Identification of a Novel Inhibitor of Nuclear Factor-kappa B, RelA-associated Inhibitor*

Jian-Ping YangDagger , Mayumi Hori, Takaomi Sanda, and Takashi Okamoto§

From the Department of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-kappa B subunit p65 (RelA) and inhibits its transcriptional activity. RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait. We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay. RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain. RAI mRNA was preferentially expressed in human heart, placenta, and prostate. Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay. RAI inhibited the action of NF-kappa B p65 but not that of p53 in transient luciferase gene expression assays. Similarly, RAI inhibited the endogenous NF-kappa B activity induced by tumor necrosis factor-alpha . RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells. RAI protein appeared to be located in the nucleus and colocalized with NF-kappa B p65 that was activated by TNF-alpha . These observations indicate that RAI is another inhibitor of NF-kappa B in addition to Ikappa B proteins and may confer an alternative mechanism of regulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa  B (NF-kappa B)1 is a sequence-specific DNA-binding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus, and a variety of cellular genes, particularly those involved in immune and inflammatory responses (1-4). The members of the NF-kappa B family in mammalian cells include the proto-oncogene c-Rel, p50/p105 (NFkappa B1), p65 (RelA), p52/p100 (NFkappa B2), and RelB. All of these proteins share a conserved 300-amino acid region known as the Rel homology domain which is responsible for DNA binding, dimerization, and nuclear translocation of NF-kappa B (5, 6). In most cells, Rel family members form hetero- and homodimers with distinct specificities in various combinations. A common feature of the regulation of the NF-kappa B family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as Ikappa Bs (2, 7). Treatment of cells with a variety of inducers such as phorbol esters, interleukin 1, and tumor necrosis factor-alpha (TNF-alpha ) results in dissociation of the cytoplasmic complexes and translocation of NF-kappa B to the nucleus (8, 9). The dissociation of NF-kappa B·Ikappa B complexes is known to be triggered by the phosphorylation and subsequent degradation of Ikappa B proteins (10-12). This exposes the nuclear localization sequence in the remaining NF-kappa B heterodimer, leading to nuclear translocation and subsequent binding of NF-kappa B to DNA regulatory elements of the target genes. The p65 subunit is a major component of NF-kappa B complexes and is responsible for trans-activation (13).

Here we report the identification, cloning, and characterization of a novel RelA-binding protein, called RAI for "RelA-associated inhibitor." The RAI cDNA encodes a protein with high structural homology to the C-terminal 200 amino acid segments of 53BP2, containing four ankyrin repeats and an SH3 domain that are known to be involved in specific protein-protein interactions. We confirmed the interaction between p65 and RAI in vitro using the bacterially expressed fusion proteins and in vivo using immunoprecipitation/Western blot assay. We demonstrate that RAI inhibited NF-kappa B-dependent transcription through interfering with its DNA binding activity of NF-kappa B.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Plasmids-- Plasmids were constructed by standard methods (14). The human p65 fragment (amino acids 176-405) was cloned into pAS2-1 (CLONTECH, Palo Alto, CA) by PCR amplification of p65 cDNA using 5'- and 3'-oligonucleotides containing BamHI restriction sites (forward: 5'-GGCGGATCCCTCCGCCTGCCGCCTGTC-3', and reverse: 5'-GCTGGATCCGGGGCAGGGGCTGGAGCC-3') and was used for the yeast two-hybrid screening. The resulting plasmid was named pAS2-1-p65 (176-405).

A clone encoding the largest RAI insert obtained from yeast two-hybrid screening was cloned into EcoRI-XhoI-digested vector pGEX-5X-2 (Pharmacia). The full-length RAI was amplified using oligonucleotides 5'-ACGCGAATTCAATGTGGATGAAGGACCCT-3' and 5'-GCCGGATCCTCTAGACTTTACTCCTTTG-3' containing EcoRI and BamHI restriction sites, respectively, and cloned into plasmid pEGFP-C1 (CLONTECH), yielding plasmid pEGFP-RAI in-frame with GFP. pEGFP-RAI (1-146) was generated from pEGFP-RAI by cutting with BssHII and BamHI, blunt ended with Klenow enzyme and ligated into pEGFP-C1. The pEGFP-RAI (132-351) was generated by amplifying the corresponding RAI fragment by PCR using the oligonucleotides 5'-GCTTCGAATTCTGTGCTGCGGAAGGCG-3' and 5'-GCCGGATCCTCTAGACTTTACTCCTTTG-3' containing EcoRI and BamHI sites, and inserted into pEGFP-C1 vector. pFLAG-RAI was created for expression in the cultured cell by inserting the full-length RAI fragment from pEGFP-RAI into pFLAG-CMV-2 vector. pFLAG-RAI (1-134) was generated by amplifying the corresponding RAI fragment by PCR using the oligonucleotide primers 5'-ACGCGAATTCAATGTGGATGAAGGACCCT-3' and 5'-GTCCGGATCCTACAGCACAGAGCGCATCTC-3' containing EcoRI and BamHI sites, and inserted into pFLAG-CMV-2 vector. pFLAG-RAI (132-351) was generated by inserting the EcoRI-BamHI fragment of RAI from pEGFP-RAI (132-351) to pFLAG-CMV-2 vector.

