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Originally published In Press as doi:10.1074/jbc.M300730200 on June 4, 2003

J. Biol. Chem., Vol. 278, Issue 34, 32195-32203, August 22, 2003
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Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1*

Larbi Amazit {ddagger}, Youssef Alj, Rakesh Kumar Tyagi § , Anne Chauchereau ||, Hugues Loosfelt, Christophe Pichon, Jacques Pantel **, Emmanuelle Foulon-Guinchard {ddagger}{ddagger}, Philippe Leclerc §§, Edwin Milgrom and Anne Guiochon-Mantel ¶¶

From the INSERM U135, Hormones, Gènes et Reproduction, IFR Bicêtre, Laboratoire d'Hormonologie et Biologie Moléculaire, AP-HP, Hôpital Bicêtre, 78 rue du Général Leclerc, 94275-Le Kremlin-Bicêtre cedex, France and the §§Institut Fédératif de recherche Bicêtre, 80 rue du Général Leclerc, 94276 Le Kremlin-Bicêtre cedex, France

Received for publication, January 22, 2003 , and in revised form, June 3, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Steroid hormone receptors belong to the superfamily of nuclear receptors (1, 2). Although these proteins are mainly localized in the nucleus, they are constantly shuttling between the nucleus and the cytoplasm (3–5). Their nuclear localization signals (NLS)1 have been characterized as complex amino acid sequences encompassing the DNA binding domain and part of the hinge region in the ligand binding domain (6–11). Nuclear receptors activate transcription by recruiting coactivators and cointegrators (12, 13), which in turn recruit histone acetyltransferases to acetylate histones. Acetylation of the nucleosomes generates a loose chromatin form that allows the assembly of the transcription initiation complex on the target gene promoter (14–16). On the contrary, corepressor binding leads to deacetylation of histones and inhibition of gene transcription (15–18).

Little is known about the subcellular localization and dynamic changes in subcellular distribution of steroid receptor coregulators. Available data are still contradictory, and these variations may be attributed to different cell culture conditions, cell differentiation state, and different detection methods (19–24). Most of these observations were obtained by expressing fluorescent chimeric proteins in live cells (23, 25). The advantage of this approach is to allow in vivo detection of the protein and thus the study of intracellular trafficking dynamics. However, the data obtained with this method need to be substantiated with immunodetection experiments to avoid potential pitfalls due to the fluorescent tag. Indeed, the nature of the fluorescent tag (EGFP, DsRed1, DsRed2, etc.) and its position in the expressed protein (N- or C-terminal) can modify the conformation of the protein and thus interfere with its subcellular localization, trafficking, and transcription function (26).

The present study documents the subcellular localization and the dynamics of intracellular trafficking of the steroid receptor coactivator 1 (SRC-1) as a prototype of nuclear receptors coactivators (27, 28). It belongs to the p160 family of coactivators that also includes SRC-2/GRIP1/TIF2 (29, 30) and SRC-3/AIB1/ACTR/RAC3/TRAM1/p/CIP (31–36). This family of coactivators is characterized by the presence of several conserved functional domains: a bHLH-PAS N-terminal domain, a CBP interacting domain (AD1), a glutamine-rich region, a C-terminal activation domain (AD2), and several LXXLL boxes involved in nuclear receptor binding. Isoforms of SRC-1 have been described, SRC-1a and SRC-1e, which differ in their C-terminal region (37–39). Presently, the minor amount of information on the subcellular localization of SRC-1 is controversial (20, 23, 40). We have used several fluorescent tags for live cell imaging in addition to molecular and immunological approaches. In this report we show that SRC-1 is trafficking between the nucleus and the cytoplasm. We postulate that nucleocytoplasmic trafficking of SRC-1 has a role in modulating the transcriptional activity of PR in particular and steroid receptors in general, thereby controlling molecular events of hormone action.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids: Nomenclature—Plasmid derivatives denoted with the {Delta} symbol lack the coactivator segment delineated by the numbered amino acids. Plasmids encoding the wild-type human SRC-1 (pSG5-SRC-1 and pSG5-HA-SRC-1) and deletion mutants (pSG5-HA-SRC-1{Delta}(1–567), pSG5-HA-SRC-1{Delta}(1–781)) have been previously described (41). Expression vectors pSG5-HA-SRC-1{Delta}(1–1198) and pSG5-HA-SRC-{Delta}(1–177) were obtained by ligation after digestion of pSG5-HA-SRC-1 by NotI/MscI and XmaI/PstI, respectively. Expression vectors pSG5-HA-SRC-1{Delta}(216–1440) and pSG5-HA-SRC-1{Delta}(784–1440) were obtained after insertion of a stop codon in the 5'HindIII and 5'BamHI sites of pSG5-HA-SRC-1, respectively. PCR was used to generate the expression vector for the SRC-1 mutant pSG5-HA-SRC-1{Delta}(785–1038) by inserting a BamH1 site in position 2343 corresponding to the amino acid 782. For the generation of the mutant pSG5-HA-SRC-1{Delta}(988–1440) a PCR fragment was prepared containing BamHI/MscI extremities and a stop codon in the position 2962 corresponding to the amino acid 988. For these two constructs, the deletion was obtained by replacing the wild-type fragment BamHI/MscI by the BamHI/MscI-digested PCR fragment. PCR-based site-directed mutagenesis of pSG5-HA-SRC-1 was used to create deletion mutants: pSG5-HA-{Delta}(18–36)-SRC-1 (named {Delta}(NLS)SRC-1 in the text), pSG5-HA-SRC-1{Delta}(865–876), pSG5-HA-SRC-1{Delta}(948–960), pSG5-HA-SRC-1{Delta}(948–969), pSG5-HA-SRC-1{Delta}(865–876, 948–960), and pSG5-HA-SRC-1{Delta}(990–1060). pSG5-HA-GFP-SRC-1 and pSG5-HA-GFP-{Delta}(NLS)-SRC-1 plasmids were generated by PCR amplification of the EGFP sequence from pEGFP-C1 vector (Clontech, Palo Alto, CA) using the primers 5'-EGFP (5'-ACTGGCGCGCCTATGGTGAGCAAGGGCGAGGA-3') and 3'-EGFP (5'-AGCGCGGCCGCCGGACTTGTACAGCTCGTCCA-3') to create an EGFP encoding fragment with 5'AscI and 3'NotI sites used for subcloning into pSG5-HA-SRC-1 and pSG5-HA-{Delta}(NLS)-SRC-1 vectors, respectively. HA-DsRed-PR was generated by PCR amplification of the DsRed1 sequence from pDsRed1-C1 vector (Clontech) using the primers 5'DsRed (5'-ACTGGCGCTAGCATGGTGCGCTCCTCCAAGAA-3') and 3'DsRed (5'-GATGTCGAATTCGAGCAGGAACAGGTGGTGGC-3') to create a DsRed1 encoding fragment with 5'NheI and 3'EcoRI sites used for subcloning into pSG5-HA-rPR vector, encoding rabbit progesterone receptor. Each construct was verified by sequencing analysis. PR encoding vector and (PRE)2-TATA-CAT reporter plasmids have been previously described (42). pKSV-PR{Delta}(25–103, 547–662) expression vector encodes a cytoplasmic PR mutant (named {Delta}(NLS)PR in the text) and has been previously described (43).

