p105·Ikappa Bgamma and Prototypical Ikappa Bs Use a Similar Mechanism to Bind but a Different Mechanism to Regulate the Subcellular Localization of NF-kappa B*

Anu K. Moorthy and Gourisankar GhoshDagger

From the Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, California 92093-0359

Received for publication, July 25, 2002, and in revised form, October 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p105, also known as NF-kappa B1, is an atypical Ikappa B molecule with a multi-domain organization distinct from other prototypical Ikappa Bs, like Ikappa Balpha and Ikappa Bbeta . To understand the mechanism by which p105 binds and inhibits NF-kappa B, we have used both p105 and its C-terminal inhibitory segment known as Ikappa Bgamma for our study. We show here that one Ikappa Bgamma molecule binds to NF-kappa B dimers wherein at least one NF-kappa B subunit is p50. We suggest that the obligatory p50 subunit in Ikappa Bgamma ·NF-kappa B complexes is equivalent to the N-terminal p50 segment in all p105·NF-kappa B complexes. The nuclear localization signal (NLS) of the obligatory p50 subunit is masked by Ikappa Bgamma , whereas the NLS of the nonobligatory NF-kappa B subunit is exposed. Thus, the global binding mode of all Ikappa B·NF-kappa B complexes seems to be similar where one obligatory (or specific) NF-kappa B subunit makes intimate contact with Ikappa B and the nonobligatory (or nonspecific) subunit is bound primarily through its ability to dimerize. In the case of Ikappa Balpha and Ikappa Bbeta , the specific NF-kappa B subunit in the complex is p65. In contrast to Ikappa Balpha ·NF-kappa B complexes, where the exposed NLS of the nonspecific subunit imports the complex to the nucleus, p105·NF-kappa B and Ikappa Bgamma ·NF-kappa B complexes are cytoplasmic. We show that the death domain of p105 (also of Ikappa Bgamma ) is essential for the cytoplasmic sequestration of NF-kappa B by p105 and Ikappa Bgamma . However, the death domain does not mask the exposed NLS of the complex. We also demonstrate that the death domain alone is not sufficient for cytoplasmic retention and instead functions only in conjunction with other parts in the three-dimensional scaffold formed by the association of the ankyrin repeat domain (ARD) and NF-kappa B dimer. We speculate that additional cytoplasmic protein(s) may sequester the entire p105·NF-kappa B complex by binding through the death domain and other segments, including the exposed NLS.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The NF-kappa B family of transcription factors plays an important role in a large number of cellular processes including immune response and inflammation, cellular development, and differentiation (1-4). This family comprises of five distinct members, p50, p65, p52, c-Rel, and RelB, that exhibit a high degree of sequence homology at their N termini. This region, known as the Rel homology region, is responsible for important functions like DNA binding, dimer formation, nuclear localization, and Ikappa B binding (1-4).

NF-kappa B dimers are regulated by inhibitor Ikappa B proteins, which include Ikappa Balpha , Ikappa Bbeta , Ikappa Bepsilon , Ikappa Bgamma , p105 (NF-kappa B1), p100 (NF-kappa B2), Bcl-3, Ikappa Bzeta , and MAIL (4-21). In most cells NF-kappa B remains inactive as a complex with Ikappa B. In response to a variety of extracellular signals, the Ikappa B molecule is phosphorylated by Ikappa B kinases, which leads to the ubiquitination and subsequent degradation of Ikappa B by the proteosome machinery within the cell (22).

It has long been thought that all Ikappa B proteins inhibit NF-kappa Bs by masking the nuclear localization signal (NLS)1 of NF-kappa B and thereby sequestering them in the cytoplasm (1-4, 22). However, recent studies have indicated that the Ikappa B proteins differ with regard to their regulation of NF-kappa B subcellular localization. The Ikappa Balpha ·NF-kappa B complex exhibits dynamic shuttling between the cytoplasm and the nucleus (23-27). However, despite its transient presence in the nucleus, NF-kappa B remains bound to Ikappa Balpha , and this prevents DNA binding and activation of transcription. Ikappa Bbeta , on the other hand, sequesters NF-kappa B in the cytoplasm of resting cells (26-28). The detailed mechanism of this process is not clear. It has been suggested that other ancillary proteins may be required for this function.2

p105 and p100 are the precursors of NF-kappa B subunits p50 and p52, respectively, which are located in their N termini. Both p105 and p100 have similar structural organizations (3) (Fig. 1). The central portion of these molecules has a glycine-rich region that has been shown to play a critical role in processing of the precursor (29, 30). The C termini resemble other Ikappa B molecules that possess ankyrin repeats (AR) (14, 15, 31). These two proteins also contain a death domain immediately C-terminal to the ankyrin repeat domain (ARD). The Ikappa B kinase phosphorylation sites are located further downstream within a region called the destruction box (see Fig. 1). A separate gene also encodes the C-terminal part of p105. This gene product, known as Ikappa Bgamma , has been shown to exist only in certain cell types, like mouse pre-B cells (12) (Fig. 1).


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Fig. 1.   Domain organization of p105 and Ikappa Bgamma . A schematic representation of various domains in p105·Ikappa Bgamma with key regions indicated by arrows is shown. Some of the constructs used in this study, p105 (1-971), p105Delta N (245-971), Ikappa Bgamma (365-971), and ARD-Ikappa Bgamma (500-971), are shown. The corresponding C-terminal domain-deleted forms are p105Delta C (1-800), p105Delta NDelta C (245-800), Ikappa Bgamma Delta C (365-800), and ARD-Ikappa Bgamma Delta C (500-800), and the destruction box-deleted form is p105Delta DB (1-887).

