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Experimental Cell Research 293 (2004) 14–21

Cytoplasmic mislocalization of BRCA1 caused by cancer-associated

mutations in the BRCT domain

Jose Antonio Rodriguez,1 Wendy W.Y. Au, and Beric R. Henderson*

Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, NSW 2145, Australia

Received 3 June 2003, revised version received 17 September 2003

Abstract

BRCA1 is inactivated by gene mutations in >50% of familial breast and ovarian cancers. BRCA1 is primarily a nuclear protein,

although others previously reported cytoplasmic staining in breast tumor cells. In this study, we demonstrate the cytoplasmic

mislocalization of BRCA1 caused by a subgroup of clinically relevant cancer mutations. We show that mutations that disrupt or delete the

C-terminal BRCT domains, but not other regions of BRCA1, caused significant relocalization of BRCA1 from nucleus to cytoplasm. Two

of the BRCT mutations tested (M1775R and Y1853X) are known to adversely affect BRCA1 protein folding and nuclear function. The

BRCT mutations reduced BRCA1 nuclear import by a mechanism consistent with altered protein folding, as indicated by the restoration

of nuclear staining by more extensive C-terminal deletions. Furthermore, we observed increased cytoplasmic staining of both the ectopic

and endogenous forms of the BRCA1-5382insC mutant (deleted BRCT domain) in HCC1937 breast cancer cells. Unlike wild-type

BRCA1, the BRCA1-5382insC mutant failed to form DNA damage-inducible foci when targeted to the nucleus by BARD1. We propose

that BRCT mutations alter nuclear targeting of BRCA1, and that this may contribute to the inhibition of nuclear DNA repair and

transcription function.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Nuclear transport; Breast cancer; BRCA1 mutations; BRCT domains; DNA repair

Introduction

The BRCA1 tumor suppressor gene is mutated in the

germ-line of women who suffer a genetic predisposition to

breast and ovarian cancer [1,2]. BRCA1 mutations are

found in approximately 50% of patients with inherited

breast cancer, and up to 90% of families with breast and

ovarian cancer susceptibility [1,2]. The BRCA1 protein

comprises 1863 amino acids [1], and has proposed roles

in DNA repair, transcriptional activation, cell cycle regula-

tion and apoptosis [3–5]. BRCA1 has two main types of

protein interaction domains that are frequently targeted by

genetic mutations: an N-terminal RING finger domain and

two tandem BRCT domains at the C-terminal end. The

0014-4827/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2003.09.027

* Corresponding author. Westmead Millennium Institute, Darcy Road,

PO Box 412, Westmead, NSW 2145, Australia. Fax: +61-2-9845-9102.

E-mail address: beric_henderson@wmi.usyd.edu.au (B.R. Henderson).1 Present address: Department ofMedical Oncology, Academic Hospital

Vrije Universiteit Amsterdam, 1081HV, Amsterdam, The Netherlands.

RING domain mediates an enzymatic ubiquitin E3 ligase

activity when BRCA1 forms a stable heterodimer with its

binding partner, BARD1 [6,7]. The C-terminal BRCT

domains are thought to mediate the transcriptional activity

of BRCA1 [8,9]. As a tumor suppressor, inherited mutations

affect certain vital functions of BRCA1. The most frequent

published mutations occur within the BRCT domains and

these have been reported to adversely affect BRCA1 nuclear

functions including DNA repair [10,11] and transcriptional

activity [9,12,13].

Several years ago, Chen et al. [14] published controver-

sial findings which claimed that BRCA1 was mislocalized

almost exclusively to the cytoplasm in breast cancer tissues,

but remained nuclear in normal tissue and in other cancer

cell types. However, this finding was not validated by others

[15], and it was later concluded that antibody specificity was

the main problem, with most agreeing that BRCA1 in

general displayed a ‘‘primarily’’ nuclear localization in

different breast cancer cell lines and in histological breast

tumor tissue slices [16]. Nonetheless, BRCA1 subcellular

localization is well known to vary between nucleus and

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–21 15

cytoplasm in both patient tumor samples [17,18] and in

breast cancer cell lines [19,20]. Our laboratory recently

discovered that BRCA1 is a nuclear-cytoplasmic shuttling

protein, and that its nuclear localization is regulated by the

combined action of nuclear localization (NLS; Refs.