The luciferase reporter constructions of 4kappa Bw-Luc and 4kappa Bm-Luc have been described previously (15). pCMV-p65 plasmid was derived from human RelA cDNA. pEBVHis-Ikappa B-alpha plasmid was constructed by inserting Ikappa B-alpha cDNA in-frame into pEBVHisA vector (Invitrogen). PG13-Luc, containing a generic p53 response element, and pCMV-p53wt, expressing wild type human p53, were generous gifts from Dr. B. Vogelstein.

All PCR amplification reactions used ExpandTM high fidelity system (Roche Molecular Biochemicals, Mannheim, Germany). All the constructs were confirmed by dideoxynucleotide sequencing using ABI PRISMTM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer, Foster City, CA) on an Applied Biosystems 313 automated DNA sequencer.

Yeast Two-hybrid Screen-- pAS2-1-p65 (176-405) was used as bait to screen human placenta and brain cDNA libraries following the Matchmaker Two-Hybrid System protocol (CLONTECH). Positive yeast clones were selected by prototrophy for histidine and by expression of beta -galactosidase. Library-derived plasmids were rescued from positive clones and transformed into Escherichia coli HB101. Subsequent two-hybrid assays were carried out by introducing the plasmids into yeast strain Y187 carrying a Gal1-regulated reporter gene and detected the expression of beta -galactosidase to confirm the specific binding. Plasmids containing cDNA clones that specifically interact with p65 (176-405) were identified by restriction mapping, PCR using gene specific primers, and DNA sequencing of both strands. Sequence Network BLAST searches were conducted by using the National Center for Biotechnology Information (NCBI) on-line service.

Cell Culture and Transfection-- HeLa, 293, Saos-2, and COS-1 cells were grown at 37 °C in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, 1 mM glutamate, 100 units of penicillin, and 100 µg/ml streptomycin. Jurkat cells were maintained in RPMI 1640 with 10% fetal bovine serum plus antibiotics. Cells were transfected using SuperFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's recommendations.

Recombinant Proteins-- pGEX-5X-2 plasmid encoding glutathione S-transferase (GST) fusion proteins were transformed in E. coli strain BL21 (DE3) pLysS following induction with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside at 28 °C overnight. Recombinant GST fusion proteins were purified by incubating the bacterial extracts in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 0.1 mM EGTA, 100 mM NaCl, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosylphenylalanyl chloromethyl ketone, 0.25% (v/v) Nonidet P-40) with glutathione-Sepharose beads (Pharmacia Biotech). The beads were then pelleted, washed five times with ice-cold buffer A, and suspended in 1 ml of buffer A. The purity and quantity of bound GST fusion proteins were examined by 5-20% SDS-PAGE and stained using Coomassie Brilliant Blue.

In Vitro Binding Assays-- [35S]Methionine-labeled p65 was synthesized by using the TNT T7/SP6 wheat germ extract-coupled system (Promega, Madison, WI) according to the manufacturer's protocols. For in vitro protein-protein interaction studies, an equal amount of the in vitro-translated [35S]methionine-labeled p65 was incubated with 5 µg of purified GST-RAI fusion proteins or GST alone (as a negative control) that were bound to glutathione-Sepharose beads in 250 µl of buffer A at 25 °C for 1 h. The beads were then washed by resuspension and centrifugation five times with 1 ml of ice-cold binding buffer A containing 0.1% Nonidet P-40. Bound proteins were eluted with an equal volume of SDS loading buffer, boiled for 3 min, resolved by 5-20% SDS-PAGE, and visualized by autoradiography.

Isolation of Full-length RAI cDNA-- A fragment encoding the longest RAI from the yeast two-hybrid screen was used as a hybridization probe to screen a human placenta 5'-STRETCH PLUS cDNA library (CLONTECH). The probe was labeled by 32P using the Prime-It Random Primer Labeling Kit (Stratagene). Hybridization was performed according to the manufacturer's recommendations. The filters were washed to a final stringency of 0.1 × SSC-0.5% SDS at 65 °C and exposed to x-ray film (X-Omat AR; Eastman Kodak, Rochester, NY) overnight at -80 °C with intensifying screens. The films were developed and plaques hybridizing on filters were identified and isolated. Phages were eluted from agarose plugs in SM buffer and stored at 4 °C (primary plaque pools). Secondary and tertiary plaque purifications were performed in similar fashion to that for the primary pools on dilutions from the primary pools until single plaques could be isolated.

Western Blotting-- Full-length RAI was cloned into a CMV promoter expression vector designed to place a FLAG epitope tag at the 5' end of the open reading frame. 293 cells were transfected using SuperFect transfection reagent as described above. Proteins were isolated by extraction of whole cells in a SDS-containing buffer. Western blotting was performed by a standard technique, using anti-FLAG antibody (Santa Cruz) at a dilution of 1/1,000. Secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG antibody, was used at a dilution of 1/2,500, and protein bands were visualized by enhanced chemiluminescence (Amersham).