Monoclonal and Polyclonal Anti-SRC-1 Antibodies—Rabbit polyclonal and murine monoclonal antibodies were produced against the N-terminal amino acids (1–361) of the human SRC-1 according to a previously described protocol (44). Two polyclonal antibodies and five monoclonal antibodies (anti-SRC-1 S1 to S5) were generated and analyzed by enzyme-linked immunosorbent assay. The specificity of antibodies was studied as previously described (44), including detection of SRC-1 expressed by transfection of mammalian cells, using Western blot and immunocytochemical techniques. The antibodies generated a very weak signal against the endogenous protein in non-transfected cells. In these studies, we used the higher titer polyclonal antibody (1:4000) and the S4 monoclonal antibody (IgG1K) at the concentration of 5 µg/ml.

Cell Culture—COS-7, CV1, HeLa, and L cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Gaithersburg, MD) containing 10% fetal calf serum (FCS) (Invitrogen), and supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), and antibiotics (Invitrogen). BHK21 cells were grown under the same conditions except for temperature (33 °C) and atmosphere (10% CO2 and 90% air). The generation of mouse L cells stably expressing SRC-1 was performed as described previously (45). For hormonal regulation experiments, cells were grown in the presence of 10% steroid-depleted FCS. Where indicated cycloheximide (CHX) (Sigma) was used at a concentration of 10 µg/ml. This concentration suppressed 95% of protein synthesis (46–48). Progesterone (Sigma) and R5020 (17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione) were used at a concentration of 10 nM. Leptomycin B (LMB) was used at a concentration of 40 nM (43, 49). L cell lines permanently expressing pSG5-HA-SRC-1 and pSG5-GFP-SRC-1 were obtained as previously described (45).

Cell Cycle—BHK21 cells transfected in duplicate with SRC-1-encoding vector were first maintained for 36 h in isoleucine-deficient minimum essential medium supplemented with 5% charcoal-stripped and dialyzed FCS to arrest cells in the G1 phase (50). Cells were then released from cell cycle arrest and switched to complete DMEM containing 1.5 mM hydroxyurea for 12 h to resynchronize the cells in early S phase (51). For G2 synchrony (52), cells were released from hydroxyurea and cultured in DMEM containing Hoechst 33342 (Calbiochem) at 1.5 µg/ml for 12 h. One set of cells was analyzed by immunochemistry (see below), and another was used for cell cycle analysis. Cell cycle analysis was carried out by flow cytometry. Briefly, cells were collected by trypsinization, pelleted, and resuspended in phosphate buffered saline (PBS) to a final concentration of ~106 cells/ml. Two volumes of cold absolute ethanol were added, and the samples were stored at –20 °C until the day of analysis. During analysis, cells were pelleted and resuspended in PBS. These ethanol-fixed cells were incubated in a staining solution containing 50 µg/ml propidium iodide and 100 µg/ml RNase A (Stratagene) in PBS as described previously (53). The cells were analyzed after 1 h of incubation in darkness at 4 °C.