In resting cells, p105 is partially processed, generating p50. The exact mechanism of this limited processing event is not known, although co-translational processing events have been proposed (32, 33). The unprocessed p105 functions as an inhibitor molecule and nonspecifically inhibits almost all NF-kappa B subunits, including p50 (34-36). An earlier study has shown that p105 retains itself, as well as other NF-kappa B molecules, in the cytoplasm (37). The mechanism of inhibition is, however, still unclear. The p105 molecule, like the classical Ikappa B inhibitors, can also undergo complete degradation in response to signals and the sequential action of Ikappa B kinases, ubiquitin ligases, and the 26 S proteasome (38-41). Both the death domain and the destruction box have been shown to be important for Ikappa B kinase phosphorylation (41, 42). In contrast to p105, the mechanism of p100 processing is very different. In resting cells, most of p100 remains unprocessed, and in response to appropriate signals all of it gets processed into p52 (43, 44).

In this study, we address the functional properties of p105 by asking the following questions. How does p105 regulate subcellular distribution of NF-kappa B subunits? How does p105 nonspecifically inhibit other NF-kappa B proteins? What is the relationship between p105·NF-kappa B and Ikappa Bgamma ·NF-kappa B complexes? We find that although Ikappa Bgamma resembles other Ikappa Bs in exhibiting a 1:1 stoichiometry of binding to NF-kappa B dimers, it has a unique specificity for NF-kappa B dimers that contain at least one p50 subunit. This obligatory p50 subunit in Ikappa Bgamma ·NF-kappa B complexes is structurally equivalent to the N-terminal p50 segment of p105 in p105·NF-kappa B complexes. Interestingly, although Ikappa Bgamma fails to mask one NF-kappa B NLS, it still retains NF-kappa B in the cytoplasm. We observe that the death domain of p105 is necessary but not sufficient for this cytosolic retention. We suggest that p105 and Ikappa Bgamma inhibitors may require an as yet unknown cellular factor(s) to sequester NF-kappa B in the cytoplasm.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein Expression and Purification from Escherichia coli-- The cloning, expression, and purification of the NF-kappa B subunits has been described previously (45, 46). Full-length and truncated glutathione S-transferase-Ikappa Bgamma were made by cloning into the pGEX-2T vector (Amersham Biosciences). The fusion protein was expressed in E. coli BL21 DE3 and purified by glutathione-agarose column chromatography following the manufacturer's protocol (Amersham Biosciences).

Native Polyacrylamide Gel Electrophoresis-- Proteins and protein complexes were diluted in 10 mM Tris (pH 7.5), 200 mM NaCl, 4% glycerol, and 2 mM beta -mercaptoethanol. The reactions were allowed to equilibrate at room temperature for 1 h. Native gel loading dye (50 mM Tris, pH 7.5, 0.1% bromphenol blue, 10% glycerol, and 1.25 mM beta -mercaptoethanol) was then added to each sample. 10% native polyacrylamide gels were prepared with 0.25× Tris-borate-EDTA buffer. The samples were loaded on the gel and run in Tris-borate-EDTA buffer for 2 h at a constant current (3 mA). The protein bands were visualized by Coomassie staining.

Fluorescence Polarization Competition Assay-- Fluorescence polarization competition assays were done as described previously (47). Briefly, varying concentrations of Ikappa Bgamma were mixed with constant amounts of p50 homodimer pre-equilibrated with fluorescein-labeled DNA. The competition assay binding curves were analyzed for IC50 values, defined as the concentration of Ikappa Bgamma at 0.5 fractional occupancy.

Plasmids, Cell Culture, and Transfections-- cDNAs encoding full-length p50 and full-length/truncated p105 were cloned into a pcDNA vector (Invitrogen) containing either an N-terminal FLAG (p50 constructs) or an N-terminal HA (p105·Ikappa Bgamma constructs) tag. HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics and transfected with plasmid DNA using the LipofectAMINE Plus reagent (Invitrogen). Protein expression was checked by Western blot.

Immunofluorescence-- HeLa cells were grown on 8-well chamber slides (Lab Tek). The cells were transfected with a total of 0.2 µg of plasmid DNA. After 24 h, the cells were washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. The 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 containing 0.1% Tween 20 at room temperature for 30 min. Fluorescent detection was done by incubating the cells with monoclonal antibody 12CA5 (against HA), M2 (against FLAG), and H-286 (against p65) in PBS containing 5 mg/ml bovine serum albumin and 0.2% Nonidet P-40 at room temperature for 2 h. The cells were washed three times with buffer containing 0.2% Nonidet P-40 in PBS and incubated with fluoresceinated secondary antibody at room temperature for 1 h. Finally, the cells were washed three times with buffer containing 0.1% Tween 20 in PBS, and the slide was mounted with Vectashield (Vector Laboratories).