[21,22]) and nuclear export signals (NES; Ref. [23]). In

most cases, however, cellular and ectopically expressed

BRCA1 are primarily nuclear due to nuclear import medi-

Fig. 1. Impact of BRCA1 mutations on nuclear localization. The position of di

known cancer-associated mutations (see right-hand column with reference given)

to published BRCA1 variants [24]. Many of these mutations are known to dis

replication (R), or protein folding (F) as shown in the far-right-hand column (refe

expression plasmids encoding BRCA1 mutants were generated by PCR-based sit

and the ectopic proteins were detected by immunostaining (using monoclonal an

in >200 cells by fluorescence microscopy, and the % cells with nuclear (N), nuc

graphs. Values are mean of two experiments with <20% variation.

ated by the two NLSs and interaction with the RING

domain binding protein, BARD1, which can carry BRCA1

into the nucleus and trap it there by masking its nuclear

export signal [20].

We decided to revisit the question of BRCA1 subcellular

localization, and more specifically to determine whether any

of the more commonly studied gene mutations affect

BRCA1 localization by regulating nuclear import or export.

fferent BRCA1 mutations is shown. The various mutations correspond to

or, in the case of the C-terminal deletions D1751 to D773, are very similar

rupt specific functions such as transcription (T), DNA repair (D), DNA

rence in superscript) (nt, not tested). To test for alterations in localization,

e-directed mutagenesis of wild-type BRCA1, transfected into MCF-7 cells,

tibody Ab-1, Oncogene Research). The subcellular distribution was scored

lear/cytoplasmic (NC), or cytoplasmic (C) staining of BRCA1 is shown in

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–2116

We demonstrate that out of the ten mutations tested, only the

five which target the BRCT domains altered BRCA1

localization, causing it to be excluded from the nucleus.

This nuclear exclusion was not due to increased nuclear

export, but to reduced nuclear import. Similar findings were

observed for both the overexpressed and endogenous forms

of the BRCT mutant, BRCA1(5382insC). Since the BARD-

dependent import pathway [20] is still active for the BRCT

mutants, their overall nuclear entry is reduced but not fully

negated. Our new findings may explain some of the dispar-

ity in the literature concerning BRCA1 localization and

movement.

Results

BRCA1 mutations within the BRCT domain cause

cytoplasmic mislocalization

To assess the impact of BRCA1 cancer mutations on

subcellular localization, we first used PCR-based muta-

genesis to create a series of single-site or truncated

BRCA1 mutant cDNAs corresponding to naturally occur-

ring breast and ovarian cancer-linked mutations [24–27].

These mutant BRCA1 cDNAs were cloned into the

mammalian expression vector, pFLAG-CMV2 (see Ref.

[23]), for transfection and expression in human breast

cancer cells. As summarized in Fig. 1, many of the

Fig. 2. Effect of blocking BRCA1 nuclear export on the localization of BRCA1 mu

MCF-7 cells showing predominantly nuclear (N), nuclear/cytoplasmic (NC), or cy

CRM1-dependent nuclear export. BRCA1 nuclear export was blocked by LMB tr

nuclear export signal (Leu86Ala/Ile90Ala, see Ref. [23]). Bars represent the mean

of transfected MCF-7 cells expressing each of the BRCA1 variants bearing an ac

BRCA1 mutations selected for testing were previously

reported to disrupt BRCA1 nuclear transcription

[8,9,28,29], DNA repair [10,11,30] or DNA replication

[31] activities, and some affect folding of the BRCA1

carboxy terminus [32].

When transiently expressed in MCF-7 cells and exam-

ined for localization by fluorescence microscopy, most

BRCA1 mutants displayed an altered subcellular distribu-

tion compared to that of wild-type BRCA1 (Fig. 1).