Transfection and Analysis of CAT Activity—Transfections of CV1 cells were performed in 6-well dishes by the standard calcium phosphate precipitation method as described previously (41). CAT activity was measured with the CAT enzyme-linked immunosorbent assay kit (Roche Applied Science). Protein concentrations were determined by using the BCA protein assay (Pierce), and CAT activity was corrected for protein content. The results are the means ± S.E. of three separate experiments.

Immunodetection and Confocal Microscopy—Cells were seeded on glass coverslips placed in 6-well dishes (Costar). Transfections were performed with the indicated expression vectors using LipofectAMINE according to the manufacturer's recommendations (Invitrogen). For GFP-{Delta}(NLS)-SRC-1/DsRed-PR and {Delta}(NLS)-PR/HA-SRC-1 cotransport experiments, the corresponding expression plasmids were transfected at a ratio of 1:5. Cells were fixed and processed as previously described (7). For PR detection, we used the Mi60 monoclonal antibody (54) at a final concentration of 8 µg/ml. Rat monoclonal anti-HA epitope antibody 3F10 (Roche Diagnostic) was used at 2 µg/ml. All secondary antibodies were used at a dilution of 1:400. The conjugated secondary antibodies goat Alexa 488, goat Alexa 546, and goat Alexa 594 (Molecular Probes) and sheep Cy3 (Sigma) were used to detect anti-PR Mi60 monoclonal, anti-SRC-1 polyclonal, anti-HA (3F10) monoclonal, and anti-SRC-1 (S4) monoclonal antibodies, respectively. In some experiments, a nucleic acid marker was added during secondary antibody incubation (SYTOX Green, Molecular Probes) at 200 nM.

The subcellular localization of the coactivator was scored in at least 100 cells under each experimental condition. Staining was considered as nuclear when it was exclusively nuclear (N) or stronger in the nucleus than in the cytoplasm (N > C). In all other cases, it was considered cytoplasmic (if C = N, if C > N and if C). Fluorescent cells were observed and scanned with the LSM410 system (Carl Zeiss, Jena, Germany) assembled on an inverted microscope Axiovert 135M. Digitalized pictures were analyzed by Zeiss LSM application.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SRC-1 Is Localized in Both the Cytoplasm and the Nucleus— We transfected COS-7 cells with an expression vector encoding SRC-1 to examine its subcellular distribution. Immunocytochemical detection (40 h post-transfection) using a specific anti-SRC-1 antibody showed the protein to be present in both the cytoplasm and the nucleus in about 50% of cells (Fig. 1, a–c). In the other cells, labeling was restricted either to the nucleus or to the cytoplasm. In both compartments, SRC-1 was associated with corpuscular structures. Localization of transcriptional regulators in nuclear speckles has previously been described, but the presence of similar structures in the cytoplasm is an unusual observation (55, 56). Furthermore, some detection techniques have been shown to produce artifactual data. Thus, we examined the possibility that our observation might be an artifact due to fixation, immunocytochemistry, or transfection. Identical localization of SRC-1 was observed even when we varied the fixation methods (ethanol, paraformaldehyde, and methanol), antibodies (monoclonal or polyclonal anti-SRC-1 antibodies, anti-HA antibody detection of a HA-SRC-1 construct), cell types (CV1, L, SW13, and C33), and transfection methods (calcium phosphate and LipofectAMINE). The same pattern of localization was also observed in living cells after transfection with GFP-SRC-1 (Fig. 1b). The possibility that overexpression of the protein might be responsible for this pattern has also been considered. However, by varying the amount of the vector (from 1 to 2000 ng), we did not observe difference in the localization of SRC-1 (Fig. 2A). Similar results were also observed with established L cell lines permanently expressing either SRC-1 or GFP-SRC-1 fusion protein (Fig. 2B, panel a).



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  		FIG. 1.
SRC-1 is localized in both the nucleus and the cytoplasm. COS-7 cells were transiently transfected with vectors encoding SRC-1 (a) and GFP-SRC-1 (b). a, after 40 h of expression, the cells were fixed, treated for immunodetection with anti-SRC-1 antibody, and observed by confocal microscopy. b, live cells were directly observed by confocal microscopy. c, subcellular distribution of SRC-1 was scored in at least 100 cells (see "Experimental Procedures"). The results are means ± S.D. of three independent determinations. Bar, 10 µm.

 


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  		FIG. 2.
SRC-1 subcellular localization is not affected by its expression level. A, COS-7 cells were transiently transfected with different amounts of vector encoding SRC-1: 1 ng (a), 5 ng (b), 100 ng (c), 500 ng (d), and 2000 ng (e). Forty hours post-transfection, the cells were fixed, treated for immunodetection with anti-SRC-1 antibody, and observed by confocal microscopy. Bar, 5 µm. B, L cells were fixed, treated for immunodetection, and observed by confocal microscopy. a, L cells permanently expressing SRC-1 were analyzed by immunofluorescence with anti-SRC-1 antibody. b, L cells were treated for immunodetection with a non-specific antibody. c, L cells were treated for immunodetection with anti-SRC-1 antibody. SRC-1 can be either nuclear or nuclear and cytoplasmic in different cells. Bar, 10 µm.

 

Detection of endogenous SRC-1 in non-transfected cells was at the limit of sensitivity of the immunocytochemical method. Diffuse nucleocytoplasmic labeling was observed (Fig. 2B, panel c). Thus, based on our observations, SRC-1 localizes either in the nucleus or in the cytoplasm or in both compartments in different cells.

