INTRODUCTION

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Transcriptional induction of genes driven by members of the NF-B/Rel family of activators plays a crucial role in many processes of the immune response and under a number of cellular stress conditions including viral infection (2, 5, 40, 44). The activation of NF-B through different pathways is controlled primarily by its release from the interaction with inhibitory IB molecules. Five different mammalian NF-B/Rel proteins are known, p50, p52, p65, c-Rel, and RelB, which form homo- and heterodimers. These are sequestered by the IB molecules IB, IB, and IB and by p105 and p100, the precursor molecules for p50 and p52, respectively (2, 42). Best understood is the activation of NF-B from complexes with IB by diverse inducers like tumor necrosis factor alpha (TNF-), lipopolysaccharide (LPS), phorbol myristate acetate, (PMA) or human T-cell leukemia virus type 1 Tax (7, 8, 38, 43). These lead to phosphorylation at Ser 32 and 36. Phosphorylated IB is then polyubiquitinated and subsequently degraded by the proteasome (3, 9, 34). In addition to the amino-terminal signal response box containing Ser 32 and 36, signal-dependent degradation is more efficient in the presence of the carboxy-terminal PEST sequence of IB (7, 8, 38, 43), although the mechanistic need of the latter is not understood. The high turnover of IB in the resting state is also driven by the proteasome but is independent of the amino-terminal serines and of the PEST sequence and does not involve ubiquitin conjugation of the molecule (19). Processing of p105 is, at least in some cell lines, signal inducible and involves the same steps as IB breakdown, including phosphorylation, ubiquitin conjugation, and degradation by the proteasome (14, 29, 31). An endoprotease activity initially cleaves p105 into p50 and the carboxy-terminal IB domain (21). A multisubunit kinase, which phosphorylates IB at Ser 32 and 36 and which requires ubiquitination of one of its subunits, has been identified biochemically (10). Recently, two IB kinases which phosphorylate IB at Ser 32 and 36 and mediate NF-B activation in response to TNF- have been cloned (24, 35).

The overlapping functions of the various IB and NF-B proteins contrast with their specific physiological activities, as revealed by gene-targeting experiments (2). These specific physiological activities imply that functional specificity may be imposed by the differential response of individual IB or NF-B/Rel proteins to particular signaling pathways. Therefore, potential regulatory differences between diverse IBs, such as IB and IB, are of interest.

IB was first identified by Zabel and Baeuerle (45), and murine IB was cloned with probes derived from peptides of the purified protein (37). Human cDNAs encoding IB were isolated by using the two-hybrid system (20) (see below). A notable difference between IB and IB is the basal phosphorylation of IB, which is needed for interaction with NF-B (22). Recent reports have shown that IB and IB have properties in common. Murine IB is proteolytically degraded after stimulation with LPS or interleukin-1 (IL-1) (37). In contrast to IB, IB is not resynthesized immediately after stimulation and is degraded with delayed kinetics compared to IB (37). The degradation of murine IB after stimulation with TNF-, IL-1, or human T-cell leukemia virus type 1 Tax depends on Ser 19 and Ser 23 (12, 25). These residues are embedded in a sequence similar to the one surrounding Ser 32 and 36 in IB, and a recently identified IB kinase phosphorylates both proteins at these residues (13, 33). Furthermore, the carboxy-terminal PEST sequence of IB is needed for efficient proteolysis (16, 41). Inducible degradation of IB is blocked by inhibitors of the proteasome and possibly requires ubiquitination (12, 25). Unphosphorylated recombinant IB, but not IB, can associate with Rel factors, and latent cytoplasmic complexes of IB and Rel factors are sensitive to phosphatase treatment. Major sites of constitutive phosphorylation of IB have been mapped to Ser 313 and Ser 315, which are phosphorylated by casein kinase II (CKII) (11). Phosphorylation at these residues is required for efficient complex formation between IB and c-Rel (11). Recombinant unphosphorylated IB can bind to p65 but not to c-Rel (11). Carboxy-terminal phosphorylation by CKII of IB increases its affinity to NF-B (39). Tax-induced degradation of IB leads to the induction of c-Rel-containing complexes (15), further indicating that c-Rel-IB complexes are physiologically important.

Newly synthesized IB first accumulates as an underphosphorylated species, which forms a stable complex with NF-B p65 (36). It has been shown that in contrast to the phosphorylated form, underphosphorylated IB interacts with p65 without shielding the nuclear localization signal region. De novo-translated, underphosphorylated IB may sequester NF-B, thereby protecting it from being withdrawn into cytoplasmic complexes with IB, and it can be transported with NF-B into the nucleus (36).

