From the Department of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
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ABSTRACT |
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Here we report the identification and
characterization of a novel protein, RelA-associated inhibitor (RAI),
that binds to the NF- Nuclear factor 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- 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 4
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 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- 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
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 I 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 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- 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 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 Isolation of RAI cDNA Clones as a p65-binding Protein--
We
applied the yeast two-hybrid system to identify proteins that interact
with NF-
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 I
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).
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.
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 NF- 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 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).
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- RAI Inhibited NF-
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-
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
RAI Interferes with the DNA Binding of NF- In an attempt to identify novel proteins capable of interacting
with the p65 subunit of NF- 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 I 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 I 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 I Taken together, our findings provide evidence of a novel NF-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-
B p65 but not that of p53 in transient luciferase gene
expression assays. Similarly, RAI inhibited the endogenous NF-
B
activity induced by tumor necrosis factor-
. 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-
B p65 that was activated by TNF-
. These
observations indicate that RAI is another inhibitor of NF-
B in
addition to I
B proteins and may confer an alternative mechanism of regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
(NF-
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-
B
family in mammalian cells include the proto-oncogene c-Rel, p50/p105
(NF
B1), p65 (RelA), p52/p100 (NF
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-
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-
B
family is their sequestration in the cytoplasm as inactive complexes
with a class of inhibitory molecules known as I
Bs (2, 7). Treatment
of cells with a variety of inducers such as phorbol esters, interleukin
1, and tumor necrosis factor-
(TNF-
) results in dissociation of
the cytoplasmic complexes and translocation of NF-
B to the
nucleus (8, 9). The dissociation of NF-
B·I
B complexes
is known to be triggered by the phosphorylation and subsequent
degradation of I
B proteins (10-12). This exposes the nuclear
localization sequence in the remaining NF-
B heterodimer, leading to
nuclear translocation and subsequent binding of NF-
B to DNA
regulatory elements of the target genes. The p65 subunit is a major
component of NF-
B complexes and is responsible for trans-activation
(13).
B-dependent
transcription through interfering with its DNA binding activity of
NF-
B.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bw-Luc and 4
Bm-Luc have
been described previously (15). pCMV-p65 plasmid was derived from human
RelA cDNA. pEBVHis-I
B-
plasmid was constructed by inserting
I
B-
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.
-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
-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.
-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.
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.
B-
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-
B p65 (C-20) antibody (Santa Cruz).
-actin fragment.
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.
B-dependent or mutant reporter plasmid (4
Bw-Luc or
4
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-
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.
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
[
-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
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
-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.
B-
/MAD-3 (47 clones), I
B-
/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.
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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.
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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.
-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.
Specificity testing of RAI interactions with multiple genes in the
yeast two-hybrid system
-Galactosidase activity was assayed by using a standard
filter assay. Growth on 3-AT plates and
-galactosidase activity are
scored as a range from no growth on 3-AT plates and no
-galactosidase activity (
) to activity generated by the strong
positive control (++++).
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 I
B-
(pEBVHis-I
B-
) 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-I
B-
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-I
B-
antibody in
the cells overexpressing p65 and I
B-
(lane 6). A
control murine monoclonal antibody did not demonstrate an
immunoreactive band of p65 (lane 5). The amount of p65
associated with I
B-
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-I B-
. 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.
-actin probe. Similar amounts of
-actin
transcript of 2 kilobases were detected in all tissues, along with an
additional lower band that might represent
-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 -actin cDNA (lower
panel) as a probe. Molecular weight markers are shown on the
left.
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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- .
HeLa cells were transfected with GFP-RAI expressing plasmid and after
24 h cells were left untreated or treated with TNF-
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.
. 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
I
B proteins without stimulation with TNF-
. TNF-
induced
nuclear translocation of p65 that was colocalized with RAI in the
nucleus (Fig. 5, J-L).
B-dependent Gene
Expression--
Since RAI was shown to bind p65 through its ankyrin
repeats and SH3 domain as similarly to I
B family proteins, we asked
whether RAI had any effect on the
B-dependent
transcription. The luciferase reporter plasmid (4
Bw-Luc) containing
four tandem copies of the
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
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
B sites (4
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-
B-binding motifs.
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Fig. 6.
