INTRODUCTION

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The Rel/NF-B family of transcription factors plays a central role in the immune, inflammatory, and acute-phase responses and in the control of cell proliferation (reviewed in references 5-7, 52, 68 and 72). The vertebrate proteins p50/NF-B1, p52/NF-B2, RelA, c-Rel, and the viral oncoprotein v-Rel belong to this family (7, 72). These proteins share an N-terminal Rel homology domain that participates in their DNA binding, dimerization, and nuclear localization. The formation of Rel homodimers and heterodimers enables their binding to the major groove of decameric B DNA sites and the regulation of gene expression (reviewed in references 5-7, 52, 68, and 72).

Rel factors share common pathways of activation that involve their rapid release from inhibitory IB proteins in response to various stimuli (reviewed in references 7, 32, 52, and 72). IB factors associated with cytoplasmic Rel dimers mask the Rel nuclear localization signal (NLS), thereby impeding Rel translocation to the nucleus (10, 28, 77). In response to stimuli, IB undergoes phosphorylation and rapid degradation via the 26S proteasome (1, 15, 16, 20, 43, 50, 58, 59, 62, 69, 73). These processes allow the nuclear translocation of Rel proteins and the activation of gene expression. In turn, nuclear Rel factors trigger the resynthesis of IB, giving rise to an autoregulatory loop that terminates the activation process (17, 21, 46, 64, 66). The transient accumulation of newly synthesized IB in the nucleus of stimulated cells correlates with the repression of Rel/NF-B-dependent gene expression (3, 56). Accumulating evidence supports a model in which the entry of IB into the nucleus triggers the relocalization of Rel complexes from the nucleus to the cytoplasm to terminate the activation process (4, 11, 41). The recent identification of a nuclear export signal in IB is consistent with this model (4). In light of these findings, it appears that IB functions as a dual regulator of Rel proteins by controlling their activation in the cytoplasm and by terminating their activity in the nucleus.

The N-terminal domain of IB contains serine and lysine residues that undergo phosphorylation and ubiquitination, respectively, and that are critical for its inducible degradation (1, 15, 16, 20, 24, 43, 50, 58, 59, 62, 69, 73). Mutation or deletion of this domain stabilizes the inhibitor under inducing conditions (2, 15, 43, 65). A central domain of six ankyrin repeats is essential for the interaction of IB with Rel factors (34-37). The C-terminal 35 amino acids of IB contain a PEST region, rich in negatively charged residues. While this portion of the protein is dispensable for its inducible degradation and for its interaction with Rel, phosphorylation of this portion regulates the constitutive degradation of the protein (2, 8, 48, 51, 63, 65, 71). This portion of IB also has been implicated in the inhibition of RelA and c-Rel DNA binding (26, 61). Of the different Rel domains that contact IB, the Rel NLS region appears to be the main site for interaction with the inhibitor. Mutation of the NLS decreases the affinity of Rel for IB and enables Rel to escape the inhibitory function of IB (10, 28, 45, 77).

Among the different Rel/NF-B dimers, p50-RelA complexes are the most extensively studied. Much less is known, however, about the regulation and function of the transactivating subunit c-Rel. Studies with transgenic mice and knockout animals demonstrated an essential role for c-Rel in lymphocyte proliferation, in immune and inflammatory responses, and in T-cell development (12, 42). As c-rel is the cellular homolog of the viral oncogene v-rel, it is not surprising to find a correlation between the amplification and overexpression of the c-rel gene and oncogenesis (reviewed in references 31 and 49). The activity of c-Rel therefore must be tightly regulated.

In this study, we characterized the functional domains of IB involved in the cytoplasmic and nuclear regulation of c-Rel. We show that IB can function in vivo to regulate the transcriptional activity of c-Rel in the nuclear compartment. Our studies indicate that the central ankyrin domain of IB, together with a few negatively charged C-terminal acidic amino acids, is necessary and sufficient to regulate c-Rel in the nucleus. While the C-terminal domain of IB is dispensable for the cytoplasmic retention of c-Rel, we show that its N-terminal domain and ankyrin repeats are essential for this function. We also provide evidence that the N-terminal region of IB is required to mask the Rel NLS. Our results indicate that the determinants for the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin domains of IB, while those necessary for nuclear Rel protein regulation map to its central ankyrin domain and to a few acidic amino acids that immediately follow in the C-terminal IB PEST region.