Immunoprecipitation-- For immunoprecipitation from whole cell lysates, HeLa cells were harvested 24 h post-transfection and lysed with buffer containing 20 mM Tris (pH 7.5), 0.2 M NaCl, 1% Triton X-100, 1 mM EDTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Immunoprecipitation was carried out by the protein A pull-down method. Briefly, 0.1-0.5 mg equivalent of protein from transfected cell extract was diluted with 300 µl of lysis buffer. The appropriate antibodies were added to the extract and incubated overnight with protein A-Sepharose 4B beads (Sigma) at 4 °C. The beads were pulled down by brief centrifugation and washed three times with the lysis buffer. The immunoprecipitates were then eluted from the beads with 2× Laemmli buffer devoid of beta -mercaptoethanol by heating at 95 °C for 5 min. The bound proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The bound proteins were then identified by Western blot. In vitro immunoprecipitation was carried out with a similar protocol using purified E. coli proteins in place of whole cell extract.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interactions between NF-kappa B Dimers and Ikappa Bgamma -- Unprocessed p105 functions as an inhibitor of NF-kappa B, although its mode of inhibition appears to be quite different from the prototypical Ikappa B proteins such as Ikappa Balpha and Ikappa Bbeta . As opposed to Ikappa Balpha and Ikappa Bbeta that bind through p65, p105 inhibits all NF-kappa B members nonspecifically. To understand the biochemical basis of NF-kappa B inhibition by p105, we wanted to first test how the p105 C terminus (Ikappa Bgamma ) binds to NF-kappa B dimers. We have characterized the interactions between Ikappa Bgamma and various NF-kappa B dimers by protein-protein gel shift assays under native conditions. To simplify the assay we generated a truncated Ikappa Bgamma containing only the ARD fused to a poly-His peptide. This construct will be referred to as ARD-Ikappa Bgamma Delta C. We observe that ARD-Ikappa Bgamma Delta C binds strongly to the p50·p50 homodimer and p50·p65 heterodimer but only weakly to the p65·p65 and c-Rel·c-Rel homodimer (Fig. 2A). Although no stable complexes are formed between Ikappa Bgamma and p65·p65 or c-Rel·c-Rel homodimers, we do not observe free Ikappa Bgamma in these lanes. It is likely that these complexes are weaker and smear during electrophoresis. Based on these results we suggest that complexes between Ikappa Bgamma and non-p50 containing NF-kappa B dimers are highly unstable.


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Fig. 2.   Ikappa Bgamma binds to p50 containing NF-kappa B dimers with a 1:1 stoichiometry. A, native gel mobility shift assay of Ikappa Bgamma binding to different NF-kappa B dimers. Lane 1, ARD-Ikappa Bgamma Delta C; lane 2, p50·p50; lane 3, p50·p65; lane 4, p65·p65; lane 5, c-rel·c-rel; lane 6, ARD-Ikappa Bgamma Delta C+p50·p50; lane 7, ARD-Ikappa Bgamma Delta C+p50·p65; lane 8, ARD-Ikappa Bgamma Delta C+p65·p65; lane 9, ARD-Ikappa Bgamma Delta C+c-rel·c-rel. The free and complexed proteins are indicated by arrows. B, schematic representation of the p105·p50 and Ikappa Bgamma ·p50·p50 complex. The structural equivalence of p105-p50 (right) and the Ikappa Bgamma ·p50·p50 (left) complex is shown. C, stoichiometry of the Ikappa Bgamma ·NF-kappa B complex. Native gel electrophoresis shows the 1:1 stoichiometry of binding in the Ikappa Bgamma ·NF-kappa B complex. Lane 1, ARD-Ikappa Bgamma Delta C; lane 2, FL-Ikappa Bgamma (365-971); lane 3, p50·p50; lane 4, ARD-Ikappa Bgamma Delta C+p50·p50; lane 5, FL-Ikappa Bgamma (365-971)+p50·p50; lane 6, ARD-Ikappa Bgamma Delta C+FL-Ikappa Bgamma (365-971)+p50·p50. The free and complexed proteins are indicated by arrows.

The fact that p105 binds to all NF-kappa B proteins and Ikappa Bgamma binds only to p50 dimers (any NF-kappa B dimer that contains at least one p50 subunit) suggests that the required p50 subunit in Ikappa Bgamma ·NF-kappa B complexes is likely the N-terminal p50 segment of p105 in p105·NF-kappa B complexes. Thus, the p105·p50 (or p105·p65) complex can be considered to be structurally equivalent to the Ikappa Bgamma ·p50·p50 (or Ikappa Bgamma ·p50·p65) complex (Fig. 2B).

The binding data presented above suggest that Ikappa Bgamma prefers p50 containing NF-kappa B dimers. However, these experiments do not reveal whether one or two molecules of Ikappa Bgamma bind to one molecule of NF-kappa B dimer. This is important considering that Bcl-3, which contains seven AR like Ikappa Bgamma , has been proposed to bind to p50·p50 and p52·p52 homodimers in a 2:1 molar ratio (48-50). To elucidate Ikappa Bgamma ·NF-kappa B binding stoichiometry, we have used two different Ikappa Bgamma constructs, one is the full-length Ikappa Bgamma expressed as a glutathione S-transferase fusion protein (FL-Ikappa Bgamma ), and the other is the previously described ARD-Ikappa Bgamma Delta C. As seen in Fig. 2C (lanes 4 and 5), both of these proteins bind p50·p50 homodimer efficiently, and the resulting complex migrates to different positions in the native gel. In a reaction mixture containing both Ikappa Bgamma s and the p50 homodimer, binding of two molecules of Ikappa Bgamma to the homodimer would result in a distinct, additional complex composed of these Ikappa Bgamma molecules of different lengths and p50. However, when equivalent amounts of FL-Ikappa Bgamma , ARD-Ikappa Bgamma Delta C, and p50·p50 homodimer were mixed, only two distinct complexes, one corresponding to the FL-Ikappa Bgamma ·p50·p50 complex and the other to the ARD-Ikappa Bgamma Delta C·p50·p50 complex, were seen (Fig. 2C, lane 6). Thus, we can conclude that one molecule of Ikappa Bgamma binds to one p50·p50 homodimer.

The Role of the NF-kappa B NLS in Ikappa Bgamma Binding-- The presence of one free NLS is a prime reason for the nucleocytoplasmic shuttling of the Ikappa Balpha ·NF-kappa B complex. To elucidate the status of the NF-kappa B NLS(s) in its complex with Ikappa Bgamma , p50·p50 homodimers of different lengths, both with and without the NLSs, were prepared, and their binding to ARD-Ikappa Bgamma Delta C was tested using native gel shift assays. Fig. 3A shows that all three of the p50·p50 dimers were capable of binding to ARD-Ikappa Bgamma Delta C, although the dimer with both NLSs deleted (p50-350(-NLS)) seems to bind Ikappa Bgamma relatively weakly (Fig. 3A, lanes 5-7). This suggests that Ikappa Bgamma may not use the NLS sequence for p50 binding, or at least this region does not seem to contribute significantly. Similar binding patterns were also observed with FL-Ikappa Bgamma (data not shown).


