From the Department of Molecular Medicine and Institute of Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78245-3207
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ABSTRACT |
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Active transport of proteins into the nucleus is
mediated by interaction between the classical nuclear localization
signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a
fragment of BRCA1 containing the two NLS motifs as a bait for yeast
two-hybrid screening, we have isolated four clones, one of which is
importin . Here we characterize one of the other clones identified,
BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in
human mammary epithelial cells and some but not all tissues of mice.
The isolated full-length cDNA encodes a novel protein containing
600 amino acid residues with pI 6.04. Characteristic motifs of C2H2
zinc fingers and leucine heptad repeats are present in the middle and
C-terminal regions of the protein, respectively. BRAP2 also shares
significant homology with a hypothetical protein from yeast
Saccharomyces cerevisiae, especially in the zinc finger
region. Antibodies prepared against the C-terminal region of BRAP2
fused to glutathione S-transferase specifically recognize a
cellular protein with a molecular size of 68 kDa, consistent with the
size of the in vitro translated protein. Cellular BRAP2 is
mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar
specificity to that of importin
in both two-hybrid assays in yeast
and glutathione S-transferase pull-down assays in
vitro. Other motifs such as the SV40 large T antigen NLS motif
and the bipartite NLS motif found in mitosin are also recognized by
BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS
motifs in vitro. These results imply that BRAP2 may
function as a cytoplasmic retention protein and play a role in
regulating transport of nuclear proteins.
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INTRODUCTION |
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The passage of macromolecules between the nucleus and the
cytoplasm occurs through nuclear pores. Small macromolecules can diffuse through the nuclear pores at a rate inversely proportional to
their mass. Proteins with molecular masses greater than 40-60 kDa are
actively transported through the nuclear pores. To be transported into
the nucleus, the protein must either contain a nuclear localization
signal or, if not, be bound to another protein that does (1, 2). This
process requires at least four different factors acting in two distinct
steps. The first step is mediated by importin (also termed
karyopherin
) and importin
(also termed karyopherin
). The
subunit is primarily responsible for
NLS1 recognition, whereas the
subunit appears to mediate docking to the nuclear pore complex. The
second translocation step requires the small G protein Ran/TC4 and an
interacting partner, p15 (3-13).
The presence of a nuclear localization signal may not be sufficient to direct nuclear import. The target efficiency of NLS motifs can be modified by the presence of multiple NLS motifs within a protein, by modifications of the flanking sequences, and by the accessibility of the NLSs to the import machinery (14-17). These regulatory mechanisms provide for delicate control over the transport of many nuclear proteins. Failure of such control can have profound effects on the cells (18, 19).
Several tumor suppressor genes encode nuclear proteins, the correct nuclear localization of which is critical to their function. For example, the tumor suppressor p53 is ordinarily a nuclear protein. However, wild-type p53 has been shown to be mislocated in the cytoplasm of several different cancer cells, whereas mutant p53 remains in the nucleus (20). Such aberrant localization of wild-type p53 implicates its functional inactivation and reflects its importance in carcinogenesis. Similarly, BRCA1 is also a nuclear protein in normal breast epithelial cells and is mislocated to the cytoplasmic compartment of many advanced breast cancer cells, such as established cell lines, cancer cells from malignant pleural effusions, and some primary breast cancer specimens (21, 22). This suggests that the aberrant subcellular localization of BRCA1 may be responsible for its inactivation in some sporadic breast cancers. Recently, the WT1 tumor suppressor has also been shown to be mislocated in the cytoplasm of breast cancer cells (23).
Previously, we have shown that BRCA1 contains two functional NLS motifs
that direct its transport into the nucleus (24). In an attempt to
elucidate mechanisms that might regulate this process, we used a
fragment of BRCA1 containing both nuclear localization signals as a
bait for a yeast two-hybrid screen. Several interacting proteins were
identified. One of these is importin , which interacts specifically
with the NLS motifs of BRCA1 (24). In this communication, we
characterize BRAP2, which is a cytoplasmic protein that binds to the
two functional NLS motifs of BRCA1. These results suggest that BRAP2
may serve as a cytoplasmic retention protein for regulating nuclear
targeting.
