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Mol Cell Biol. Oct 2007; 27(19): 6913–6932.
Published online Jul 23, 2007. doi:  10.1128/MCB.01695-06
PMCID: PMC2099228
Regulation of SRC-3 Intercompartmental Dynamics by Estrogen Receptor and Phosphorylation[down-pointing small open triangle]
Larbi Amazit,1,2 Luigi Pasini,1 Adam T. Szafran,1 Valeria Berno,1 Ray-Chang Wu,1 Marylin Mielke,1 Elizabeth D. Jones,1 Maureen G. Mancini,1 Cruz A. Hinojos,1 Bert W. O'Malley,1 and Michael A. Mancini1*
Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030,1 INSERM 693, Récepteurs Stéroïdiens, Physiopathologie Endocrinienne et Métabolique, Faculte de Medecine Paris Sud, 63 rue Gabriel Peri, 94276 Le Kremlin Bicetre Cedex, France2
*Corresponding author. Mailing address: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-8952. Fax: (713) 798-3175. E-mail: mancini/at/
Present address: European Molecular Biology Laboratory, Mouse Biology Unit, 00016 Monterotondo, Italy.
Received September 9, 2006; Revised October 19, 2006; Accepted May 23, 2007.
The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor α (ERα) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERα, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERα, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERα-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.
Nuclear receptors (NRs) are transcription factors that regulate the expression of genes in a tissue-specific and temporally specific manner and control crucial physiological processes including reproduction, development, and cellular homeostasis (46, 74). NRs act in cooperation with molecules collectively referred to as coregulators (28, 44). The steroid receptor coactivator 3 gene (SRC-3) (also known as AIB1, ACTR, RAC3, TRAM-1, and p/CIP) is a member of the SRC/p160 gene family and was independently identified as encoding a coactivator of NRs (2, 9, 40, 68, 69, 72). In addition to SRC-3, other members of the p160 family include SRC-1 (52) and SRC-2 (GRIP1/TIF2) (77).
SRC-3 has been characterized as an oncogene. Transgenic overexpression of SRC-3 in mice leads to mammary gland tumorigenesis, and its genetic deletion confers resistance to chemically induced mammary tumorigenesis (34, 73). Consistent with this, SRC-3 is amplified and/or overexpressed in a variety of human cancers including breast, ovarian, pancreatic, prostate, gastric, endometrial, and esophageal cancers (2, 19, 21, 61, 85). Results from cell culture systems and targeted gene disruption experiments in mice demonstrated that SRC-3 also plays important roles in female reproductive function, puberty, cytokine signaling, and vasoprotection (86, 89). Moreover, SRCs have been shown to coactivate an increasing number of additional transcription factors such as STATs, E2F1, Ets, and NF-κB (3, 22, 45, 80, 83).
SRC-3 activity is regulated by a variety of posttranslational modifications including methylation, phosphorylation, acetylation, ubiquitination, and sumoylation (10, 17, 43, 47, 82, 83). The phosphorylation of SRC-3 at distinct sites creates a combinatorial code that directs the various phosphorylated forms into different pathways (84). These events have been shown to be important for its interaction with CBP/p300, CARM1, and NRs and for its oncogenic potential (18, 84). Several extracellular signaling molecules, including steroid hormones, growth factors, and cytokines, induce SRC-3 phosphorylation (18, 84). These actions are mediated by a wide range of kinases including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase, p38 mitogen-activate protein kinase (MAPK), and IκB kinases (IKKs) (18, 20, 55, 83, 84). The roles of individual phosphorylation sites and their role in fine-tuning SRC-3 activity remain incompletely defined.
Estrogen receptor α (ERα) is a founding member of the NR superfamily that regulates the transcription of specific target genes in response to hormones (74). All p160 family members associate with ER as coactivators and enhance its transcriptional activity (29). The ER spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution to very short-lived (seconds) foci following the addition of either agonists or antagonists (65, 67). Curiously, while agonist-induced foci of ER colocalize with the coactivator SRC-1, overlap with sites of transcription is rare (65). Furthermore, even in the presence of agonist, the intranuclear mobilities of ER and SRC-1 are significantly different (89). These observations suggest that NR associations are not only transient but also not directly functional in the context of gene regulation.
While a wealth of studies revealed the molecular functions of both NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in a cellular context. It is still not clear how transcriptional complexes (defined as stable from biochemical analyses of cell lysates) containing NRs/coactivators (and other components) are organized in terms of nuclear architecture. What is clear, using live-cell studies, is that interactions between NRs and DNA, or NRs and cofactors, are extremely dynamic, with half-lives (interaction times) measured in seconds and not tens of minutes, as reported for previous chromatin immunoprecipitation analyses (58, 62). Interestingly, toward an endpoint of linking cellular mapping and trafficking of gene regulators to function, considerably disparate results have been reported. For example, the SRC-3 cellular localization remains controversial despite the presumption that it is present in the nucleus (at least in part) while functioning as a gene regulator (5, 24, 56, 68, 70).