Nuclear Cytoplasmic Trafficking of SRC-1—We next tried to understand the heterogeneous localization of SRC-1. One possibility was that SRC-1 localization varied in a cell cycle phase-dependent manner. BHK21 cells were transfected with an SRC-1 expression vector and arrested at different stages of the cell cycle (G1, S, and G2/M). Fluorescence-activated cell sorting analysis of the arrested cells failed to demonstrate a correlation between SRC-1 subcellular localization and cell cycle phase (Fig. 3).



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  		FIG. 3.
SRC-1 subcellular localization is not modified during the cell cycle. BHK21 cells transfected with SRC-1 encoding vector were synchronized in G1 and cultured in defined conditions to observe the different cell cycle phases as described under "Experimental Procedures." Cell cycle phases were determined by cytofluorimetry (G1, early S, and G2). At each point, the subcellular localization of SRC-1 was scored by immunodetection as described under "Experimental Procedures." Nuclear labeling: open bars. Cytoplasmic labeling: closed bars. The results are means ± S.D. of three independent determinations.

 

Another possible explanation for the heterogeneity of immunolabeling of SRC-1 was that it reflected a dynamic situation, the protein being localized initially in one of the cellular compartments and secondarily moving to the other. We could thus observe cells at different stages of such a cycle. To investigate this possibility, we transfected COS-7 cells with SRC-1 expression vector and fixed the cells at different time points after transfection (Fig. 4). A short time after transfection (13 h), SRC-1 was exclusively nuclear and concentrated into speckles. With increasing time following transfection (20, 26, and 32 h), a growing proportion of cells with primarily cytoplasmic labeling of SRC-1 was observed (14, 22, and 64%, respectively). Forty hours after transfection, SRC-1 was mostly located in cytoplasmic speckles (78% of cells) (Fig. 4c).



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  		FIG. 4.
SRC-1 initially localizes in the nucleus and secondarily in the cytoplasm. COS-7 cells were transiently transfected with a vector encoding SRC-1 and fixed at different time points after transfection (13, 20, 26, 32, and 40 h). After immunodetection for SRC-1, the cells were observed by confocal microscopy. a, cells were treated for immunodetection with anti-SRC-1 antibody. b, nuclei of corresponding cells shown in a were observed after labeling with SYTOX Green. c, subcellular distribution of SRC-1 was scored in at least 100 cells for each time point (see "Experimental Procedures"). The results are means ± S.D. of three independent determinations. Bar, 5 µm. SRC-1 is localized in the nucleus after 13 h of transfection and begins to appear progressively in the cytoplasm when increasing time after transfection.

 

This result could be explained by two mechanisms. Either SRC-1 is initially localized in the nucleus and thereafter transported into the cytoplasm or the high levels of transfected protein are overwhelming the nuclear transport system at the later time points.

To distinguish between these two possibilities, cells were transfected with an SRC-1 expression vector, treated with cycloheximide to block new protein synthesis 16 h after transfection, and then fixed at different time periods. At the time points that we examined, protein synthesis was blocked in conditions where SRC-1 was nuclear, and thus we could track any changes in its distribution. We found that 40 h after transfection, SRC-1 was almost completely localized in the cytoplasm (Fig. 5). This implied that, after its translation, SRC-1 was imported into the nucleus and consequently exported into the cytoplasm.



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  		FIG. 5.
SRC-1 continues to accumulate in the cytoplasm in the presence of cycloheximide. COS-7 cells were transiently transfected with a vector encoding SRC-1. After 16 h of transfection, the cells were incubated either in the absence (–CHX) or in the presence (+CHX) of cycloheximide (10 µm/ml). The cells were fixed at different time points after transfection (13, 20, 32, and 40 h), processed for immunodetection with anti-SRC-1 antibody, and observed by confocal microscopy. Bar, 5 µm. The presence of cycloheximide does not influence the accumulation of SRC-1 in the cytoplasm. This accumulation is thus due to protein export from the nucleus and not to neosynthesized SRC-1.

 

We used another experimental approach to verify this conclusion. Most proteins that exit the nucleus use the crm1/exportin-dependent pathway, which is specifically inhibited by leptomycin B. If SRC-1 is indeed exported from the nucleus through this pathway, leptomycin B should induce a nuclear accumulation of the protein.

To address this, COS-7 cells were transfected with the GFP-SRC-1 expression vector and cultured in the presence or absence of leptomycin B (see "Experimental Procedures"). The subcellular localization of the fusion protein was observed at different times after transfection (20–40 h). Leptomycin B treatment resulted in an exclusively nuclear localization of SRC-1, irrespective of time. SRC-1 was either homogeneously distributed in the nucleoplasm or found in speckles (Fig. 6A, panels f–i). In contrast, in untreated cells SRC-1 progressively appeared in cytoplasmic speckles (Fig. 6A, panels a–e). Similar results were obtained when SRC-1 (non-GFP construct) was transfected and observed by indirect immunodetection (data not shown). Finally, we attempted to verify if the same phenomenon could be observed in the case of endogenous SRC-1. For this purpose, non-transfected HeLa cells were cultured in the presence or absence of leptomycin B, and SRC-1 subcellular localization was analyzed by indirect immunofluorescence methods using anti-SRC-1 antibodies. In the absence of leptomycin B, endogenous SRC-1 was weakly detectable in both the cytoplasm and the nucleus (Fig. 6B, panel a). Its concentration significantly increased in the nucleus of leptomycin-treated cells and many nuclear speckles could be observed (Fig. 6B, panel b).