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Fig. 3.   The NF-kappa B NLS is not completely masked in Ikappa Bgamma ·NF-kappa B complexes. A, p50 homodimer binding by Ikappa Bgamma . Native gel mobility shift assay of Ikappa Bgamma binding to different p50 dimers. Lane 1, ARD-Ikappa Bgamma Delta C; lane 2, p50-376(+NLS); lane 3, p50-363(+NLS); lane 4, p50-350(-NLS); lane 5, ARD-Ikappa Bgamma Delta C+p50-376(+NLS); lane 6, ARD-Ikappa Bgamma Delta C+p50-363(+NLS); lane 7, ARD-Ikappa Bgamma Delta C+p50-350(-NLS). p50-376, p50- 363, and p50-350 represent p50 homodimers up to residue numbers 376, 363, and 350, respectively. The presence or absence of the NLS is indicated within brackets. The free and complexed proteins are also indicated. B, co-immunoprecipitation of Ikappa Bgamma with NF-kappa B. Top panel, Western blot (WB) showing the co-immunoprecipitation (IP) of ARD-Ikappa Bgamma Delta C with p50·p65 heterodimer using the p65 NLS antibody. Bottom panel, control experiment with Ikappa Balpha and p50·p65 heterodimer. The p65 band is indicated by arrows.

Co-immunoprecipitation experiments were performed to further confirm the presence of at least one free NLS in the Ikappa Bgamma ·NF-kappa B complex. If the p65 NLS in the Ikappa Bgamma ·p50·p65 complex were free, then anti-p65 NLS antibody would interact with the p65 NLS. Here, the p50·p65 heterodimer was used because of the availability of the p65 NLS-specific monoclonal antibody. Free NF-kappa B p50·p65 heterodimer and the Ikappa Bgamma ·p50·p65 heterodimer complex were incubated with p65 NLS antibody. It was seen that the antibody was able to pull down NF-kappa B in both the free and Ikappa Bgamma complexed form (Fig. 3B, top panel). This demonstrates that the p65 NLS in the p50·p65 heterodimer is not protected by Ikappa Bgamma . As a parallel control, the Ikappa Balpha ·p50·p65 complex was also tested. The p65 NLS antibody was unable to pull down NF-kappa B in the Ikappa Balpha ·p50·p65 complex (Fig. 3B, bottom panel). This corroborates with earlier results that have shown that the p65 NLS is masked in the Ikappa Balpha ·p50·p65 complex (26, 27, 52, 53). Thus, these experiments show that at least one NF-kappa B NLS remains unmasked in the Ikappa Bgamma ·NF-kappa B complex.

Ikappa Bgamma ·NF-kappa B and p105·NF-kappa B Complexes Are Cytoplasmic-- The above observation that at least one NF-kappa B NLS is free in the Ikappa Bgamma ·NF-kappa B complex suggests that these complexes should localize to the nucleus. The subcellular distribution of the complex was tested by simultaneously transfecting HeLa cells with Ikappa Bgamma , p50, and p65. Immunostaining shows that free p50 and p65, which are most likely to be present as a heterodimer, are nuclear (Fig. 4A, left panel), but co-expression of Ikappa Bgamma leads to cytoplasmic retention (Fig. 4A, right panel). Thus, although the Ikappa Bgamma ·p50·p65 has at least one NLS free, the complex does not localize to the nucleus.


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Fig. 4.   Ikappa Bgamma ·NF-kappa B and p105·NF-kappa B complexes are cytoplasmic. A, immunofluorescence showing the distribution of the p50·p65 (left panel) and the Ikappa Bgamma ·p50·p65 complexes (right panel) within the cell. HeLa cells were co-transfected with p50 and p65 or Ikappa Bgamma , p50, and p65. Immunostaining was done using N-terminal p65 and FLAG antibody to detect p65 and p50, respectively. The p50·p65 complex was localized in the nucleus, whereas the Ikappa Bgamma ·p50·p65 complex was cytoplasmic. B, immunofluorescence showing the distribution of the p105·p50 (top panel) and the Ikappa Bgamma ·p50 complexes (bottom panel) within the cell. HeLa cells were co-transfected with HA-tagged p105 or Ikappa Bgamma (green) and FLAG-tagged p50 (red). Both p105 and p50 were mainly retained in the cytoplasm. Similarly, both Ikappa Bgamma and p50 were also retained in the cytoplasm. C, immunofluorescence showing the distribution of the p105·p50-NLS and the Ikappa Bgamma ·p50-NLS complexes. HeLa cells were co-transfected with HA-tagged p105 or Ikappa Bgamma (green) and FLAG-tagged p50-NLS (red). Left panels, in the absence of LMB, both p105·p50-NLS (top panel) and the Ikappa Bgamma ·p50-NLS (bottom panel) complexes are retained in the cytoplasm. Right panels, in the presence of LMB also, both p105·p50-NLS (top panel) and the Ikappa Bgamma ·p50-NLS (bottom panel) complexes are retained in the cytoplasm.

The subcellular distribution of p105 and Ikappa Bgamma complexes with p50 was also tested. Immunofluorescence studies show both these complexes to be cytoplasmic (Fig. 4B). Free p50, as expected, is localized in the nucleus (data not shown).