In the present study, we used both live- and fixed-cell approaches, including quantitative high-throughput microscopy (HTM), to elucidate several aspects of SRC-3 function in a cellular context. We demonstrate that SRC-3 is in fact a primarily nuclear protein and that a fraction of the cellular pool can shuttle between the nucleus and the cytoplasm. Furthermore, its phosphorylation state and interactions with ER, a prototypical NR, regulate the intranuclear dwell time and the subnuclear dynamics of SRC-3. Finally, we show that transient intranuclear ER-SRC-3 complex formation is dependent on the phosphorylation state of SRC-3 and that the inhibition of SRC-3 phosphorylation results in an altered residency at an ER-regulated promoter.
The expression plasmids pCMV-Flag-SRC-3 and pCMV-Flag-SRC3-AAA, in which all three LXXLL motifs have been mutated to LXXAA, were generated as described previously (84, 90). pCMV-Flag-SRC3(ΔNES) codes for a mutant lacking residues 1031 to 1130 and was generated by PCR. The expression plasmids for green fluorescent protein (GFP)-SRC-3, yellow fluorescent protein (YFP)-SRC-3, and hemagglutinin (HA)-SRC-3 were generated by inserting the SRC-3 fragment from pCMV-Flag-SRC-3 (83) into pEGFP-C3, pYFP-C3, and pTRE2hyg2-HA vectors, respectively (Clontech Laboratories, Inc.). The six constructs GFP-SRC-3(A1) to GFP-SRC-3(A6) (individual alanine mutations of the six identified SRC-3 phosphorylation sites) (84) as well as the GFP-SRC3(A1-6) construct (expression plasmid in which all six identified phosphorylation sites have been mutated to alanine) (90) were generated by subcloning the SRC-3(A1-6) fragment from pCMV-Flag-SRC3(A1-6) into pEGFP-C3. The expression plasmids GFP-ER, cyan fluorescent protein (CFP)-ER, YFP-SRC-1, and GFP-SRC-1 were made as described previously (65). GFP-ER(ΔNLS) was described previously (90). GFP-ER(S118A) was generated by inserting the ER(S118A) fragment from pCMV5-ER(S118A) (38) into pEGFP-C3.
Hormones and inhibitors.
In all experiments, estradiol (E2) was added to a final concentration of 10−8 M. Stock solutions of U0126 MEK kinase inhibitor (Promega) were made in dimethyl sulfoxide (10 mM), stored at −20°C, and used within 7 days. In Fig. Fig.66 to to8,8, where indicated, transfected cells were first pretreated with 45 μM U0126 for 1 to 3 h and then treated throughout the duration of the experiment. SB202190, a p38 MAPK inhibitor (Upstate Biotechnology), was used at a final concentration of 20 μM. Epidermal growth factor (EGF) (Invitrogen) was dissolved in ethanol and used at 100 ng/ml. Leptomycin B (Sigma) was used at 40 nM, a concentration efficient to inhibit nuclear export of a nuclear export signal (NES)-containing protein (75).
FIG. 6.
FIG. 6.
EGF-mediated phosphorylation of SRC-3 enhances its nuclear localization. (A) Real-time in vivo monitoring of GFP-SRC-3, GFP-SRC3(A1-6), and GFP-SRC-1 upon the addition of EGF (100 ng/ml). HeLa cells were transfected with the indicated vectors and incubated (more ...)
FIG. 8.
FIG. 8.
Promoter recruitment of SRC-3 is abrogated in the presence of U0126. (A) The PRL array-HeLa cell line contains multiple genomic integrations of an ER element, which is spatially confined and visualized by the accumulation of fluorescently tagged ERα. (more ...)
Cell culture and heterokaryon assays.
Endogenous SRC-3 and exogenously expressed SRC-3 (XFP-SRC-3) were examined under various conditions in several cell types including HeLa, MCF-7, T47D, GH3 (rat pituitary tumor cells, which have endogenous ER), NHDF-Ad (adult normal human dermal fibroblast cells), CV-1 (African green monkey kidney transformed fibroblast cells), and CGF (primary nontumor human foreskin fibroblasts) cells. HEK293 and mouse 3T3L1 cells were obtained from the ATCC. SRC-3−/− mouse embryonic fibroblasts (MEFs) were described previously (84). For routine culture, cells were maintained in complete Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (FBS). For transfection experiments, cells were plated 1 day before in DMEM medium without red phenol (Invitrogen) containing 5% dextran charcoal-stripped-dialyzed FBS (named “free-hormone medium” throughout the paper). Heterokaryons were obtained as previously described (26).
Immunoprecipitation and Western blotting analysis.
