Identification of multiple nuclear export sequences
in Fanconi anemia group A protein that contribute
to CRM1-dependent nuclear export
Miriam Ferrer1, Jose A. Rodrı ´guez1, Ellen A. Spierings1, Johan P. de Winter2,
Giuseppe Giaccone1and Frank A.E. Kruyt1,*
1Department of Medical Oncology and2Department of Clinical Genetics, VU University Medical Center, Amsterdam,
Received January 13, 2005; Revised March 7, 2005; Accepted March 21, 2005
The Fanconi anemia (FA) pathway plays an important role in maintaining genomic stability, and defects in
this pathway cause cancer susceptibility. The FA proteins have been found to function primarily in a nuclear
complex, although a cytoplasmic localization and function for several FA proteins has also been reported. In
this study, we investigated the possibility that FANCA, FANCC and FANCG are subjected to active export out
of the nucleus. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, the
accumulation of epitope-tagged FANCA in the nucleus increased, whereas FANCC was affected to a lesser
extent and FANCG showed no response. CRM1-mediated export of FANCA was further confirmed using
CRM1 cotransfection, which led to a dramatic relocalization of FANCA to the cytoplasm. Five functional
leucine-rich nuclear export sequences (NESs) distributed throughout the FANCA sequence were identified
and characterized using an in vivo export assay. Simultaneous inactivation of three of these NESs resulted
in a discrete but reproducible increase of FANCA nuclear accumulation. However, these NES mutations
did not affect the ability of FANCA to complement the mitomycin C or cisplatin sensitivity of FA-A lympho-
blasts. Surprisingly, mutations in the other two NESs resulted in an almost complete relocation of the protein
to cytoplasm, suggesting that these motifs overlap with domains that are crucial for nuclear import. Taken
together, these findings indicate that FANCA can be actively exported out of the nucleus by CRM1, revealing
a new mechanism to regulate the function of the FA protein complex.
Fanconi anemia (FA) is a genetically heterogeneous disorder
characterized by congenital malformations, bone-marrow
failure and cellular hypersensitivity to cross-linking agents
such as mitomycin C (MMC) and cisplatin (CDDP) (1–3).
Till date, 11 complementation groups have been described
and nine FA genes (FANCA, -B, -C, -D1/BRCA2, -D2, -E,
-F, -G and -L) have been cloned (4–11). FANCA, -C, -E,
-F, -G and -L proteins, as well as the recently cloned
FANCB, form a nuclear multiprotein complex that leads to
the activation of the downstream FANCD2 protein by monou-
biquitination (11–13). Monoubiquitinated FANCD2 is tar-
geted to the sites of DNA damage where it interacts with
BRCA1 and BRCA2 (FANCD1) (8,14,15). As the monoubi-
quitination of FANCD2 is controlled by the FA core
complex, a tight regulation of the core complex itself is criti-
cal. In this regard, recent studies have shown that several sub-
complexes are formed. These subcomplexes localize to
different compartments, and several groups have described
interactions between FA proteins and cytoplasmic com-
ponents, such as proteins involved in the regulation of oxi-
dative stress (16–20). Regardless of the possibility of a role
in the cytoplasm of some of these subcomplexes, they are
known to interact and relocalize to the chromatin during
the S-phase of the cell cycle and upon DNA damage (21–23).