To further test the ability of these complexes to be retained in the cytoplasm, a mutant p50 with an additional NLS (p50-NLS) was made. Cells co-transfected with p50-NLS and p105 or Ikappa Bgamma reveal that the complexes are retained in the cytoplasm (Fig. 4C, left panels). To test whether the p105·p50-NLS complex shuttles between the nucleus and cytoplasm, HeLa cells were treated with the nuclear export inhibitor leptomycin B (LMB), and the subsequent changes in localization were monitored. LMB treatment does not appear to alter the localization of the p105·p50-NLS or the Ikappa Bgamma ·p50-NLS complexes (Fig. 4C, right panels), implying that these complexes do not shuttle. This result is in contrast to that observed for Ikappa Balpha ·NF-kappa B complexes where addition of the inhibitor confines both proteins within the nucleus (26). Thus, the p105·NF-kappa B complexes seem to be similar to the Ikappa Bbeta ·NF-kappa B complexes, which are primarily cytoplasmic. Free p50-NLS, as expected, is localized within the nucleus (data not shown).

The Death Domain Is Necessary for Cytoplasmic Retention of NF-kappa B-- To assess the possible role of the death domain and the C-terminal tail containing the destruction box in the cytoplasmic retention of NF-kappa Bs, deletion mutants of both p105 and Ikappa Bgamma were constructed. HeLa cells were co-transfected with p50 and various truncated forms of p105 or Ikappa Bgamma . Subcellular localization of these complexes was monitored by immunofluorescence. As mentioned in the previous results section, the p105·p50 complex is cytoplasmic (Fig. 5A, left panel). Removal of the last ~100 residues, which includes the destruction box, p105Delta DB, did not alter the localization of the complex (Fig. 5A, right panel). However, deletion of the C-terminal 171 residues (encompassing the death domain), p105Delta C, localizes the complex exclusively in the nucleus (Fig. 5A, middle panel). These results suggest that the death domain of p105·Ikappa Bgamma plays a role in the retention of NF-kappa B complexes within the cytoplasm.


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Fig. 5.   Death domain is necessary for cytoplasmic retention. A, co-transfection of full-length p105 with p50 leads to cytoplasmic localization of both proteins (left panel). Co-transfection of death domain and destruction box truncated p105 (p105Delta C) with p50 leads to nuclear accumulation of both proteins (middle panel). Co-transfection of destruction box truncated p105 (p105Delta DB) with p50 leads to cytoplasmic accumulation of both proteins (right panel). B, co-transfection of N-terminally truncated p105, p105Delta N, with p50 leads to cytoplasmic localization of both proteins (left panel). Co-transfection of death domain and destruction box truncated p105Delta N (p105Delta NDelta C) with p50 leads to nuclear accumulation of both proteins (right panel). C, co-transfection of full-length Ikappa Bgamma with p50 leads to cytoplasmic localization of both proteins (left panel). Co-transfection of death domain and destruction box truncated Ikappa Bgamma (Ikappa Bgamma Delta C) with p50 leads to nuclear accumulation of both proteins (right panel). D, co-transfection of ARD-Ikappa Bgamma with p50 leads to cytoplasmic localization of both proteins (left panel). Co-transfection of death domain and destruction box truncated ARD-Ikappa Bgamma (ARD-Ikappa Bgamma Delta C) with p50 leads to nuclear accumulation of both proteins (right panel). E, co-immunoprecipitation of the C-terminally truncated p105 and Ikappa Bgamma with p50. Western blot (WB) showing the binding of HA-tagged, death domain-deleted, and destruction box-deleted p105 (and Ikappa Bgamma ) to FLAG-tagged p50. HeLa cells were co-transfected with p50 and various truncated forms of p105 or Ikappa Bgamma s. The p105·p50 or the Ikappa Bgamma ·p50 complex was co-immunoprecipitated (IP) by incubating with anti-FLAG antibody. Western blotting was done to detect p105 or Ikappa Bgamma using anti-HA antibody. Control experiments were done with cells transfected with only p105 or Ikappa Bgamma s in the absence of p50. All of the C-terminally truncated p105 and Ikappa Bgamma seem to form a stable complex with p50.

p50 is again retained in the cytoplasm when co-expressed with a truncated p105, p105Delta N (Fig. 1), where the N-terminal, DNA-binding immunoglobulin domain (residues 1-244) is deleted (Fig. 5B, left panel). However, when both the N- and C-terminal domains (p105Delta NDelta C) are deleted, these proteins localize to the nucleus (Fig. 5B, right panel).

Similar results were obtained with various deletion mutants of Ikappa Bgamma . Co-expression of full-length Ikappa Bgamma and p50 lead to cytoplasmic retention of the complex (Fig. 5C, left panel). Deletion of the C terminus, Ikappa Bgamma Delta C, leads to nuclear localization of both proteins (Fig. 5C, right panel). Also, a shorter construct of Ikappa Bgamma comprising the ARD and all residues downstream, ARD-Ikappa Bgamma (Fig. 1) and the corresponding C terminally deleted form, ARD-Ikappa Bgamma Delta C, showed a similar retention pattern (Fig. 5D).

It is known that NF-kappa B dimers are nuclear proteins, and it is only in complex with Ikappa B that they are retained in the cytoplasm. As described above, co-expression of p50 and death domain-deleted p105 or Ikappa Bgamma leads to nuclear localization of both components. To test whether these proteins localize independently or are present as a complex in the nucleus, co-immunoprecipitation experiments were done. FLAG antibody (M2) was used to immunoprecipitate FLAG-tagged p50 from whole cell lysates that had been co-transfected with FLAG-p50 and HA-p105 or HA-Ikappa Bgamma . Subsequent Western blotting was done using the HA antibody. The results clearly show that these proteins form a complex because the FLAG antibody was able to pull down both p50 and p105 (or Ikappa Bgamma ) (Fig. 5E). In all, the above experiments suggest that the death domain of p105·Ikappa Bgamma is necessary for cytoplasmic retention.