MCF-7 or HEK293 cells were lysed in lysis buffer (20 mM Tris-HCl [pH 8.0], 125 mM NaCl, 0.5% NP-40, 2 mM EDTA, 0.2 mM NaF, 0.2 mM Na3VO4, protease inhibitor cocktail) for 15 min, and the debris was cleared by centrifugation at 13,400 × g for 15 min at 4°C. Lysates were incubated overnight (4°C) with the indicated antibodies (anti-ER clone 60C [Upstate Biotechnology]or anti-Flag M2 [Sigma]). The antibody was allowed to bind to protein A/G beads (Santa Cruz Biotechnologies) for 30 min and then washed extensively with lysis buffer. For Western blot analysis, the samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Bio-Rad). The indicated antibodies were diluted in TBST buffer (5% nonfat dry milk, 50 mM Tris-HCl, 150 mM NaCl [pH 7.5], 0.1% [vol/vol] Tween 20) and added to the membranes for 1 h at room temperature (RT) or overnight at 4°C followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies for 30 min at RT (Sigma). All proteins were detected with ECL Plus detection reagents (Amersham) and visualized by chemiluminescence.
Transfection and luciferase reporter gene assay.
Cells were transfected using TransFectin lipid reagent (Bio-Rad Laboratories) according to the manufacturer's instruction. In cotransport experiments (see Fig. 4D to E), cells were cotransfected with a total of 500 ng of plasmids in a ratio of NR to coactivator of 1/10; briefly, 45 ng of μg of the plasmid DNA encoding GFP-ER(ΔNLS) was cotransfected with 450 ng the plasmid encoding Flag-tagged SRC3 or Flag-tagged SRC3(ΔNES) or the empty vector pCMVTag2B (Stratagene, La Jolla, CA). For the luciferase assay, HeLa cells were cotransfected in 24-well plates with an estrogen or 5× upstream activation sequence (UAS)-thymidine kinase (TK) (GAL4-TK) response element/luciferase reporter gene (150 ng/well) and the corresponding expression plasmids: ER (5 ng/well), SRC-3 (150 ng/well), or Gal4-SRC-3 (50 ng/well), respectively. Cells were cultured for 24 h, and where indicated, E2 (at 10−8 M) was added, and the cells were incubated for an additional 16 h. Whole-cell lysates were prepared and assayed for luciferase activity according to instructions provided by the manufacturer (Promega), and the activity was normalized against the total amount of protein.
FIG. 4.
FIG. 4.
SRC-3 rapidly shuttles between the nucleus and cytoplasm. (A) Automated quantification of SRC-3 nuclear localization after treatment with leptomycin B. T47D cells were cultured for 48 h in hormone-free medium and then incubated with leptomycin B (LB) (more ...)
RNA-mediated interference.
MAPK1 (p42/ERK2) and MAPK3 (p44/ERK1) small interfering RNAs (siRNAs) were purchased from the Validated Stealth RNA interference (RNAi) collection (Invitrogen). Cells were seeded in free-hormone medium 1 day before transfection at 60% confluence in a six-well plate (for Western blotting) containing glass coverslips (for HTM analysis). One hundred picomoles of RNAi per well was transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Seventy-two hours after transfection, coverslips were removed from the wells and processed for immunochemistry, while the remaining cells were collected in lysis buffer for Western blot analysis.
Generation of stable cell lines.
The HeLa tet-off cell line expressing HA-SRC-3 was generated by transfecting a HeLa tet-off cell line (Clontech Laboratories, Inc.) with the expression vector pTRE2hyg2-HA-SRC-3. Stable transformants were selected in hygromycin (200 μg/ml) and neomycin (100 μg/ml), and resistant clones were screened for expression of SRC-3 by Western blotting.
The production of the physiologically regulated, visible prolactin (PRL) array-HeLa cell line was performed as described previously for a similar cell line (63). Briefly, to establish a stable line bearing a chromosomally integrated array of plasmid p52X-PRL-DS-Red2-SKL, HeLa cells were plated in a 100-mm dish to a density of ~60% confluence in OptiMEM medium supplemented with 4% FBS. The cells were cotransfected with p52X-PRL-DS-Red2-SKL and pTK-Hygro (Clontech) at a 100:1 ratio using a threefold excess (by volume) of Fugene6 (Roche) reagent to DNA. After ~18 h of incubation with the DNA-lipid mixture, the cells were rinsed, detached, and split into 10 100-mm dishes. From this point, the cells were selected in OptiMEM medium supplemented with 4% FBS plus 400 μg/ml hygromycin. Approximately 100 colonies were picked and expanded directly onto 24-well dishes and then into a 35-mm dish containing a 12-mm-diameter coverslip. To assess the integration of p52X-PRL-dsRED2-SKL, coverslips were transiently cotransfected with GFP-ER and GFP-Pit-1 (both were tagged with GFP to maximize the fluorescent signal) and examined microscopically. A substantial percentage of dsRED2-SKL-positive cells with low diffuse levels of GFP signal in the nucleus had a single (sometimes two) bright intranuclear focus of fluorescence. These data suggested ER and/or Pit-1 interactions with an integrated 52X-PRL array (presumably multicopy) and provided encouragement for the next step, which was designed to separate cells based on reporter expression. To this end, we expanded several heterogeneous colonies of 52X-PRL HeLa cells. The clone (HeLa/52X-DM66-Red2-PTS 19) shown in this study was one of them and was named “PRL-HeLa clone 19.” Note that in contrast to the previously described HeLa/52X-DM66-Red2-PTS 23 clone (i.e., clone 23) (63), the clone 19 used in this study was purified from a second-generation clonal cell line and showed much less basal activation in the absence of hormone (see Fig. S5 in the supplemental material).
