Identification and Characterization of BCL-3-binding Protein
IMPLICATIONS FOR TRANSCRIPTION AND DNA REPAIR OR RECOMBINATION*
Nobumasa Watanabe,
Sumiko Wachi and
Takashi Fujita
From the
Department of Tumor Cell Biology, The Tokyo Metropolitan Institute of
Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan
Received for publication, April 4, 2003
, and in revised form, May 1, 2003.
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ABSTRACT
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A putative oncogene bcl-3 was originally identified and cloned at
the breakpoint in the recurring chromosome translocation t(14;19) found in
some cases of B cell chronic lymphocytic leukemia. Studies of
bcl-3-deficient mice demonstrated a critical role for bcl-3
in the development of a normal immune response and the formation of germinal
centers in secondary lymphoid organs. However, the molecular mechanism that
underlies B cell leukemogenesis and the knockout mouse phenotype remains
unclear. Here we have identified and characterized BCL-3-binding protein
(B3BP) as a protein interacting specifically with the bcl-3 gene
product (BCL-3) by a yeast two-hybrid screen. We found that B3BP associates
with not only BCL-3 but also p300/CBP histone acetyltransferases. The
N-terminal region of B3BP that contains the ATP-binding site is important for
the interaction with BCL-3 and p300/CBP. Homology searches indicate that the
ATP-binding region of B3BP, which contains a typical Walker-type ATP-binding
P-loop, most resembles that of 2',3'-cyclic nucleotide
3'-phosphodiesterase of mammals and polynucleotide kinase of T4
bacteriophage. In fact B3BP shows intrinsic ATP binding and hydrolyzing
activity. Furthermore, we demonstrated that B3BP is a 5'-polynucleotide
kinase. We also found a small MutS-related domain, which is thought to be
involved in the DNA repair or recombination reaction, in the C-terminal region
of B3BP, and it shows nicking endonuclease activity. These observations might
help to gain new insights into the function of BCL-3 and p300/CBP, especially
the coupling of transcription with repair or recombination.
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INTRODUCTION
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bcl-3 was originally identified at the breakpoint in the t(14; 19)
chromosome translocation in some cases of chronic B cell lymphocytic leukemia
and shown to be up-regulated transcriptionally in peripheral blood lymphocytes
from patients with the corresponding translocation. Therefore, its involvement
in the pathogenesis of chronic B cell lymphocytic leukemia has been strongly
suggested (1). It was also
reported that bcl-3 gene expression is induced by many cell growth-
or survival-promoting factors in lymphocytic cell lines
(24),
suggesting the close involvement of bcl-3 in cell proliferation and
survival. Subsequent studies showing a correlation of bcl-3 induction
with mouse skin carcinogenesis
(5), human breast cancer
(6), and hepatocyte
proliferation (7) have
indicated its involvement in carcinogenesis and the growth of cells other than
B lymphocytes. In fact, ectopic expression of bcl-3 blocked
interleukin-4 deprivation-induced apoptosis of a T cell line in vitro
and also increased the survival rate of activated T cells in vivo
(2,
8,
9). Transgenic mice in which
bcl-3 is expressed under the control of an Ig heavy chain enhancer
showed an expansion of the B cell population
(10). Nevertheless, there is
no experimental evidence of a role for bcl-3 in cell
transformation.
Extensive biochemical study has revealed some of the molecular functions of
bcl-3. First, amino acid sequence alignment showed that BCL-3
contains seven repeats of an ankyrin-like unit and belongs to the I
B
family of proteins, which modulate the DNA binding activity and subcellular
localization of the transcription factor NF-
B
(11). Subsequently, it was
demonstrated that BCL-3 physically associates with the p50 and p50B homodimers
of NFKB1 and NFKB2, respectively, and confers transcriptional activation to
the otherwise inert complex; hence it functions as a transcriptional
co-activator (12,
13). We have previously
demonstrated that BCL-3 induces the nuclear translocation of the p50 homodimer
generated via reorganization from cytoplasmic p50/p105
(14). This BCL-3-induced p50
homodimer formation has been observed in vivo; that is, ectopic
expression of BCL-3 in thymocytes induced the formation of the p50 homodimer
(15), and stimulation of
cultured T cells with interleukin-9 leads to the induction of BCL-3
expression, which is followed by p50 homodimer generation
(3). Moreover, p50 homodimer
has been implicated in cell survival or the inhibition of apoptosis
(3,
16). Subsequently, it has been
shown that BCL-3 interacts with general transcription factors
(17), other transcriptional
co-activators (18,
19), and also other
DNA-binding factors (17,
18), all of which indicates a
general role for BCL-3 in the transcriptional activation. Recently, it has
also been demonstrated that a putative BCL-3 ortholog of Caenorhabditis
elegans interacts with MRT-2 cell cycle checkpoint protein and indeed is
involved in the DNA damage response by protein-protein interaction screening
combined with a large scale phenotypic analysis
(20).
Above all, knockout mouse studies gave rise to important information on the
biological role of BCL-3
(2123).
The BCL-3-deficient mouse was susceptible to certain kinds of pathogens.