Dynamic nuclear-cytoplasmic trafficking of proteins is a
common regulatory mechanism for many cellular processes
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*To whom correspondence should be addressed at: Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amster-
dam, The Netherlands. Tel: þ31 204443374/1738; Fax: þ31 204443844; Email: email@example.com
Human Molecular Genetics, 2005, Vol. 14, No. 10
Advance Access published on March 24, 2005
by guest on May 31, 2013
such as cell-cycle progression and signal transduction (24). In
fact, several proteins involved in DNA repair pathways and
maintenance of genome integrity, such as BRCA1 and p53,
were recently shown to be regulated, at least in part, by shut-
tling between the nucleus and the cytoplasm (25). The active
transport of molecules across the nuclear envelope is com-
monly mediated by nuclear import and export receptors of
the karyopherin family. Interaction with the transport recep-
tors is usually mediated by specific sequences in the cargo
protein termed nuclear localization signals (NLSs) or nuclear
export signals (NESs) (26). Classical NLSs, such as those in
SV40 large T antigen or nucleoplasmin, are defined as a
short sequence that contains several critical basic amino
acids (27). Typical leucine-rich NESs, such as the NES
found in the HIV Rev protein, consist of motifs containing
large hydrophobic residues (often leucines) with a character-
istic spacing among them (26,28,29). The best-characterized
nuclear export mechanism for proteins is mediated by the
nuclear export receptor CRM1 (chromosome maintenance
region 1/exportin 1) (30,31). CRM1 directly binds to leucine-
rich NESs to translocate the cargo protein through the nuclear
pore from the nucleus to the cytoplasm.
In this study, we sought to find whether nuclear-cytoplasmic
shuttling mechanisms are involved in the regulation of the
subcellular distribution of three FA proteins, FANCA,
FANCC and FANCG. Although FANCA is known to contain
a bipartite NLS and is predominantly localized in the nucleus
(9,12,32–39), we show here that this protein has the ability to
shuttle between the nucleus and the cytoplasm using the
CRM1-mediated nuclear export pathway. Five functional
NESs were identified in FANCA and inactivation of several
of these NESs reduced its CRM1-dependent export. However,
NES-inactivation did not affect FANCA functionality with
respect to the protection against DNA cross-linking agents
in FA-A lymphoblasts.
We can conclude from this study that the regulation of the
subcellular localization of FANCA is a complex process invol-
ving active nuclear import and CRM1-dependent nuclear
export, and that multiple transport elements located in differ-
ent regions of the protein contribute to this process.
FANCA shuttles between the nucleus and the cytoplasm
through CRM1-mediated export
It has been established that the components of the FA core
complex FANCA, FANCC and FANCG proteins localize
mainly in the nucleus, although a small fraction of the proteins
is present in the cytoplasm (12,35,38). In accordance with this,
the localization of Flag-tagged FANCA, FANCC and FANCG
in MCF-7 cells was either nuclear or both nuclear and cyto-
tion of a significant fraction of the FA proteins in the cytoplasm
led ustotestwhetheran active nuclear exportmechanism could
be involved in mediating their cytoplasmic localization.
Treatment with leptomycin B (LMB), a specific inhibitor of
the nuclear export receptor CRM1, prevents the CRM1/NES
interaction and thus induces the accumulation of shuttling
proteins into the nucleus (40). MCF-7 cells transfected with
Flag-tagged versions of FANCA, FANCC and FANCG were
exposed overnight to LMB. Increased nuclear accumulation
of FANCA-Flag and, to a lesser extent, of Flag-FANCC was
observed, whereas LMB did not significantly change the sub-
cellular distribution of FANCG-Flag (Fig. 1B). Similar results
were obtained in HeLa cells (data not shown). To further
characterize the effect of blocking CRM1-mediated export
on the localization of FANCA and FANCC, we carried out
a time course analysis. LMB treatment resulted in a time-
dependent accumulation of FANCA-Flag in the nucleus,
with an increase in the percentage of cells showing nuclear
FANCA staining from 40 to 60% only 2 h after addition of
LMB and reaching a maximum ?80% after 16 h exposure
(Fig. 1C). The nuclear accumulation of Flag-FANCC after
LMB treatment occurred at slower rate and was less pro-
nounced than for FANCA.
As a complementary strategy to LMB treatment, given the
predominantly nuclear steady state localization of the FA
proteins, Flag-tagged FANCA, FANCC and FANCG were
cotransfected with YFP–CRM1 or YFP alone as a negative
control. Overexpression of CRM1 has been shown to
induce nuclear export of NES-containing proteins that are
predominantly localized in the nucleus (41). The localization
of FANCA co-expressed with YFP was similar to that of
FANCA expressed alone (Fig. 2A). However, co-expression
with the export receptor led to a dramatic change in the
nucleocytoplasmic distribution of FANCA with an exclusively
cytoplasmic localization in .90% of transfected cells. In con-
trast, distribution of FANCC (Fig. 2B) and FANCG (data not
shown) did not significantly change upon co-expression with
Taken together, these findings indicate that the CRM1
pathway plays an important role in determining the nucleo-
cytoplasmic localization of FANCA. Therefore, we focused
on further characterizing CRM1-mediated export of FANCA.