Cells were grown on acid-etched and poly-d-lysine-coated glass coverslips (12-mm diameter). After the specified treatment, cells were removed from the incubator, immediately rinsed in ice-cold phosphate-buffered saline (PBS), fixed with a solution of 4% formaldehyde in PEM buffer {0.1 M PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], 2 mM EGTA, 3 mM MgCl2}, permeabilized in 0.5% Triton X-100 (in PEM buffer for 30 min), and then washed and quenched in sodium borohydride (0.5 mg/ml) (10 min in PEM buffer). The cells were then washed and incubated for 1 h (RT) in 5% nonfat dry milk in TBST before incubation overnight with primary antibody (at 4°C) and subsequently with fluorophores conjugated to appropriate secondary antibodies (goat anti-mouse antibody-Alexa 555; Molecular Probes). The primary antibodies used were anti-HA (clone 3F10) (200 ng/ml), SRC-1 (1 μg/ml), and AIB1/SRC-3 (0.5 μg/ml) (obtained from Roche Molecular Biochemicals, Upstate Biotechnology, and BD Biosciences, respectively). After primary and secondary antibody labeling, cells were postfixed and quenched (as described above), counterstained with DAPI (4′,6′-diamidino-2-phenylindole) (0.5 mg/ml for ~1 min), rinsed quickly in water, and then mounted onto slides (ProLong Gold; Molecular Probes).
Cells were imaged using the Cell Lab IC 100 image cytometer from Beckman Coulter, Inc., with a Nikon 40× Plan S fluor 0.90-numerical-aperture (NA) objective, except for the experiment shown in Fig. 7B, D, and F, where a Nikon 63× Plan Apo 1.20-numerical aperture (NA) objective was used. Three channels were imaged (channel [DAPI] was used to find the focus and nuclei): channel 1 was used for imaging GFP-SRC-3, GFP-SRC-3(A1-6), GFP-ER, and GFP-ER(S118A), and channel 2 was used for imaging endogenous SRC-3, HcRed, or HcRed-ER. A correlated channel segmentation algorithm was used to identify and quantify the localization and level of fluorescence of SRC-3/ER in the nucleus and the cytoplasm (6). After image acquisition and application of the correlated channel segmentation algorithm, the total cell populations for each treatment were progressively filtered (gated) using the following criteria: nontransfected cells, nuclei clusters, mitotic cells, and apoptotic cells were filtered out from the total cell population using an intersection of DNA content gate, a DNA cluster gate, and a “keep-two-colors” gate. Filtration of low-level expression of GFP-SRC-3 gates was generated as described previously (6) and applied to produce the final cell population to be analyzed. The images and masks were visually inspected for accuracy.
FIG. 7.
FIG. 7.
SRC-3 phosphorylation by MAPK is necessary for its interaction with ER. (A) HeLa cells expressing GFP-SRC-3 and HcRed-ER were incubated in hormone-free medium for 48 h. Cells were then incubated for 2 h with or without 10 nM E2 (vehicle or E2). Where (more ...)
Biochemical cell fractionation and live-cell monitoring of in situ extraction.
In situ biochemical fractionation was performed on HeLa cells using a well-established protocol (65, 71). Briefly, cells were grown on poly-d-lysine-coated 40-mm glass coverslips and extracted with hypotonic (HYPO) buffer (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and protease inhibitors) or cytoskeletal (CSK) buffer (10 mM PIPES [pH 6.8], 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, and protease inhibitors). For the Western blot analysis shown in Fig. Fig.2A,2A, equivalent lysate samples of whole cells, CSK extraction buffer supernatant, a second CSK extraction buffer washout, or the resuspended CSK pellet were probed with a rabbit polyclonal anti-SRC-3 antibody (1:1,000).
FIG. 2.
FIG. 2.
SRC-3 extractability is modulated by ER. (A) SRC-3 is not tightly bound within the nucleus. An antibody against SRC-3 was used to probe a Western blot of HeLa cell lysates. Whole-cell extract, CSK extraction buffer supernatant (CSK-S), a second CSK buffer (more ...)