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  		FIG. 6.
SRC-1 export is inhibited by leptomycin B. A, GFP-SRC-1 export is inhibited by leptomycin B in transfected cells. COS-7 cells were transfected with a vector encoding GFP-SRC-1. Thirteen hours after transfection, the cells were incubated in the absence (–LMB) or in the presence (+LMB) of leptomycin B (40 nM). The cells were observed after 13 h (a), 20 h (b and f), 26 h (c and g), 32 h (d and h), and 40 h (e and i) of transfection. Bar, 5 µm. B, endogenous SRC-1 export is inhibited by leptomycin B. HeLa cells were incubated for 15 h in the absence (a) or in the presence (b) of leptomycin B (40 nM). The cells were then fixed, and immunodetection was performed with anti-SRC-1 antibody. Bar, 10 µm. In the presence of leptomycin B, SRC-1 is trapped in the nucleus.

 

We conclude that SRC-1 is physiologically directed into the nucleus after its synthesis and is then exported back to the cytoplasm. If nuclear export of SRC-1 is blocked, the protein is concentrated in the nucleus.

Characterization of Nuclear Localization and Nuclear Export Signals in SRC-1—We studied SRC-1 nuclear localization mechanisms by analyzing the subcellular distribution of a series of deletion mutants (Fig. 7A). The deletion of amino acids 1–177 was found to inhibit the nuclear localization of SRC-1 (Fig. 7A). Analysis of the primary sequence shows that the 1–177 region contains a stretch of basic amino acids (aa 18–36) similar to a bipartite NLS (Fig. 7B). Only cytoplasmic localization was observed after deletion of this region in the {Delta}(NLS)-SRC-1 mutant (Fig. 7, A and C, panel b). A similar result was observed in live cells with an EGFP fusion protein containing the {Delta}(NLS)-SRC-1 (Fig. 7C, panel c). Thus, amino acids 18–36 constitute a bipartite signal necessary for SRC-1 nuclear localization. We also observed that {Delta}(NLS) mutants were localized in cytoplasmic speckles even after a short period of transfection (8 h). It is thus not necessary for SRC-1 to pass through the nucleus prior to formation of cytoplasmic speckles.



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  		FIG. 7.
Characterization of SRC-1 nuclear localization signal (NLS). A, determination of SRC-1 nuclear localization signal by the study of cytoplasmic deletion mutants. The wild-type SRC-1, 1440 amino acids in length, is schematically represented at the top. Boxes corresponding to main functional domains are indicated above. AD1 and AD2, activation domains 1 and 2; CID, CBP interacting domain; NRID, nuclear receptor-interacting domain; Q, glutamine-rich domain. *, LXXLL motif. The wild-type SRC-1 and cytoplasmic deletion mutants are represented below with a thick line interrupted by a gap corresponding to the deleted amino acids. B, SRC-1 NLS sequence and comparison with some previously published sequences of bipartite NLS. Basic amino acids are shown in bold. The position of the last amino acid in the sequence of the protein is indicated in exponent. x, Xenopus; h, human. C, subcellular localization of {Delta}(NLS)-SRC-1 mutants. COS-7 cells were transfected with HA-SRC-1 (a), HA-{Delta}(NLS)-SRC-1 (b), or GFP-{Delta}(NLS)-SRC-1 (c) encoding vectors. After 40 h of expression, the cells were fixed and treated for immunodetection with anti HA (a and b) antibody and observed by confocal microscopy. Live cells were directly observed by confocal microscopy (c). Bar, 10 µm. {Delta}(NLS)-SRC-1 shows an exclusively cytoplasmic localization.

 

We then studied the mechanism of nuclear export of SRC-1. Leptomycin B inhibition of SRC-1 nuclear export suggests that crm1/exportin1 pathway is involved and that this export is NES-mediated. Study of subcellular localization of SRC-1 deletion mutants showed that the deletion of amino acids 785–1038 produces an exclusively nuclear protein (Fig. 8A). We examined the amino acid sequence of this region and found two clusters of hydrophobic amino acids homologous to classic leucine-rich NES (aa 865–876 and 948–969) (Fig. 8B). However, when we produced mutants deleted of amino acids 865–876 and 948–960, they still showed nuclear export (Fig. 8A). Because it was possible that these two putative NES were cooperative, we thus combined the two deletions. However, the double mutant deleted for both amino acid sequences 865–876 and 948–960 was still exported to the cytoplasm. In addition, because there are hydrophobic amino acids downstream from amino acid 960 that might be involved in nuclear export, we produced a {Delta}948–969 mutant. However, this mutant did not show any inhibition of nuclear export (Fig. 8A). These sequences, although very homologous to classic NES, are thus non-functional in the context of the full-length SRC-1. Hence, we considered the possibility that SRC-1 nuclear export occurred through one or more signals differing from classic NES. Because the deletion of amino acids 785–1038 produced an exclusively nuclear protein, deletions mutants were constructed to dissect this region. Mutants {Delta}988–1440 as well as {Delta}990–1060 did not show any nuclear export. Overall, these results suggest that the region between amino acids 990 and 1038 is involved in SRC-1 export mechanism.