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EXPERIMENTAL PROCEDURES |
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Yeast Two-hybrid Screen and Isolation of Full-length BRAP2 cDNA-- The plasmid pAS-BRCA3.5 (24), which contains the GAL4 DNA-binding domain (25, 26) fused to BRCA1 (amino acids 1-1142), was used as the "bait" for screening a cDNA library prepared from human B lymphocytes as described previously (27). To isolate the full-length BRAP2 cDNA, a 0.8-kb XhoI-digested cDNA fragment derived from the original clone was labeled by random priming and used to screen a fibroblast cDNA library (28). The longest cDNA clones were subcloned into pBSK (Stratagene) and sequenced by the dideoxynucleotide termination method (29). Sequences were analyzed with the computer program DNASTAR (DNASTAR, Madison, WI).
RNA Blotting Analysis--
Total RNA was extracted by the
guanidine isothiocyanate-CsCl method (30). Total RNAs from T47D and
HBL100 cells were then used to prepare poly(A)+ RNA using
the poly(A) Tract mRNA isolation system (Promega, Madison, WI).
About 10 µg of total RNA or 2 µg of poly(A)+ RNA was
denatured in 50% formamide, 2.2 M formaldehyde, 1 × MOPS (0.2 M MOPS (pH 7.0), 0.5 M sodium
acetate, 0.01 M EDTA), and analyzed by 1.2% agarose gel
electrophoresis (30). The RNA was then transferred to Hybond paper
(Amersham, Buckinghamshire, United Kingdom), and immobilized by UV
cross-linking. Prehybridization and hybridization were carried out in
40% formamide, 10% polyethylene glycol 8000, 0.25 M
Na2HPO4/NaH2PO4 (pH
7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS, 100 µg
of salmon sperm DNA per ml. The 32P-labeled 0.8-kb BRAP2
cDNA was used to probe the blot at 42 °C for 18 h. The
1.0-kb G-like cDNA was also used as a probe to serve as a
control for RNA quality. This gene is ubiquitously expressed at a
relative high level in most mouse tissues
(31).2 The initial washing
was done with 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS at room temperature, and the
final washing was done with 0.1× SSC, 0.1% SDS at 65 °C for 30 min.
Plasmid Construction and Expression of Fusion Proteins in
Escherichia coli--
The glutathione S-transferase (GST)
fusion system was used to generate fusion proteins (32). Several
expression plasmids were constructed: GST-BRAP2N (encoding amino acids
12-291); GST-BRAP2C (encoding amino acids 455-570); GST-BRAP2
(encoding amino acids 343-524); GST-importin (encoding amino acids
12-529); GST-T (containing the SV40 large T antigen NLS: PKKKRKV);
GST-mitosin (encoding amino acids 2927-3113); GST-BRCA1Bgl (encoding
amino acids 341-748); GST-BRCA1 (encoding amino acids 762-1315).
Expression of the fusion proteins was induced by addition of
isopropyl-
-D-thiogalactopyranoside to a final
concentration of 0.1 mM in an exponentially growing bacteria culture at 30 °C. After a 3 h incubation, bacteria
were collected and lysed as described previously (31). Fusion proteins were purified with glutathione-agarose beads. Purified proteins were
then used for in vitro binding assays, as described below or
to generate antibody, as described previously (28).
Immunoprecipitation-- To identify endogenous protein synthesized in vivo, about 5 × 106 HBL100 cells were metabolically labeled with [35S]methionine for 2 h and subsequently lysed in ice-cold lysis 250 buffer. The clarified lysate was incubated with anti-BRAP2C antibody at 4 °C for 1 h, then protein A-Sepharose beads were added, and the mixture was incubated for 1 h with constant rotation. After washing extensively with lysis 250 buffer, the beads were boiled in SDS sample buffer, and the immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis. For double immunoprecipitation, the immunoprecipitates were boiled in 200 µl of dissociation buffer (20 mM Tris-Cl (pH 7.4), 50 mM NaCl, 1% SDS, and 5 mM dithiothreitol), and the denatured proteins were diluted with 1 ml of lysis 250 buffer and re-immunoprecipitated with other antibodies.
Cell Fractionation-- The protocols to separate membrane, nuclear, and cytoplasmic fractions were adapted from those previously published (22, 33). All three fractions and total cell lysate were then assayed for p84 and BRAP2 by immunoprecipitation subsequently as described above. The same fractions were also incubated with glutathione-agarose beads, then separated by SDS-polyacrylamide gel electrophoresis, and stained with Coomassie Blue to detect endogenous GST.