The Death Domain Is Not Sufficient for Cytoplasmic Retention-- Next, we wanted to test whether the death domain was the exclusive retention signal in these Ikappa B molecules. Full-length Ikappa Bgamma when expressed in HeLa cells exhibits a nucleocytoplasmic distribution (Fig. 6A, left panel). The death domain-truncated Ikappa Bgamma Delta C, as expected, was localized within the nucleus (Fig. 6A, right panel). Both ARD-Ikappa Bgamma and ARD-Ikappa Bgamma Delta C, by themselves, were present in the nucleus (Fig. 6B). Thus, the presence of the death domain is not sufficient for cytoplasmic localization.


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Fig. 6.   Death domain is not sufficient for cytoplasmic retention. A, transfection of full-length Ikappa Bgamma leads to nucleocytoplasmic localization of the protein (left panel). Transfection of death domain and destruction box truncated Ikappa Bgamma (Ikappa Bgamma Delta C) leads to nuclear accumulation of the protein (right panel). B, transfection of ARD-Ikappa Bgamma leads to nuclear localization of the protein (left panel). Transfection of death domain and destruction box truncated ARD-Ikappa Bgamma (ARD-Ikappa Bgamma Delta C) also leads to nuclear accumulation of the protein (right panel). C, transfection of the C-terminal death domain-destruction box of p105 (p105 (800-971)) shows nuclear accumulation of the protein (left panel). Transfection of the death domain of p105 (p105 (800-887)) also leads to nuclear accumulation (right panel).

To further test the role of the death domain, two more constructs were made. One containing only the death domain, p105 (800-887), and the other with all the segments further downstream, p105 (800-971). Both of these proteins localized within the nucleus (Fig. 6C). This suggests that neither the death domain by itself nor its presence in the Ikappa Bgamma molecule provides the retention signal.

The Death Domain Does Not Participate in NF-kappa B Binding-- Because the death domain of p105·Ikappa Bgamma seems to be important for cytoplasmic localization of NF-kappa B, a possible model of the complex could be one in which the death domain binds and masks the exposed NLS of NF-kappa B, thereby preventing nuclear import. If this were true, then full-length Ikappa Bgamma must interact with the p50·p50 homodimer with higher affinity than the death domain-truncated Ikappa Bgamma . Although the native gel shift assays showed that the death domain was not essential for NF-kappa B binding, these experiments were of a qualitative nature. Fluorescence polarization competition assays were done to determine the binding affinity of NF-kappa B for both full-length and death domain-truncated Ikappa Bgamma . In a solution-based competition assay, fixed amounts of fluorescein-labeled kappa B DNA bound to the p50·p50 dimer was incubated with increasing amounts of Ikappa Bgamma . In this experiment, the Ikappa Bgamma -dependent dissociation of the p50·p50 dimer from DNA is accompanied by a distinct change in fluorescence polarization. The DNA binding inhibition constant at equilibrium was then calculated. As shown in Fig. 7, both Ikappa Bgamma s inhibit NF-kappa B DNA binding to a similar extent. Thus, the death domain does not seem to play a major role in NF-kappa B binding to Ikappa Bgamma .


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Fig. 7.   Death domain is not involved in NF-kappa B binding. The fluorescence polarization competition assays were performed to determine the binding affinities of the Ikappa Bgamma ·p50 complexes. The affinity of full-length Ikappa Bgamma ·p50 was found to be 38 nM (green) and that of the C-terminally deleted Ikappa Bgamma Delta C·p50 was found to be 45 nM (red).

Limited Processing of p105: a Possible Model of the Cytoplasmic Complex-- In addition to testing the localization of p50 complexes with the full-length and truncated forms of p105, we have also expressed just p105 and its truncated forms in HeLa cells and tested their individual subcellular distribution. p105 is present exclusively in the cytoplasm (Fig. 8A, left panel), whereas the C-terminally truncated p105, p105Delta C, was exclusively nuclear (Fig. 8A, right panel). The N-terminally truncated p105, p105Delta N, also localized to the cytoplasm (Fig. 8B, left panel). The corresponding C-terminal deleted p105, p105Delta NDelta C, was nuclear (Fig. 8B, right panel). Localization patterns of p105 and its derivatives are thus identical to that observed previously for the p105·p50 complexes in HeLa cells, expressing both p105 (or its derivatives) and p50.


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Fig. 8.   Subcellular localization of full-length and N-terminally truncated p105. A, transfection of full-length p105 leads to cytoplasmic localization of the protein (left panel). Transfection of death domain- and destruction box-truncated p105 (p105Delta C) leads to nuclear accumulation of the protein (right panel). B, transfection of N-terminally truncated p105, p105Delta N, leads to cytoplasmic localization of the protein (left panel). Transfection of death domain and destruction box truncated p105 (p105Delta NDelta C) leads to nuclear accumulation of the protein (right panel). C, processing of p105 in the presence and absence of co-expressed p50. Western blot showing the processing of various p105 mutants. HeLa cells were transfected with C-terminally (p105Delta C), N-terminally (p105Delta N), and both C- and N-terminally (p105Delta NDelta C) truncated p105 in the presence and absence of p50. As seen, all three mutants undergo processing (lanes 1-3), and this is abrogated in the presence of co-expressed p50 (lanes 4-6). D, N-terminally deleted p50 binding by ARD-Ikappa Bgamma Delta C. Native gel mobility shift assay of ARD-Ikappa Bgamma Delta C binding to N-terminally deleted p50 dimers. Lane 1, ARD-Ikappa Bgamma Delta C; lane 2, p50Delta N-376; lane 3, p50Delta N-363; lane 4, ARD-Ikappa Bgamma Delta C+p50Delta N-376; lane 5, ARD-Ikappa Bgamma Delta C+p50Delta N-363. p50Delta N-376 and p50Delta N-363 represent p50 (245-376) and p50 (245-363), respectively. The free and complexed proteins are indicated by arrows.