Interestingly, we tested a single point mutation (R507I)

that lies within the first BRCA1 nuclear localization signal

(503-KRKRRP, Ref. [21]), but this had only a very minor

effect on nuclear localization. By contrast, five different

BRCA1 constructs (P1749R, M1775R, Y1853X, 5382insC

and D1751) that contain single amino acid mutations or

short deletions (including removal of only the last 11 amino

acids in Y1853X) within the C-terminal tandem BRCT

domains, shifted BRCA1 from the nucleus to the cytoplasm

(Fig. 1). Similar results were observed in T47D breast

cancer cells, for both untagged and Yellow Fluorescent

Protein (YFP)-tagged versions of these constructs (data not

shown). Two of the C-terminal mutations (M1775R and

Y1853X) that restricted nuclear localization are identical to

mutations that disrupt BRCA1 C-terminal folding [32] (Fig.

1), suggesting that the conformational changes they elicit

might be deleterious to BRCA1 nuclear transport. Indeed,

the cytoplasmic mislocalization was specific to changes in

the BRCT region, as more extensive C-terminal deletions

tants C61G, D1751 and Y1853X. Graphs show the proportion of transfected

toplasmic (C) BRCA1 (wild-type or mutant) in the presence or absence of

eatment (6 ng/ml for 12 h) or by introducing an inactivating mutation in the

F SE of at least two experiments. Panels show confocal microscopy images

tive (NESwt) or inactive (NESmut) export signal.

Fig. 3. Nuclear-cytoplasmic distribution of endogenous BRCA1(5382insC).

Subcellular localization of the endogenous BRCA1(5382insC) mutant in

HCC1937 cells was determined by two approaches. (A) Immunofluor-

escence microscopy using two-well characterized primary monoclonal

antibodies, Ab1 (binds N terminus) and Ab4 (binds exon 11) (Oncogene

Research), and a fluorescein anti-mouse secondary antibody. (B) Western

blot analysis of full-length BRCA1 using Ab4, where the nuclear/

cytoplasmic (N/C) ratio was determined by densitometry. Reprobing the

blot with anti-Topoisomerase II antibody provided a fractionation control.

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–21 17

had the opposite effect and caused a progressive increase in

the nuclear localization of BRCA1 (Fig. 1).

BRCT mutations reduce BRCA1 nuclear import

BRCA1 is a nuclear-cytoplasmic shuttling protein [23],

and contains sequences that facilitate both nuclear import

(NLS) and export (NES; reviewed in Ref. [33]). Conse-

quently, the increased cytoplasmic localization of BRCT-

targeted mutations could result from either a blockage to

nuclear import, or from enhanced nuclear export. To

distinguish between these possibilities, we evaluated the

change in localization of different BRCA1 mutants when

nuclear export was blocked. Inhibition of CRM1-depen-

dent nuclear export was achieved by treatment with the

export inhibitor leptomycin B (LMB; see Ref. [23]), or by

the introduction of a site-directed mutation into the

BRCA1 nuclear export signal (as first described for

wild-type BRCA1 in Ref. [23]). We found that blocking

nuclear export by either method caused wild-type BRCA1

to shift even more prominently to the nucleus of trans-

fected MCF-7 cells (Fig. 2). A similar nuclear shift was

observed for the N-terminal RING mutation, C61G. In

contrast, the BRCA1 C-terminal mutants D1751 and

Y1853X remained trapped in the cytoplasm, even when

nuclear export was inhibited (Fig. 2). These novel findings

reveal that the nuclear import of the BRCT mutants is

severely impaired.

A BRCT mutation affects nuclear localization of

endogenous BRCA1

Currently there is only one well-studied cell line,

HCC1937 [34], reported to express a form of BRCA1

mutated within the C-terminal BRCT domain. We exam-

ined mutant BRCA1 localization in HCC1937 cells by

immunofluorescence microscopy and by Western blotting

of fractionated extracts. Consistent with our transient

expression data, the localization of endogenous BRCA1-

5382insC was significantly more cytoplasmic than the

wild-type BRCA1 expressed in HBL-100 cells (Fig. 3).