Antigen-specific antibody production was severely impaired because germinal
center formation in secondary lymphoid organs was markedly inhibited. Such a
phenotype was at least to some extent similar to that of NFKB1 and NFKB2
knockout mice
(2326),
suggesting physiological significance of the interaction of BCL-3 with these
proteins. During the development of germinal centers, B cell-specific genetic
recombination of the Ig gene, class switch recombination, and somatic
hypermutation proceed to produce a large amount of Ig that has a much higher
affinity for the antigen. One hypothesis is that BCL-3 directly regulates
class switch recombination and somatic hypermutation because these genetic
alterations have a close correlation with the transcriptional activation of
the Ig gene itself and the intronic switch region
(2729).
In this study we identified B3BP as a protein that specifically interacts
with BCL-3. It was shown that B3BP also interacts with histone
acetyltransferase p300/CBP and that the ATP-binding site of B3BP is important
for the association with BCL-3 or p300/CBP. Biochemical analysis revealed that
B3BP has polynucleotide kinase activity to transfer a phosphate group to the
5' end of DNA and RNA substrates. Moreover, a small MutS-related
(Smr)1 domain found in
the C-terminal region of B3BP exhibits nicking endonuclease activity, which is
postulated to have a role in mismatch repair or genetic recombination
(30,
31). These findings suggest
that B3BP plays a role connecting transcriptional activation and genetic
recombination of the Ig gene.
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EXPERIMENTAL PROCEDURES
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Yeast Two-hybrid ScreeningA fragment of mouse BCL-3 cDNA
encoding amino acids 16440 cloned into the LexA DNA-binding domain
vector pBTM116 and a human spleen cDNA library cloned into the GAL4 activation
domain vector pGAD10 (Clontech) were sequentially used to transform L40 yeast
cells according to the protocol described on the Yeast Transformation Home
Page
(www.umanitoba.ca/faculties/medicine/units/biochem/gietz/Trafo.html).
Transformants were plated on selection medium (lacking tryptophan, leucine,
and histidine) containing 10 mM 3-amino-1,2,4-triazole. After
incubation for 7 days at 25 °C, the clones that allowed growth were
identified and confirmed to express
-galactosidase. The plasmids were
recovered according to Matchmaker protocols (Clontech) and retransformed into
yeast cells containing either pBTM116-BCL-3 or pBTM116 empty vector. cDNA
inserts from plasmids that allowed the yeast cells containing pBTM116-BCL-3 to
grow on selection medium were identified and further characterized by DNA
sequencing. cDNA Cloning and Plasmid ConstructionTo obtain a
full-length B3BP cDNA, a human T cell cDNA library carried in
ZAP
Express (Stratagene) was screened under stringent conditions using the cDNA of
a
4.7-kb insert isolated in the two-hybrid screen as a probe. The cDNA
inserts from positive phage clones were excised in vivo to generate
subclones in the pBK-CMV phagemid and confirmed by sequencing. The ORF
containing full-length B3BP cDNA was assembled on a mammalian expression
vector, pEF-BOS (32). To
express GST fusion proteins in Escherichia coli JM109, DNA fragments
encoding amino acids 2631, 11711770, 394630, and
16881770 of B3BP were cloned into pGEX-4T (Amersham Biosciences) to
construct pGEX-B3BP(N), pGEX-B3BP(C), pGEX-B3BP(M), and pGEX-B3BP(Smr),
respectively. A cDNA encoding full-length mouse BCL-3 was cloned into pGEX-4T
to express GST-BCL-3 fusion protein. cDNA fragments encoding B3BP, BCL-3,
BCL-3 derivatives, and NFKB1 were subcloned into pCS2+
(33) to transcribe and
translate in rabbit reticulocyte lysate (TNT SP6 Quick System;
Promega). HA-tagged expression vectors were constructed by inserting the DNA
fragment encoding MGYPYDVPDYASLGG for pEF-HA-B3BP and pEF-HA-BCL-3 in the
N-terminal end of B3BP and BCL-3, respectively. HA-tagged p300 and FLAG-tagged
CBP constructs were described previously
(34). FLAG-tagged constructs
were obtained by inserting the DNA fragment encoding GPGDYKDDDKGDYKDDDK for
pEF-B3BP-FLAG and pEF-BCL-3-FLAG in the C-terminal end of B3BP and BCL-3,
respectively. To construct expression vectors encoding B3BP derivatives,
B3BP(K/A) and B3BP(K/R), site-directed mutagenesis of the Lys453
residue within the ATP-binding site of B3BP was conducted using
QuikChangeTM (Stratagene) according to the manufacturer's instructions.
The authenticity of the substitutions and the absence of any undesired
mutations were confirmed by sequence analysis.
AntibodiesThe monoclonal antibodies against the HA epitope
(12CA5; Roche Applied Science), FLAG epitope (M2; Sigma-Aldrich), and p300/CBP
(mixed monoclonal antibodies; Upstate Biotechnology, Inc.) were obtained
commercially. The monoclonal antibody against NFKB1 was described previously
(35).
