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Human Molecular Genetics Advance Access originally published online on March 24, 2005
Human Molecular Genetics 2005 14(10):1271-1281; doi:10.1093/hmg/ddi138
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Identification of multiple nuclear export sequences in Fanconi anemia group A protein that contribute to CRM1-dependent nuclear export

Miriam Ferrer1, Jose A. Rodríguez1, Ellen A. Spierings1, Johan P. de Winter2, Giuseppe Giaccone1 and Frank A.E. Kruyt1,*

1Department of Medical Oncology and 2Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands

* To whom correspondence should be addressed at: Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel: +31 204443374/1738; Fax: +31 204443844; Email: kruyt{at}vumc.nl

Received January 13, 2005; Revised March 7, 2005; Accepted March 21, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 lymphoblasts. 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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) (1Go–3Go). 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 (4Go–11Go). 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 monoubiquitination (11Go–13Go). Monoubiquitinated FANCD2 is targeted to the sites of DNA damage where it interacts with BRCA1 and BRCA2 (FANCD1) (8Go,14Go,15Go). As the monoubiquitination of FANCD2 is controlled by the FA core complex, a tight regulation of the core complex itself is critical. In this regard, recent studies have shown that several subcomplexes are formed. These subcomplexes localize to different compartments, and several groups have described interactions between FA proteins and cytoplasmic components, such as proteins involved in the regulation of oxidative stress (16Go–20Go). 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 (21Go–23Go).

Dynamic nuclear-cytoplasmic trafficking of proteins is a common regulatory mechanism for many cellular processes such as cell-cycle progression and signal transduction (24Go). 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 shuttling between the nucleus and the cytoplasm (25Go). The active transport of molecules across the nuclear envelope is commonly mediated by nuclear import and export receptors of the karyopherin family. Interaction with the transport receptors is usually mediated by specific sequences in the cargo protein termed nuclear localization signals (NLSs) or nuclear export signals (NESs) (26Go). 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 (27Go). 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 characteristic spacing among them (26Go,28Go,29Go). The best-characterized nuclear export mechanism for proteins is mediated by the nuclear export receptor CRM1 (chromosome maintenance region 1/exportin 1) (30Go,31Go). 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 (9Go,12Go,32Go–39Go), 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 involving active nuclear import and CRM1-dependent nuclear export, and that multiple transport elements located in different regions of the protein contribute to this process.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (12Go,35Go,38Go). In accordance with this, the localization of Flag-tagged FANCA, FANCC and FANCG in MCF-7 cells was either nuclear or both nuclear and cytoplasmic in 75–95% of the cells (Fig. 1). The reproducible detection of a significant fraction of the FA proteins in the cytoplasm led us to test whether an active nuclear export mechanism could be involved in mediating their cytoplasmic localization.



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  		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.

 
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 (40Go). 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 subcellular 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 pronounced 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 (41Go). 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 contrast, distribution of FANCC (Fig. 2B) and FANCG (data not shown) did not significantly change upon co-expression with YFP–CRM1.



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  		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.

 
Taken together, these findings indicate that the CRM1 pathway plays an important role in determining the nucleocytoplasmic localization of FANCA. Therefore, we focused on further characterizing CRM1-mediated export of FANCA.

Identification of multiple functional leucine-rich NESs in FANCA
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 (28Go,42Go). By visual and computer-assisted inspection of the FANCA sequence, we identified nine potential leucine-rich NESs in FANCA (Table 1).


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  		Table 1. Candidate NES motifs in FANCA
 
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 (43Go). 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 positive control, insertion of the FANCA cNESs [518–534] and [1013–1043] resulted in a complete cytoplasmic relocalization of Rev(1.4)–GFP protein. The FANCA cNESs [54–80], [263–284] and [860–880] also induced cytoplasmic 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. Actinomycin D prevents Rev nuclear import, thus allowing the detection of weaker NESs that otherwise cannot overcome the import activity exerted by the strong Rev-NLS (43Go). As expected, the nuclear export activity of these five cNESs was efficiently blocked after LMB addition to the cells, confirming 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).





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  		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 (43Go). 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.

 

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  		Table 2. Subcellular distribution of candidate FANCA-NESs tested in the Rev(1.4)-GFP vector for export assay
 
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 (43Go). The FANCA-NES1 (residues 54–80), -NES2 (residues 263–284) and -NES4 (residues 860–880) were rated as weak (score 2+/3+); whereas the FANCA-NES3 (residues 518–534) and -NES5 (residues 1013–1043) were rated as strong (score 9+).