For real-time extraction experiments, a HYPO solution (10 nM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol) was washed over live cells (via a small peristaltic pump) directly on the microscope to remove loosely soluble proteins and extract the cytoplasmic fraction. This solution was used in the real-time live-cell extraction experiments, as it is a commonly used fractionation buffer and would directly relate real-time single-cell experiments to the bulk biochemical methods previously used to study SRC-3 fractionation (56, 83). Cells were grown in 35-mm Delta T dishes (Bioptechs) and secured to a stage adapter for temperature control. HEPES-buffered medium was circulated through the Delta T dish using a Bioptechs peristaltic pump and inflow/outflow tubing. The temperature was controlled at 37°C (±0.1°C). A Bioptechs objective heating collar was also used (at 37°C). In order to minimize photo damage, cells were imaged with neutral-density filters to allow only 50% of the total light and identical 2-s exposure times.
For imaging, HeLa cells were plated and transfected in Delta T dishes (Bioptechs). Live-cell microscopy was performed using a Zeiss 510 confocal microscope utilizing the 488-nm (GFP), 458-nm (CFP), or 514-nm (YFP) laser lines of the argon laser set at 75% of maximal power. In all experiments, cells were maintained at 37°C, and fresh medium containing the appropriate ligand (i.e., EGF) was cycled over the cells. All imaging was done with a 100×/1.4-NA objective with pinhole set at 4 airy units. Scanning was bidirectional at the highest possible rate using a 3.5× zoom with laser power attenuated to 1% of bleach intensity. For all experiments, cells were selected for low levels of GFP-SRC-3 expression to limit experimental artifacts. For fluorescence recovery after photobleaching (FRAP) experiments, five prebleach images were acquired followed by 20 iterative bleach pulses of a total duration of 200 ms using a bleach region of interest rectangle covering approximately 25% of the nucleus. Single-section images were collected every 500 ms for 60s. The relative fluorescence signal in the bleached region was determined by two sequential normalization steps using the mean bleach region of interest (It) and the mean nucleus (Nt) signals as follows:
equation M1
equation M2
Prebleach values are not used in the second normalization step. The half-time of maximal recovery was determined by fitting a logarithmic equation to the recovery curve and determining the value of t when y was 0.5 using the Excel curve-fitting function. Control and test cells were matched for passage number, transfected, and treated in unison in order to obtain consistent results. Means ± standard errors (SE) of the means (SEM) for the half-life (t1/2) results from each experimental group were compared using appropriate statistical tests and considered significantly different if the P value was <0.05. For dual FRAP experiments (see Fig. Fig.3D),3D), both fluorescent proteins were bleached with the same laser wavelength of 458 nm; subsequently, simultaneous images corresponding to the CFP and YFP fluorescence were obtained using the multitracking function of the microscope.
FIG. 3.
FIG. 3.
Intranuclear mobility and subnuclear localization of SRC-3 are influenced by ER in live cells. (A) Cells were transfected with GFP-SRC-3 (bottom) or GFP-SRC-1 (top). A region corresponding to ~25% of the size of the nucleus was photobleached. (more ...)
Fluorescence deconvolution microscopy and quantitative colocalization.
Deconvolution microscopy was performed with a Zeiss AxioVert S100 TV microscope and a DeltaVison restoration microscopy system (Applied Precision, Inc.). Cells were imaged using either the 63× or 100× objective lens (both 1.40 NA). A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images.
Quantitative colocalization analyses (see Fig. Fig.8B)8B) were done with the Colocalizer Pro Software using the Pearson's correlation coefficient [R(r)]. The estimation of R(r) is one of the standard techniques applied for matching one image to another in order to describe the degree of overlap between the two patterns (i.e., the array transgene). The R(r) coefficient accounts for only the similarity of shapes between the two images and does not depend upon image pixel intensity values. Briefly, 10 images for each condition were acquired and analyzed by deconvolution microscopy. The R(r) coefficient was calculated for each array transgene in each image, and the average calculated values for each condition were plotted on the histogram (error bars represent SE).
Fluorescent in situ hybridization.
The methods used here, including procedures for nonisotopic probe preparation and fluorescent in situ hybridization, have been described previously in numerous publications. Briefly, coverslips with adherent cells were rinsed twice in PBS, dipped in CSK buffer (17a), extracted on ice for 5 min in CSK buffer containing 0.5% Triton X-100 and 2 mM vanadyl-ribonucleoside complex (Gibco-BRL), rinsed in CSK-vanadyl-ribonucleoside complex, fixed in 4% paraformaldehyde-PBS for 10 min, rinsed again in PBS, and stored in 0.4% paraformaldehyde at 4°C until use. Hybridization to RNA was carried out at 37°C in standard buffers containing 5 μg/ml probe and 50% formamide overnight. After incubation, samples were rinsed in a series of SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) buffers, detected for biotin using a streptavidin-Alexa Fluor 594 conjugate (Molecular Probes, Eugene, OR), and rinsed in a series of PBS washes.