We show here that in human cells the IB gene gives rise to different gene products, which elicit differential responsiveness to several inducers tested. Two of these, termed IB1 and IB2, have been analyzed in detail. Compared to IB, IB1 is most similar in responsiveness to inducing agents whereas IB2, which has a carboxy-terminal sequence substitution truncating a PEST domain, is either degraded incompletely or inert in response to some inducers. Both IB1 and IB2 bind with highest affinity to p65 homodimers and with lower affinity to heterodimers containing c-Rel or p65. The affinity depends largely on constitutive phosphorylation at the carboxy-terminal PEST sequences of both molecules. IB1, but not IB2, is partially localized in the nucleus in B cells, indicating different roles of the two isoforms for modulating or maintaining constitutive NF-B activation. The resistance of IB2, the predominant form in human cells, to a number of inducers tested suggests that this isoform may serve to limit the fraction of cytoplasmic NF-B released into the nucleus upon cellular stimulation.

	MATERIALS AND METHODS

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Cell culture. Namalwa and IM9 cells were grown in RPMI 1640 (BRL/GIBCO)-4 mM glutamine-100 U of penicillin per ml-100 µg of streptomycin per ml-7.5% fetal calf serum; suspension HeLa cells were kept in Joklik modified minimum essential medium (BRL/GIBCO)-1% nonessential amino acids, 100 U of penicillin per ml-100 µg of streptomycin per ml-10% fetal calf serum. Jurkat cells were grown in RPMI 1640 (BRL/GIBCO)-4 mM glutamine-100 U of penicillin per ml-100 µg of streptomycin per ml-10% fetal calf serum. Where indicated, cells were stimulated with 10 ng of TNF- (Biomol) per ml, 50 ng of PMA (Sigma) per ml-5 µg of ionomycin (Sigma) per ml, or 50 U of IL-1 (Biomol) per ml for the indicated times.

Genomic PCR and reverse transcription-PCR. PCR was performed by standard methods with IB-specific primers and HeLa cell-derived nucleic acids.

Library screening. A HeLa cell cDNA library was screened with the insert of the TRIP9 clone isolated by two-hybrid screening (20) as a probe. Positive cDNAs were sequenced on both strands by the dideoxynucleotide method.

DNA constructs for prokaryotic expression. pETIB2 (encoding amino acids 1 to 338) was generated from pCDM8IB(351/21) by PCR with the appropriate oligonucleotides with the insert cloned into the BamHI site of pET3c (Novagen). Protein expression in Escherichia coli BL21(DE3)pLysS, and purification was carried out as described previously (27).

DNA constructs for eukaryotic cell expression. pECEp50, pECEp65, pECEc-rel, and pcDNA3IB have been described previously (19, 28). FLAG-tagged pcDNA1IB2 (amino acids 1 to 338), FLAG-tagged pcDNA1IB2N (amino acids 55 to 338), FLAG-tagged pcDNA1IB2C (amino acids 1 to 307), and FLAG-tagged pcDNA1IB2NC (amino acid 55 to 307) were generated from pCDM8IB(351/21) by PCR and cloning into the BamHI site of FLAG-tagged pcDNA1, which contained a FLAG epitope inserted into the HindIII and BamHI sites of pcDNA1. FLAG-tagged pcDNA1IB1 was generated by cloning the IB1 cDNA from pCDM8IB(351/1) into pcDNA1. The NF-B reporter plasmid 2X B-Luc contained the annealed tandem Ig enhancer sequence 5'-CAGTTGAGGGGACTTTCCCAGATCTAGTTGAGGGGACTTTCCCAG-3' and the 45 to +83 fragment of the human immunodeficiency virus type 1 promoter (EcoRI and HindIII sites) inserted into KpnI-HindIII of pGV-B (Toyo Ink Co., Tokyo, Japan).