Identification of multiple functional leucine-rich NESs
We searched for sequence motifs in FANCA that could
mediate CRM1-dependent export. CRM1 is known to bind
to NESs that are characterized by the presence of leucines
and other large hydrophobic amino acids with a defined
spacing between them (28,42). By visual and computer-
assisted inspection of the FANCA sequence, we identified
nine potential leucine-rich NESs in FANCA (Table 1).
Short FANCA amino acid segments containing each of the
candidate-NESs (cNESs) were tested for export activity, using
an in vivo export assay. Two cNESs located in close proximity
were tested in a single segment. This assay is based on the
ability of functional NESs to restore the nuclear export of
the Rev(1.4)–GFP fusion protein, an NES-deficient mutant
of the HIV Rev protein fused with GFP (43). As expected,
Rev(1.4)–GFP was located in the nucleus, and the positive
control containing Rev-NES was completely localized in
the cytoplasm of MCF-7 cells (Fig. 3A). Similar to the posi-
tive control, insertion of the FANCA cNESs [518–534] and
[1013–1043] resulted in a complete cytoplasmic relocaliza-
tion of Rev(1.4)–GFP protein. The FANCA cNESs [54–80],
1272Human Molecular Genetics, 2005, Vol. 14, No. 10
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relocalization of Rev(1.4)–GFP, although to a lesser extent.
The activity of these NESs was more clearly observed after
treatment of the transfected cells with actinomycin D. Actino-
mycin D prevents Rev nuclear import, thus allowing the detec-
tion of weaker NESs that otherwise cannot overcome the
and [860–880]also induced cytoplasmic
import activity exerted by the strong Rev-NLS (43). As
expected, the nuclear export activity of these five cNESs
was efficiently blocked after LMB addition to the cells, con-
firming thus CRM1 dependence. The other four cNESs did
not show any export activity, as illustrated by FANCA-
cNES [1291–1325] (Fig. 3A and Table 2).
Figure 1. Subcellular localization of Flag-tagged FANCA, FANCC and FANCG in absence or presence of LMB. (A) Immunofluorescence microscopy images of
transfected MCF-7 cells expressing Flag-tagged FANCA, FANCC and FANCG. Cells were counterstained with Hoechst to show the nuclei and arrows indicate
transfected cells. (B) Subcellular distribution of Flag-tagged FA proteins in MCF-7 cells untreated and after overnight treatment with 6 ng/ml leptomycin B
(LMB). The percentage of cells with predominantly nuclear (N), nuclear and cytoplasmic (NC) or predominantly cytoplasmic (C) FA protein localization is
depicted. (C) LMB time-course experiment in MCF-7 cells transfected with FANCA-Flag and Flag-FANCC. The proportion of cells with nuclear (filled
circles), nuclear-cytoplasmic (open circles) and cytoplasmic (inverse filled triangles) staining is depicted. Values represent the mean+SD of three independent
experiments. A minimum of 200 cells were counted per sample.
Human Molecular Genetics, 2005, Vol. 14, No. 101273
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The five functional NESs in FANCA, hereafter named
NES1 to NES5, are evenly distributed throughout the
FANCA sequence (Fig. 3B). The strength of the export
activity of the functional NESs in the nuclear export assay
was established according to a scoring system defined in a
recent comparative study of nuclear export sequences (43).