	MATERIALS AND METHODS

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Plasmids and mutagenesis. CCR encodes the wild-type chicken c-Rel protein (18). The CCR.SVNLS mutant was generated by insertion of a double-stranded oligonucleotide (5'-CTAGAGATCACGCCACCAAAGAAGAAAAGGAAAGTGGAGGACCCT-3') encompassing the simian virus 40 (SV40) large T antigen NLS at the unique HincII site of the chicken c-rel gene. Mutant CCR.KO.SVNLS was derived by site-directed mutagenesis of CCR.SVNLS to inactivate the Rel NLS (KRQR to NWLT). A 1.7-kb EcoRI DNA fragment containing the chicken ib gene (23) was cloned into the EcoRI site of the pAlter-1 plasmid (Promega). IB deletion mutants were generated by introducing stop codons at defined positions by use of the Altered Sites mutagenesis system (Promega). Mutants 267, 282, 76-284, 285, 287, and 301, respectively, lack 52, 37, 34, 34, 32, and 18 amino acids from the C terminus of IB. Mutants 76-318 and 76-284 were generated by deletion of the first 75 amino acids from the N terminus of full-length IB (76-318) or of mutant 285 (76-284), followed by the introduction of an ATG codon at position 75 by site-directed mutagenesis. Mutations were confirmed by DNA sequence analysis with Sequenase (U.S. Biochemical).

Cell transfection and CAT assays. Cos-7 SV40-transformed African green monkey kidney cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were maintained at 37°C in an atmosphere of 5% CO₂. Transfections were carried out by a modified calcium phosphate procedure (19). Cells (5 × 10⁵) in 60-mm dishes were transfected with 5 µg of CMV rel plasmid DNA in the presence or absence of wild-type or mutant CMV ib DNA and 3 µg of pIL6CAT. The total amount of plasmid DNA transfected (13 µg) was kept constant by the addition of the CMV vector. The calcium phosphate precipitate was removed after 16 to 24 h of incubation, and the cells were washed and refed with medium containing 3% fetal bovine serum. Cell extracts were prepared 36 to 48 h after transfection, and the protein concentration was measured by the method of Bradford (14). CAT activities were determined within the linear range of the assay as described previously (29). Assays were performed with 10 µg of total cellular protein for 20 min. Normalized CAT activity from the average of three independent experiments is shown.

Nuclear and cytoplasmic extracts. Nuclear and cytoplasmic extracts were prepared as described previously (55). Briefly, cells were harvested and resuspended in buffer A (10 mM HEPES [pH 7.8], 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 0.1% Nonidet P-40). Extracts were incubated for 10 min on ice and fractionated by centrifugation at 10,000 × g. Supernatants containing the cytoplasmic fractions were recovered. The pellets were resuspended in buffer A and centrifuged again at 10,000 × g. The nucleus-containing pellets were lysed in 20 µl of buffer C (20 mM HEPES [pH 7.8], 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) and incubated for 20 min on ice. Nuclear extracts were separated from cell debris by centrifugation at 10,000 × g and diluted in 80 µl of buffer D (20 mM HEPES [pH 7.8], 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF).

Immunofluorescence. Cells were seeded onto glass coverslips and transfected as described above. Coverslips were harvested at 48 h posttransfection. The cells were fixed in 4% paraformaldehyde and permeabilized in phosphate-buffered saline-0.2% Triton X-100. Coverslips were incubated with rabbit polyclonal antibody Ab1801, specific for the C terminus of c-Rel (45), or with anti-p40/IB antibody (39) followed by fluorescein-conjugated goat anti-rabbit antibodies. Coverslips were mounted in the presence of 0.2% para-phenylenediamine (Sigma) and photographed at a magnification of ×600.

Coimmunoprecipitation assays. Cos-7 cells were harvested at 48 h posttransfection, lysed by sonication in 50 mM Tris HCl (pH 7.5)-150 mM sodium chloride-1% sodium deoxycholate-1% Triton X-100-10 µg of leupeptin per ml-10 mM sodium pyrophosphate-50 mM sodium fluoride-0.5 mM sodium orthovanadate (57), and quantitated for protein concentration by the method of Bradford (14). Equal amounts of whole-cell extracts (400 to 600 µg) were immunoprecipitated with anti-c-Rel antibody Ab1801 (45) or with antibody Ab1507, specific for the c-Rel NLS (26), and protein A-Sepharose (Pharmacia). Samples were washed extensively with ELB buffer (50 mM HEPES [pH 7.0], 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40) (26) supplemented with Complete protease inhibitor cocktail (Boehringer Mannheim Biochemicals) and resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis.