Characterization of the sequence requirements of FANCA-NESs
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 conserved and most critical for the export activity (44Go,45Go). 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 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 full-length FANCA
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 proteins and wild-type FANCA-Flag were transiently expressed in MCF-7 cells and their subcellular localization was determined 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 accumulation of wild-type FANCA and the NES-mutants. Unexpectedly, FANCA-NES2m-Flag and FANCA-NES4m-Flag 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).



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  		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 independent experiments. At least 200 cells per experiment were scored using an unbiased method.

 
Hypothesizing that a more efficient inhibition of FANCA nuclear export would be achieved by simultaneously inactivating 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.



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  		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.

 
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 corrected by stable transfection with wild-type FANCA. Similarly, 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 cytoplasmic localization of FANCA-NES4m-Flag, no complementation was observed with this mutant.



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  		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 contrast 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 apoptotic cells after 72 h exposure to 0.2 and 2 µM CDDP. Data shown are representative experiments.

 
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 corrected 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 concentrations of CDDP. Thus, increased nuclear retention of FANCA by mutation of the identified NESs does not affect its complementation function.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The subcellular distribution of the FA proteins and their functional role in the different compartments have been somewhat controversial. However, it has been recently clearly demonstrated that the FA proteins are involved in homologous recombination repair of chromosomal double-strand breaks produced by cross-linking agents in the nucleus (46Go–49Go). 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 cell cycle (22Go,23Go,50Go). Up to now, the mechanisms that contribute 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 expressed 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 (51Go–53Go), although the relative contribution of each of these signals to the nuclear export of APC is not yet fully understood (54Go). Inactivation of three of these NESs in FANCA reduced, but did not completely abolish the nuclear export of the protein. Two potential mechanisms, not mutually exclusive, may underlie the incomplete 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 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 (55Go), and patient-derived mutations outside the NLS interfering with the nuclear accumulation of FANCA have been described (56Go,57Go). 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 (25Go). 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 conformational 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 subcomplexes in the nucleus that may interfere with cell-cycle progression 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 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 limitations. On the one hand, the available antibodies against FANCA do not detect the endogenous protein in immunofluorescence 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 experiments.

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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture and cell lines
Cells were grown at 37°C in a 5% CO2 incubator. 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 (36Go). The previously described plasmid pCDNA3-FANCA-Flag (37Go) 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 (43Go) (discussed subsequently).

The full-length FANCA-NESs mutants FANCA-NES1m (L72A/L74A), FANCA-NES2m (F276A/L278A), FANCA-NES4m (F868A/F870A) and FANCA-NES5m (L1028A/L1030A), were generated using a standard PCR-based mutagenesis procedure, whereas FANCA-NES3m (M528A/L530A) was generated using the QuikChange-XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA), following manufacturer's instructions. The full-length FANCA-NESs mutants containing combinations of the different 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 (58Go) 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 (37Go) with the fragments containing the corresponding NESs mutants.

DNA transfection and LMB treatment
For transient transfections, 2.5x105 MCF-7 or HeLa cells were seeded onto sterile glass coverslips in six-well plates and transfected with 0.5–1 µg of plasmid DNA using Lipofectamine 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 37°C 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 monoclonal 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 Vectashield (Vector Laboratories, Inc., Burlingame, CA, USA). 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 (400x 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 (43Go). MCF-7 cells were plated in duplicate and transfected with empty pRev(1.4)–GFP (negative 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 µg/ml cycloheximide either alone or plus 5 µg/ml actinomycin D (Sigma). Cycloheximide treatment ensures that cytoplasmic GFP arises from nuclear export and not from newly synthesized protein, whereas actinomycin D prevents Rev-NLS-mediated nuclear import, allowing the detection of weaker NESs. The subcellular localization 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 (43Go). CRM1-dependence of the functional NESs was further confirmed by LMB treatment of the transfected cells (6 ng/ml for 3 h). Mutated FANCA-NESs were also tested for functionality using the in vivo export assay as described for the wild-type sequences.

MMC and CDDP sensitivity assay
HSC72 (FA-A) lymphoblastoid cells were stably transfected with pCEP4-FANCA-Flag (wild-type or NES-mutated) by electroporation using an ECM830 electrosquareporator (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 (36Go,59Go).


   ACKNOWLEDGEMENTS
 
We are very grateful to B. Meussen for expert technical assistance 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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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