The FANCA-NES1 (residues 54–80), -NES2 (residues 263–
284) and -NES4 (residues 860–880) were rated as weak
(score 2þ/3þ); whereas the FANCA-NES3 (residues 518–
534) and -NES5 (residues 1013–1043) were rated as strong
Characterization of the sequence requirements of
To further characterize the identified FANCA-NES, mutational
analyses were performed in the context of the nuclear export
assay. The last two hydrophobic residues of the consensus
NES sequences, usually leucines, are generally the most con-
served and most critical for the export activity (44,45). Site-
directed mutagenesis was used to generate alanine substitutions
of these amino acids in the five FANCA-NESs (Fig. 3C).
Mutations in all NESs resulted in a loss of nuclear export
Figure 2. FANCA-Flag is a bona fide CRM1-dependent nuclear shuttling protein. MCF-7 cells were cotransfected with vectors expressing FANCA-Flag (A) or
Flag-FANCC (B) in combination with YFP negative (control) or YFP–CRM1. Subcellular distribution of FA proteins was determined by fluorescence
microscopy and DNA was visualized with Hoechst. Arrows indicate transfected cells. Data represent the mean+SD of three independent experiments. A
minimum of 200 cells were counted per sample.
1274 Human Molecular Genetics, 2005, Vol. 14, No. 10
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activity as evidenced by the localization of the GFP-fusion
proteins in the nucleus of 81–91% of the transfected cells,
even after treatment with actinomycin D (Fig. 3C).
Mutational inactivation of the NESs in the context of
To assess the role of the NESs in the localization of full-length
FANCA, we used site-directed mutagenesis to generate a
series of five constructs each containing inactivating mutations
in one of the FANCA-NESs. These single NES-mutant pro-
teins and wild-type FANCA-Flag were transiently expressed
in MCF-7 cells and their subcellular localization was deter-
mined in the absence or presence of LMB. Figure 4 shows
that inactivation of NES1, NES3 and NES5 had almost no
effect on FANCA distribution. Inhibition of CRM1-export
by LMB resulted in comparable levels of nuclear accumu-
lation of wild-type FANCA and the NES-mutants. Unexpec-
mutants were almost completely excluded from the nucleus
and were retained in the cytoplasm, even after LMB treatment.
Therefore, these mutants appear to be unable to enter the
nucleus (Fig. 4).
Hypothesizing that a more efficient inhibition of FANCA
nuclear export would be achieved by simultaneously inacti-
vating several NESs, double- and triple-NES mutants
FANCA-Flag constructs were generated, and their subcellular
distribution was analyzed. The double-NES mutants FANCA-
NES1/3m-Flag, FANCA-NES1/5m-Flag and FANCA-NES3/
5m-Flag displayed an increase of ?15% in the proportion of
cells with only nuclear staining when compared with wild-
type FANCA (Fig. 5). The combined inactivation of NES1,
NES3 and NES5 (FANCA-NES1/3/5m-Flag) did not further
enhance nuclear localization and still showed some LMB
response, an indication that CRM1-export was not completely
abrogated. In contrast, we noted that the nuclear entry of the
triple mutant was reduced when compared with the double-
NESs mutants, suggesting that multiple mutations may impair
import of the protein.
Inactivation of FANCA-NESs does not affect
complementing activity of FANCA
To determine whether inactivation of FANCA-NESs altered
its function, we used a cross-linking agent complementation
assay to test the ability of the different FANCA-NESmutant-
Flag constructs to correct the MMC sensitivity of FA-A
cells. As shown in Figure 6A, sensitivity of the FA-A cell
line HSC72 to MMC in growth-inhibition assays was cor-
rected by stable transfection with wild-type FANCA. Simi-
larly, stable expression of the triple NES mutant FANCA-
NES1/3/5m-Flag also restored MMC resistance in HSC72
cells. However, as predicted from the almost complete cyto-
plasmic localization of FANCA-NES4m-Flag, no complemen-
tation was observed with this mutant.
The complementing activity of the FANCA-NES mutants in
HSC72 cells was also studied by FACS analysis of PI-stained
cells after treatment with another cross-linking agent, CDDP.