p105 is known to undergo limited processing to generate p50 molecules. To test whether the other truncated forms of p105 were also capable of similar processing, Western analysis was done using HA monoclonal antibody. We observe that like full-length p105, its truncated forms were also processed in a limited manner within the cell. The appearance of an additional band of lower molecular weight indicates a processed band, derived from the C-terminal processing of the intact protein. The lengths of the processed products vary according to the truncations of p105. Cells transfected with the C-terminally truncated form, p105Delta C, show a processed band corresponding to full-length p50, and cells transfected with the smaller, N-terminally truncated form, p105Delta N and p105Delta NDelta C, generate a shorter p50 (Fig. 8C). On the other hand, in cells transfected with both truncated p105 and p50, no processed p50 band is detected. This observation corroborates with that seen earlier for full-length p105 and p50 (41). These results lead us to suggest that p105 is first processed in a limited manner and that the resulting complex formed between the unprocessed p105 and the processed p50 is identical to the one in which both proteins are expressed within the same cell.

A minimal complex formed between p105 and p50 seems to require just the dimerization domain of p50. Therefore, the smallest p105 derivative, p105Delta NDelta C, can be partially processed into a truncated p50 comprising of only the dimerization domain (p50Delta N), and this can associate with unprocessed p105Delta NDelta C. Such a complex would be equivalent to the complex between the p50 dimerization domains and the ARD-Ikappa Bgamma Delta C. Indeed, native gel shift assays show that the dimerization domain of p50 (p50Delta N-376 and p50Delta N-363) can form a stable complex with ARD-Ikappa Bgamma Delta C (Fig. 8D). However, the p105Delta NDelta C·p50Delta N complex is not cytoplasmic because the death domain is absent. In contrast, the p105Delta N·p50Delta N complex is cytoplasmic.

These results indicate that in addition to the death domain, p105·NF-kappa B or the equivalent Ikappa Bgamma ·NF-kappa B complex formation is necessary for cytoplasmic retention. In the retention process, only the N-terminal domain (residues 1-244) and the C-terminal region (residues 885-971) of p105 are dispensable.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Earlier studies using competition assays have shown that Ikappa Bgamma preferentially inhibits p50·50 homodimer (18, 31, 51). In the present study we have performed direct binding assays to demonstrate that Ikappa Bgamma interacts stably with both p50·p65 and p50·p50 dimers but not with p65·p65 or c-Rel·c-Rel dimers. This suggests that Ikappa Bgamma may have a preference for p50 containing NF-kappa B dimers. We and others have previously shown that Ikappa Balpha interacts strongly with p65 containing dimers (23, 24, 26). The x-ray structure of Ikappa Balpha ·p50·p65 heterodimer showed that the p65 subunit makes extensive contacts with Ikappa Balpha (52, 53). Thus, a central theme seems to govern all Ikappa B·NF-kappa B binding wherein the stability of the interaction is dictated by the association of an Ikappa B molecule with one specific NF-kappa B subunit. The second subunit, which we refer to as the nonspecific subunit, associates with the specific subunit through its dimerization domain. This is illustrated in Fig. 9A. The nature of the specific subunit is directly correlated to the nature of the binding Ikappa B molecule. For Ikappa Balpha , p65 serves as the specific subunit. Ikappa Bbeta also exhibits similar dimer specificity, requiring p65 as the primary binding partner. On the other hand, in Ikappa Bgamma , the specific subunit appears to be p50. In p105·NF-kappa B complexes, by the very nature of the molecule, the docking occurs intramolecularly with the N-terminal p50 arm serving as the specific subunit. It is possible that in Ikappa B·NF-kappa B complexes the nonspecific subunit can be exchanged with other nonspecific subunits, and this may dictate the cellular pool of active NF-kappa Bs.


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Fig. 9.   Model of the Ikappa B·NF-kappa B complex. A, schematic representation of NF-kappa B binding to Ikappa B. One Ikappa B molecule binds to one NF-kappa B dimer comprising of two types of subunits: a specific subunit and a nonspecific subunit. Left panel, prototypical Ikappa B·NF-kappa B complexes (Ikappa Balpha ·NF-kappa B and Ikappa Bbeta ·NF-kappa B) where the specific subunit is p65. Right panel, atypical Ikappa B·NF-kappa B complexes (p105·NF-kappa B) where the specific subunit is p50. B, a possible model of the cytoplasmic p105·NF-kappa B complex. Transfected p105 is capable of undergoing limited processing, which is coupled to a dimerization event leading to the formation of a p105·p50 complex. Co-expression of both p105 and p50 can lead to a similar complex formation without processing. The NLS in this complex seems to be exposed, and yet it is retained in the cytoplasm. A possible mode of sequestration could be binding to another cytoplasmic protein(s) through the scaffold formed because of the association of p105 and p50 and the death domain of p105.

Sequestration of NF-kappa B in the cytoplasm can be considered to be one of the most simplistic ways by which Ikappa B molecules exhibit their inhibitory function. They can do this by blocking the NLS of NF-kappa B dimers. However, both Ikappa Balpha and Ikappa Bbeta , upon binding to the specific NF-kappa B dimers, mask only the NLS of the specific NF-kappa B subunit. As expected therefore, Ikappa Balpha ·NF-kappa B complexes are not truly cytoplasmic but dynamic nucleocytoplasmic (23-26). We observe that in the Ikappa Bgamma ·p50·p65 complex the p65 NLS is free. Although no structural evidence exists to confirm that the NLS of the specific subunit is completely masked, two lines of evidence indicate this to be most likely. First, we show that when both NLSs are removed the affinity of the p50·p50 homodimer for Ikappa Bgamma appears to be reduced, and second, we show that the NLS of the nonspecific p65 subunit of the Ikappa Bgamma ·p50·p65 complex is free. Therefore, reduced binding affinity of p50·p50 homodimer for Ikappa Bgamma is likely due to the removal of the NLS of the p50 subunit that makes specific contacts with Ikappa Bgamma .