DNA-binding assays. DNA-binding assays of in vivo-synthesized c-Rel proteins were performed with nuclear (3 µg) or whole-cell (20 µg) extracts from transfected cells prepared as described for coimmunoprecipitation assays. Extracts were incubated with 3 × 10⁴ cpm of a ³²P-labeled IL6-B oligonucleotide probe in 12.5 mM HEPES (pH 7.9)-12% glycerol-5 mM MgCl₂-60 mM KCl-0.2 mM EDTA-1 mM DTT-1 µg of bovine serum albumin per µl-1 µg of poly(dI-dC) per µl (74) and analyzed on 5% native polyacrylamide gels. In supershift assays, extracts were preincubated with 0.5 to 2 µl of antisera specific for c-Rel (45) or IB (39) prior to DNA-binding reactions.

	RESULTS

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c-Rel mutants that escape cytoplasmic regulation by IB. To bypass cytoplasmic regulation by IB, c-Rel was constitutively targeted to the nucleus by inserting the NLS of the SV40 large T antigen 3' to the Rel homology domain of the chicken c-rel gene (38) (CCR.SVNLS; Fig. 1A). As a negative control, the Rel NLS was inactivated by mutagenesis of CCR.SVNLS (CCR.KO.SVNLS; Fig. 1A). The effect of IB on the subcellular localization of wild-type and mutant c-Rel proteins was examined by immunofluorescence. As anticipated, c-Rel localized to the nucleus of transiently transfected Cos-7 cells, and the coexpression of the chicken IB protein led to the sequestration of c-Rel in the cytoplasm (Fig. 1B, panels a and b) (26, 61). In contrast, CCR.SVNLS and CCR.KO.SVNLS constitutively localized to the nucleus irrespective of the presence or absence of IB (Fig. 1B, panels c to f). This result demonstrated the ability of these proteins to escape cytoplasmic regulation by IB.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   c-Rel mutants that bypass cytoplasmic regulation by IB. (A) Structures of the CCR.SVNLS and CCR.KO.SVNLS mutants of c-Rel. The Rel NLS and the SV40 NLS are depicted as white and black boxes, respectively. Mutation of the Rel NLS in CCR.KO.SVNLS is indicated by X. WT, wild type. (B) Subcellular localization of c-Rel and c-Rel-derived mutants. Cos-7 cells cotransfected with wild-type or mutant c-rel genes, together with CMV or CMV ib expression plasmids, were analyzed by indirect immunofluorescence with an anti-c-Rel antibody. (C) Transcriptional activity of wild-type and mutant c-Rel proteins. Cos-7 cells were transfected with CMV vector DNA or with expression plasmids encoding c-Rel or c-Rel-derived mutants, alone or together with an expression vector for IB. The pIL6CAT reporter plasmid, containing three B DNA sites upstream of the interleukin-6 promoter, was included in the transfection. The average CAT activity from three independent experiments was normalized to that of wild-type c-Rel.