Antibodies for immunoblots and immunoprecipitation. We used anti-p65 (rabbit) (Santa Cruz Biotechnology Inc., sc-109), anti-p50 (rabbit) (Rockland), anti-c-Rel (rabbit) (Santa Cruz Biotechnology Inc., sc-272), anti-relB (rabbit) (Santa Cruz Biotechnology Inc., sc-226), anti-p52 (mouse) (Upstate Biotechnology Inc., 05-361), anti-IB/MAD3 (rabbit) (Santa Cruz Biotechnology Inc., sc-203), anti-FLAG (rabbit) (Santa Cruz Biotechnology Inc., sc-807), anti-IB (rabbit) (Santa Cruz Biotechnology Inc., sc-969, against the N-terminal sequence of IB1 and IB2 [N-20]), anti-IB (rabbit) (Santa Cruz Biotechnology Inc., sc-945, against the C-terminal sequence of IB1 [C-20]), and the antibody against the C-terminal sequence of IB2 [b2(C)], raised against the peptide VSQEERQGSPAGGSG (amino acids 324 to 338 of IB2, synthesized by Eurogentec S.A.).

Preparation of cell extracts. For whole-cell extracts, cells were washed with phosphate-buffered saline (PBS) twice and incubated on ice for 15 min in 20 mM HEPES (pH 7.9)-350 mM NaCl-1 mM MgCl₂-0.5 mM EDTA-0.1 mM EGTA-1% Nonidet P-40 (NP-40)-0.5 mM dithiothreitol-0.4 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Boehringer Mannheim)-50 mM sodium fluoride-1 mM sodium orthovanadate. After centrifugation at 14,000 rpm for 20 min in an Eppendorf centrifuge, the supernatant was used as a whole-cell extract. For preparation of cytoplasmic and nuclear extracts, the cells were washed and resuspended in buffer A and 0.125% NP-40 was added. The cells were left for 5 min on ice and centrifuged at 1,000 × g for 10 min. The supernatant was used as the cytoplasmic extract, and the pellet was treated with buffer C for 15 min to yield the nuclear extract as described previously (27).

Immunoblots. Cell extracts (40-µg samples) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and prior to transfer, the gels were equilibrated in ice-cold blotting buffer (25 mM Tris-HCl [pH 8.3], 0.01% SDS, 20% methanol). The proteins were transferred to a polyvinylidene difluoride membrane (Millipore) as described previously (28). Western blots were analyzed by chemiluminescence (Tropix) as described previously (19).

Immunoprecipitation. Antibodies (10-µl samples) were coupled to protein A-Sepharose (Pharmacia) in PBS for 2 h. The whole-cell extract or cytoplasmic extract was incubated for 2 h with antisera or antibodies coupled to protein A-Sepharose. The precipitated proteins were washed several times with ice-cold PBS, boiled, separated by SDS-PAGE, and transferred to a PVDF membrane as described previously (27).

Electrophoretic mobility shift assay (EMSA). Gel retardation assays were performed with an H2K oligonucleotide probe. DNA binding reactions were performed in 20 µl of binding buffer [20 mM HEPES (pH 8.4), 60 mM KCl, 4% Ficoll, 5 mM dithiothreitol 1 µg of bovine serum albumin, 2 µg of poly(dI-dC)] for 20 min at 30°C. The reaction mixture was loaded onto a 4% nondenaturing polyacrylamide gel containing 1× Tris-borate-EDTA (TBE). The gels were run at 250 V for 1 h, dried, and visualized by autoradiography.

Immunoprecipitation-EMSA (IP-shift) assay. The precipitated proteins, coupled to protein A beads, were washed several times with ice-cold PBS and incubated in 20 µl of 0.8% deoxycholate on ice for 10 min. After centrifugation, NP-40 (final concentration, 1%) was added to the supernatant, which was then used for EMSA.

Phosphatase treatment of cytoplasmic extracts. Cell extract proteins (40 µg) were incubated with 2 U of shrimp alkaline phosphatase (United States Biochemical Co.) at 37°C for 30 min. As a control, shrimp alkaline phosphatase was inactivated at 65°C for 30 min.

Transient transfection. Transfection of suspension HeLa cells was performed by electroporation with a Bio-Rad Gene Pulser. Cells were grown at 5 × 10⁵ cells/ml, harvested 5 to 6 h after the last feeding, and resuspended at 2 × 10⁷ cells/ml. Then 0.4 ml of cells was mixed with 20 µg of DNA at room temperature, electroporated at 250 V and 950 µF, and immediately transferred to small culture flasks containing 10 ml of prewarmed medium. At 48 h later, the transfected cells were harvested. For reporter gene assays, HeLa cells were transfected by electroporation with 2× B-Luc (10 µg), p65 expression vector (5 µg), and expression vectors containing IB, IB1, or IB2 (1 or 5 µg). Luciferase activities were measured with a Luminometer Lumat LB9501 (Berthold).