Co-immunoprecipitation-- After transfection, 293 cells were cultured for 24 h and then harvested. After washing with PBS, cells were lysed in 350 µl of ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.2% Nonidet P-40, 10 mM sodium fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 µg/ml pepstain A) for 30 min. The lysate was cleared by centrifugation. The supernatants were incubated with anti-FLAG M2 murine monoclonal antibody (Kodak) or control mouse monoclonal antibody (Dako, Glosrup, Denmark) or rabbit polyclonal Ikappa B-alpha antibody (Santa Cruz) overnight at 4 °C and then with protein A-Sepharose (Pharmacia) for 4 h. The beads were washed six times with 1 ml of lysis buffer. Bound proteins were eluted with an equal volume of SDS loading buffer and resolved on 10% SDS-PAGE. Western blot was conducted as described above using anti-NF-kappa B p65 (C-20) antibody (Santa Cruz).

Northern Blot Hybridization-- Northern blot analysis of the human multiple tissue blots (CLONTECH) were performed according to the manufacturer's recommendations. A full-length of RAI cDNA was used as a probe. The blots were also probed with an beta -actin fragment.

Microscopic Examination-- HeLa cells were cultured in 4-well chamber slides and transfected with plasmids expressing various GFP fusion proteins using SuperFect transfection reagent (Qiagen). After 24 h, cells were fixed for 15 min in 4% paraformaldehyde and stained with the DNA-binding dye Hoechst-33342 at room temperature for 15 min followed by washing in PBS. The intracellular locations were examined by fluorescence microscopy. In order to examine colocalization of p65 and RAI, pEGFP-RAI (0.1 µg) was transfected into HeLa cells cultured in 4-well chamber slides. Twenty-four hours after transfection, cells were untreated or treated with 5 ng/ml TNF-alpha for 30 min, then immunostained as described previously (16). Briefly, cells were fixed with 4% (w/v) paraformaldehyde/PBS for 10 min at room temperature and then permeabilized by 0.5% Triton X-100/PBS for 20 min at room temperature. They were then incubated with rabbit polyclonal antibody against p65 (Santa Cruz) for 1 h at 37 °C. After washing with PBS, the cells were incubated with tetramethylrhodamine B isothiocyanate-conjugated goat anti-rabbit IgG antibody (Cappel Organon Teknika, Durham, NC) for 30 min at 37 °C. These cells were then stained with Hoechst-33342 at room temperature for 15 min to view the nuclear morphology.

Transient Luciferase Assay-- HeLa, Saos-2, and 293 cells were cultured in 12-well plates and transfections were performed with SuperFect transfection reagent (Qiagen). For each transfection, 50 ng of kappa B-dependent or mutant reporter plasmid (4kappa Bw-Luc or 4kappa Bm-Luc) or 250 ng of p53-dependent luciferase reporter plasmid (PG13-Luc) and 30 ng of internal control plasmid pRL-TK were used. The empty vector pEBVHisB or pFLAG-CMV-2 was used to adjust the total amount of DNA transfected to 1.5 µg. Twenty-four hours post-transfection, cells were harvested and measured the luciferase activity and Renilla luciferase activity as described (15). Jurkat cells were transfected with the indicated plasmids. Twenty-four hours post-transfection, the cells were induced by 5 ng/ml TNF-alpha for 24 h and harvested for luciferase assay. The luciferase activity was normalized by the Renilla luciferase activity used as an internal control for transfection efficiency.

EMSA-- 293 cells were transfected for 24 h and nuclear extracts were prepared as described previously (17). The nuclear pellet was lysed in 35 µl of extraction buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 350 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol). The protein content was measured by the DC protein Assay (Bio-Rad). The electrophoretic mobility shift assay (EMSA) was performed as described previously (18, 19) using the kappa B sequence taken from the human immunodeficiency virus long terminal repeat. Oct-1 consensus oligonucleotide (5'-TGTCGAATGCAAATCACTAGAA-3') was used for the effect of RAI on the Oct-1 DNA binding. Oligonucleotides were labeled using DNA polymerase Klenow fragment (Takara Biomedicals, Japan) and [alpha -32P]dATP (3000 Ci/mmol, ICN Pharmaceuticals Inc.). DNA binding reactions were performed at 30 °C for 15 min in 10-µl reaction volume containing 5 µg of nuclear extract proteins. Analysis of binding complexes was performed by electrophoresis in 5% native polyacrylamide gels with 0.5 × Tris-borate-EDTA buffer, followed by autoradiography. For DNA competition experiments, unlabeled competitor oligonucleotides were added into the reaction mixture at 50-fold molar excess over the probe.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of RAI cDNA Clones as a p65-binding Protein-- We applied the yeast two-hybrid system to identify proteins that interact with NF-kappa B p65. The cDNA fragment encoding amino acids 176-405 of p65, containing the dimerization domain, nuclear localization signal, and the proline-rich motif, was cloned into pAS2-1 containing a DNA-binding domain of yeast GAL4 in-frame. The resulting plasmid, pAS2-1-p65 (176-405), was used as bait to screen human cDNA libraries (placenta and brain) that had been constructed in GAL4 transcriptional activation domain vector pACT2. From approximately 1.6 × 106 Y190 yeast transformants, 78 colonies grew on selective medium and turned blue when tested in a filter lift beta -galactosidase assay. After re-screening using yeast strain Y187, 63 of these plasmid clones were found to contain cDNAs, which specifically interacted with the p65 bait. These clones were then characterized by DNA sequence analysis of both strands.