Measurement of CDDP-induced cell death, represented by
cells appearing in the sub-G1 fraction, showed that expression
of both wild-type FANCA and FANCA-NES1/3/5m-Flag cor-
rected for drug sensitivity (Fig. 6B). In line with the results
obtained after MMC treatment, expression of FANCA-
NES4m-Flag did not complement for sensitivity to low con-
centrations of CDDP. Thus, increased nuclear retention of
FANCA by mutation of the identified NESs does not affect
its complementation function.
The subcellular distribution of the FA proteins and their func-
tional role in the different compartments have been somewhat
controversial. However, it has been recently clearly demon-
strated that the FA proteins are involved in homologous
recombination repair of chromosomal double-strand breaks
produced by cross-linking agents in the nucleus (46–49).
Nonetheless, some components of the FA core complex are
also found in the cytoplasmic compartment and, in addition,
it has been recently shown that the subcellular localization
of some core complex components, such as FANCA,
FANCC and FANCG, changes during different stages of the
Table 1. Candidate NES motifs in FANCA
aBold letters mark the ‘core’ residues that fit the NES consensus sequence, and hydrophobic residues have been italicized.
bThis single fragment contains two candidate NESs.
Human Molecular Genetics, 2005, Vol. 14, No. 10 1275
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cell cycle (22,23,50). Up to now, the mechanisms that contri-
bute to the nucleocytoplasmic distribution of FA proteins have
remained poorly characterized.
In this study, we report that FANCA is a nuclear-
cytoplasmic shuttling protein that can actively enter and exit
the nucleus. We provide two lines of evidence indicating
that FANCA can be exported out of the nucleus by a CRM1-
dependent mechanism. First, FANCA showed a rapid nuclear
accumulation after treatment with the CRM1-inhibitor LMB
and secondly, co-expression with CRM1 relocated the major
proportion of FANCA into the cytoplasm. In contrast, neither
FANCC nor FANCG relocated to the cytoplasm when expres-
sed together with CRM1, although a slow and limited response
of FANCC to LMB treatment was noted.
In an attempt to further dissect the mechanisms responsible
for FANCA nuclear export, we performed a comprehensive
search for leucine-rich NESs in FANCA responsible for
the interaction with CRM1. Out of nine candidate-NESs
tested, we identified five functional sequences that function
as autonomous NESs in an in vivo export assay. The presence
of multiple functional NESs in a protein is unusual but not
unprecedented. For example, the colon cancer-associated
protein APC contains multiple NES (51–53), although the
relative contribution of each of these signals to the nuclear
export of APC is not yet fully understood (54). Inactivation
of three of these NESs in FANCA reduced, but did not com-
pletely abolish the nuclear export of the protein. Two potential
mechanisms, not mutually exclusive, may underlie the incom-
plete nuclear accumulation of FANCA after NES inactivation.
The first possibility is that the presence of additional sequences
different from leucine-rich NESs can mediate FANCA nuclear
export, perhaps through an indirect mechanism (e.g. binding to
Figure 3. Identification and characterization of five functional leucine-rich NESs in FANCA. (A) In vivo export assays using nuclear localized Rev(1.4)–GFP
(negative control) and plasmids containing FANCA-candidate leucine-rich NESs to be tested for export activity by fluorescence microscopy. Rev-NES was used
as positive control. The addition of actinomycin D (ActD) facilitates the detection of weak NES motifs. Treatment with LMB confirms the CRM1-dependence of
the functional NESs. The level of export activity was established according to the assay scoring system (43). The five functional NESs were named NES1 to
NES5. (B) Schematic representation of FANCA showing the localization of the five functional NESs. (C) Mutation and inactivation of the FANCA-NESs.
Although only residues located in the ‘NES core’ (sequence that fits consensus NES) are depicted for clarity, the same flanking residues indicated in
Table 1 were cloned. The last two hydrophobic residues from the wild-type sequence ‘wt’ (underlined) were substituted ‘mt’ for alanines (in bold) and
export activity was monitored. The percentage of cells with nuclear (Nuc) staining (average of at least three experiments with ,10% variation) is indicated.