	RESULTS

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Human IB is expressed as a set of differentially spliced isoforms. We previously isolated a human homolog of IB by using the human thyroid receptor as a bait in the two-hybrid system (20). Rescreening HeLa cell cDNA libraries with the original yeast isolate yielded additional clones with two distinct 3' ends. Sequence alignment with murine IB (37) demonstrates that one of these encodes a protein, IB1, that is most similar to the murine protein. The variant IB2 isoform differs from IB1 by a substitution of new C-terminal and 3' untranslated sequences, which results in truncation of the C-terminal PEST motif (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   (Top) Schematic presentation of human IB1 and IB2 compared to murine IB. The position of the peptides used for raising the antibodies is indicated. (Bottom) Sequence alignment of the carboxy-terminal PEST sequences of human IB1 and IB2 and murine IB. a.a., amino acids.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Protein expression of IB isoforms. (A) Western blot analysis of Namalwa cell cytosol with an amino-terminal antibody recognizing a common epitope (N-20) or carboxy-terminal antibodies raised against isoform-specific sequences [C-20 for IB1 and (C) for IB2], as indicated (lanes 1 to 3). Specific bands at 43 and 41 kDa were absent after peptide competition (lane 4 and 5) or probing with preimmune serum (lane 6). (B) Immunoprecipitation. Namalwa cell cytosolic extracts were subjected to immunoprecipitation with N-20, and the precipitates were immunoblotted with C-20 or (C) antibodies, as indicated. (C) Equal cell equivalents of human and murine cell lines were analyzed by Western blotting for IB isoform expression with the amino-terminal antibody, as indicated. (Left) Human cell lines; (right), murine cell lines; (bottom), summary of the relative expression levels of IB1 and IB2. N.D., not detectable. The signals around 29 kDa are cross-reacting proteins. The positions of IB1 and IB2 are indicated by solid and open arrowheads, respectively.

Both cellular IB isoforms associate predominantly with p65 homodimers but also with heteromers containing c-Rel or p65; equal inhibition of NF-B-dependent transcription. To investigate the preferential association of IB1, IB2, and IB with Rel factors, cellular proteins immunoprecipitated with antibodies against the five mammalian NF-B/Rel subunits were blotted and subsequently probed with IB-specific antibodies (Fig. 3A). IB1 and IB2 associated predominantly with p65 and only weakly with p50 or p52, presumably through their heteromeric complexes with p65 (lanes 1, 2, 5, 11, 12, and 15). In contrast, almost no RelB and only little c-Rel associated with IB1 or IB2 (lanes 3, 4, 13, and 14), although both Rel proteins were efficiently precipitated (Fig. 3A, bottom). Similarly, IB interacted predominantly with complexes containing p65 (lanes 6 to 10). IB was found to a larger extent in complexes with p50 or p52 than were IB1 and IB2 (lanes 6 and 10). Similarly, in primary and transformed murine T cells, IB and IB (IB1) were associated primarily with p65 and to a lesser extent with c-Rel (11).

View larger version (59K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   View larger version (68K): [in this window] [in a new window]   View larger version (64K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   FIG. 3.   Interaction and functional interference of IB1, IB2, and IB with NF-B/Rel factors (A) Coimmunoprecipitation of IBs with NF-B/Rel. Whole-cell Namalwa extracts were subjected to immunoprecipitation with antibodies (Ab) directed against p50, p65, c-Rel, RelB, or p52. (Top) Precipitates were separated by SDS-PAGE, and the blot was sequentially probed with antibodies against IB1 (lanes 1 to 5), IB (lanes 6 to 10), and IB2 (lanes 11 to 15). Specific signals are indicated by solid arrowheads; open arrowheads indicate residual signals from the preceding staining. (Bottom) Precipitates from anti-Rel immunoprecipitations were probed with anti-Rel antibodies, as indicated. The position of precipitated immunoglobulin is indicated by open arrowheads. (B) Coimmunoprecipitation/supershift (IP-shift) analysis of IB-bound Rel factors. (Top) Namalwa cells; (bottom) HeLa cells. Immunoprecipitation was carried out with whole-cell extracts and antibodies directed against IB, IB1, or IB2. Bound NF-B/Rel factors were eluted from the pellets by deoxycholate treatment and analyzed for subunit composition by EMSA and supershifting/inhibition with NF-B/Rel-specific antibodies. Antibodies against IB, IB1, or IB2 precipitated NF-B/Rel DNA binding activity (lanes 3, 8, and 13), which was strongly reduced or absent when the epitope-containing peptides were included in the precipitation reaction mixture or when the preimmuneserum was used (lanes 2, 7, and 12). DNA binding activity obtained from IB (lanes 2 to 6), IB1 (lanes 7 to 11), or IB2 (lanes 12 to 16) was challenged with anti-p65 antibody (lanes 4, 9, and 14), with anti-p65 antibody and peptide competition (lanes 5, 10, and 15), or with anti-p50 antibody (lanes 6, 11, and 16), as indicated. In lanes 1, no protein was added. Free DNA is not shown. Supershifted complexes are indicated (S). (C) Phosphorylation-dependent interaction of IB1 and IB2 with NF-B in HeLa cells. Whole-cell extracts were subjected to immunoprecipitation with antibodies against IB, IB1, or IB2 (lanes 1 to 3, 4 to 6, and 7 to 9, respectively). The beads were treated with shrimp alkaline phosphatase (SAP; lanes 2, 5, and 8), heat-inactivated phosphatase (hi SAP; lanes 3, 6, and 9), or buffer alone (lanes 1, 4, and 7), and the supernatants were tested by EMSA with the H2K binding-site probe. (D) IB, IB1, and IB2 equally inhibit NF-B reporter gene activation by p65. HeLa cells were transfected with 2×B-Luc reporter without (open bars) or with (solid bars) p65 expression construct. IB, IB1, or IB2 expression constructs were cotransfected in increasing amounts, as indicated. The mean values from four independent transfections are shown.