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  		FIG. 8.
Characterization of SRC-1 region involved in its nuclear export. A, study of nuclear deletion mutants. The wild-type SRC-1 and nuclear deletion mutants are represented below with a thick line interrupted by a gap corresponding to the deleted amino acids. B, putative hydrophobic NES sequences in SRC-1 and comparison with known NES. Conserved leucines (or hydrophobic amino acids) are shown in bold. The consensus sequence derived is shown below. C, subcellular localization of the {Delta}(990–1060)-SRC-1 mutant. COS-7 cells were transfected with the vector encoding {Delta}(990–1060)-SRC-1. After 40 h of expression, the cells were fixed, treated for immunodetection with anti SRC-1 antibody, and observed by confocal microscopy. Bar,10 µm. Mutant {Delta}(990–1060)-SRC-1 localizes exclusively in the nucleus.

 

We conclude that the SRC-1 nuclear localization signal is a classic bipartite signal located in the N-terminal region of the protein. Moreover, the region necessary for SRC-1 nuclear export is present in the C-terminal region of the protein and does not contain any consensus leucine-rich NES. Therefore, this region can either function as a non-classic export signal or as a region of interaction with a carrier protein involved in the export.

Subcellular Localization of SRC-1: Effect on PR-regulated Gene Transcription—To compare the transcriptional activities of the cytoplasmic mutant {Delta}(NLS)-SRC-1 with the wild-type SRC-1, CV1 cells were cotransfected with the wild-type or mutated SRC-1 expression vectors, a PR expression vector, and a reporter gene and treated with hormone. Unexpectedly, the cytoplasmic mutant provoked the same enhancement of transcriptional activity as the wild-type SRC-1 (Fig. 9A). Because we have previously shown that PR shuttles between the nucleus and the cytoplasm (45), we considered the possibility that the {Delta}(NLS)-SRC-1 could interact with the hormone-PR complex in the cytoplasm and then be actively transported into the nucleus.



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  		FIG. 9.
Comparison of transcription-enhancing properties of wild-type SRC-1 and SRC-1 cytoplasmic mutant. A, CV1 cells were transfected with a vector encoding PR, the (PRE)2-TATA-CAT reporter gene, and increasing amounts of pSG5-SRC-1 (closed bars) or pSG5-{Delta}(NLS)-SRC-1 (open bars). 24 h after transfection, the cells were treated with hormone for 24 h, and CAT activity was measured. The results are means ± S.D. of three independent determinations. B, COS-7 cells were cotransfected with vectors encoding DsRed-PR and GFP-{Delta}(NLS)-SRC-1. 24 h after transfection, the cells were cultured for 4 h either in the absence of hormone (a–c) or in the presence of hormone and cycloheximide (10 µg/ml) (d–f). Live cells were directly observed by confocal microscopy. a and d, DsRed-PR; b and e, GFP-{Delta}(NLS)-SRC-1; c and f, DsRed-PR and GFP-{Delta}(NLS)-SRC-1 overlay. C, COS-7 cells were cotransfected with vectors encoding {Delta}(NLS)PR and HA-SRC-1. 24 h after transfection, the cells were cultured for 4 h either in the absence of hormone (a–c) or in the presence of hormone and cycloheximide (10 µg/ml) (d–f). The cells were fixed, treated for immunodetection with the appropriate monoclonal antibody (see "Experimental Procedures"), and observed by confocal microscopy. a and d, {Delta}(NLS)PR; b and e, HA-SRC-1; c and f, {Delta}(NLS)PR and HA-SRC-1 overlay. {Delta}(NLS)SRC-1 is transported into the nucleus by the hormone-PR complex. This observation supports the conserved enhancing properties of hormone driven transcription of {Delta}(NLS)SRC-1.

 