SRC-3 is a nuclear protein.
HTM-based automated cytometry delivers rapid and accurate fluorescence imaging under similar acquisition parameters (i.e., type of optics, number of channels, exposure times, and number of fields) (see reference 6) and allows the quantitative study of variation in hundreds of cellular parameters (49). We used HeLa cells immunolabeled with a monoclonal anti-SRC-3 antibody to examine endogenous SRC-3 localization. The quantitative “fractional localized intensity in the nucleus” (FLIN) value is a ratio measurement and is independent of cell-to-cell variations in signal intensities. Measurements of thousands of cells expressing endogenous SRC-3 gave an average FLIN value of 96%, thus indicating that in an average cell, 96% of detectable SRC-3 fluorescence is nuclear (Fig. (Fig.1A;1A; see Fig. S1A in the supplemental material). We observed similar results when we used MCF-7 cells and, in previous manual image analysis studies, GH3, COS-1, CV-1, and CGF cell lines (data not shown).
FIG. 1.
FIG. 1.
SRC-3 is a predominantly nuclear protein. (A and B) Automated quantification of SRC-3 subcellular localization using high-resolution HTM. HeLa cells were grown for 48 h in hormone-free medium. Shown are filled-area histograms of FLIN obtained for the (more ...)
To confirm these results, we repeated the experiments using transiently expressed GFP-SRC-3 in HeLa cells. It was noted in these studies that numerous small and large cytoplasmic and nuclear foci (from <1 to >5 μm) were observed in cells expressing higher-than-endogenous levels of GFP-SRC-3 (see reference 6). To avoid abnormal cellular localization and/or potential toxicity due to a gross overexpression of GFP-SRC-3, we excluded all cells having more than three times the level of endogenous SRC-3 found in HeLa cells from our analyses (see reference 6). When gated for this lower expression level, which can exclude over 90% of the transfected cells, exogenously expressed GFP-SRC-3 also showed FLIN values similar to those of the endogenous protein (average FLIN value of 95.6%) (Fig. (Fig.1B;1B; see Fig. S1B in the supplemental material).
To eliminate concerns associated with the transient transfection of GFP-fused proteins, we also generated a HeLa cell line stably expressing HA-SRC-3 under tetracycline control. Immunolabeling with anti-HA antibodies further confirmed that the coactivator is localized in the nucleus (Fig. 1C and D). Taken together, these results show a steady-state nuclear occupancy of the coactivator when expressed at approximately physiological levels across multiple cell lines, with no indication of nuclear foci as reported previously for several overexpression-based cytological studies (5, 59).
SRC-3 is a readily extractable protein.
Several NRs and other transcription factors have been reported to remain associated with the insoluble nuclear matrix fraction after various types of extraction (71, 76). To determine the strength of the SRC-3 association within the nuclear structure, we performed biochemical fractionation of HeLa cells. Upon exposure to a buffer containing detergent (CSK buffer and 0.5% Triton X-100), virtually all of the endogenous SRC-3 was released into the supernatant, suggesting that the vast bulk of unstimulated SRC-3 does not tightly associate with extraction-resistant nuclear structures (Fig. (Fig.2A2A).
By coupling a biochemical extraction and an in situ imaging approach, we were able to differentially measure the regulation of SRC-3 nuclear affinity in situ using fluorescent microscopy. HYPO buffer was used to extract the coactivators (SRC-3 and SRC-1) bound to the nucleus with low affinity. Cells expressing low levels of YFP-SRC-3 or YFP-SRC-1 or coexpressing YFP-SRC-3 and CFP-ER (±10 nM E2) were examined. Bright-field images were used to show that cells remained intact and within the focal plane. When SRC-3 was expressed alone, only ~6% of the initial nuclear SRC-3 remained after a 5-min exposure to the ice-cold HYPO solution (Fig. (Fig.2B,2B, left). In contrast, ~10-fold more (55%) nuclear SRC-1 remained under the same conditions (Fig. (Fig.2B,2B, right), indicating differences in nuclear affinity between these two coactivators. However, coexpression of ER with SRC-3 (±E2) significantly increased the nuclear retention of SRC-3 so that ~84% now remained after extraction with HYPO buffer (Fig. (Fig.2C2C).
To measure extractability over many cells (thousands), we extended this technique with HTM analysis. Equivalent numbers of fields were randomly acquired, and the average nuclear intensity was determined for each condition and normalized to the total number of cells. The results show that for cells expressing GFP-SRC-3, HYPO buffer is effective in extracting the coactivator, since after 3 min of incubation, the average fluorescence intensity per cell dramatically dropped from ~1.1 to ~0.1 (Fig. (Fig.2D,2D, left). In contrast, when cells were cotransfected with GFP-SRC-3 and HcRed-ER, extractability was reduced (from a value of 1.7 in nontreated cells to 0.5 for HYPO) (Fig. (Fig.2D,2D, middle). With E2 present, extractability was reduced further and not significantly different from that of unextracted controls (Fig. (Fig.2D,2D, right). Similar results were obtained with CSK buffer, which was used to extract tightly bound coactivators.