1276 Human Molecular Genetics, 2005, Vol. 14, No. 10
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another NES-containing protein). This hypothesis is supported
by the observation that CRM1 co-expression still leads to the
relocalization of the major part of the NES-inactivated
FANCA proteins (data not shown). The second mechanism
that could explain incomplete nuclear accumulation after
NES-inactivation is that the introduction of multiple mutations
in FANCA may interfere with the ability of the protein to enter
or be retained in the nucleus. In support of this view, we noted
that the LMB response of the multiple NES mutant was
reduced with respect to wild-type FANCA.
The nucleocytoplasmic localization of FANCA is probably
a complex process in which different regions of the protein are
involved. Several regions of FANCA other than the defined
N-terminal bipartite NLS are necessary for the nuclear entry
of the protein (55), and patient-derived mutations outside the
NLS interfering with the nuclear accumulation of FANCA
have been described (56,57). Interestingly, some of these
FANCA mutations are located in close proximity to two of
the weaker NESs identified in this study, NES2 and NES4.
Consistent with a role of these regions in nuclear import, we
found that point mutations in NES2 or NES4 abrogated the
nuclear accumulation of FANCA, even in the absence of a
functional CRM1 pathway.
Nuclear–cytoplasmic shuttling has emerged in the last few
years as an important regulatory mechanism for proteins
involved in DNA damage, such as BRCA1 or p53 (25). In
this study, we show that a similar transport mechanism may
regulate the subcellular distribution of FANCA, one of the
components of the FA core complex, indispensable for the
functionality of the pathway. We propose that nuclear export
mechanisms may be involved in regulation of the formation
of the FA nuclear core complex. In this model, the binding
of FANCA to FANCG, FANCB and/or FANCL induces con-
formational changes and may mask several NESs and/or
expose the NLS, allowing the import of FANCA and attached
proteins into the nucleus. In the nucleus, this complex can bind
to the other FA proteins, which may further block NES
activity and stabilize the nuclear localization of the core
complex. Following the repair of the cross-linker-induced
DNA lesions, the complex will dissociate thereby uncovering
the NESs in FANCA leading to its export out of the nucleus, in
this way avoiding the accumulation of immature FA subcom-
plexes in the nucleus that may interfere with cell-cycle pro-
gression or perhaps additional functions of the FA proteins
in other subcellular compartments. In this model, the presence
of multiple NESs in FANCA allows a fine-tuning of the
Table 2. Subcellular distribution of candidate FANCA-NESs tested in the Rev(1.4)-GFP vector for export assay
Test sequenceUntreated 3 h ActD 3 h LMBActivity
Numbers represent the mean +SD of at least three independent experiments.
aThis single test sequence contains two candidate-NESs.
Human Molecular Genetics, 2005, Vol. 14, No. 101277
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localization of the FA complex that may be required for
optimal functioning of the pathway.
We realize that our data obtained with overexpressed
FANCA can lead to the expression of free uncomplexed
FANCA that cannot enter the nucleus and cover the real
effect of NES-inactivation. Unfortunately, the lack of tools
to perform endogenous mutagenesis studies with FA proteins
makes the use of overexpression systems the best available
tool nowadays to dissect the mechanisms regulating FANCA
localization. Moreover, our attempts to demonstrate the effect
of LMB treatment on the trafficking of the endogenous
FANCA protein unfortunately failed due to technical limit-
ations. On the one hand, the available antibodies against
FANCA do not detect the endogenous protein in immunofluo-
rescence assays and, on the other, we found repeatedly that
LMB treatment negatively affects the quality in various ways
generated nuclear and cytoplasmic cell fractions in different
cell lines, thus hampering conclusive western blotting
Further studies on patient-derived mutations of FANCA that
interfere with the normal subcellular distribution of the protein
could help in understanding the relevance on the nuclear-
cytoplasmic properties of this protein in the context of the
whole core complex.
