer makes similar contacts to those observed between IB␣ and the p65 subunit NLS polypeptide (residues 291-319) in the x-ray crystal structures. We speculate that a second p65 NLS or the p50 NLS of the p50/p65 heterodimer partially contacts IB␤ in a manner that might expose some flanking sequences to proteases (Fig. 8). There is little doubt, however, that IB␣ does not contact the second NLS in either of the two complexes.
Analogy to the NF-B p50/p65 heterodimer⅐IB␣ complex x-ray crystal structure places the second p65 homodimer NLS polypeptide within the vicinity of the first and second ankyrin repeats of IB. This is a region of relatively high sequence homology between IB␣ and IB␤. IB␤, however, contains a unique 47-amino acid insertion between its third and fourth ankyrin repeats. IB␣-based modeling of the IB␤ ankyrin repeats places the IB␤ insertion on the same side of IB␤ as the second NF-B p65 subunit NLS polypeptide. 2 Therefore, we suggest that the IB␤ insert region could be involved in masking the second NF-B NLS polypeptide.
Compartmentalization of Free and NF-B-complexed IB in Resting Cells-IB␣ has long been characterized as a strictly cytoplasmic inhibitor of transcription factor NF-B. It functions as such by masking the NLSs of the NF-B dimers (43). After crystallographic analysis of the NF-B p50/p65 heterodimer⅐IB␣ complex revealed that only the NF-B p65 subunit NLS is masked via direct contacts with the first two ankyrin repeats of IB␣, several groups reported that NF-B⅐IB␣ complexes are dynamic in nature and distribute between the nucleus and cytoplasm (23)(24)(25)(26). Here, we have shown that the free p50 subunit NLS is responsible for transport of NF-B⅐IB␣ complexes to the nucleus. Interestingly, nuclear detection of NF-B⅐IB␣ occurs only when the nuclear export receptor CRM1 is blocked by treatment with LMB. These observations suggest that the nuclear export of the complex is a more efficient process than entry (Fig. 8). This is perhaps due to the combined effect of multiple nuclear export signals identified in IB␣ and the activation domain of p65, which cancel the opposing effect of the one free NLS (44).
Here we report that free IB␤ is a strictly cytoplasmic protein in p65 Ϫ/Ϫ cells. It is not clear why IB␤, in the absence of any nuclear export sequence, remains cytoplasmic, while IB␣, by contrast, can enter the nucleus freely. We further report that complexes between NF-B and IB␤ remain primarily within the cell cytoplasm. We propose that the combined effects of three different factors, the masking of both NF-B NLSs by IB␤, the absence of any inherent IB␤ nuclear localization potential, and the putative p65 activation domain nuclear export signal, results in the strict cytoplasmic retention of NF-B⅐IB␤ complexes. This cytoplasmic NF-B in complex with IB␤ represents a unique pool of transcriptional activators that could be regulated by a subset of signaling pathways and other similarly localized effector molecules.
Unique Functional Roles for IB␣ and IB␤-Substitution of the IB␣ gene with the gene encoding IB␤ restores the wild type phenotype to IB␣ knockout mice (45,46). This finding has contributed to the belief that IB␤ plays a functionally redundant role in regulating NF-B activity. However, altered tissue distribution patterns and expression levels of IB␤ under the IB␣ promoter could account for the observed rescue phenotype. It remains impossible to assign a precise function to IB␤ in the absence of IB␤ Ϫ/Ϫ mice.
Based on our study, we propose that although both IB␣ and IB␤ are capable of inhibiting NF-B activity, they do so by differing mechanisms. The most striking of these differences involves the ability of IB␤ to mask both NLS polypeptide sequences of the NF-B p65 homodimer as opposed to IB␣, which masks one of the two. As a result of this difference, NF-B⅐IB␣ complexes shuttle between the nucleus and cytoplasm, whereas NF-B⅐IB␤ complexes remain principally in the cell cytoplasm. On a final note, during the preparation of this manuscript, Tam and Sen (47) reported that unlike IB␣, IB␤ and IB⑀ inhibit basal nucleocytoplasmic shuttling of NF-B p65 and c-Rel dimers.