Primary nucleotide sequencing revealed four independent clones of the same unidentified gene encoding a protein not previously described in the GenBank data base in addition to the genes already identified as p65-binding proteins including Ikappa B-alpha /MAD-3 (47 clones), Ikappa B-beta /Trip9 (4 clones), p50/p105 (3 clones), p65/RelA (2 clones), and c-Rel (1 clone). All of these four clones were isolated from human placenta cDNA library not from human brain cDNA library, sharing the same 3' end, but contained different insert sizes. We called it RAI based on its biological functions as described below.

To confirm the direct interaction between p65 and RAI, we performed in vitro binding assay between recombinant RAI and p65 proteins. The cDNA insert from plasmid pACT2-RAI was cloned in-frame into the GST expression plasmid for production of the GST-RAI fusion protein. The GST fusion proteins were expressed in E. coli and purified by glutathione-Sepharose beads (Fig. 1, lanes 1 and 2). [35S]Methionine-labeled p65 was synthesized by in vitro transcription and translation protocol using wheat germ extract (Fig. 1, lane 3). The purified GST fusion proteins bound to glutathione-Sepharose beads were incubated with the in vitro synthesized p65. The beads were washed extensively and the bound materials were analyzed by SDS-PAGE followed by autoradiography. As shown in Fig. 1, p65 interacted strongly with RAI (lane 5). Binding between p65 and RAI was considered specific because no interaction of p65 was observed with the control GST protein (lane 4).


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Fig. 1.   Binding of RAI to p65 in vitro. GST-RAI fusion proteins were produced in E. coli. and purified by incubation with glutathione-Sepharose beads. The beads were washed and the bound proteins were resolved on SDS-PAGE and stained with Coomassie Brilliant Blue (lanes 1 and 2). [35S]Methionine-labeled p65 translated in vitro was incubated with glutathione-Sepharose beads loaded with GST or GST-RAI. After washing the beads, the eluted proteins were analyzed by SDS-PAGE and autoradiography. 1/10 of the labeled p65 translated in vitro was also run (lane 3). The p65 band is indicated by an arrow.

Isolation of Full-length Clones of RAI-- The four independent clones isolated from the yeast two-hybrid screen were partial-length cDNA derived from the same gene. Plaque hybridization of a human placenta cDNA library with the partial cDNA fragment of RAI isolated from the two-hybrid screen was performed to obtain a full-length clone. About 106 plaques were screened and nine cDNA clones were isolated with approximately 1 to 6 kilobase pairs in length. The two longest cDNAs were sequenced on both strands and were found to contain the same complete open reading frame for RAI. However, these clones differed at their 5'-untranslated regions and each contained all of the sequences present in pACT2-RAI. The likely translation initiation codon of RAI was preceded by an in-frame stop codon located 27 base pairs upstream in both clones. Although the nucleotide sequence 5' of the first ATG (CTGGCGATG) does not resemble the consensus initiation sequence, as more than 90% of the translation in vertebrates starts from the first methionine (20), we designate this ATG as the putative translation start site. DNA sequence analysis revealed an open reading frame predicted to encode a putative protein of 351 amino acid (aa) residues (Fig. 2A) with a predicted molecular mass of 40 kDa. Interestingly, a BLAST search revealed that the C-terminal of this protein has extensive homology to the C-terminal region of 53BP2, previously reported as p53-binding protein in a yeast two-hybrid screen (21). RAI protein was predicted to contain four consecutive ankyrin repeats and an SH3 domain at its C terminus as that of 53BP2 (21, 22), with 52% amino acid identity with 53BP2 at the C-terminal of 223 amino acid residues (Fig. 2B). Apart from the ankyrin repeats and SH3 domain, the RAI sequence is unrelated to that of any protein in current data bases using BLAST research.


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Fig. 2.   RAI protein. A, amino acid sequence of RAI. Ankyrin repeats (ank) are underlined and the SH3 domain (sh3) is double underlined. The start sites of the clones isolated in the yeast two-hybrid screen are indicated by a open triangle (one clone encompassing amino acids 57-351) and a filled triangle (three identical clones encompassing amino acids 78-351). The nucleotide sequences encoding human RAI are available as AF078036 and AF078037 in GenBank. B, homology between RAI and 53BP2. Amino acid sequence alignments of C-terminal regions of human RAI and human 53BP2. * indicates identical residues. · shows conserved amino acid residues. The putative amino acid residues of human 53BP2 that are known to interact with p53 are indicated by the filled triangle. C, expression of RAI protein. HeLa cells were transfected with the construct expressing FLAG-tagged RAI, empty vector, or mock transfected. Protein extracts were analyzed by Western blotting with anti-FLAG tag antibody. Sizes are indicated in kilodaltons.

To characterize the predicted protein product of RAI, the full-length clone was inserted into pFLAG-CMV2 vector (pFLAG) containing an FLAG epitope tagged onto the N terminus (pFLAG-RAI). pFLAG-RAI was transiently transfected into 293 cells and the cell lysates were prepared and separated by SDS-PAGE followed by immunoblotting with anti-FLAG antibody. The FLAG epitope-tagged RAI protein migrated as a single band with an apparent molecular mass of 46 kDa which was significantly slower than its predicted size on an SDS-PAGE gel (Fig. 2C).