In Vitro Binding Assay-- For in vitro transcription and translation of BRCA1, either wild-type BRCA1 containing amino acids 303-701 or the same fragment containing the KLP, KLS, KLN, or KLP + KLS mutations (24) was cloned into the pBSKF vector translationally in frame with the flag epitope. The flag epitope in the vector additionally provides the first methionine for the proteins. For in vitro transcription and translation of importin, the PstI fragment of the importin cDNA (encoding amino acids 64-529) was cloned into the PstI site of pBSKF. For in vitro transcription and translation of the yeast BRAP2 homolog, the full-length yeast BRAP2 cDNA was cloned from yeast genomic DNA by polymerase chain reaction into pBSK under the control of the T3 promoter. Glutathione-Sepharose beads containing about 20 µg of GST or GST fusion proteins were preincubated with Tris-buffered saline-bovine serum albumin buffer (25 mM Tris-HCl (pH 8.0), 120 mM NaCl, 10% bovine serum albumin, 1 µg/ml of protease inhibitors including leupeptin, antipain, aprotinin, and pepstatin) for 30 min at room temperature with rotation. The beads were then incubated with an equal amount of in vitro translated products in standard lysis buffer (100 mM NaCl, 50 mM Tris (pH 7.4), 5 mM EDTA, 0.5% Nonidet P-40, and protease inhibitors) for 1 h at room temperature with rotation. Complexes were washed extensively with the standard lysis buffer, boiled in protein loading buffer, separated by SDS-polyacrylamide gel electrophoresis, and detected by fluorography. Quantitation of binding efficiency was done using the personal densitometer SI (Molecular Dynamics, Sunnyvale, CA).
Transfection and Immunostaining-- Cells were transfected using the standard calcium phosphate precipitation protocol with expression vectors for either green fluorescent protein (GFP) alone, GFP fused to full-length BRAP2, or flag-tagged full-length BRAP2. After transfection, cells were trypsinized and replated on coverslips in a tissue culture dish. 24 h after transfection, the cells were either observed directly for fluoresence under a fluoresence microscope or prepared for immunostaining as follows. The cells were washed in phosphate-buffered saline (PBS) and fixed for 30 min in 4% paraformaldehyde in PBS with 0.5% Triton X-100. After treating with 0.05% saponin in water for 30 min and washing extensively with PBS, cells were blocked in PBS containing 10% normal goat serum. The cells were then incubated with M2 anti-flag monoclonal antibody (Eastman Kodak Co.) for 1 h, followed by three washes with PBS. They were then incubated with Texas red-conjugated secondary anti-mouse antibody (Amersham) for 1 h. After washing extensively in PBS with 0.5% Nonidet P-40, cells were further stained with the DNA specific dye 4, 6-diamidino-2 phenylindole and mounted in Permafluor (Lipshaw-Immunonon, Inc., Pittsburgh, PA).
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RESULTS |
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Expression of BRAP2 mRNA in Breast Epithelial Cells and Adult Mouse Tissue-- As described previously, BRAP2 is the strongest binding clone isolated by a yeast two-hybrid screen for BRCA1-associated proteins (24). To determine the size of the full-length mRNA for BRAP2 and its expression profile in breast epithelial cells and mouse tissues, the 0.8-kb cDNA insert of the original clone was prepared as a probe for RNA hybridization. Initially, poly(A) selected RNA from the breast epithelial cell line, HBL100, and breast cancer cell line, T47D, were used for RNA blotting, and a single 2.0-kb mRNA was detected (Fig. 1A). This suggests that the isolated cDNA contains only a partial sequence of BRAP2 and that it is expressed in breast epithelial cells. The expression pattern of BRAP2 in adult mouse tissues was also examined by the same method. As shown in Fig. 1B, BRAP2 is abundantly expressed in testis and detectable in other tissues such as kidney, lung, liver, and brain. This transcript is expressed at very low levels in spleen, thymus, and small intestine. As with the cell lines analyzed, only a single 2.0-kb mRNA was detected in mouse tissues. The biological significance of the varied expression level in different tissues has yet to be explored.
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Isolation and Sequence Analysis of the Full-length BRAP2 cDNA-- To obtain the full-length cDNA, the 0.8-kb cDNA was used as a probe to screen a human fibroblast cDNA library. Two overlapping clones were isolated, which together defined a cDNA of about 2.0-kb, consistent with the mRNA size. The full-length cDNA was sequenced completely, and the longest open reading frame was found to encode a protein of 600 amino acid residues with pI 6.04 (Fig. 2). A termination codon was found prior to the first initiation codon, indicating that the 5'-coding sequence is within the cloned cDNA. A computer-assisted homology search of GenBankTM at the National Center for Biotechnology Information found the predicted protein to be novel. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats were identified in the middle and C-terminal regions of the protein, respectively. Interestingly, this protein shares significant homology (23.7% overall similarity) with a hypothetical 585 amino acid protein from Saccharomyces cerevisiae (Fig. 3, GenBankTM accession number P38748). In particular, the zinc finger region (amino acids 317-389) shares 62% homology with the yeast protein (amino acids 301-378). These results suggest that BRAP2 encodes a novel protein that is conserved in yeast.