Mapping of C-terminal IB sequences necessary for the cytoplasmic and nuclear regulation of c-Rel. The C-terminal PEST domain of IB was implicated in the inhibition of Rel DNA binding, and the phosphorylation of serine and threonine residues in this region was proposed to be required for this activity (26, 61). The C-terminal sequences of IB necessary for the nuclear regulation of c-Rel were mapped by deletion analysis (Fig. 2). The serine and threonine residues in the PEST domain were mutated to alanine to address the role of phosphorylation in the nuclear regulation of c-Rel (A5; Fig. 2). Mutants were assayed for association with c-Rel and CCR.SVNLS, for effects on their subcellular localization, and for inhibition of their DNA-binding and transcriptional activities.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Structures and properties of IB mutants. The structure of IB, comprised of an N-terminal domain (stippled box), ankyrin repeats (hatched boxes), and a C-terminal region (open box) containing a PEST domain (black box), is depicted (not to scale). C-terminal deletion mutants 267, 282, 285, 287, 301, and 76-284 were generated by introducing stop codons. Translation start codons were introduced at position 75 in mutants 76-318 and 76-284. Mutant A5 contains alanine substitutions for the serines at positions 287, 292, 293, and 295 and the threonine at position 300 of IB. Mutant 287-295 has an internal deletion of nine amino acids in the C-terminal PEST domain of IB. Mutants 285-Tail 4 Glu, 285-Tail 2 Asp, and 285-Tail 4 Ala, respectively, have Glu, Asp, and Ala tails added to the C terminus of mutant 285. nd, not determined.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Interaction of c-Rel and CCR.SVNLS with wild-type or mutant IB. Extracts from Cos-7 cells transfected with CMV vectors expressing c-Rel (lanes 2 to 10) or CCR.SVNLS (lanes 11 to 19), alone (lanes 2 and 11) or together with expression plasmids for IB (lanes 3 and 12) or IB mutants (lanes 4 to 10 and lanes 13 to 19), were immunoprecipitated with an anti-c-Rel antibody. Complexes were resolved on 12% polyacrylamide-SDS gels and analyzed by immunoblotting with antibodies specific for c-Rel (top panels) or IB (bottom panels). The positions of c-Rel, CCR.SVNLS, and wild-type or mutant IB proteins are indicated. Control immunoblots verified that equivalent amounts of wild-type and mutant IB proteins were expressed in the cells (data not shown).

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Subcellular localization of c-Rel and CCR.SVNLS in the presence of wild-type or mutant IB proteins. Cos-7 cells were transfected with expression plasmids encoding c-Rel or CCR.SVNLS, together with control CMV vector DNA or CMV expression plasmids for IB or IB mutants. Cells were analyzed by immunofluorescence with anti-c-Rel or anti-IB antibodies (). A minimum of 80 positively staining c-Rel-expressing cells were examined. Representative fields are shown.

View larger version (31K): [in this window] [in a new window]   View larger version (60K): [in this window] [in a new window]   View larger version (75K): [in this window] [in a new window]   FIG. 5.   Effects of IB mutants on the transcriptional and DNA-binding activities of c-Rel. (A) Differential effects on c-Rel- and CCR.SVNLS-mediated transcription. Cos-7 cells were transfected with CMV vector DNA or CMV expression plasmids encoding c-Rel or CCR.SVNLS, alone (0) or together with expression plasmids for IB or IB mutants. The pIL6CAT reporter plasmid was included in the transfections. The average CAT activity from three independent experiments was normalized to that of wild-type c-Rel. (B) Inhibition of c-Rel and CCR.SVNLS DNA binding. Whole-cell extracts (20 µg) from Cos-7 cells transfected with CMV expression vectors for c-Rel (lanes 3 to 12) or CCR.SVNLS (lanes 14 to 23), alone (lanes 3 and 14) or together with CMV ib (lanes 4 and 15) or ib mutants (lanes 5 to 11 and 16 to 22), were analyzed in gel retardation assays. Extracts were incubated with a 32P-labeled IL6-B DNA probe and resolved in 5% native acrylamide gels. DNA-bound complexes containing c-Rel or CCR.SVNLS were supershifted with an anti-c-Rel antibody () (lanes 12 and 23). The bracket on the left indicates the position of DNA-bound c-Rel and CCR.SVNLS complexes. The bracket on the right indicates the position of the supershifted Rel complexes. (C) Formation of complexes containing IB C-terminal deletion mutants, c-Rel, and DNA. Whole-cell extracts (20 µg) from Cos-7 cells transfected with vector DNA (lane 1) or CMV c-rel alone (lanes 2, 6, and 9) or together with vectors expressing IB (lane 3) or IB C-terminal deletion mutant 285 (lanes 4, 7, and 10) or 287 (lanes 5, 8, and 11) were analyzed for binding to an IL6-B DNA probe in gel retardation assays. DNA-bound complexes were supershifted with anti-c-Rel (lanes 6 to 8) or anti-IB (lanes 9 to 11) antisera (). Electrophoresis was carried out three times longer than usual to allow the separation of the different complexes. The brackets indicate the positions of DNA-bound Rel proteins (left) and of supershifted complexes (right).