Specificity of RAI Interaction in Yeast Two-hybrid Assay-- As the ankyrin repeats and SH3 domain of RAI are highly homologous to that of 53BP2 and this region of 53BP2 has been shown to mediate the interaction with p53 (23, 24), we performed a yeast two-hybrid assay to test the ability of RAI to interact with p53. As shown in Table I, the interaction of p65 (176-405) and itself and that between p53 and T-antigen, as positive controls, were evident both for the colony growth on 3-AT plate and for beta -galactosidase activity. Interestingly, p65 bound to RAI as strong as to p65 itself. However, despite its similarity to 53BP2, RAI failed to interact with p53 in the yeast two-hybrid assay.

                              
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Table I
Specificity testing of RAI interactions with multiple genes in the yeast two-hybrid system
Yeast Y190 cells were cotransformed with expression vectors encoding various GAL4 DNA-binding domain (BD) and GAL4 transcription activation domain (AD) fusion proteins. Colonies of equal size were replated in the presence of 25 mM 3-AT and allowed to grow for 3 days at 30 °C. beta -Galactosidase activity was assayed by using a standard filter assay. Growth on 3-AT plates and beta -galactosidase activity are scored as a range from no growth on 3-AT plates and no beta -galactosidase activity (-) to activity generated by the strong positive control (++++).

NF-kappa B p65 Co-immunoprecipitates with RAI in Vivo-- In order to examine whether p65 binds to RAI in vivo, 293 cells were transiently transfected with the plasmid expressing FLAG epitope-tagged RAI (pFLAG-RAI) or His-tagged Ikappa B-alpha (pEBVHis-Ikappa B-alpha ) and/or p65 expression plasmid (pCMV-p65). Cell extracts from these transfected cells were immunoprecipitated with a murine monoclonal anti-FLAG or a rabbit polyclonal anti-Ikappa B-alpha antibodies. The immunoprecipitates were fractionated by SDS-PAGE and immunoblotted with rabbit polyclonal anti-p65 antibody. As shown in Fig. 3, after immunoprecipitation with anti-FLAG antibody, we were able to demonstrate an immunoreactive band of 65 kDa that was recognized by anti-p65 antibody in a cell lysate prepared from 293 cells overexpressing RAI and p65 (lane 4). This band was identical to that observed by immunoprecipitation with anti-Ikappa B-alpha antibody in the cells overexpressing p65 and Ikappa B-alpha (lane 6). A control murine monoclonal antibody did not demonstrate an immunoreactive band of p65 (lane 5). The amount of p65 associated with Ikappa B-alpha was greater than that associated with RAI although it may be due to the different affinity of antibodies (compare lanes 4 with lane 6). Collectively, these results demonstrated in Figs. 1 and 3 suggested that an interaction between p65 and RAI occurs in intact cells as well as in vitro.


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Fig. 3.   Association of RAI with p65 in 293 cells. Whole cell lysates were prepared from 293 cells 24 h after transfection with the indicated plasmids and were precipitated with the following antisera: lanes 1-4, anti-FLAG; lane 5, anti-IgG; lane 6, anti-Ikappa B-alpha . Immune complexes were collected and subjected to SDS-PAGE followed by Western blotting with anti-p65 antibody. The position of the p65 proteins (filled arrow) and Ig heavy chain proteins (open arrow) are indicated.

Tissue Distribution of RAI-- We performed a Northern blot analysis on a human multitissue blot to determine the expression pattern of RAI. As shown in Fig. 4, the RAI probe detected two transcripts of approximately 3.4 and 6 kilobases in most cases. The expression of RAI mRNA was significantly high in heart, placenta, and prostate while it was markedly reduced in brain, liver, skeletal muscle, testis, and peripheral blood leukocyte. As a control for the amount of RNA present in each lane, we reprobed the Northern blot with an beta -actin probe. Similar amounts of beta -actin transcript of 2 kilobases were detected in all tissues, along with an additional lower band that might represent alpha -actin in heart and muscle tissue.


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Fig. 4.   Northern blot analyses of RAI mRNA. Northern blot analyses were carried out using a human multiple tissue blot (CLONTECH) with either a labeled RAI cDNA (upper panel) or a labeled beta -actin cDNA (lower panel) as a probe. Molecular weight markers are shown on the left.

Subcellular Localization of RAI-- To analyze the subcellular distribution of RAI protein, we constructed various plasmids expressing various RAI proteins fused to GFP: pEGFP-RAI (full-length), pEGFP-RAI (residues 1-146), pEGFP-RAI (residues 132-351), and pEGFP-p65 (residues 176-405). After 24 h of transfection of these plasmids in HeLa cells, cells were fixed and examined under a fluorescent microscopy. As shown in Fig. 5, A-F, both GFP-RAI and GFP-RAI (132-351) located mainly in the nucleus of the cells, predominantly in the nucleoplasm (Fig. 5, B and D) in contrast to GFP alone, which is distributed diffusely both in the cytoplasm and the nucleus (Fig. 5E). It was noted that the N-terminal region of RAI (1-146) that was fused to GFP localized in the nucleus, particularly in the nucleolus (Fig. 5C), suggesting a potential of RAI to move into the nucleolus. As expected, pEGFP-p65 (176-405) also localized in the nucleus (Fig. 5F). The same cellular localization pattern of these proteins was also observed in COS-1 cells (data not shown).