Unlike the ectopically expressed 5382insC mutant, how-

ever, which showed partial nuclear staining in only 30% of

transfected cells, the endogenous mutant displayed partial

nuclear staining in most cells. This may partly reflect its

association with endogenous BARD1, given that BARD1

can act as a BRCA1 nuclear chaperone independent of the

BRCA1 NLSs [20]. In support of this possibility, we found

that co-expression of a BARD1 expression plasmid caused

ectopically expressed BRCA1(5382insC) to shift strongly

into the nucleus (Fig. 4A), as recently described for wild-

type BRCA1 [20]. These observations indicate that

BRCA1 nuclear import is reduced, but not abolished, by

BRCT mutations, and are consistent with the reduced

nuclear accumulation of mutant BRCA1 previously ob-

served in some breast tumors [18].

Nuclear localization of BRCT mutants is not increased by

proteasome blockade

Some BRCT mutants are known to display altered folding

and turnover [32]. To exclude the possibility that reduced

nuclear staining of these mutants was due to degradation in

the nuclear compartment, we treated transfected MCF-7 cells

with the proteasome inhibitor MG132, and compared the

distribution of wild-type or mutant BRCA1. As shown in

Fig. 4, using a concentration of MG132 effective in stabi-

lizing endogenous beta-catenin (data not shown), we ob-

served a general increase in YFP-BRCA1 staining but did

not observe a strong increase in nuclear staining of the BRCT

Fig. 4. Cytoplasmic localization of BRCA1 C-terminal mutants is not due to nuclear degradation. MCF-7 cells were transfected with wild-type or mutant

BRCA1 plasmids and treated for 16 h with 20 AM MG132 (a proteasome inhibitor) before fixation and mounting. The subcellular distribution of the YFP

fusions was scored (shown for wild-type and 5382insC mutant) and found to be very similar to that of untreated cells (see Fig. 1).

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–2118

mutants. The same result was seen in three separate experi-

ments. This suggests that the cytoplasmic localization ob-

served is due more to reduced nuclear import than to nuclear

degradation of BRCA1.

BRCT mutations disrupt BRCA1 intranuclear localization

Within the nucleus, cellular BRCA1 co-localizes with

BARD1 and specific DNA repair factors in S-phase-associ-

ated dots [35,36] and redistributes into DNA repair-associat-

ed nuclear foci following DNA damage [37]. DNA damage-

induced foci are presumed to be intranuclear sites for DNA

repair [11,37]. Unlike wild-type BRCA1, the cellular form of

BRCA1(5382insC) expressed in HCC1937 cells does not

form nuclear foci before [10] or after DNA damage [38].

However, this observation has not yet been validated by

transfection experiments using the exogenous form of the

mutant, presumably because the transiently expressed mutant

protein displays such poor nuclear staining (Fig. 1). To

circumvent this problem, we co-expressed the BRCA1(5382-

insC) mutant with BARD1 to ensure its nuclear entry (see

Fig. 5A), and because BARD1 promotes recruitment of

BRCA1 to nuclear foci [20].

YFP fusions of wild-type and 5382insC mutant BRCA1

were used to distinguish the ectopic protein from endoge-

nous BRCA1. When co-expressed in asynchronous MCF-7

cells, both ectopic BRCA1 and BARD1 co-localized in

distinct ‘‘S-phase’’ dots, and following treatment with the

DNA damaging agent, methyl methanesulfonate, redistrib-

uted to form many discrete foci as previously observed for

cellular BRCA1 and BARD1 [35–37]. We scored the

proportion of BRCA1-positive nuclei that displayed foci

before and after DNA damage, and found that wild-type

BRCA1 formed >10 foci in over 40% of transfected cells

(Fig. 5B). In striking contrast, the 5382insC mutant dis-

played a severe loss in its ability to form nuclear foci, even

when targeted to the nucleus by BARD1 (Fig. 5B). This is

the first direct demonstration that BRCT mutations adversely

affect both BRCA1 nuclear localization and recruitment to

DNA repair-associated nuclear foci.

Discussion

In this study, we identified a specific subset of C-

terminal mutations (those localized to the BRCT domain)

that cause the partial or near-complete nuclear exclusion of

BRCA1. Similar findings were made for both transiently

expressed and endogenous BRCA1. The observed cyto-

plasmic mislocalization may partly explain why BRCT

mutations reduce BRCA1 nuclear DNA repair [10,11],

replication [27] and transcriptional activities [8,9,28,29].