MATERIALS AND METHODS
Cell culture and cell lines
Cells were grown at 378C in a 5% CO2incubator. Human
MCF-7 breast cancer cells and HeLa cervical carcinoma
cells were grown in DMEM supplemented with 10% heat-
inactivated fetal calf serum (FCS) and antibiotics. HSC72
(FA-A) mutant lymphoblasts were cultured in RPMI 1640
medium supplemented with 10% FCS and antibiotics. Media,
serum and antibiotics were all purchased from Invitrogen
(Invitrogen BV, Breda, The Netherlands). Stable transfectants
derived from HSC72 cells were cultured as the parental cell
lines in medium supplemented with Hygromycin B (Roche
Diagnostics Nederland BV, The Netherlands).
Plasmid construction. The constructs pCDNA3-Flag-FANCC
and pCDNA3-FANCG-Flag have been described elsewhere
Figure 4. Effect of NES-inactivating mutations on the localization of
FANCA-Flag. Immunofluorescence microscopy images (left) of MCF-7
cells transfected for 48 h with constructs encoding wild-type and single
FANCA-NES mutants. Localization of FANCA-NES1m, -NES3m and
-NES5m proteins do not change when compared with wild-type FANCA. In
contrast, NES2m and NES4m are retained in the cytoplasm, even after
LMB treatment. Cells were counterstained with Hoechst to show the nuclei.
Arrows indicate transfected cells. Graphs (right) show the percentage of
cells that display predominantly nuclear (N), nuclear-cytoplasmic (NC) and
predominantly cytoplasmic (C) staining in the absence and presence of
LMB (6 ng/ml for 6 h). Data represent the mean+SD of at least three inde-
pendent experiments. At least 200 cells per experiment were scored using an
Figure 5. Effect of combined NES inactivation on FANCA-Flag localization.
Subcellular distribution of double and triple FANCA-NES mutants in the
absence and presence of LMB (6 ng/ml for 6 h). Graphs show the percentage
of cells that display predominantly nuclear (N), nuclear-cytoplasmic (NC) and
predominantly cytoplasmic (C) staining. Data represent mean +SD of at least
three independent experiments. At least 200 cells per experiment were scored
using an unbiased method.
1278 Human Molecular Genetics, 2005, Vol. 14, No. 10
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(36). The previously described plasmid pCDNA3-FANCA-
Flag (37) was used as a template for PCR amplification of
FANCA DNA sequences encoding candidate NES motifs
and short flanking regions. PCR products were cloned as
BamHI/PinAI fragments into pRev(1.4)–GFP plasmid (43)
(L1028A/L1030A), were generated using a standard PCR-
(M528A/L530A) was generated using the QuikChange-XL
Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA),
FANCA-NESs mutants containing combinations of the differ-
ent single mutants were constructed by replacing the wild-type
DNA fragments with the corresponding mutated fragment. All
constructs were verified by DNA sequencing. Oligonucleotide
sequences and detailed protocols used in cloning and site-
directed mutagenesis are available upon request. The plasmids
pEYFP–C1 (Clontech, Palo Alto, CA, USA) and pEYFP–
CRM1 (58) were used in cotransfection experiments.
For stable transfection and functional analysis, FANCA-
NES1m/3m/5m-Flag and FANCA-NES4m-Flag mutants were
subcloned into the expression vector pCEP4 (Clontech) by
replacing the wild-type fragments of pCEP4-FANCA-Flag
(37) with the fragments containing the corresponding NESs
DNA transfection and LMB treatment
For transient transfections, 2.5 ? 105MCF-7 or HeLa cells
were seeded onto sterile glass coverslips in six-well plates
and transfected with 0.5–1 mg of plasmid DNA using Lipo-
fectamine Plus (Invitrogen BV), according to manufacturer’s
guidelines. When indicated, LMB (LMB, LC Laboratories,
Woburn, MA, USA) was added to the culture medium to a
final concentration of 6 ng/ml and cells were incubated at
378C for the indicated period of time.
Fluorescence microscopy analysis
Transfected cells were fixed with 3.7% formaldehyde in PBS
for 30 min and permeabilized with 0.2% Triton in PBS for
10 min. Following a blocking step with 3% BSA (Sigma,
St Louis, MO, USA) in PBS for 1 h, the anti-Flag M2 mono-
clonal antibody (Stratagene) was diluted 1:350 in blocking
solution and applied for 1 h. After washing with PBS, cells
were incubated with either a FITC-conjugated goat anti-
mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA)
or an Alexa Fluor 594-conjugated goat anti-mouse secondary
antibody (Molecular Probes, Eugene, OR, USA) for 45 min.