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Fig. 5.   Subcellular distribution of RAI. A, schematic diagram of RAI protein. B-F, subcellular localization of various GFP-RAI fusion proteins, GFP and GFP-p65 (176-405) in the transiently transfected HeLa cells. Various GFP fusion proteins were visualized by fluorescence microscopy. G-L, colocalization of RAI with p65 induced with TNF-alpha . HeLa cells were transfected with GFP-RAI expressing plasmid and after 24 h cells were left untreated or treated with TNF-alpha for 30 min. Immunofluorescence microscopic examinations were carried out with anti-p65 antibody. Cell nuclei were stained with Hoechst-33342. The cells in G, H, and I, or those in J, K, and L were the same cells, respectively.

We then examined whether RAI was colocalized with p65 in the nucleus. HeLa cells were transfected with pEGFP-RAI plasmid expressing GFP-RAI fusion protein and cells were left untreated or treated for 30 min with TNF-alpha . These cells were stained with p65 antibody. As shown in Fig. 5, G-I, transfection with pEGFP-RAI by itself did not change the distribution of p65 which was sequestered in the cytoplasm bound to Ikappa B proteins without stimulation with TNF-alpha . TNF-alpha induced nuclear translocation of p65 that was colocalized with RAI in the nucleus (Fig. 5, J-L).

RAI Inhibited NF-kappa B-dependent Gene Expression-- Since RAI was shown to bind p65 through its ankyrin repeats and SH3 domain as similarly to Ikappa B family proteins, we asked whether RAI had any effect on the kappa B-dependent transcription. The luciferase reporter plasmid (4kappa Bw-Luc) containing four tandem copies of the kappa B sequence was co-transfected with an expression vector for FLAG-RAI (pFLAG-RAI) and/or p65 expression vector (pCMV-p65) into HeLa cells. Twenty-four hours after transfection, luciferase activities were determined. As shown in Fig. 6A, co-transfection with pCMV-p65 strongly activated the reporter gene expression (Fig. 6A, lane 9). Co-transfection with pFLAG-RAI markedly inhibited the p65-induced luciferase gene expression in a dose-dependent manner for the amount of pFLAG-RAI plasmid DNA (Fig. 6A, lanes 11, 13, and 15). Although to a lesser extent, we also observed suppression of the basal level of kappa B-dependent luciferase expression by pFLAG-RAI (Fig. 6A, lanes 3, 5, and 7). This inhibitory effect of RAI was considered not due to the differences in transfection efficiency since an internal control plasmid pRL-TK containing a constitutive promoter of the herpes simplex virus thymidine kinase was unaffected by the expression of RAI protein (data not shown). Moreover, when a luciferase reporter construct harboring four mutated inactive kappa B sites (4kappa Bm-Luc) was used, no significant effects of co-transfection of pFLAG-RAI were observed with or without co-transfection of pCMV-p65 (black lanes). These results suggested that the inhibitory action of RAI was dependent on the intact NF-kappa B-binding motifs.


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Fig. 6.   Inhibition of NF-kappa B-dependent gene expression by RAI. A, HeLa cells were transfected with either 4kappa Bw-Luc (containing wild type NF-kappa B binding sites) or 4kappa Bm-Luc (containing mutated NF-kappa B binding sites) together with pCMV-p65 and various amounts of pFLAG-RAI plasmids. B, Saos-2 cells, a p53-null cell line, were transfected with p53-dependent luciferase reporter (PG13-Luc) together with pCMV-p53wt and pFLAG-RAI plasmids. Cells were lysed and the luciferase activities were measured 24 h after transfection. C, Jurkat cells were transfected with 4kappa Bw-Luc together with increasing amounts of pFLAG-RAI plasmid. After 24 h of transfection, cells were treated with 5 ng/ml TNF-alpha and harvested after an additional incubation for 24 h. D, responsible region of RAI in inhibiting NF-kappa B-dependent gene expression. 293 cells were transfected with 4kappa Bw-Luc together with pCMV-p65 and various RAI expression plasmids. Results of RAI inhibition were observed in all cell lines examined. The representative data are demonstrated. The luciferase activity was normalized by Renilla luciferase activity that was co-transfected as an internal control. The data are presented as the fold increase in luciferase activities (mean ± S.D.) relative to control transfection of three independent experiments.

The finding of RAI being highly homologous to 53BP2 prompted us to test whether RAI affects transcriptional activation by p53. Saos-2 cells, a p53-null human osteosarcoma cell line, were transfected with a reporter plasmid (PG13-Luc) under the control of p53 together with p53-expressing plasmid (pCMV-p53) and increasing amounts of RAI expression plasmid (pFLAG-RAI). Although the luciferase expression was increased 10-fold by the p53 expression plasmid, no significant effect of RAI on the p53-dependent transcription was observed (Fig. 6B).