Fig. 5. BRCA1(5382insC) can be targeted to the nucleus by BARD1 but

does not form nuclear foci. (A) Nuclear entry of the C-terminal cancer

mutant (5382insC) was stimulated by BARD1 co-expression in MCF-7

cells (as described in Ref. [20] for wild-type BRCA1). Staining of

transiently expressed BRCA1(5382insC) and BARD1 is described in

Methods. (B) Comparison of BRCA1 nuclear focus formation. YFP

fusions of BRCA1 were co-transfected with a BARD1 expression vector

into MCF-7 cells, and BRCA1 nuclear foci were then scored before or

after a 1-h treatment with 0.01% MMS. The proportion of cells displaying

<10 or >10 BRCA1 nuclear foci was determined by fluorescence

microscopy (100� oil immersion objective) and graphed. Values shown

are mean F SE of two independent experiments scoring about 100

BRCA1-positive cells per sample. Representative cell images were

collected using a SPOT digital camera and are shown in the bottom

panel. Of the images shown for wild-type BRCA1, �MMS images are

typical of those with 1–10 foci, whereas +MMS images shown are typical

of DNA damaged cells with >10 foci.

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–21 19

Significantly, even when the mutant BRCA1(5382insC)

was forced into the nucleus by the co-expression of

BARD1, it differed from wild-type BRCA1 in that it was

not recruited to nuclear foci which are thought to be the

sites of DNA repair. Therefore, altered intranuclear traf-

ficking may further contribute to the reduced DNA repair

activity of BRCT-mutated forms of BRCA1 [10,11].

Our findings predict enhanced cytoplasmic staining of

BRCA1 in familial breast cancers that carry similar BRCT

mutations. There are a few published immunohistochemical

reports observing increased cytoplasmic BRCA1 in tumors

from patients with BRCA1 germ-line mutations (e.g. Refs.

[14,17,18,39]); however, most have lacked suitable controls

for antibody cross-reactivity and/or classification of the

mutations involved. Furthermore, it is important to distin-

guish between full-length BRCA1 and the exon 11-splice

variant form, as we have done here by using the exon 11-

specific antibody Ab4 (see Fig. 3), because the shorter

splice variants often lack the NLSs and are therefore

intrinsically more cytoplasmic.

The abnormal subcellular distribution of the BRCT

mutants correlates with their ability to disrupt BRCA1 C-

terminal folding [32]. Interestingly, some BRCT mutations

are known to reduce BRCA1 expression in tumors [18], and

we also observed a decreased expression of these mutants in

transient assays. The expression level of both wild-type and

mutant BRCA1 was moderately increased following treat-

ment with the proteasome inhibitor, MG132. However, the

BRCT mutants did not relocalize back to the nucleus in

MG132-treated cells (Fig. 4), suggesting that their misloc-

alization does not reflect nuclear degradation.

It is important to note that the cytoplasmic mislocaliza-

tion observed was specific to changes in the BRCT region,

and that more extensive deletions of the carboxyl terminus

actually had the opposite effect, causing a progressive

increase in the nuclear localization of BRCA1 (Fig. 1).

We interpret these findings to suggest that C-terminal

deletion beyond the tandem BRCT repeats, which pack

together in a defined head-to-tail arrangement [32], at least

partially restores correct folding in the remainder of the

protein including accessibility of the central nuclear local-

ization signals. Put more simply, we propose that BRCT

mutations impair accessibility and function of the BRCA1

NLS. Indeed, the strong cytoplasmic shift caused by the

BRCT mutations Y1853X, 5382insC and D1751 was

similar to that previously observed in the same system

for NLS-mutated BRCA1 [20]. The mutations did not

seem to prevent the alternative RING/BARD1-dependent

nuclear import pathway [20], as co-expression of BARD1

caused almost complete relocalization of mutant BRCA1 to

the nucleus. In conclusion, it will be of interest to deter-

mine whether the altered localization of BRCT mutants

results in a gain of function, especially given that BRCA1

has previously been shown to associate with centrosomes

[40].