Recombinant Proteins and in Vitro Binding AssayAll of the
pGEX-based bacterial expression vectors were transformed into E. coli
strain JM109, and GST fusion proteins were expressed and purified using
glutathione-Sepharose resin according to the manufacturer's directions
(Amersham Biosciences). For the in vitro biochemical analysis, GST,
GST-B3BP(M), and GST-B3BP(Smr) were eluted from the column with buffer (50
mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM
dithiothreitol, and 20 mM glutathione) and dialyzed against
phosphate-buffered saline. Subsequently, GST-B3BP(Smr) was treated with
thrombin protease, and the Smr domain was further purified by ion-exchange
chromatography on a Mono S column (Amersham Biosciences). For the GST-based
interaction assay, GST fusion proteins attached to glutathione matrix beads
were incubated with rabbit reticulocyte lysate containing
35S-radiolabeled protein in Nonidet P-40 binding buffer (150
mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA,
1% (v/v) Nonidet P-40, and 10% (v/v) glycerol) for 2 h at 4 °C. The beads
were subsequently washed five times with Nonidet P-40 binding buffer, and the
bound proteins were fractionated by SDS-PAGE and visualized by autoradiography
or Coomassie Brilliant Blue (CBB) staining. Reticulocyte lysate used in
Fig. 5B was
ATP-depleted by passing through the Sephadex G-50 (Amersham Biosciences)
column.

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FIG. 5. The involvement of ATP-binding site of B3BP in the interaction with
BCL-3 and p300/CBP. A, the effect of Lys453
substitution on the interaction with BCL-3 and p300/CBP. 293T cells were
transfected with a series of HA-tagged and FLAG-tagged expression vectors
indicated at the top of the figure. The interaction was analyzed as
described in Fig. 3. The
molecular mass markers are shown on the right. B, the effect of ATP
on the interaction of B3BP with BCL-3 in vitro. GST-based interaction
assay was performed as described in the legend to
Fig. 2 except that reticulocyte
lysate containing 35S-labeled B3BP was ATP-depleted (and also small
compounds) by gel filtration and then subjected to the assay in the presence
or absence of ATP.
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FIG. 3. Interaction of B3BP with BCL-3 and histone acetyltransferase p300/CBP in
293T cells. 293T cells were transiently transfected with each combination
of HA-tagged and FLAG-tagged expression vectors shown on the top of
the panel. Total cell lysates (input) and proteins bound to the
anti-FLAG matrix (antiFLAG ppt.) were subjected to Western blot
analysis using anti-FLAG antibody (anti FLAG blot) and anti-HA
antibody (anti HA blot). Molecular mass markers are shown on the
left. The positions of the HA-tagged proteins are shown on the
right. The asterisk shows nonspecific signals.
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FIG. 2. In vitro binding of B3BP to BCL-3. A, B3BP binds
to BCL-3 in vitro. Equal volumes of reticulocyte lysate that contain
35S-labeled products were run on 520% SDS-polyacrylamide gel
and visualized by autoradiography as shown in lanes 13
(input). Lane 1, B3BP; lane 2, BCL-3; lane
3, luciferase. Luciferase was used as a negative control in this
experiment. A GST pull-down assay was performed using equal amounts of the
reticulocyte lysate and purified GST (lane 4), GST-B3BP(N) (lane
5), GST-B3BP(C) (lane 6), or GST-BCL-3 (lane 7). The
bound proteins were subjected to SDS-PAGE and visualized by autoradiography.
Lanes 47 were exposed 10 times longer than lanes
13. Molecular mass markers are shown on the right of each
panel. B, integrity of BCL-3 ankyrin repeat domain is important for
binding with B3BP. Structures of BCL-3 and its deletion mutants used in the
experiment are shown. The black box represents the ankyrin repeat
domain, which contains seven ankyrin-like units. BCL-3 C has the entire
ankyrin repeat domain but lacks the C-terminal region. BCL-3 ank lacks
the entire sixth and a part of the fifth and seventh ankyrin repeat. Equal
volumes of reticulocyte lysate containing 35S-labeled products were
run on an SDS-polyacrylamide gel and visualized by autoradiography (lanes
14, input). Lane 1, BCL-3; lane 2,
BCL-3 C; lane 3, BCL-3 ank; lane 4, NFKB1. Equal
amounts of reticulocyte lysate and purified GST (lanes 5, 7, 9, and
11) or GST-B3BP(N) (lanes 6, 8, 10, and 12) were
mixed, and the proteins precipitated with glutathione-Sepharose were analyzed
(lanes 512, GST pulldown). Exposure was as in A. The
molecular mass markers are shown on the right of each panel.
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Cell Culture, DNA Transfection, Immunoprecipitation, and Western
Blotting293T cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum and transfected by the calcium
phosphate method. 48 h after transfection, 293T cells (
2 x
106 cells/6-cm dish) were harvested, washed with phosphate-buffered
saline, and suspended in 400 µl of the extraction buffer (20 mM
Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM EDTA, 0.5% (v/v)
Triton X-100, 100 µg of leupeptin/ml, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 mM dithiothreitol, 1
mM sodium orthovanadate, 10% (v/v) glycerol, and 10 mM
benzamidine). Subsequently, the suspensions were allowed to stand on ice for
30 min and clarified by centrifugation (356,000 x g, 10 min),
and the resulting supernatant was subjected to immunoblot analysis and
immunoprecipitation. For the FLAG-based interaction assay, typically 100 µl
of cell lysate prepared as described above was incubated with 10 µl of
anti-FLAG M2-agarose (Sigma-Aldrich) in the extraction buffer for 2 h at 4
°C. Immunoprecipitates were washed five times with the extraction buffer,
resuspended in 1x Laemmli's sample buffer, and subjected to SDS-PAGE.