IB1 and IB elicit a similar signal responsiveness, whereas IB2 is less responsive or even resistant to signal-induced degradation. The sequence difference between IB1 and IB2 affects the PEST sequence, which plays a role in signal-dependent degradation in human IB (7, 8, 38, 43). To investigate potential differences in signal inducibility, HeLa, Namalwa, or Jurkat cells were stimulated with TNF-, PMA/ionomycin, or IL-1 for various times and analyzed for nuclear NF-B accumulation and degradation of IB, IB1, and IB2 (Fig. 4). An image quantitation of the steady-state amounts of IB shown in the Western blots is depicted graphically (Fig. 4, bottom panels). TNF- or IL-1 stimulation led to a rapid translocation of NF-B p50-p65 and of p65 homodimers, irrespective of the cell type. In contrast, PMA acted rapidly in Namalwa and Jurkat cells but only slowly in HeLa cells, indicating cell type differences in the PMA/Ca²⁺ induction mechanism. Similarly, the induction of NF-B by PMA required 30 to 45 min and was dependent on de novo protein synthesis in Hl60 cells but was fast in 70Z/3 cells (18). The kinetics of nuclear translocation of NF-B was most often paralleled by a synchronous degradation of IB and of IB1. The relative decrease in steady-state amounts, however, was greatest for IB, closely followed by IB1. In contrast, IB2 was much less responsive and in some cases even resisted degradation. The identical degradation kinetics suggest that all three inhibitors were degraded by the same mechanism. In line with this conclusion, the delayed activation of NF-B in PMA-stimulated HeLa cells coincided with simultaneous degradation of IB and IB1 at the same delayed time point, while IB2 seemed not to respond (Fig. 4A). Thus, the relative amount of IB2 produced by alternative splicing provides a means of limiting the fraction of IB-bound NF-B that is released after cellular stimulation.

View larger version (58K): [in this window] [in a new window]   View larger version (41K): [in this window] [in a new window]   View larger version (40K): [in this window] [in a new window]   FIG. 4.   Different efficiencies of signal-induced proteolytic degradation of IB1 and IB2. EMSA (top) and Western blots (whole-cell extracts) (middle) are shown. (A) HeLa cells; (B) Namalwa cells; (C) Jurkat cells. Stimulation of cells with TNF-, PMA plus ionomycin, or IL-1 was carried out for 0, 15, 30, 90, 180, or 300 min, as indicated. The positions of IB, IB1, and IB2 in the Western blots are indicated. (Bottom) Densitometric quantitation of IB1 (open circles), IB2 (triangles), and IB (dots). The steady-state amounts of the three IBs after increasing times of stimulation are plotted, with the amounts at 0 min arbitrarily set at 1.0. Activated NF-B consisted of p65 homodimers and p50-p65 as determined by antibody supershifting (data not shown).