Negatively charged residues at the beginning of the IB PEST domain are essential for regulation of the activity of c-Rel in the nucleus. The acidic nature of the IB PEST domain and the important role of amino acids immediately C terminal to the ankyrin repeats in the inhibition of CCR.SVNLS-mediated transcription led us to investigate the effect of acidic amino acids C terminal to residue 285 on the nuclear regulation of c-Rel. IB mutants with tails of aspartate or glutamate residues added to the C terminus of mutant 285 were analyzed for inhibition of c-Rel- or CCR.SVNLS-mediated transcription in transient CAT assays (285-Tail 2 Asp and 285-Tail 4 Glu; Fig. 2 and 6A). A neutral tail of alanines was added as a control (285-Tail 4 Ala; Fig. 2). In agreement with the findings described above, mutant 285 inhibited c-Rel-mediated transcription via cytoplasmic retention but had no significant inhibitory activity toward the constitutively nuclear CCR.SVNLS protein (Fig. 6A). In sharp contrast, the addition of as little as two aspartate or four glutamate residues to the C terminus of IB mutant 285 fully restored its ability to inhibit the transcriptional activity of CCR.SVNLS. In contrast, the neutral tail of alanine residues had no inhibitory effect, despite the efficient association of the 285-Tail 4 Ala protein with c-Rel and CCR.SVNLS in coimmunoprecipitation assays (Fig. 6A and data not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 6.   Effects of negatively charged residues in the C terminus of IB on the transcriptional and DNA-binding activities of c-Rel. (A) Effects on Rel-mediated transcription. Cos-7 cells were transfected with vector DNA or CMV plasmids encoding c-Rel or CCR.SVNLS, alone (0) or in combination with expression vectors for IB or IB mutants. Reporter plasmid pIL6CAT was included in the transfections. The normalized CAT activity from the average of three independent experiments is shown. (B) Effects on Rel DNA binding. Shown is a gel retardation analysis of whole-cell extracts (20 µg) from Cos-7 cells transfected with CMV vectors expressing c-Rel (lanes 2 to 7 and 14) or CCR.SVNLS (lanes 8 to 13), alone (lanes 2, 8, and 14) or together with vectors encoding wild-type (lanes 3 and 9) or mutant (lanes 4 to 7 and 10 to 13) IB proteins. Proteins were assayed for binding to a 32P-labeled IL6-B DNA probe. The bracket indicates the position of ectopically expressed Rel-DNA adducts. The arrowhead indicates c-Rel-DNA complexes supershifted with an anti-c-Rel antiserum () (lane 14). The lower band present in all lanes corresponds to a DNA-bound complex of endogenous proteins.

The N terminus of IB is required to retain c-Rel in the cytoplasm. We complemented our study of IB by characterizing the activity of mutants lacking its N terminus or both its N- and C-terminal domains (mutants 76-318 and 76-284; Fig. 2). Mutants were analyzed for their effect on the subcellular localization of c-Rel by immunofluorescence. As expected, the coexpression of c-Rel with wild-type IB led to its confinement to the cytoplasm (Fig. 7A, compare panels c and a). However, the coexpression of c-Rel with N-terminal IB mutant 76-318 or 76-284 resulted in weak cytoplasmic and strong nuclear staining for c-Rel (Fig. 7A, compare panels e and g with panel c). This result suggested that deletion of the N terminus of IB impaired its capacity to retain c-Rel in the cytoplasm.

View larger version (71K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   View larger version (90K): [in this window] [in a new window]   FIG. 7.   Effect of IB N-terminal deletion mutants on the subcellular localization and transcriptional and DNA-binding activities of c-Rel. (A) Effects on subcellular localization. The subcellular localization of c-Rel and IB proteins was assessed by immunofluorescence analysis of Cos-7 cells expressing c-Rel alone (0) or together with IB or its mutants 76-318 and 76-284, with anti-c-Rel or anti-IB antibodies (). (B) Effects on Rel-mediated transcription. Cos-7 cells were transfected with pIL6CAT together with vector DNA or expression plasmids for c-Rel or CCR.SVNLS, alone (0) or in combination with plasmids encoding wild-type IB or mutant 76-318 or 76-284. The average CAT activity from three independent experiments was normalized to that of wild-type c-Rel. (C) Effects on Rel DNA binding. Whole-cell extracts (20 µg) from Cos-7 cells transfected with vector DNA (lane 1) or with CMV c-rel alone (lanes 2 and 6) or together with expression plasmids for IB or mutant 76-318 or 76-284 (lanes 3 to 5) were analyzed for binding to a 32P-labeled IL6-B DNA probe in gel retardation assays. As a control, the DNA-bound c-Rel complex was supershifted with an anti-c-Rel antibody (). The bracket indicates the position of c-Rel-DNA complexes. The arrowhead indicates supershifted c-Rel complexes. The lower band present in all lanes corresponds to a DNA-bound complex of endogenous proteins.