Finally, coverslips were mounted onto microscope slides with
Hoechst 33342 (Sigma) was used to counterstain the cell
nuclei. Slides were examined under UV light on an inverted
Leica DMIRB/E fluorescence microscope (Leica Heidelberg,
Heidelberg, Germany). Images (400? magnification) were
collected using Leica Q500MC Quantimet software V01.01
(Leica Cambridge Ltd, Cambridge, UK). To determine the
subcellular localization of epitope-tagged proteins, at least
200 transfected cells per sample were scored after coding
and mixing the slides to ensure unbiased results.
In vivo Rev(1.4)–GFP nuclear export assay
Several candidate NES sequences were identified in FANCA
by visual inspection of the protein sequence or using a
computer program kindly provided by Dr M. Fornerod
(NKI, Amsterdam, The Netherlands). The export activity of
these sequences was tested using the Rev(1.4)–GFP in vivo
nuclear export assay (43). MCF-7 cells were plated in
duplicate and transfected with empty pRev(1.4)–GFP (nega-
tive control) and pRev(1.4)–GFP containing the Rev-NES
(positive control) or the constructs containing each of
the FANCA candidate NESs. At 48 h post-transfection, cells
were treated for 3 h with 10 mg/ml cycloheximide either
alone or plus 5 mg/ml actinomycin D (Sigma). Cycloheximide
treatment ensures that cytoplasmic GFP arises from nuclear
export and not from newly synthesized protein, whereas
Figure 6. Increased nuclear retention of FANCA-NES1/3/5m-Flag does not
affect its ability to restore MMC or CDDP resistance in FA-A cells. MMC
growth inhibition assays (A) and CDDP PI-staining assays (B) of FA-A
HSC72 cells stably transfected with FANCA-Flag and derived NES
mutants. FANCA-NES1/3/5m-Flag corrects for MMC hypersensitivity in con-
trast to inactivation of NES4 that disrupts FANCA nuclear import, rendering
the protein inactive in complementing sensitivity. For MMC sensitivity test,
the percentage of surviving cells compared with the untreated control is
depicted. For CDDP sensitivity test, graph represents the percentage of apop-
totic cells after 72 h exposure to 0.2 and 2 mM CDDP. Data shown are repre-
Human Molecular Genetics, 2005, Vol. 14, No. 10 1279
by guest on May 31, 2013
actinomycin D prevents Rev-NLS-mediated nuclear import,
allowing the detection of weaker NESs. The subcellular locali-
zation of the GFP-fusion proteins was determined in at least
200 cells per sample (unbiased counting), and the activity of
the functional NESs was rated according to the scoring
system described by Henderson and Eleftheriou (43).
CRM1-dependence of the functional NESs was further con-
firmed by LMB treatment of the transfected cells (6 ng/ml
for 3 h). Mutated FANCA-NESs were also tested for function-
ality using the in vivo export assay as described for the wild-
MMC and CDDP sensitivity assay
HSC72 (FA-A) lymphoblastoid cells were stably transfected
with pCEP4-FANCA-Flag (wild-type or NES-mutated) by
(BTX, San Diego, CA, USA). Expression of the proteins in
the stable cell lines was confirmed by immunoblotting with
FANCA-specific antibodies (data not shown). The MMC-
induced growth inhibition assays and the CDDP-induced cell
death analysis were performed as described earlier (36, 59).
We are very grateful to B. Meussen for expert technical assis-
tance and to Dr B. Henderson (Westmead Institute for Cancer
Research, Sydney, Australia) for providing the pRev(1.4)–
GFP plasmid. We would also like to thank Dr A. Medhurst
for helpful discussions and technical advice. This work was
supported by The Dutch Organization for Scientific Research
(NWO), Grant VUA 9-02-21-221.
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