The proteins were electrophoretically transferred onto polyvinylidene
difluoride membranes (Immobilon; Millipore Corp.) and incubated with anti-HA
(12CA5) and anti-FLAG (M2) monoclonal antibodies. Subsequently they were
visualized with appropriate secondary antibodies conjugated with horseradish
peroxidase and an ECL+plus Western blotting detection system (Amersham
Biosciences).
Assay for ATP Binding and Hydrolyzing ActivityB3BP-FLAG and
its derivatives were expressed in 293T cells and purified using anti-FLAG
M2-agarose resin as described above. For the ATP binding assay, proteins
attached to the beads were suspended in buffer (20 mM Tris-HCl, pH
7.5, 50 mM NaCl, 5 mM MgCl2, and 0.1% (v/v)
Tween 20) and subjected to UV irradiation in the presence of
-32P-labeled 8-azidoadenosine-5'-triphosphate
(8-azido-ATP). After the washing out of the uncross-linked 8-azido-ATP, the
proteins were separated by SDS-PAGE and visualized by autoradiography or CBB
staining. For the ATP hydrolyzing assay, the proteins were cross-linked with
-32P-labeled 8-azido-ATP and incubated in the presence or
absence of 10 µg of yeast transfer RNA/ml for 60 min at 30 °C in the
buffer described above. After a wash, the proteins were separated by SDS-PAGE,
visualized by autoradiography, and quantified using a Bio Image Analyzer
BAS-2500 (FUJIFILM). The amount of protein subjected to the assay was
confirmed by CBB staining.
Assay for Polynucleotide Kinase ActivityRecombinant
proteins, GST and GST-B3BP(M), or FLAG-tagged B3BP and its derivatives
expressed in 293T cells and absorbed onto anti-FLAG resin were incubated in
buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 5
mM MgCl2) at 37 °C for 1 h in the presence of
-32P-labeled ATP with the following substrates:
single-stranded DNA,
3'OH-GGAATTCAGAATGTTTTAGCCCTCCATGGGCTGCATGTGG-5'OH;
double-stranded DNA, 40 bp annealed with
3'OH-GGAATTCAGAATGTTTTAGCCCTCCATGGGCTGCATGTGG-5'OH and
3'OH-GCTTCATCCACATGCAGCCCAGCGAGGTCTAAAACATTCTG-5'OH, yeast
transfer RNA (Roche Applied Science). The samples were separated on a
denaturing 10% polyacrylamide gel and visualized by autoradiography. The
proteins subjected to the assay were run on a NuPAGE 412% Bis-Tris gel
with SDS-containing MES buffer (Invitrogen) and visualized by CBB
staining.
Assay for Nicking Endonuclease ActivityRecombinant proteins
were incubated in buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and 50
µg of bovine serum albumin/ml) at 37 °C for 2 h with a supercoiled
circular DNA (pEF-BOS) as a substrate. The samples were separated on 1%
agarose gel and visualized by ethidium bromide staining. The proteins
subjected to the assay were run on a NuPAGE 412% Bis-Tris gel with
SDS-containing MES buffer (Invitrogen) and visualized by CBB staining.
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RESULTS
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Identification of B3BP as a Protein Interacting with BCL-3
We sought to identify proteins other than NFKB1 and NFKB2 that interact
specifically with BCL-3. We set up a yeast two-hybrid system with full-length
BCL-3 as bait and screened a GAL4 activation domain-tagged cDNA library
derived from the human spleen. Although full-length BCL-3 activates
transcription substantially in yeast when tethered to DNA via the LexA
DNA-binding domain, a competitive inhibitor of His3p, 3-amino-1,2,4-triazole,
completely abrogated its intrinsic activity, and screening worked properly
under these conditions. From a screen of
6 x 106
colonies, 38 clones grew on the selective medium and showed
-galactosidase activity. One clone had a 4.7-kb insert, and a protein of
corresponding molecular mass (
200 kDa) was detected by anti-GAL4
activation domain antibody in the yeast lysate (data not shown). Subsequent
sequence analysis revealed that it contains a large in-frame ORF, and a data
base search indicated that a portion of its cDNA has been registered as
KIAA1413 with unknown function. We obtained full-length cDNA by screening a
human T cell cDNA library using the 4.7-kb insert as a probe. The largest
clone was isolated and sequenced. It contained a 6626-bp cDNA insert and
extended 132 bp 5' of a Kozak consensus sequence for the predicted start
of translation. The predicted polypeptide specified by the ORF comprised 1770
amino acids, which was calculated to be
200 kDa. Recently, a 374-amino
acid fragment near the C-terminal region of this predicted polypeptide was
identified by yeast two-hybrid screening and reported as N4BP2
(Nedd4 ubiquitin ligase-binding partner 2)
(36), and its full-length
protein was referred to as flN4BP2. It was demonstrated that the corresponding