Methods

Cell culture and transfection

MCF-7, T47D human breast cancer cells and HBL100

immortalized human breast epithelial cells were maintained

J.A. Rodriguez et al. / Experimental Cell Research 293 (2004) 14–2120

in Dulbecco’s modified Eagle’s media (DMEM) supple-

mented with 10% fetal calf serum (FCS). HCC1937 cells

were grown in RPMI and 10% FCS. All cells were grown at

37jC in a humidified 5% CO2 atmosphere. Cells were

seeded onto sterile glass coverslips and transfected at 50–

60% confluency with 1–2 Ag of plasmid DNA using Lip-

ofectamine Reagent (Life Technologies) according to the

manufacturer’s instructions. At 6 h post-transfection, the

transfection mix was removed and replaced with DMEM

containing 10% FCS. Cells were fixed and processed 30

h post-transfection for fluorescence microscopy. When re-

quired, transfected cells were treated with LMB at a final

concentration of 6 ng/ml for 12 h before fixation.

Plasmid construction

A series of mutations, most of which correspond exactly

to published cancer-linked mutations, were introduced into

the BRCA1 cDNAwithin the expression vector pF-BRCA1

[23]. The C61G mutant was previously described [20]. The

introduction of all other point mutations and truncating

mutations involved a combination of PCR mutagenesis

and cloning, and specific details of the individual cloning

strategies and primer sequences are available upon request.

Immunofluorescence microscopy

Immunostaining was carried out as described [20]. Cells

expressing YFP-tagged proteins were fixed in 3.7% forma-

lin/PBS for 15 min at room temperature, washed and then

mounted for direct detection of the autofluorescent protein.

Untagged ectopic BRCA1 was detected by immunofluores-

cence using monoclonal antibodies Ab-1 and Ab-4 (Onco-

gene Research), which recognise an epitope in the amino

terminus and the central portion of BRCA1, respectively.

BRCA1-bound antibody was detected with fluorescein iso-

thiocyanate-conjugated goat anti-mouse secondary antibody

(Sigma). BARD1 was detected with polyclonal antibody

699D [20] diluted 1:800 in blocking solution, followed by

biotin-conjugated secondary antibodies (Santa Cruz) and

Texas Red–avidin D (Vector Laboratories). Cell nuclei were

counterstained with the chromosome dye Hoechst 33285

(Sigma). The subcellular localization of each ectopic protein

was determined by scoring cells using an Olympus BX40

epifluorescence microscope, and the proportion of cells

displaying nuclear foci was determined by scoring nuclei

using a 100� objective. Digital images were collected using

a SPOT camera or an Optiscan confocal microscope.

Cell fractionation and Western blotting

HCC1937 and HBL100 cells were separated into nuclear

and cytoplasmic fractions using the NE-PER extraction kit

(Pierce) according to the manufacturer’s instructions. Pro-

tein concentrations in nuclear and cytoplasmic fractions

were determined with the Bio-Rad dye-binding assay. Cell

extracts were denatured, separated by 8% SDS-polyacryl-

amide gel electrophoresis and transferred to PVDF mem-

branes. The Western blot filters were blocked in blocking

buffer (1% fetal calf serum, 5% dried milk in phosphate-

buffered saline containing 0.1% Tween 20) and probed with

the primary antibody. BRCA1 was detected with either the

monoclonal antibody Ab-1 diluted 1:200 (data not shown)

or Ab-4 diluted 1:200, followed by incubation with the

horseradish peroxidase-conjugated secondary antibody

(1:1000). As a fractionation control, the blot was also

probed with Topoisomerase II antibody Ab1 (Santa Cruz

Biotech). Blotted proteins were visualized using the ECL

detection system (Amersham Pharmacia Biotech). Rainbow

colour markers (Amersham Pharmacia Biotech) were used

as molecular size standards.

Acknowledgments

We thank Dr. Jeff Holt and Richard Baer for providing

BRCA1 and BARD1 plasmids and antibodies. This work

was supported in part by grants to BRH from the National

Health and Medical Research Council of Australia and the

CureCancer Australia Foundation.

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