To verify this hypothesis, we performed cotransfection experiments with expression vectors encoding the DsRed1 protein fused to wild-type PR (DsRed-PR) and a GFP-SRC-1 deleted of the NLS (GFP-{Delta}(NLS)-SRC-1). In the absence of hormone, the two proteins were separated: PR was nuclear, whereas {Delta}(NLS)-SRC-1 was cytoplasmic (Fig. 9B, panels a–c). After administration of hormone (and cycloheximide to prevent PR neosynthesis), PR remained nuclear, whereas the {Delta}(NLS)-SRC-1 was shifted into the nucleus (Fig. 9B, panels d–f). The two molecules colocalized in nuclear speckles. We also performed experiments in which an expression vector for {Delta}(NLS)PR was co-transfected with wild-type SRC-1 encoding vector. We used conditions in which previous experiments have shown SRC-1 to be exclusively localized in the nucleus (20 h after transfection). In the absence of hormone, {Delta}(NLS)PR was cytoplasmic and SRC-1 was nuclear (Fig. 9C, panels a–c). In the presence of hormone, SRC-1 remained in the nucleus, whereas {Delta}(NLS)PR was now detected in the nucleus (Fig. 9C, panels d–f). The two molecules again colocalized in nuclear speckles. These observations suggest that the initial nuclear localization of SRC-1 is not mandatory for its coactivator activity and that it can be imported into the nucleus by a "piggy-back" mechanism through protein-protein interaction with one of its shuttling nuclear partners such as PR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Available data on the subcellular localization of p160 family members are quite divergent. SRC-1 has been reported to be localized either in the nucleus (23, 25, 40) or in the cytoplasm (20) depending on the cell type and on whether it is endogenous or transfected. In the presence of p300, it was shown to be located in punctuate nuclear complexes (57). Similar divergent data have been described for GRIP1/SRC-2, which has been found either in nuclear speckles (55) or in cytoplasmic speckles as well as in the nucleus (20). Nuclear or cytoplasmic localization of TRAM1 and p/CIP (SRC-3) has also been observed (19, 22, 55). Similar nuclear or cytoplasmic punctuate labeling has also been described for corepressors such as silencing mediator of retinoic and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (NCoR) (56, 58) as well as other transcriptional coregulators (21, 24, 59). These discrepancies could be explained by our present observations. Like SRC-1, other members of this family of coregulators could be in a dynamic state trafficking between the nucleus and the cytoplasm. For this reason and depending on experimental conditions, they could be observed in either of these two compartments. All these observations suggest that nucleocytoplasmic trafficking is a general phenomenon for nuclear receptors and their coregulators. Taken together, the emerging interpretation is that the localization of coactivators and corepressors in the nucleus and the cytoplasm might underlie a common physiological mechanism to regulate their function.

We have characterized the nuclear localization signal of SRC-1. It corresponds to a classic bipartite NLS and is localized at the extreme N-terminal part of the protein, specifically in the basic region of the bHLH. The conservation of this amino acid sequence in the other members of p160 family suggests that this sequence may correspond to a common NLS (Fig. 10A, panel a).



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  		FIG. 10.
A, comparison of SRC-1 NLS sequence with corresponding sequences in the p160 family and in the bHLH/PAS family. Basic amino acids are shown in bold. The position of the last amino acid in the sequence of the protein is indicated in superscript. h, human. a, SRC-1 NLS alignment with putative NLS of other human p160 family members. Localization in the bHLH domain of the protein. b, SRC-1 NLS alignment with NLS of other bHLH-PAS family members. This signal is localized in the N-terminal region of the protein. B, comparison of SRC-1 nuclear export domain with corresponding sequences in the p160 family of different species. Leucines are shown in bold. The position of the last amino acid in the sequence of the protein is indicated in superscript. h, human; m, mouse; r, rat.

 

SRC-1 belongs to the bHLH-PAS family of proteins, which also includes AhR, ARNT (HIF1{beta}), hypoxia-inducible factors (HIF-1{alpha}, 2{alpha} and 3{alpha}), and Sim. The mechanism for the subcellular localization of these proteins has been studied (60–63). The ARNT translocator is localized in the nucleus and possesses a NLS in its N-terminal region (61) (Fig. 10A, panel b). AhR, HIF1{alpha}, and HIF2{alpha} are found in the cytoplasm and the nucleus in the absence of stimulus, but they shift completely to the nucleus after activation and heterodimerization with ARNT. AhR NLS is also a bipartite amino acid sequence localized in the N-terminal region of the protein (64, 65) (Fig. 10A, panel b). Based on sequence homology, the NLS of HIF1{alpha} has been assigned to its N-terminal region but found to be inactive in the context of the full-length protein (62). A C-terminal motif responsible for hypoxia-induced nuclear translocation of HIF2{alpha} and HIF1{alpha} has been identified (63). This motif corresponds to a bipartite NLS containing an unusually long spacer. Homologous sequences are found in the other HIF family members. Nuclear localization mechanisms are thus heterogeneous for this subfamily of proteins. SRC-1 behaves as ARNT with a constitutive nuclear localization mediated by an N-terminal NLS.

The ability of leptomycin B to block nuclear export of SRC-1 implicates the involvement of the crm1/exportin1-dependent pathway. Crm1/exportin1 is involved in the rapid nuclear export of proteins having regulatory functions in the nucleus such as nuclear kinases (66), cell cycle proteins (67), transcription factors (68), and chromatin remodeling factors (69). Our results show that a transcriptional coactivator activity may also be controlled by an exportin-mediated pathway.

Crm1/exportin1-mediated export usually requires the presence of short hydrophobic, leucine-rich signals (NES) in the exported proteins (70), which are present in SRC-1. The first candidate residues (aa 865–876) are homologous to the classic NES consensus sequence (Fig. 8B). In contrast, this is not conserved in other members of the p160 family. Surprisingly, its deletion had no consequence on SRC-1 subcellular localization. The second sequence (aa 948–969) is also homologous to the classic NES consensus (Fig. 8B) and is highly conserved in SRC-2 and SRC-3. However, its deletion had no consequence on SRC-1 subcellular localization. This suggested that these putative NES might not be exposed in the native conformation of the protein rendering them inaccessible to interact with exportin1. However, it remains possible that they become accessible under some specific situations. Such a situation has been described in the case of AhR, which is exported back to the cytoplasm after inactivation (65, 71).