region specifically associates with Nedd4 in vitro and in
vivo and was ubiquitinated by Nedd4 in vitro. There has been no
biochemical characterization of flN4BP2 so far; therefore we refer to it as
BCL-3-binding protein in this paper. Salient features of B3BP include a
consensus nucleotide-binding site, the Walker A motif, at residues
447454, GLPGSGKS. Homology searches using the BLAST algorithm indicate
that the nucleotide binding motif and its neighboring sequence most resemble
those of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)
of mammals and polynucleotide kinase (PNK) of bacteriophage T4 (PNK domain;
Fig. 1, A and
B), both of which possess 5'-polynucleotide kinase
activity. The other motif shown by the analysis is the Smr domain found in the
C-terminal region of B3BP (Fig. 1,
A and C). The Smr domain has been described as a
highly conserved sequence in the C-terminal region of the bacterial MutS2
family and phylogenetically speculated to be involved in DNA mismatch repair
and meiotic chromosome crossing-over
(30,
31). We also isolated a mouse
homolog of B3BP cDNA that encodes a protein
70% identical to the human
protein (data not shown). Notably, the N- and C-terminal regions of 300 amino
acids containing the PNK and Smr domains, respectively, are more than 88%
identical, suggesting functional conservation of these domains. Furthermore,
in a survey of the Drosophila melanogaster genome data base, we found
a putative ORF, LD21293, that encodes a polypeptide containing both the PNK
and Smr domains at its N and C termini, respectively. However, the region
between these domains exhibits no significant similarity. There is no ORF that
encodes a single polypeptide containing these domains in the C.
elegans or Saccharomyces cerevisiae genome, although stand-alone
ORFs that encode the Smr domain are found in many lineages of eukaryotic and
prokaryotic genomes (30,
31).

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FIG. 1. Sequence and domain organization of B3BP. A, structure of
human B3BP. The ATP-binding motif with homology to those of CNP and T4 PNK and
the Smr domain are shown by black and gray boxes,
respectively. The arrows depict the region that corresponds to the
original cDNA isolated in the two-hybrid screening. B, a sequence
alignment of ATP-binding regions of B3BP, CNP, and T4 PNK. Identical and
similar amino acids are indicated by asterisks and dots,
respectively. C, a sequence alignment of Smr domains identified by
PSI-BLAST searches in eukaryotic species. The asterisks indicate
identical residues. The colons and dots indicate conserved
and semi-conserved substitutions, respectively. The organisms and the protein
accession numbers are indicated.
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B3BP Interacts with BCL-3 in VitroThe interaction between
B3BP and BCL-3 was confirmed in vitro. GST fusion proteins containing
the N-terminal (amino acids 2631; GST-B3BP(N)) or C-terminal (amino
acids 11711770; GST-B3BP(C)) regions of B3BP were expressed in E.
coli and affinity-purified on glutathione-Sepharose beads. These proteins
were then incubated with 35S-labeled BCL-3 translated in rabbit
reticulocyte lysate. After extensive washing, the bound proteins were
separated on an SDS-polyacrylamide gel and visualized by autoradiography. As
shown in Fig. 2A,
BCL-3 bound to the N-terminal portion but not the C-terminal portion of B3BP
(lanes 5 and 6). In a converse experiment, GST-BCL-3 fusion
protein bound to full-length B3BP (lane 7). The coprecipitation of
BCL-3 and GST-BCL-3 is due to homophilic interaction
(37,
38). We used luciferase, which
is unrelated to BCL-3 or B3BP, as a negative control and found that it bound
neither to GST nor to GST fusion proteins.
It has been shown that the ankyrin repeat domain of BCL-3 is important for
the interaction of the homodimer of the NFKB1 p50 subunit
(38). Therefore, we next
examined the involvement of the ankyrin repeat domain in the association with
B3BP (Fig. 2B). Wild
type BCL-3 and the C-terminal deletion mutant, BCL-3(
C), which lacks
the C-terminal portion (amino acids 353437) but retains the entire
ankyrin repeat domain, specifically associated with GST-B3BP(N) (lanes
58). However, another mutant BCL-3(
ank), which lacks a part
of the ankyrin repeat (amino acids 260339) did not bind to GST-B3BP(N)
(lanes 9 and 10), indicating the importance of the ankyrin
repeat domain. BCL-3(
ank) failed to interact with the NFKB1 p50 subunit
as well (data not shown). Although the ankyrin repeat domains of BCL-3 and
NFKB1 and NFKB2 are homologous, no significant association was detected
between NFKB1 and GST-B3BP(N) (lanes 11 and 12).