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Identification of Cellular BRAP2 Protein-- To identify the cellular protein encoded by BRAP2, we prepared two specific antibodies that recognize either the N-terminal region (amino acids 12-291) or the C-terminal region (amino acids 455-570) of BRAP2. Purified GST fusion proteins encoding these regions served as antigens for immunizing mice. The antibodies generated were used to immunoprecipitate the in vitro translated, [35S]methionine-labeled BRAP2 protein. As shown in Fig. 4, the anti-BRAP2C antibody, but not pre-immune serum, immunoprecipitates the in vitro translated BRAP2 protein (Fig. 4, lanes 2 and 3). When metabolically [35S]methionine-labeled HBL100 cells were used for immunoprecipitation, anti-BRAP2C antibody recognized a 68-kDa protein that was absent in the immunoprecipitates of the same cell lysates brought down by pre-immune serum (Fig. 4, compare lanes 4 and 5). Depletion of the antibody by preincubation with GST-BRAP2C fusion protein abolished the specific immunoprecipitation, whereas incubation with GST alone had no effect (Fig. 4, lanes 6 and 7). Specificity of the antibody for the 68-kDa protein was further confirmed by reimmunoprecipitation of proteins obtained from the first immunoprecipitates following their recovery by denaturation (Fig. 4, lane 8). The cellular BRAP2 detected by anti-BRAP2C antibody migrates as two major bands, the higher form of BRAP2 may represent a posttranslational modification and is marked by an asterisk (Fig. 4, lane 8). Similar results were also obtained with the other polyclonal antibody, anti-BRAP2N (data not shown). The size of the protein translated in vitro from the full-length cDNA is identical to that of the cellular protein immunoprecipitated by the anti-BRAP2C antibody (Fig. 4, compare lanes 1 and 5), suggesting that the 68-kDa protein is the authentic gene product of BRAP2.
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BRAP2 Is a Cytoplasmic Protein-- To explore its potential function, the subcellular localization of BRAP2 was examined by both biochemical fractionation and epitope-tagged expression. HBL100 cells were biochemically fractionated into nuclei, cytoplasm, and membrane components as described previously (33), and these fractions were immunoblotted with anti-BRAP2N antibody to detect BRAP2. It was found that BRAP2 distributed mainly in the cytoplasm, including membrane and cytosolic fractions, but was not detected in nuclei (Fig. 5A). p84, a nuclear matrix protein (34) and glutathione S-transferase, a cytoplasmic protein (35) served as controls for the fractionation procedure (Fig. 5A). Since the currently available anti-BRAP2 antibodies are not suitable for immunostaining, an alternative method to confirm the biochemical fractionation results was needed. For this purpose, an expression plasmid containing the full-length BRAP2 cDNA fused in-frame with the flag epitope was constructed under the regulation of the CMV immediate early gene promoter (CEPF-BRAP2), permitting detection of exogenous protein using the anti-flag monoclonal antibody M2 (Kodak). By this method, flag-tagged BRAP2 was mainly localized in the cytoplasm when the plasmid was transfected into Saos2 cells (Fig. 5B). Cells transfected with the control plasmid containing the flag-epitope without BRAP2 did not show any staining (data not shown).
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BRAP2 Binds to NLS Motifs of BRCA1--
The idea that BRAP2 may
bind to the NLS motifs of BRCA1 was based on two considerations. First,
the BRCA1 bait used for screening contains two functional NLS motifs (a
third potential NLS motif was found to be nonfunctional). Second, one
of the other interacting clones is importin (24). To test this
hypothesis, either the wild-type BRCA1 cDNA fragment encoding amino
acids 1-1142 (BRCA3.5) or the same region containing one of the three
mutated NLS sequences (KLP, KLS, and KLN) as described previously (24),
was used in a yeast two-hybrid assay with BRAP2 (Fig.