Our results indicate that SRC-3 is a readily extractable protein and that the presence of ER (or ER plus E2 more so) dramatically increases the affinity of SRC-3 for an insoluble intranuclear structure. These data also show that, in the absence of ER or E2, SRC-3 is a more readily extractable protein than SRC-1 and provide a technical rationale for why protocols that separate cytoplasmic and nuclear proteins using HYPO suggest that SRC-3 is cytoplasmic at a basal state (56, 83).
Intranuclear dynamics of SRC-3.
Since the biochemical extractions revealed SRC-3 to have a lower affinity for the nuclear compartment than SRC-1, it was suggested that they might also show differences in nuclear mobility. Therefore, we used FRAP analysis to examine GFP-SRC-3 and GFP-SRC-1 mobility in live cells. In these studies, we briefly bleached (200 ms) a small region of the nucleus and measured fluorescence recovery into the region (Fig. 3A and B). An extremely high mobility of SRC-3 is indicated by the immediate loss of fluorescence from the entire nuclear pool of GFP-SRC-3 (Fig. (Fig.3A,3A, bottom) and a short recovery time (t1/2 = 0.7 ± 0.27 s) (Fig. (Fig.3B).3B). Photobleaching of GFP-SRC-1 revealed a more distinct bleach area (Fig. (Fig.3A,3A, top) immediately after bleaching and a significantly longer recovery time (t1/2 = 1.39 ± 0.19 s), indicating that although SRC-1 is also dynamic, it is less mobile than SRC-3. These results are consistent with our previous work (67) and the observation that SRC-3 is more extractable than SRC-1 and further explain the discrepancy observed between biochemical fractionation and in situ localization.
To explain the effect of ER on SRC-3 mobility/nuclear affinity, we investigated the impact of ER expression on SRC-3 subnuclear location. Even though transcriptional activation by NRs involves interactions with coactivators, the exact location of these interactions is not readily visible except through image-based approaches. To examine the ER influence upon SRC-3 distribution, we analyzed the subnuclear localization of YFP-SRC-3 coexpressed with CFP-ER in HeLa cells. In control experiments (without E2), both SRC-3 and ER appeared in a diffuse distribution within the nucleus (vehicle) (Fig. (Fig.3C).3C). As previously described for ER and SRC-1 (65), the presence of E2 (2 h) changed the distribution of SRC-3 and ER to a nuclear hyperspeckled pattern, where SRC-3 and ER colocalize within hundreds of distinct foci in an optical plane (E2) (Fig. (Fig.3C).3C). These data indicate that the ligand induces a dynamic reorganization of ER and SRC-3. It is important to note that our previous work has shown that overlaps between these foci and sites of active transcription are uncommon (65). To evaluate the stability of the colocalizing ER-SRC-3 foci, we performed FRAP analyses on cells expressing both YFP-SRC-3 and CFP-ER. The results showed that the recovery t1/2 of SRC-3 is significantly higher in the presence of ER (t1/2 = 0.72 ± 0.12 s) (Fig. (Fig.3D).3D). Upon the addition of 10 nM E2 (1 h), SRC-3 mobility in the presence of ER was further reduced (t1/2 = 1.58 ± 0.14 s) (Fig. (Fig.3D).3D). Thus, although distinct, colocalizing foci form after ligand administration, their nature remains transient.
SRC-3 shuttles between the nucleus and cytoplasm.
The ability of coactivators to regulate gene expression is dependent not only upon their subnuclear dynamics (as defined by FRAP) but also upon their general cellular compartmentalization. Whether or not a coactivator can move rapidly between compartments to gain access to transcription sites impacts the feasibility of the hypotheses to explain the coactivator's mechanism of action. In order to first determine if a fraction of SRC-3 shuttles between the nucleus and cytoplasm, we used an indirect approach by incubating cells with a specific inhibitor of nuclear export (leptomycin B) (81) and then analyzing its effect upon nuclear compartmentalization. For these experiments, we used T47D cells cultured for at least 48 h in hormone-free medium and expressing endogenous SRC-3, which showed a slightly lower average FLIN value (87%) (Fig. (Fig.4A,4A, upper panel) than HeLa cells (Fig. (Fig.1A).1A). When T47D cells were incubated with leptomycin B for 4 h, average FLIN levels increased to 97% (Fig. (Fig.4A,4A, lower panel), suggesting that the small pool of cytoplasmic SRC-3 is in part the result of active nuclear export.