IB1 and IB2 are basally phosphorylated but differ in their nuclear accumulation in B cells. Since IB1 and IB2 differ in their PEST sequence, which is a target of constitutive phosphorylation of IB and of IB (4, 11), we analyzed the effect of alkaline phosphatase treatment on the electrophoretic mobility of both isoforms in SDS-PAGE (Fig. 5A). Both IB1 and IB2 revealed increased mobility after treatment with alkaline phosphatase but not after treatment with heat-inactivated enzyme. The same result was obtained with HeLa or Namalwa cell-derived proteins. Thus, both IB isoforms are constitutively phosphorylated. This finding is also in agreement with the phosphatase-mediated release of NF-B activity from both isoforms (Fig. 3C).

View larger version (48K): [in this window] [in a new window]   View larger version (51K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   View larger version (38K): [in this window] [in a new window]   FIG. 5.   (A) Constitutive phosphorylation of IB1 and IB2. Cytosolic extracts of Namalwa (lanes 1 to 3) or HeLa (lanes 4 to 6) cells were either untreated (lanes 1 and 4), treated with shrimp alkaline phosphatase (SAP; lanes 2 and 5), or heat-inactivated phosphatase (hi SAP; lanes 3 and 6) and subjected to Western blot analysis with anti-IB (N-20) antibody. (B) Nuclear localization of IB1, but not of IB2, in B cells. Whole-cell extracts (WCE), S100 extracts (S100), or nuclear extracts (NE) of Namalwa cells (top) or HeLa cells (bottom) were analyzed in Western blots with antibodies against IB1 or IB2 as indicated. (C) Nuclear IB1 in B cells is bound to dimers containing p65. IP-shift analysis (as described in the legend to Fig. 3B) with nuclear extracts of Namalwa (lanes 1 to 4) or IM9 (lanes 5 and 6) B cells. An antibody specific for IB1 (C-20) precipitated an NF-B activity which was challenged with antibodies directed against p65 or p50 (lanes 2 and 3). An antibody directed against IB2 [(C)] did not precipitate NF-B (lane 4). C-20, but not (C), precipitated NF-B activity from IM9 cells (lanes 5 and 6). (D) Nuclear IB1 is constitutively phosphorylated. (Left) Namalwa cells, either untreated or stimulated with PMA for 5 h, were fractionated into S100 extracts and nuclear extracts (NE) and immunoblotted with anti-IB1 (lanes 1 to 4) or anti-IB2 antibodies (lanes 5 to 8), as indicated. (Right) Nuclear extracts of unstimulated (lanes 1 to 3) or PMA-stimulated (lanes 4 to 6) Namalwa cells were treated with shrimp alkaline phosphatase (SAP; lanes 2 and 5) or heat-inactivated SAP (hi SAP; lanes 3 and 6) or left untreated (lanes 1 and 4) and analyzed for IB1 migration in a Western blot.

Role of the amino-terminal and carboxy-terminal domains of IB for NF-B/Rel interaction, DNA binding inhibition, and basal phosphorylation. In both IB and NF-B-p105, an acidic sequence, which is part of the PEST sequence, is required for a high-affinity interaction with NF-B (17). IB has two acidic regions, one after the third ankyrin repeat and one as part of the PEST sequence (20, 37). To address the role of the carboxy-terminal PEST sequence in the interaction with NF-B, FLAG-tagged wild-type IB or IB mutants lacking either amino-terminal, carboxy-terminal, or both sequences (N, C, and NC) were cotransfected with p65 into HeLa cells (Fig. 6A). Immunoprecipitation with an anti-p65 antibody and blotting with an anti-FLAG antibody revealed that none of the sequences before the first and after the sixth ankyrin repeat is required for binding to NF-B (lanes 1 to 5). This is in contrast to IB, which requires carboxy-terminal sequences for high-affinity interaction (19). Despite equal affinities under immunoprecipitation conditions, marked differences were observed in the ability of the IB mutants to inhibit the DNA binding of NF-B (Fig. 6B). Whereas wild-type IB and IBN effectively inhibited DNA binding of p65 homodimers and heterodimers of transfected p65 and endogenous p50 (compare lane 3 with lanes 4 and 5), IBC inhibited only p65 dimers but not heteromeric NF-B (lane 6). Surprisingly, IBNC was even unable to inhibit either homodimeric or heterodimeric NF-B (lane 7). Since IBC and IBNC efficiently bound to NF-B (Fig. 6A), a folding problem in these mutants is unlikely. None of the IB mutants was able to inhibit the DNA binding of transfected p50 homodimers (data not shown). IB or IBN, but not IBC, was able to inhibit the DNA binding of transfected p50-c-Rel (data not shown). Thus, efficient DNA binding inhibition with less favored NF-B heteromers requires the presence of the carboxy-terminal sequences of IB.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   (A) Interaction of IB deletion mutants with p65 in transfected HeLa cells as shown by immunoprecipitation. p65 was transfected alone (lane 1) or together with wild-type IB2 or IB2 mutants lacking either the sequences before the first ankyrin repeat (N) or after the sixth repeat (C) or both (NC) (lanes 2 to 5). All constructs were equally well expressed (data not shown). After immunoprecipitation with an anti-p65 antibody, coprecipitated IB molecules were detected by immunoblotting. (B) DNA binding inhibition assay. Whole-cell extracts of mock-transfected (M) HeLa cells (lane 2), cells transfected with p65 alone (lane 3), or cells cotransfected with p65 and either wild-type IB2, IB2N, IB2C, or IB2NC (lanes 4 to 7) were subjected to EMSA. Lane 1 contains DNA probe assayed alone. The position of DNA complexes containing p65 homodimers and p50-p65 is indicated. (C) IB2 is constitutively phosphorylated in the carboxy-terminal domain. S100 extracts of HeLa cells that were either mock transfected (lanes 1 to 3), transfected with FLAG-tagged IB2 (lanes 4 to 6), IB2N (lanes 7 to 9), or IB2C (lanes 10 to 12) were left untreated (lanes 1, 4, 7, and 10) or incubated with shrimp alkaline phosphatase (SAP; lanes 2, 5, 8, and 11) or heat-inactivated enzyme (hi SAP; lanes 3, 6, 9, and 12) and blotted with anti-FLAG antibody.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