The inhibitory effect of RAI on NF-kappa B was also examined in response to a physiological inducer of NF-kappa B, TNF-alpha . Jurkat cells were co-transfected with NF-kappa B-dependent reporter plasmid (4kappa Bw-Luc) and increasing amounts of RAI or Ikappa B-alpha expression plasmid. Then the cells were stimulated by TNF-alpha to activate NF-kappa B. As expected, co-transfection with pEBVHis-Ikappa B-alpha resulted in strong inhibition on transactivation by TNF-alpha in the transfected Jurkat cells (Fig. 6C, lanes 6-8). Similarly, pFLAG-RAI inhibited the TNF-alpha -induced NF-kappa B activation, although the inhibitory action by RAI was not as efficiently as Ikappa B-alpha (Fig. 6C, compare lanes 3-5 with lanes 6-8). These results indicated that RAI could inhibit the activity of endogenous NF-kappa B that was induced by TNF-alpha as well as the exogenously transduced p65.

The interaction between p65 and RAI required the C terminus of RAI containing ankyrin repeats and SH3 domain as demonstrated by the results of yeast two-hybrid screen and the in vitro binding assay (Fig. 1). We thus tested whether the inhibitory effect of RAI on kappa B-dependent transactivation required these binding domains. The plasmid construct expressing the full-length, the N terminus, or the C terminus of RAI was transfected into 293 cells and the cell lysate was confirmed for protein expression by Western blot using anti-FLAG antibody (data not shown). We then co-transfected these constructs into 293 cells together with NF-kappa B-dependent reporter plasmid (4kappa Bw-Luc) and the luciferase assay was carried out as described above. As shown in Fig. 6D, full-length RAI inhibited the p65-activated gene expression in 293 cells in a similar way as that in HeLa cells (Fig. 6A). The C terminus of RAI could inhibit the p65-induced luciferase gene expression in a dose-dependent manner as efficient as full-length RAI (Fig. 6D, lanes 3 and 4). In contrast, the N terminus of RAI without the p65-binding regions had no inhibitory effect (Fig. 6D, lanes 5 and 6). These findings suggested that the inhibitory effect of RAI on kappa B-dependent transactivation required the ankyrin repeats and SH3 domain of RAI.

RAI Interferes with the DNA Binding of NF-kappa B-- Since RAI shares a common feature of Ikappa Bs in that they contain multiple ankyrin repeats at its C terminus and can bind the central region of p65, we performed EMSA to investigate whether RAI could inhibit the NF-kappa B action by interfering with its specific DNA binding activity. As demonstrated in Fig. 7A, overexpression of p65 in 293 cells was accompanied by a strong induction of the nuclear kappa B binding activity (Fig. 7A, compare lanes 1 and 2). Overexpression of RAI inhibited p65-DNA binding in a dose-dependent manner (Fig. 7A, lanes 3-5). In comparison to Ikappa B-alpha (Fig. 7A, lane 6), inhibition of RAI was less efficient and consistent with the results of luciferase reporter assay (Fig. 6C). The specificity of the DNA-protein binding was confirmed by competition experiments using DNA competitors. The excess amount (50-fold) of the unlabeled wild type kappa B oligonucleotide (Fig. 7B, lane 3) but not the oligonucleotide mutated in the kappa B-binding site (Fig. 7B, lane 4) blocked the kappa B-specific DNA binding activity of the nuclear extract from cells transfected with pCMV-p65. In contrast, the DNA binding activity of the ubiquitous transcription factor Oct-1 was not affected by expression of RAI using the same nuclear extracts that were assayed for NF-kappa B DNA binding activity (Fig. 7C). These findings suggested that RAI could specifically inhibit NF-kappa B transactivation through interfering with its DNA binding.


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Fig. 7.   Inhibition of DNA binding activity of NF-kappa B by co-transfection of RAI-expressing plasmid. A, NF-kappa B DNA binding activity of the 293 cell nuclear extract after transfection with pCMV-p65 either alone or in combination with various amounts of pFLAG-RAI. After 24 h of transfection, nuclear extracts were prepared and analyzed by EMSA with a 32P-labeled oligonucleotide containing the NF-kappa B-binding site. The NF-kappa B specific complex (filled arrow) and the free probe (open arrow) are indicated. B, competition analysis. 293 cells were mock-transfected (lane 1) or transfected with pCMV-p65 (lanes 2-4). Reaction mixtures containing nuclear extracts were either left untreated (lanes 1 and 2) or incubated with a 50-fold excess of unlabeled wild-type kappa B-specific oligonucleotide (lane 3) or its mutant (lane 4) before adding the kappa B-specific DNA probe. C, Oct-1-binding activity was not affected by transfection with the RAI expressing plasmid. The same nuclear extracts as in A were analyzed with an Oct-1-specific probe in EMSA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In an attempt to identify novel proteins capable of interacting with the p65 subunit of NF-kappa B, we have isolated a cDNA encoding a protein named RAI in a yeast two-hybrid screen. We confirmed the interaction in vitro using GST pull-down assay and in vivo using immunoprecipitation and Western blot assays (Figs. 1 and 3). Amino acid sequence analyses of RAI revealed that it resembles 53BP2 in that they both contain four ankyrin repeats and an SH3, as noted earlier, with 52% amino acid identity with 53BP2 at their C termini. However, RAI could not bind to p53 in a yeast two-hybrid assay (Table I). The RAI, when overexpressed, located predominantly in the nucleus of transfected cells (Fig. 5B) and inhibited NF-kappa B-dependent gene expression, whereas the p53-dependent gene expression was essentially unaffected (Fig. 6). The direct interaction between RAI and p65 most likely contributes to the inhibition of NF-kappa B activity through interfering with its NF-kappa B DNA binding activity (Fig. 7). Thus, RAI is a novel NF-kappa B-binding protein with a specific repression activity of transcription.