6A). Importin
(amino acids
220-529) and one other BRCA1 associated protein, BRAP12, isolated from
our original two-hybrid screen (24) were used as positive and negative
controls, respectively. Both BRAP2 and importin
bind to BRCA3.5 or
the KLN mutant, but not to the KLP mutant (Fig. 6B).
Previously, we have shown that the NLS deleted in the KLP mutant is
required for nuclear transport of BRCA1. The interaction between
importin-
and the KLS mutant was decreased about 6-fold compared
with the wild-type, whereas BRAP2 interacts well with the KLS mutant
(Fig. 6B). In contrast, BRAP12 binds equally well to the
wild-type and all three mutants.
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BRAP2 Binds to Other NLS Motifs--
To test whether BRAP2 can
bind other kinds of NLS, we tested binding to the NLS motifs from the
SV40 large T antigen (36) and a bipartite NLS from mitosin (37). For
this purpose, the following GST fusion constructs were generated:
GST-T, containing the SV40 large T antigen NLS motif (PKKKRKV);
GST-mitosin, encoding the mitosin C terminus from amino acids 2927 to
3113, which contains a bipartite NLS motif
(KRQRSSGIWENGGGPTPATPESFSKKSKK); GST-BRCA1Bgl,
encoding the BRCA1 BglII fragment from amino acids 341 to
748, which contains both of the functional NLS motifs of BRCA1. Another
GST fusion construct encoding amino acids 762-1315 of BRCA1 (without
functional NLS motifs, GST-BRCA1) was also prepared as a negative
control. The purified GST and GST fusion proteins were used for binding
in vitro translated [35S]methionine-labeled
BRAP2, importin (amino acids 12-529) or the yeast BRAP2 homolog.
As shown in Fig. 8, BRAP2, importin
, and the yeast BRAP2 homolog bind to GST-T, GST-mitosin, and
GST-BRCA1Bgl efficiently (Fig. 8A, lanes 3-5;
Fig. 8B, lanes 3-5; and Fig. 8C,
lanes 3-5, respectively) but not to GST or GST-BRCA1, both lacking NLS motifs (Fig. 8A, lanes 2 and
6; Fig. 8B, lanes 2 and 6;
and Fig. 8C, lanes 2 and 6). These
data suggest that BRAP2 has general affinity for different NLS motifs,
and this function may be conserved since the yeast homolog shows
similar properties.
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DISCUSSION |
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In this paper, we have identified a novel human cytoplasmic
protein, BRAP2, that binds to the NLS motifs of BRCA1. In addition to
recognizing the NLS motifs in BRCA1, BRAP2 binds to other NLS motifs,
such as those found in SV40 large T antigen and mitosin. This suggests
that BRAP2 recognizes NLS motifs in a manner similar to that of the
known NLS receptor, importin . However, unlike importin
, which
cycles between the nucleus and the cytoplasm, BRAP2 is a cytoplasmic
protein. These results raise the possibility that BRAP2 may serve as a
cytoplasmic retention protein involved in the regulation of nuclear
protein transport.
The nuclear transport machinery has been conserved throughout
eukaryotic evolution. Interestingly, human BRAP2 shows significant homology to a hypothetical yeast protein. The highest similarity between these two proteins is in the zinc finger domain, and our preliminary results indicate that deletion of the conserved zinc finger
domain in BRAP2 diminishes its binding activity to the NLS of BRCA1 in
a yeast two-hybrid assay. Consistent with this, we have shown that
yeast BRAP2 has similar binding affinities for the various NLS motifs
as does human BRAP2, suggesting that this function is conserved. Unlike
SRP1, the yeast homolog of importin (38, 39), inactivation of BRAP2
in yeast does not result in a lethal phenotype (Refs. 18 and 19 and
data not shown). This again suggests that the role of BRAP2 may be in
regulating nuclear transport rather than being required to chaperone
proteins into the nucleus as does importin
.
Nuclear transport is a highly regulated process. The presence of a
nuclear localization signal may not be sufficient to direct nuclear
import. The accessibility of NLS motifs to the transport machinery is
crucial for the efficient targeting of nuclear proteins. A cytoplasmic
retention protein can regulate the transport of specific nuclear
proteins by masking their NLS motifs. A paradigm of this control is the
regulation of NF-B subcellular localization by I
B. NF-
B dimers
can bind to target DNA elements and activate transcription of genes
encoding proteins involved in immune or inflammation responses and cell
growth control (see Ref. 40 for review). All members of the NF-
B
family have highly conserved NLS motifs, and the integrity of their NLS
motifs is necessary for their nuclear transport (41, 42). I
B/MAD-3
protein makes direct contact with the NLS residues of both the p50 and
p65 subunits of NF-
B, and sequesters both p65 and p50 subunits of
NF-
B in the cytoplasm (17). Phosphorylation of I
B by protein
kinase C (44) upon stimulation by mitogens or cytokines dissociates I
B from NF-
B, thus allowing the free form of NF-
B to be
transported into the nucleus. Other kinases, such as casein kinase II,
p34cdc2, and protein kinase A have also been reported to be
involved in the regulation of nuclear protein transport (see Ref. 45 for review).