One possibility to account for specific physiological activities of NF-B/Rel transcription factors is differential regulation of IB proteins. In this study, we show that transcripts generated from the human IB gene are alternatively processed, yielding products which are distinct in their signal responsiveness. Two isoforms, IB1 and IB2, are found in human cells, whereas the murine cell lines tested express only IB1. IB2 differs from IB1 by a substitution that replaces the carboxy-terminal PEST sequences with intron-derived sequences. We have analyzed the functional consequence of this difference in protein structure for binding specificity and affinity to NF-B Rel proteins, for signal-responsive degradation, basal phosphorylation, nuclear localization, and inhibition of NF-B-dependent transcription.

All human cell lines tested expressed IB1 and IB2, the latter often as the predominant form, whereas murine cells expressed only IB1. We cannot fully exclude the possibility that IB2 is expressed in murine cells at a low level or is confined to specific cell types. We detected IB1 but not IB2 in murine liver, lung, lymph node, thymus, ovary, and brain by Western blotting (data not shown). It is possible that the IB2 carboxy-terminal sequence is posttranslationally modified in murine cells and thus not recognized by the antibody. Alternatively, splicing variants in murine cells may contain less highly conserved intron-derived sequences not recognized by the antibody directed against the human protein. Immunodepletion and precipitation studies indicate that murine cells may contain additional IB isoforms distinct from IB1 and IB2 (data not shown). As a further possibility, specific functions of IB splicing isoforms in human cells could be compensated for in murine cells by other modifications of IB proteins yet to be identified.

We have previously shown that IB mRNA is expressed at some level as two distinct signals (1.8 and 2.8 kb) in several human tissues analyzed, such as pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and heart tissues (20). Based on the lengths of the cloned cDNAs, IB2 should represent the longer of the two transcripts. The N-terminal anti-IB antibody recognized further signals in the transformed cell lines (Fig. 2C), suggesting the existence of still further isoforms. Several additional, independently isolated cDNA clones lacking an internal coding segment are being investigated further.