B3BP Also Interacts with Histone Acetyl Transferase p300/CBP
in Mammalian Cell LysateNext the protein-protein interaction was
investigated in mammalian cells. When HeLa cell lysate that had been
metabolically labeled with 35S was precipitated with GST-B3BP(N), a
specific association with protein of
300 kDa was detected (data not
shown). Further investigation revealed that the protein band contains p300. To
examine interactions with B3BP systemically, FLAG-tagged and HA-tagged
proteins were expressed in 293T cells by transient co-transfection
(Fig. 3). Whole cell extracts
were prepared after 36 h of transfection and mixed with anti-FLAG
antibody-conjugated resin. After extensive washing, the bound proteins were
eluted in 1x Laemmli's sample buffer and subjected to Western blot
analysis using anti-HA antibody. We found that FLAG-tagged full-length B3BP
interacted with not only HA-tagged BCL-3 (lane 8) but also p300
(lane 6) in 293T cells. Moreover, we observed homophilic interaction
with B3BP in 293T cells (lane 7). A converse experiment showed that
FLAG-tagged BCL-3 interacts with HA-tagged full-length B3BP (lane 11)
but not with p300 (lane 10). FLAG-tagged CBP interacted with
HA-tagged B3BP (lane 15) but not with BCL-3 (lane 16), a
similar result as p300.
B3BP Binds and Hydrolyzes ATPBecause B3BP possesses a
domain homologous to PNK, which binds to ATP and hydrolyzes it, we
investigated these activities for B3BP. Expression vectors encoding
FLAG-tagged B3BP (B3BP-FLAG) or its derivatives, B3BP(K/A)-FLAG and
B3BP(K/R)-FLAG in which the Lys residue within the ATP-binding motif was
substituted with Ala and Arg, respectively, were introduced into 293T cells,
and whole cell lysate was prepared at 36 h after transfection. FLAG-tagged
proteins were purified using the anti-FLAG antibody-immobilized beads and
incubated with [
-32P]8-azido-ATP. After cross-linking by UV
irradiation followed by extensive washing to remove unbound ATP, the samples
were eluted and separated on SDS-polyacrylamide gel and visualized by CBB
staining and autoradiography. Fig.
4A clearly shows that wild type B3BP has the ability to
bind ATP and that B3BP(K/A) and B3BP(K/R) exhibit reduced ATP binding,
particularly the K/A mutant. Next we examined ATP-hydrolysis by B3BP.
FLAG-tagged B3BP and B3BP(K/R) were purified and cross-linked with
[
-32P]8-azido-ATP. After extensive washing, the samples were
incubated at 30 °C in the presence or absence of yeast transfer RNA
(ytRNA) and subjected to SDS-PAGE analysis as above. CBB staining of the gel
shows that the amount of B3BP and B3BP(K/R) did not change after the
incubation (Fig. 4B),
and it was confirmed by the densitometric quantification of the gel. However,
the associated 32P at position
of the ATP cross-linked to
B3BP but not to B3BP(K/R) decreased with incubation. Interestingly, the
decrease was accelerated in the presence of ytRNA (lane 3). These
results indicate that B3BP has binding and hydrolyzing activity for ATP that
is augmented in the presence of ytRNA. As with other ATPases, the critical Lys
(Lys453) residue is important for these activities
(39,
40).

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FIG. 4. ATP-binding and hydrolyzing activity of B3BP. A, ATP
binding activity of B3BP. FLAG-tagged B3BP and its derivatives, B3BP(K/A) and
B3BP(K/R), were transiently expressed in 293T cells and adsorbed to anti-FLAG
matrix. An ATP binding assay using [ -32P]8-azido-ATP was
conducted as described under "Experimental Procedures," and the
proteins were run on an SDS-polyacrylamide gel and visualized by
autoradiography and CBB staining. B, ATP-hydrolyzing activity of
B3BP. FLAG-tagged B3BP (lanes 13) and B3BP(K/R) (lanes
46) were subjected to UV cross-linking with
[ -32P]8-azido-ATP and incubated (30 min; lanes 2, 3,
5, and 6) or not (0 min; lanes 1 and 4) in the
presence (lanes 3 and 6) or absence (lanes 1, 2, 4,
and 5) of yeast transfer RNA (ytRNA) ("Experimental
Procedures"). The proteins were separated by SDS-PAGE, stained with CBB,
and then autoradiographed. Radioactivities cross-linked with B3BP were
quantified and normalized relative to the protein amount determined by
densitometric analysis of the CBB staining (bottom panel). We
repeated the experiment and reproduced essentially the same results.
|
|
ATP Binding Activity of B3BP Is Required for the Interaction with BCL-3
and p300 Because the PNK domain coincides with the region required
for association with BCL-3 and p300, we examined the involvement of ATP
binding activity in the protein-protein interaction. FLAG-tagged B3BP,
B3BP(K/A), or B3BP(K/R) was co-expressed with HA-tagged BCL-3 or p300 and
analyzed as in Fig. 3
(Fig. 5A). The
association of B3BP with BCL-3 was impaired by the substitution of
Lys453 with Ala or Arg, the former resulting in a barely detectable
association (lanes 68). Interestingly, the addition of 2
mM ATP to the binding mixture markedly enhanced the association
between B3BP and BCL-3 in vitro
(Fig. 5B). For the
association with p300, Lys453 appears to be indispensable because
the association of p300 with either the K/A or K/R mutant was virtually absent
(lanes 1012). On the other hand, the mutations of
Lys453 exhibited little effect on the homophilic interaction of
B3BP (lanes 1416).