IB mutants with N-terminal deletions fail to mask the c-Rel NLS. The inefficient retention of c-Rel in the cytoplasm by IB mutants with N-terminal deletions suggested that the c-Rel NLS may still be exposed when c-Rel is associated with these mutants. To verify this possibility, complexes of ³⁵S-labeled c-Rel with wild-type IB or IB mutants with N-terminal deletions were analyzed by immunoprecipitation with an antibody directed against the c-Rel NLS (Ab1507; Fig. 8A). An antibody specific for the C terminus of c-Rel was used as a control (Ab1801). The antibody directed against the C terminus of c-Rel efficiently coprecipitated IB and its N-terminal deletion mutants (Fig. 8A, lanes 2 to 4). As anticipated, the anti-c-Rel NLS antibody Ab1507 failed to coprecipitate wild-type IB along with c-Rel due to masking of the Rel NLS (Fig. 8A, lane 6). In contrast, IB mutants 76-318 and 76-284 complexed with c-Rel were efficiently coprecipitated by this antibody (Fig. 8A, lanes 7 and 8). This result showed that the Rel NLS remained exposed when associated with IB N-terminal deletion mutants, whereas it was inaccessible to antibody Ab1507 when complexed with wild-type IB. Consistent with this observation, the amount of c-Rel brought down by this antibody in the presence of wild-type IB was significantly lower than that brought down in the presence of IB mutant 76-318 or 76-284 (Fig. 8A, compare lane 6 with lanes 7 and 8).

View larger version (50K): [in this window] [in a new window]   FIG. 8.   The c-Rel NLS is unmasked when associated with IB N-terminal deletion mutants. (A) Coimmunoprecipitation assay of in vitro-translated proteins. 35S-labeled c-Rel produced by in vitro translation was incubated in the presence of a threefold molar excess of in vitro-translated wild-type IB (lanes 2 and 6) or N-terminal deletion mutants of IB (lanes 3, 4, 7, and 8). c-Rel was incubated with an equal volume of a mock translation as a control (lanes 1 and 5). Reaction mixtures were immunoprecipitated with antibody Ab1801 against the C terminus of chicken c-Rel (lanes 1 to 4) or anti-c-Rel NLS antibody Ab1507 (lanes 5 to 8). Immunoprecipitated complexes were resolved in a 17% polyacrylamide-SDS gel. The open arrowhead indicates the position of c-Rel. Closed arrowheads indicate the positions of wild-type and mutant IB proteins. (B) Coimmunoprecipitation assay of in vivo-labeled proteins. Extracts from [35S]methionine- and [35S]cysteine-labeled Cos-7 cells transfected with CMV vector DNA (lanes 1 and 6) or CMV vectors expressing c-Rel alone (lanes 2 and 7) or together with wild-type or mutant IB (lanes 3 to 5 and 8 to 10) were immunoprecipitated with antibody Ab1801 against the C terminus of c-Rel (lanes 1 to 5) or anti-c-Rel NLS antibody Ab1507 (lanes 6 to 10). Reaction mixtures were resolved in a 17% polyacrylamide-SDS gel. Arrowheads indicate the positions of wild-type and mutant IB proteins. The sizes of protein molecular mass markers are indicated on the left in kilodaltons.

	DISCUSSION

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The activity of the Rel/NF-B factors is tightly regulated. While some physiological conditions require a transient Rel/NF-B response, others need prolonged activation. Integral parts of the transient activation process are the nuclear translocation of newly synthesized IB and the termination of Rel/NF-B-mediated transcription (3, 11, 41). Here we show that IB can function in vivo to inhibit the activity of c-Rel in the nucleus. As previously described for RelA (10), the introduction of an extra NLS into c-Rel helped to circumvent its cytoplasmic regulation by IB. By comparing the effects of IB on the nuclear regulation of CCR.SVNLS and on the cytoplasmic regulation of c-Rel, we demonstrated that distinct functional domains in IB are required for the cytoplasmic and nuclear regulation of c-Rel.