Our studies indicate that the region involved in SRC-1 nuclear export consists of 48 amino acids localized in the hinge region with the glutamine-rich domain (aa 990–1038). This region does not exhibit sequence homology to the consensus NES. Moreover, it is not conserved among the other members of p160 family (Fig. 10B). It is possible that this large domain is not directly involved in crm1/exportin1 interaction but is interacting with a third protein that will recruit the transporter. Alternatively, it is possible that SRC-1 harbors a non-classic NES, which may be recognized by exportin. For example, a large domain of at least 159 amino acids has been shown to be involved in nuclear export of snurportin (72). The interaction between crm1 and snurportin1 implies a tri-dimensional folding resulting in the juxtaposition of N-terminal and C-terminal domains. Other large NES have also been previously described, for example in the case of Far1 (73).

Nucleocytoplasmic shuttling of transcription factors is important in controlling their activity (74, 75). Similarly, nucleocytoplasmic trafficking of coregulators may constitute a supplementary step to control transcriptional activity of steroid receptors in response to changing physiological conditions (21, 24). A rapid export of the coactivator can terminate hormone action if the receptor export is slow, as we have observed in the case of PR (45). SRC-1 export to the cytoplasm could thus be involved in the control of hormone action.

Subcellular localization is also linked to degradation of proteins. Cyclin-dependent kinase inhibitor p27Kip1, IKB{alpha}, and p53 need to be exported to the cytoplasm to be degraded (76–78). On the contrary, some other nuclear proteins such as Far1 have been reported to be degraded in the nucleus (73). It is thus possible that SRC-1 is exported into the cytoplasm to be degraded and eliminated. The kinetics of nuclear export may also be important for protein degradation. For example, in the case of GR, a rapid nuclear export induces an increase of proteolysis (79).

Alternatively, it is possible that SRC-1 plays a role in the cytoplasm in addition to its well documented role on transcription in the nucleus. For instance, evidence suggesting a cytoplasmic activity for PR has been recently reported (80). It is thus conceivable that the nucleocytoplasmic trafficking of SRC-1 may allow some functional interactions with cytoplasmic partners. A cross-talk between SRC-1 and other signaling pathways has been previously shown in the case of AP1 proteins (81) and JAB1 (41). Therefore, nucleocytoplasmic shuttling might permit cross-talks between nuclear and cytoplasmic compartments.


   FOOTNOTES
 
* This work was supported in part by the INSERM, the Association pour la Recherche sur le Cancer, the Ligue pour la Recherche Contre le Cancer, the Faculté de Médecine Paris-Sud, and the Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by the Ministère de la Recherche et de l'Enseignement Supérieur and the Ligue pour la recherche contre le cancer. Back

§ Present address: Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India. Back

Supported by the INSERM and the Fondation pour la Recherche Medicale. Back

|| Present address: CNRS UPR9079, Oncogenèse, différenciation et transduction du signal, Institut André Lwoff, 7 rue Guy Moquet, 94800 Villejuif, France. Back

** Present address: INSERM U468, Génétique Moléculaire et Physiopathologie, Institut Mondor de Médecine Moléculaire, Hôpital Henri Mondor, 94010 Créteil cedex, France. Back

{ddagger}{ddagger} Present address: INSERM U189, Physiopathologie Subcellulaire et Régulations Métaboliques, Faculté de Médecine Lyon-Sud, BP12, 69921 Oullins cedex, France. Back

¶¶ To whom correspondence should be addressed. Tel.: 33-1-45-21-27-47; Fax: 33-1-45-21-27-51; E-mail: anne.mantel{at}bct.ap-hop-paris.fr.

1 The abbreviations used are: NLS, nuclear localization signal; NES, nuclear export signal; AD, activation domain; CAT, chloramphenicol acetyltransferase; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; DsRed, (Discosoma sp.) red fluorescent protein; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; HA, hemagglutinin; LMB, leptomycin B; PBS, phosphate-buffered saline; PR, progesterone receptor; R5020, 17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione; SRC, steroid receptor coactivator; bHLH, basic helix-loop-helix; HIF, hypoxia-inducible factor; aa, amino acid(s); PAS, Per Arnt-Sim motif. Back


   ACKNOWLEDGMENTS
 
We thank C. Boucheix (INSERM U268) for assistance with the fluorescence-activated cell sorting experiment, O. Trassard (IFR93) for help for figures, and C. Carreaud-Aumas for technical assistance. Leptomycin B was kindly provided by B. Wolff (Novartis Research Institute, Austria).

Addendum—While preparing our manuscript, p/CIP (mouse SRC-3) nucleocytoplasmic shuttling was proposed by Qutob et al. (82), based on the observation that endogenous p/CIP was localized either in the nucleus or in the cytoplasm, depending on the presence of growth factors in cell culture medium and on the effect of LMB. However, unlike SRC-1, p/CIP subcellular localization correlated with the cell cycle. In addition, we show that SRC-1 uses a different domain for nuclear export than p/CIP (Fig. 8A, mutants deleted of aa 865–876 and 948–960). Taken together, these data and recently published data on GRIP1/SRC-2 trafficking (20) show that nucleocytoplasmic trafficking is a general phenomenon for p160 family members of coactivators.



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