The domain architecture of the C-terminal inhibitory domain of p105 is different from that of Ikappa Balpha and Ikappa Bbeta . p105 does not contain a PEST sequence in its C-terminal tail as in Ikappa Balpha and Ikappa Bbeta , but it does contain a death domain. The PEST sequence in Ikappa Balpha and Ikappa Bbeta is important for NF-kappa B binding. In case of Ikappa Bgamma , we observe that the death domain does not play any role in NF-kappa B binding, suggesting that this domain is not involved in masking the exposed NLS of the nonspecific NF-kappa B subunit in p105·NF-kappa B or Ikappa Bgamma ·NF-kappa B complexes. The death domain, however, seems critical for cytoplasmic retention. When this domain is removed from p105·Ikappa Bgamma , the subcellular localization of the truncated molecules in their free form or their complexes with NF-kappa B change completely, from cytoplasmic to nuclear. This observation is consistent with earlier studies showing that removal of C-terminal 191 residues alters the localization from the cytoplasm to the nucleus (37). However, although this clearly suggested the role of the C-terminal domain of p105, this large truncation also removed most of the last AR. Therefore, it was not clear whether removal of part of the ARD contributed to altered localization of p105. Interestingly, the death domain, by itself, does not contain any retention signal because we see that the free death domain localizes to the nucleus. Full-length or N-terminally truncated Ikappa Bgamma , both of which contain the death domain, are also primarily nuclear.

We observe that free p105, as in the p105·p50 complex, is also cytoplasmic. However, p105 does not exist in its free form in cells. The molecule is partly processed generating p50, and this processed p50 forms a dimer with the precursor p105. p105·p50 complex formation is necessary for cytoplasmic sequestration of both p50 and p105. Our results show that limited processing does not produce an exactly 1:1 molar ratio of p105. It is thus possible that excess unprocessed p105 associates with other NF-kappa Bs such as p65 and c-Rel, and the p105·NF-kappa B complex is retained in the cytoplasm. Cellular localization of truncated p105 is dependent upon the presence or absence of the death domain. Our results suggest that the three-dimensional scaffold formed because of the association of p105 and p50 (or another NF-kappa B subunit), in addition to the death domain, is required for the cytoplasmic retention of the complex. Association between p105 and p50 (or any other NF-kappa B subunit) requires only the dimerization domain of p50 and the ARD of Ikappa Bgamma . However, the NLS of the nonspecific subunit is still exposed to the solvent. How is the complex then retained in the cytoplasm? We do not yet know the precise mechanism of cytoplasmic sequestration of this complex.

Earlier work in our laboratory has shown that Ikappa Bbeta ·NF-kappa B complexes are cytoplasmic, although even here, as in Ikappa Balpha ·NF-kappa B complexes, one NLS is mostly solvent-exposed. Recent studies lead us to believe that Ikappa Bbeta ·NF-kappa B complexes interact with additional cellular proteins to mask the exposed NLS, thereby sequestering them in the cytoplasm.2 It is possible that p105 employs a similar mechanism, but not necessarily the exact same mechanism. Thus, a bridging factor, which requires the death domain and the NF-kappa B scaffold, could be involved in masking the NLS. The death domain has been shown to confer various homotypic and heterotypic interactions in a large number of signaling pathways (54). It may thus play a major role in binding to the bridging factor. Consistent with this model, an earlier study demonstrated the inaccessibility or the reduced accessibility of the C-terminal tail in p105 by using antibodies raised against the C-terminal (37). This study also showed that the NLS of p105 is masked. Fig. 9B thus summarizes a likely sequence of events based on the above results. Expression of p105 or both p105 and p50 results in a p105·p50 complex that is retained in the cytoplasm. As noted earlier, the N-terminal domains and the last ~100 residues of p105 do not seem to be a part of the retention signal. Thus, a plausible model could be one in which a composite surface formed by the coming together of the relevant regions of the protein complex can interact with a putative, cytoplasmic protein that leads to its docking. This could then explain the inability of the p105·NF-kappa B or the Ikappa Bgamma ·NF-kappa B complex to move to the nucleus even with an additional NLS. We conclude that the Ikappa B family proteins employ diverse mechanisms for cytoplasmic sequestration of NF-kappa B dimers. Elucidating each of these mechanisms in detail is imperative for understanding their cellular function and participation in unique signal transduction pathways.

    ACKNOWLEDGEMENTS

We thank Prof. M. Yoshida at the University of Tokyo for providing LMB. We acknowledge Amanda Fusco for aid in performing the fluorescence polarization experiment (Fig. 7) and Tom Huxford and Rashmi Talwar for critically reading the manuscript.

    FOOTNOTES

* This work was supported by grants from NCI, National Institutes of Health and Human Frontier Science Program (to G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Chemistry & Biochemistry, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0359. Tel.: 858-822-0469; Fax: 858-534-7042; E-mail: gghosh@ucsd.edu.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M207515200

2 S. Malek, Y. Chen, and G. Ghosh, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: NLS, nuclear localization signal; AR, ankyrin repeat; ARD, ankyrin repeat domain; LMB, leptomycin B; HA, hemagglutinin; PBS, phosphate-buffered saline.

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ABSTRACT
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RESULTS
DISCUSSION
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