Role of the IB C terminus in the cytoplasmic and nuclear regulation of c-Rel. Our studies showed that the C terminus of IB, including the highly acidic PEST region, is dispensable for the cytoplasmic retention of c-Rel. Whereas deletion of this region did not significantly affect the capacity of IB to regulate c-Rel in the cytoplasm (285), our deletion studies demonstrated that an intact ankyrin domain is necessary for interaction with c-Rel and its cytoplasmic sequestration. An IB C-terminal mutant with a deletion of only three amino acids in the ankyrin domain was compromised in its interaction with c-Rel (282). Although this mutant showed some capacity to retain c-Rel in the cytoplasm, the high transcriptional activity that we observed when c-Rel was coexpressed with 282 indicated that some c-Rel dimers escaped cytoplasmic retention. This result emphasized the instability of their interaction. Thus, our failure to detect a physical association between mutant 282 and c-Rel in coimmunoprecipitation assays presumably resulted from their reduced affinity for one another and their dissociation under in vitro experimental conditions, as observed by others (26, 60, 61). Further deletion into the ankyrin domain abolished the interaction of IB with c-Rel both in vitro and in vivo (267). These findings also agree with those of others (26, 61).

Mechanism for the nuclear regulation of c-Rel. Nuclear IB factors have been shown to promote the relocalization of RelA from the nucleus to the cytoplasm, and a nuclear export sequence was implicated in this process (amino acids 265 to 277 of human IB; 4). However, our observation that IB inhibited the in vivo activity of the constitutively nuclear CCR.SVNLS protein indicates that IB must also be able to actively antagonize nuclear c-Rel function. This antagonism may occur by blocking the transcriptional activity of c-Rel and/or by promoting the dissociation of c-Rel from DNA. In this scenario, the relocalization of Rel dimers to the cytoplasm would likely constitute a second step in the inhibitory process. Reports that IB can dissociate Rel-DNA complexes in vitro and block Rel-mediated transcriptional activation agree with our data (44, 70, 76).

Role of phosphorylation of the IB PEST domain in the inhibition of c-Rel activity. The serine and threonine residues found in the IB PEST domain are highly phosphorylated by casein kinase II (8, 48, 51, 61, 63). Their mutation in chicken IB was reported to dramatically decrease its affinity for c-Rel and to abolish its inhibitory activity toward c-Rel DNA binding (61). These results are in sharp contrast to ours. In our experiments, IB mutant A5, with all of the serine and threonine residues in the PEST domain mutated to alanines, exhibited wild-type inhibition of c-Rel DNA binding. This mutant was as effective as wild-type IB in interactions with c-Rel, in the cytoplasmic retention of c-Rel, and in the inhibition of c-Rel and CCR.SVNLS DNA binding and transcriptional activation. While the discrepancy between our results and those of Sachdev et al. (61) may derive from differences in experimental conditions, our assays indicate that phosphorylation of the PEST domain is not required for the inhibitory activity of IB. Similar findings were recently obtained with human IB (9, 22, 70).

The N terminus of IB is important for masking of the c-Rel NLS. An intriguing finding from this study is that the N-terminal domain of IB led to masking of the Rel NLS. In contrast to the situation observed with wild-type IB, c-Rel associated with IB N-terminal deletion mutants was recognized by an NLS-specific antibody. This finding is consistent with the inability of these mutants to retain c-Rel in the cytoplasm. The Rel NLS was previously postulated to be essential for interactions with IB, since anti-NLS antibodies abolished the association of Rel with IB (77). Likewise, deletion or point mutations in the NLS of various Rel family members dramatically decreased their affinity for IB and impaired the inhibition of their DNA binding (10, 28). However, recent evidence suggests that the NLS itself is not required for the interaction of Rel with IB. (i) A c-Rel mutant with a deletion of its NLS efficiently interacted with IB in a yeast two-hybrid system (60). (ii) Similarly, the NLS of the Drosophila Rel homolog Dorsal is also dispensable for the interaction of Dorsal with the IB inhibitor Cactus (33). (iii) Here, we showed that the transcriptional activity of a nuclear c-Rel protein with a mutated NLS was slightly inhibited by IB but was not abolished (CCR.KO.SVNLS). These results further suggest that NLS mutations only partially interfere with c-Rel protein association with the IB inhibitor.