Inhibition of
NF- B-dependent gene expression by
RAI. A, HeLa cells were transfected with either
4
Bw-Luc (containing wild type NF-
B binding sites) or 4
Bm-Luc
(containing mutated NF-
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 4
Bw-Luc together with increasing amounts of
pFLAG-RAI plasmid. After 24 h of transfection, cells were treated
with 5 ng/ml TNF-
and harvested after an additional incubation for
24 h. D, responsible region of RAI in inhibiting
NF-
B-dependent gene expression. 293 cells were
transfected with 4
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.
B was also examined in response
to a physiological inducer of NF-
B, TNF-
. Jurkat cells were
co-transfected with NF-
B-dependent reporter plasmid
(4
Bw-Luc) and increasing amounts of RAI or I
B-
expression
plasmid. Then the cells were stimulated by TNF-
to activate NF-
B.
As expected, co-transfection with pEBVHis-I
B-
resulted in strong
inhibition on transactivation by TNF-
in the transfected Jurkat
cells (Fig. 6C, lanes 6-8). Similarly, pFLAG-RAI inhibited
the TNF-
-induced NF-
B activation, although the inhibitory action
by RAI was not as efficiently as I
B-
(Fig. 6C, compare
lanes 3-5 with lanes 6-8). These results
indicated that RAI could inhibit the activity of endogenous NF-
B
that was induced by TNF-
as well as the exogenously transduced p65.
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-
B-dependent reporter plasmid (4
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
B-dependent
transactivation required the ankyrin repeats and SH3 domain of RAI.
B--
Since RAI
shares a common feature of I
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-
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
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 I
B-
(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
B oligonucleotide (Fig. 7B, lane 3)
but not the oligonucleotide mutated in the
B-binding site (Fig.
7B, lane 4) blocked the
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-
B DNA binding activity
(Fig. 7C). These findings suggested that RAI could
specifically inhibit NF-
B transactivation through interfering with
its DNA binding.
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Fig. 7.
Inhibition of DNA binding activity of
NF- B by co-transfection of RAI-expressing
plasmid. A, NF-
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-
B-binding site. The NF-
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
B-specific oligonucleotide
(lane 3) or its mutant (lane 4) before adding the
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
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-
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-
B activity through interfering
with its NF-
B DNA binding activity (Fig. 7). Thus, RAI is a novel
NF-
B-binding protein with a specific repression activity of transcription.
B-
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.
B-
which
is localized in 19q13.1 (27). These pieces of information suggested
that RAI might have been generated from I
B-
, or vice versa, and
acquired tissue-specific expression during evolution. We do not
currently know the biological implication of restricted expression of
RAI.
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 I
B molecules have been identified with distinct and
overlapping inhibitory specificities (29-31). Although little is known
about how NF-
B selectively bind various I
B proteins, it is
hypothesized that cell-specific controls regulate interactions of the
various I
B and NF-
B (32, 33). All the I
B proteins contains
several consecutive ankyrin repeats which are essential for interaction
with NF-
B. RAI contains four consecutive ankyrin repeats at its C
terminus, however, no PEST-like region (34) responsible for the
degradation of I
B proteins was observed for RAI. It is proposed that
the cytoplasmic/nuclear partitioning of NF-
B is delicately balanced
by the concentrations of these proteins and by its relative affinities
to I
B family proteins (35). Such multiplicity of regulatory
molecules may render different cell types with distinct
susceptibilities to various signals leading to the NF-
B activation
cascade (32, 36).
B
inhibitor RAI with functional similarity to I
B proteins. It is known
that p65 directly interacts with other proteins other than the I
B
family, including p202 (37), CREB-binding protein/p300 (38), Stat 6 (39), and TAFII105 (40), which either suppress or activate NF-
B. It
is possible that a variety of proteins interacting with NF-
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-
B activation by
directly binding to p65 most probably in the nucleus. The mechanism and
the biological significance of the inhibition of NF-
B activity by
RAI should be further explored. It is intriguing to speculate that
these different inhibitory molecules may control the regulation of
NF-
B by binding in the cytoplasm or in the nucleus. The restricted
RAI tissue distribution may provide evidence for tissue-specific
regulation of NF-
B.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
TNF-
, tumor necrosis factor-
;
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.
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