The co-localization of both p65 and RAI in the transfected cells as visualized by confocal microscopy (Fig. 5, J and K) supported the interaction of both proteins in vivo (Fig. 3). The presence of ankyrin repeats and the SH3 domain in the C-terminal region of RAI may contribute to its nuclear localization. As it was recently reported, the ankyrin domains of diverse proteins such as Ikappa B-alpha and 53BP2 contain functional nuclear import signal (25). In addition, the N-terminal of RAI was also able to localize in the nucleus (Fig. 5D). However, it is still an open question as to whether the endogenous RAI shows the same subcellular distribution as the fusion protein used in this study.

All four independent RAI clones isolated from the yeast two-hybrid screen contained the C terminus of RAI including the ankyrin repeats, and the SH3 domain suggested that the C-terminal region of RAI is responsible for physical interaction with p65. Although the C terminus of RAI is highly homologous with that of 53BP2, some amino acid residues of 53BP2 that were shown to be involved in binding to p53 (23, 24) differ from RAI. The fact that RAI failed to bind p53 in the yeast two-hybrid system (Table I) and that RAI showed no significant effect on p53-dependent gene expression (Fig. 6B) suggested that this region might contain an important determinant for specific interaction.

The tissue distribution of RAI gene expression was highly restricted with high expression levels in heart, placenta, and prostate while p65 expression is observed in most of the tissues examined (data not shown). Two RAI messages of different sizes were detected (Fig. 4). DNA sequencing of the two cDNAs isolated from plaque hybridization revealed the same ORF but the 5' sequences differ in length. It is likely that RAI generates two transcripts with different 5'-untranslated regions, probably by alternative splicing. DNA data base search revealed that the partial RAI gene is localized in the 19q13.2-q13.3 region which contains the three DNA repair genes XRCC1, ERCC2 (excision repair cross-complementing rodent repair group 2), and ERCC1 (26). The locus of RAI is also near the locus of Ikappa B-beta which is localized in 19q13.1 (27). These pieces of information suggested that RAI might have been generated from Ikappa B-beta , or vice versa, and acquired tissue-specific expression during evolution. We do not currently know the biological implication of restricted expression of RAI.

It is noted that RAI was a second frequently isolated p65-binding gene in our yeast two-hybrid screen. The nuclear localization of RAI differs from that of Ikappa B family members in that most of them are cytoplasmic proteins (7, 28), suggesting their different regulation mechanisms despite structural similarity in their C termini. At least seven mammalian Ikappa B molecules have been identified with distinct and overlapping inhibitory specificities (29-31). Although little is known about how NF-kappa B selectively bind various Ikappa B proteins, it is hypothesized that cell-specific controls regulate interactions of the various Ikappa B and NF-kappa B (32, 33). All the Ikappa B proteins contains several consecutive ankyrin repeats which are essential for interaction with NF-kappa B. RAI contains four consecutive ankyrin repeats at its C terminus, however, no PEST-like region (34) responsible for the degradation of Ikappa B proteins was observed for RAI. It is proposed that the cytoplasmic/nuclear partitioning of NF-kappa B is delicately balanced by the concentrations of these proteins and by its relative affinities to Ikappa B family proteins (35). Such multiplicity of regulatory molecules may render different cell types with distinct susceptibilities to various signals leading to the NF-kappa B activation cascade (32, 36).

Taken together, our findings provide evidence of a novel NF-kappa B inhibitor RAI with functional similarity to Ikappa B proteins. It is known that p65 directly interacts with other proteins other than the Ikappa B family, including p202 (37), CREB-binding protein/p300 (38), Stat 6 (39), and TAFII105 (40), which either suppress or activate NF-kappa B. It is possible that a variety of proteins interacting with NF-kappa B are involved in a coordinate regulation of its activity and in either tissue-specific or nonspecific manner. Our results suggested that RAI might play an alternative role in controlling NF-kappa B activation by directly binding to p65 most probably in the nucleus. The mechanism and the biological significance of the inhibition of NF-kappa B activity by RAI should be further explored. It is intriguing to speculate that these different inhibitory molecules may control the regulation of NF-kappa B by binding in the cytoplasm or in the nucleus. The restricted RAI tissue distribution may provide evidence for tissue-specific regulation of NF-kappa B.

    FOOTNOTES

* This work was supported by grants-in-aid from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture of Japan, and the Japanese Health Sciences Foundation.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF078036 and AF078037.

Dagger Research fellow of the Japan Society for the Promotion of Science.

§ To whom correspondence should be addressed: Dept. of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: 81-52-853-8204; Fax: 81-52-859-1235; E-mail: tokamoto{at}med.nagoya-cu.ac.jp.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; TNF-alpha , tumor necrosis factor-alpha ; RAI, RelA-associated inhibitor; GST, glutathione S-transferase; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift assay; SH3, Src homology domain 3; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; PBS, phosphate-buffered saline; 3-AT, 3-aminotriazole.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
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