B3BP Possesses 5'-Polynucleotide Kinase
ActivityPrimary sequence alignment of the ATP-binding motif showed
a high level of conservation between B3BP and PNK. We investigated the PNK
activity of B3BP. First, the PNK domain of B3BP (amino acids 394630)
fused to GST (GST-B3BP(M)) was produced in E. coli and purified along
with the control GST. GST-B3BP(M) but not GST efficiently phosphorylated both
40-bp double-stranded DNA with blunt ends and 40-base single-stranded DNA
(Fig. 6A, lanes
4 and 6). Moreover, GST-B3BP(M) phosphorylated yeast transfer
RNA, although less efficiently (lane 8). The phosphorylation of DNA
substrate with T4 PNK using cold ATP prior to the assay totally inhibited the
incorporation of 32P into the substrates by GST-B3BP(M), indicating
that B3BP phosphorylates the 5' hydroxyl group of the substrate (data
not shown). Next we examined full-length B3BP produced in mammalian cells for
the PNK activity. FLAG-tagged B3BP and its derivatives, B3BP(K/A) and
B3BP(K/R), were expressed in 293T cells and purified using anti-FLAG antibody.
The recombinant proteins were subjected to the in vitro PNK assay
(Fig. 6B). Full-length
B3BP phosphorylated the polynucleotide substrates efficiently (lanes 2,
6, and 10); however, the activity of B3BP(K/A) and B3BP(K/R) was
below the detectable level (lanes 3, 4, 7, 8, 11, and 12).
Thus, B3BP has intrinsic 5' PNK activity for both DNA and RNA.

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FIG. 6. Polynucleotide kinase activity of B3BP. A, polynucleotide
kinase activity of ATP-binding domain of B3BP. Protein profiles of the
purified GST (lane 1) and GST-B3BP(M) (lane 2) are shown by
CBB staining. Lane M, size marker. 10 pmol of GST (lanes 3,
5, and 7) and GST-B3BP(M) (lanes 4, 6, and 8)
were subjected to polynucleotide kinase assay as described under
"Experimental Procedures" using 100 pmol of 5'-OH ends of
single-stranded DNA (ssDNA, lanes 3 and 4) or
double-stranded DNA (dsDNA, lanes 5 and 6) and 2.5 µg of
ytRNA (lanes 7 and 8) as a substrate. The polynucleotides
were run on a denaturing 10% polyacrylamide gel and subjected to
autoradiography. B, polynucleotide kinase activity of FLAG-tagged
B3BP. FLAG-tagged B3BP and its derivatives, B3BP(K/A) and B3BP(K/R), were
expressed in 293T cells and purified by anti-FLAG agarose matrix.
Polynucleotide kinase assay was performed as shown in A except the
proteins remained bound to the matrix during the assay. The polynucleotide
substrates were analyzed on a denaturing 10% polyacrylamide gel and subjected
to autoradiography as shown in the upper panel. The bound proteins
were analyzed on a NuPAGE 412% Bis-Tris gel and by CBB staining as
shown in the lower panel. The molecular mass markers are shown on the
left of the panel. Arrow, B3BP and the mutants.
Asterisk and dot, anti-FLAG Ig heavy and light chain,
respectively.
|
|
Smr Domain of B3BP Shows Nicking Endonuclease ActivityThe
Smr domain, which is found in many eukaryotes, is proposed to act as a nicking
endonuclease and participate in gene recombination
(30). Therefore, we
investigated the nicking endonuclease activity of the Smr domain. The Smr
domain of B3BP was expressed in E. coli as GST fusion protein and
purified. After the removal of the GST moiety by protease treatment, the Smr
domain was further purified by ion-exchange chromatography. The purified GST
or Smr domain was incubated with supercoiled circular plasmid DNA, and the
samples were separated by agarose gel electrophoresis.
Fig. 7 shows that the Smr
domain but not GST converted supercoiled DNA into a nicked open circular form
in a dose-dependent manner, and no linear species was detected under these
conditions. Furthermore, linearized plasmid was not detectably affected by
incubation with Smr, suggesting that it does not have exonuclease activity
(data not shown). These results indicate, for the first time, that the Smr
domain of B3BP has nicking endonuclease activity but no significant double
strand cleavage or exonuclease activity.

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FIG. 7. Nicking endonuclease activity of Smr domain of B3BP. The recombinant
GST and Smr domain were expressed and purified as described under
"Experimental Procedures." Protein profiles of the purified GST
(lane 1) and Smr domain (lane 2) are shown by CBB staining.
Lane M, size marker. 15 pmol (lanes 4 and 7), 30
pmol (lanes 5 and 8), and 60 pmol (lanes 6 and
9) of GST (lanes 36) or Smr domain (lanes
79) or buffer alone (lane 3) were incubated with 0.5
µg of supercoiled circular DNA (pEF-BOS, 5.8 kb) as substrate and analyzed
on 1% agarose gel and visualized by ethidium bromide staining as shown in the
right panel. An arrowhead and an asterisk indicate
nick-introduced open circular and supercoiled closed circular DNA molecules,
respectively.
|
|
 |
DISCUSSION
|
---|
In this study we identified B3BP, which associates specifically with BCL-3,
from a yeast two-hybrid screen of a human spleen cDNA library and
characterized it. A previous report has described the isolation of factors
interacting with BCL-3 by yeast two-hybrid screening
(19). However, only NFKB1 was
found in our study, probably because of the different screening methodology
used, e.g. a different bait (full-length versus ankyrin
domain alone) or a different cDNA library.