Our results suggest that IB2 binds to the same cellular NF-B/Rel hetero- and homodimers as IB1, which are predominantly p65 homodimers and heterodimers containing p65. In contrast, IB interacted predominantly with heterodimers containing p50, such as p50-p65. Heterodimers that were bound with lower affinity, however, such as c-Rel containing dimers, are possibly bound more efficiently by IB1 than by IB2. p65 homodimers were observed by EMSA after cellular stimulation and degradation of IB1 and/or IB2 and are probably liberated from complexes with IB (Fig. 4). Preferential association with p65 homodimers was also evident when the affinity of IB to NF-B was weakened by truncation of the carboxy terminus. In DNA binding inhibition assays (Fig. 6B), only p65 homodimers, not p65 heterodimers, were affected. Therefore, we assume that the carboxy-terminal sequences are needed to increase the binding affinity of IB to NF-B whereas the specific recognition of NF-B is achieved by the ankyrin repeat domain. As observed for IB (17), the binding affinity of IB may depend on negatively charged residues contained in the PEST sequence. Basal phosphorylation by CKII in the PEST sequence increases the negative charge and could therefore enhance the interaction strength of IB with less favored heterodimers containing c-Rel (11). Band shift assays are more stringent than immunoprecipitations under mild detergent conditions, possibly due to a competition between NF-B-DNA and NF-B-IB complex formation. This can provide the explanation why some interactions seen in one assay (precipitation) may not be detected in the other (band shift). Different stringencies in the experimental conditions may also explain conflicting results obtained by Chu et al. (11) and Suyang et al. (36). The former study reported that unphosphorylated IB can inhibit the DNA binding of p65 homodimers, whereas the latter reported that p65 forms a complex with IB that still binds to DNA. Recombinant, underphosphorylated IB cannot bind to c-Rel and inhibit its DNA binding (11). In this study, we showed that a carboxy-terminal deletion mutant of IB can bind to p65 homodimers and inhibit DNA binding but cannot inhibit the DNA binding of heterodimers. We propose that the affinity of IB to NF-B/Rel dimers decreases from p65 homodimers to p65 heterodimers or c-Rel heterodimers and that stable binding to the latter requires the negatively charged carboxy-terminal sequences of IB, which are most optimal after phosphorylation.

Previously, it has been reported that IB and IB elicit distinct responses to different inducers of NF-B and that IB is degraded only in response to LPS or IL-1 but not in response to TNF- or PMA (37). In IB-deficient mice, constitutive NF-B activity was observed only in cells of hematopoietic origin, not in fibroblasts (6). Furthermore, NF-B could still be stimulated by TNF- in fibroblasts, presumably involving degradation of IB (6). We show that TNF-, PMA/Ca²⁺ ionophore, and IL-1 can all equally induce the degradation of IB and IB1 in three different human cell lines. The extent of responsiveness varied among cell lines and stimuli, but the fact that time points of the onset of proteolysis are the same for IB and IB1 suggests that both are degraded by the same mechanism. It is therefore possible that both are phosphorylated by the same kinase(s) and degraded by a ubiquitin-dependent proteasome pathway (12, 25). In contrast, IB2 was barely degraded. This refractory response is consistent with the truncated PEST sequence in IB2, since this motif promotes efficient inducible degradation of IB. Thus, IB2 can be seen as a naturally occurring dominant negative IB isoform whose expression could generate a state of NF-B unresponsiveness to a variety of inducing agents.

An interesting and unexpected observation is the constitutive nuclear accumulation of IB1 in Namalwa and IM9 B cells which was not found in five different nonlymphoid cell lines tested. In these experiments, equal cellular ratios of cytoplasmic and nuclear fractions were used and the same fractions were analyzed for IB1 and IB2. Since IB2 was not found in the nucleus, leakage or cross-contamination of the fractions can be excluded. Nuclear IB was associated predominantly with p65, whereas the main constitutive NF-B activity in B cells consists of c-Rel-p50 (23, 26, 27). It is therefore possible that nuclear IB1 in B cells masks p65 which might have been activated constitutively by the same mechanism as c-Rel-p50. Nuclear IB has also been detected recently in mature murine WEHI 231 B cells (32). It is therefore likely that B lymphocytes in general contain nuclear IB. Consistent with the model proposed by Suyang et al. and Phillips and Ghosh (32, 36), the nuclear accumulation of IB could not only account for the prolonged NF-B activation after stimulation of non-B cells with agents such as LPS but could also directly contribute to the constitutive Rel activity in B cells.

The fact that IB2 is localized exclusively to the cytoplasm indicates that the specific carboxy-terminal sequences of IB1 are required for nuclear accumulation. Since both IB1 and IB2, are constitutively phosphorylated and contain the CKII phosphorylation sites at Ser 313 and Ser 315 (11), it is unlikely that hypophosphorylation of IB1 but not of IB2 accounts for their differential nuclear uptake. The latter mechanism has been proposed for nuclear translocation of IB1 after LPS stimulation of 70Z/3 pre-B cells (36). Therefore, mechanisms other than basal phosphorylation may account for nuclear uptake of IB1 in human cells. These may include the association and import with (modified) Rel factors or the presence of a conditional nuclear import signal in IB1.