BRCA1 is normally a nuclear protein, and we have previously identified
two NLS motifs within BRCA1 that are required for its targeting to the
nucleus (24). These NLS motifs are recognized by the NLS receptor
subunit, importin . Significantly, mutations in these sequences that
disrupt importin binding also block nuclear transport of BRCA1 (24). In
this report, we have provided data to suggest that BRAP2, a novel
cytoplasmic protein, also interacts with these two functional NLS
motifs of BRCA1. Although BRCA1 is ordinarily targeted to nuclei, we
have consistently found it to be mislocated in the cytoplasm of many
breast cancer cell lines and tumor specimens (22, 24). This
mislocalization appears to be regulatory, rather than due to mutations
in BRCA1 itself, since exogenous flag-tagged wild-type BRCA1 fails to
translocate to the nuclei of breast cancer cells while localizing to
the nuclei of other cell lines (24). Moreover, other nuclear tumor
suppressor proteins such as p53 and, most recently, WT1 have also been
observed to be mislocated in the cytoplasm of breast cancer cells (20, 23). These data suggest that a general mechanism may be responsible for
the mislocalization of these proteins in breast cancer. Such mislocalization of nuclear proteins is likely to be functionally equivalent to their inactivation, consistent with the recessive genetic
behavior of tumor suppressor genes. Whether this mislocalization is a
cause or consequence of tumor progression is a vital question to be
addressed. However, the mechanisms regulating the nuclear import of
these proteins is unknown. Identification of a novel BRCA1-associated
protein, BRAP2, as described here may provide some clues to this
process.
Interestingly, our results also point to the possibility that BRAP2
binds stronger to BRCA1 than does importin . In yeast two-hybrid
assays, the reporter gene activity resulting from the interaction
between BRAP2 and BRCA1 is about 5-fold higher than that of importin
and BRCA1 (Fig. 6B). Likewise, in the GST pull-down assay, 70% of total input BRCA1 was bound to BRAP2 and only 50% of it
was bound to importin
(Fig. 7). These results provide a scenario in
which BRAP2 may retain newly synthesized BRCA1 protein in cytoplasm.
Upon further signaling, such as phosphorylation, BRCA1 will dissociate
from BRAP2 and bind to importin
for transport into the nucleus. It
was noted that BRAP2 recognizes different kinds of NLS motifs (Fig. 8),
and it is quite likely that BRAP2 may not serve specifically to
regulate the transport of BRCA1 alone.
Whether BRAP2 indeed serves as cytoplasmic retention protein in cells remains to be established. This experiment is complicated by the scarce amount of BRCA1 protein and its modification by phosphorylation in cell. Moreover, BRAP2 may bind many cellular proteins with different NLS motifs. Nonetheless, it will be possible to address this question using a reconstituted nuclear transport system as described (43) in the future.
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ACKNOWLEDGEMENTS |
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We thank T.-T. Chen and Diane Jones for their assistance in determining the RNA expression of BRAP2 and for preparation of antiserum against BRAP2. We are also grateful to Drs. Z. Dave Sharp, and Phang-Lang Chen for reading the manuscript and for their inspiring discussions.
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FOOTNOTES |
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* This work was supported by Grants from the National Institutes of Health (P50-CA58183 and P01-CA30195).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) AF035620.
To whom correspondence should be addressed: 15355 Lambda Dr., San
Antonio, TX 78245-3207. Tel.: 210-567-7353; Fax: 210-567-7377; E-mail:
leew{at}uthscsa.edu.
1 The abbreviations used are: NLS, nuclear localization signal; BRCA1, the breast cancer 1 gene product; GST, glutathione S-transferase; PBS, phosphate-buffered saline; GFP, green fluorescent protein.
2 S. Li, C.-Y. Ku, A. A. Farmer, Y.-S. Cong, C.-F. Chen, W.-H. Lee, unpublished data.
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REFERENCES |
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