The amino acid sequence of B3BP revealed the presence of the PNK and Smr
domains near the N- and C-terminal region, respectively. Indeed, we
demonstrated that B3BP is a PNK (Fig.
6). The biological function of mammalian CNP still remains
unclear; however, T4 PNK functions in an RNA repair pathway in collaboration
with a phage-encoded RNA ligase
(41). T4 PNK is the founding
member of the family of bifunctional 5'-kinase/3'-phosphatase
enzymes that heal broken termini in RNA or DNA by converting
3'-PO4/5'-OH ends into
3'-OH/5'-PO4 ends, which are then feasible for sealing
by RNA or DNA ligases (40).
However, B3BP does not show any homology to the 3'-phosphatase domain of
the T4 PNK family. In fact we failed to detect the 3'-phosphatase
activity of full-length B3BP using an oligonucleotide with
3'-PO4 ends as substrate (data not shown). The biological
function of 5'-kinase free of 3'-phosphatase activity is totally
unknown at present; however, it is of note that 5'-phosphate is required
for small interfering RNA to function in the RNA interference pathway
(42). A functional ATP-binding
motif is required for B3BP to associate with BCL-3 and CBP/p300
(Fig. 5); notably the presence
of ATP enhanced the former association. These results suggest that the
assembly of the complex containing B3BP and its enzymatic activity are
coordinately regulated.
A BLAST survey of the genome data base indicates that B3BP could be the
only ORF encoding the Smr domain in humans. Although the biological function
of the Smr domain is totally unknown, the domain is speculated to have a role
in mismatch repair or meiotic chromosome crossing-over and expected to
function as a nicking endonuclease
(30). Indeed, we found that
the Smr domain of B3BP showed nicking endonuclease activity
(Fig. 7). Such activity is
required for various kinds of biological processes, such as the
retrotransposition of self-splicing introns, the excision repair of damaged
DNA, and replication-coupled mismatch repair. It is worth noting that there is
no gene for an eukaryotic homolog of MutH, the nicking endonuclease required
for mismatch repair in E. coli
(30). It is also reported that
some of the mismatch repair proteins are involved in transcription-coupled
excision repair of DNA (43).
Recently it was reported that p300/CBP associates with proliferating cell
nuclear antigen and thymine DNA glycosylase, suggesting a functional coupling
of transcriptional activation to DNA repair synthesis and base mismatch
repair, respectively (44,
45). Therefore, investigations
of the Smr domain and B3BP might help us to understand the mechanisms
underlying eukaryotic mismatch repair and its relation to transcriptional
activation. Nicking endonuclease activity is also supposed to be required for
the recombination of the Ig gene in germinal center B cells, i.e.
class switch recombination and somatic hypermutation, which are coupled with
transcriptional activation of the intronic switch region and Ig gene itself,
respectively (27). Moreover,
studies with gene targeted mice demonstrated that mismatch repair factors are
involved in these processes
(28).
All of the findings observed suggest that B3BP is involved in DNA or RNA
transaction processes, e.g. transcription-coupled DNA repair or
genetic recombination, because (i) B3BP interacts with not only BCL-3 but also
p300/CBP, both of which are involved in gene activation; (ii) B3BP shows
5' polynucleotide kinase activity, which is required for DNA or RNA
repair by converting 5'-OH broken termini into ligation-competent
5'-PO4 ends; and (iii) the C-terminal region of B3BP contains
the Smr domain, which is phylogenically speculated to have a role in mismatch
repair or meiotic recombination and indeed shows nicking endonuclease
activity.
 |
FOOTNOTES
|
---|
* This work was supported by grants from the Research for the Future Program;
the Japan Society for the Promotion of Science; the Ministry of Education,
Science, Sports and Culture of Japan; Nippon Boehringer Ingelheim Co., Ltd.;
and Toray Industries Inc. The costs of publication of this article were
defrayed in part by the payment of page charges. This article must therefore
be hereby marked "advertisement" in accordance with 18
U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Tel./Fax: 81-3-3823-6723; E-mail:
fujita{at}rinshoken.or.jp.
1 The abbreviations used are: Smr, small MutS-related; HA, hemagglutinin;
ORF, open reading frame; B3BP, BCL-3-binding protein; CNP,
2',3'-cyclic nucleotide 3'-phosphodiesterase; PNK,
polynucleotide kinase; GST, glutathione S-transferase; ytRNA, yeast
transfer RNA; CBP, cAMP-responsive element-binding protein; CBB, Coomassie
Brilliant Blue; 8-azido-ATP, 8-azidoadenosine-5'-triphosphate; MES,
2-[N-morpholino]ethanesulfonic acid. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Y. Kimura for critically reading the manuscript.
 |
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