To further confirm the existence of nucleocytoplasmic shuttling of SRC-3, we used interspecific heterokaryons, a method previously employed in studies of NR (26). Polykaryons were formed by the polyethylene glycol fusion of HeLa cells containing endogenous SRC-3 with MEF cells derived from homozygous SRC-3 knockout mice (86). Treatment with cycloheximide prevented new protein synthesis, and SRC-3 distribution was analyzed by immunolabeling. Mouse nuclei were identified by their specific heterochromatin pattern using DAPI (i.e., dense heterochromatic regions). One hour after fusion, SRC-3 could be readily identified in mouse nuclei at a level equivalent to that observed for human nuclei (Fig. (Fig.4B,4B, Fusion). However, in the presence of leptomycin B, the nuclear export of SRC-3 from HeLa nuclei and transfer to MEF nuclei were inhibited (Fig. (Fig.4B,4B, Fusion/LB). The same results were obtained with a HeLa cell line stably expressing HA-SRC-3 upon fusion to mouse 3T3L1 cells (data not shown). Thus, a nuclear pool of SRC-3 can migrate from one nucleus to another within about an hour, which conclusively demonstrates the nucleocytoplasmic shuttling capacity of the coactivator.
To further examine the kinetics of SRC-3 nucleocytoplasmic shuttling, HeLa cells showing intense nuclear staining and some cytoplasmic signal of GFP-SRC-3 (~5% of the cells) (see Fig. S1B in the supplemental material) were chosen for photobleaching analyses. Upon bleaching within the cytoplasmic compartment, the fluorescence intensity levels of both the cytoplasmic and nuclear compartments were monitored until a plateau was reached. Graphical analysis of the fluorescence intensity over time confirms that fluorescence recovers within the photobleached compartment (the cytoplasm) as the intensity within the unbleached compartment (the nucleus) decreases (Fig. (Fig.4C).4C). This indicates that at least a portion of the cellular pool can rapidly (within minutes) shuttle between nuclear and cytoplasmic compartments. As predicted from the previous experiments, the bleaching of cytoplasmic fluorescence in the presence of leptomycin B slowed the recovery of fluorescence (data not shown), indicating that the nuclear export of SRC-3 is regulated, at least in part, via the CRM1 export pathway.
If SRC-3 is able to shuttle from the nucleus to the cytoplasmic compartment, then a dynamic situation must exist during which the coactivator may transiently interact with cytoplasmic protein. If this hypothesis is true, and the interactions between SRC-3 and its cytoplasmic partner are strong enough, then SRC-3 may be able to return to the nucleus with its associated protein. To verify our hypothesis, an ER mutant [ERΔ(NLS)] lacking amino acids 250 to 303 (90), which encompass the ER nuclear localization signal (NLS), was used in a cotransfection assay with SRC-3. Once transfected with SRC-3, ER(ΔNLS) appeared to be cytoplasmic in the absence of hormone (Fig. (Fig.4D),4D), with an average FLIN value of 0.46 (Fig. (Fig.4E).4E). However, upon administration of E2, the receptor was shifted in the nucleus (FLIN value of 0.95) (Fig. 4D and E). Therefore, in the presence of E2, ER(ΔNLS) may be transported in the nucleus by a “piggyback” mechanism involving a cytoplasmic fraction of SRC-3. As expected, the presence of leptomycin B inhibited the cotransport of ER(ΔNLS) in the nucleus (FLIN value 0.59), indicating that the export of the coactivator was necessary for the interaction with ERΔNLS. Consequently, an SRC-3 mutant containing a deletion encompassing the NES of the coactivator [SRC-3(ΔNES)] failed to carry ER(ΔNLS) in the nucleus in the presence of E2 (FLIN value of 0.58). Taken together, these results imply that SRC-3 and its cytoplasmic partners are not separated by an intangible barrier; rather, the SRC-3 location reflects a dynamic state in which the coactivator continuously crosses the nuclear membrane, interacts with cytoplasmic proteins, and potentially shuttles them into the nucleus.
Regulation of SRC-3 localization by phosphorylation.
To further analyze the upstream effectors that may regulate the subcellular localization of SRC-3, we studied the impact of the phosphorylation status of SRC-3 at the cellular level. Previous studies have shown that the phosphorylation of SRC-3 is important for its coactivation function and that this phosphorylation can be mediated by ERK1/2 in vitro (18, 84). Thus, we treated HeLa cells expressing GFP-SRC-3 with the MEK inhibitor U0126, which is a noncompetitive inhibitor of MEK-dependent ERK phosphorylation (16). Exposure to U0126 resulted in a partial redistribution of SRC-3 to the cytoplasm (Fig. (Fig.5A,5A, top), while SRC-1 was not affected by the inhibitor (Fig. (Fig.5A,5A, middle). To further quantify the redistribution of the coregulator in response to U01216, HTM was used to establish a dose-response curve. Figure Figure5B5B shows a drop in the SRC-3 FLIN values from ~90% to ~55% over the range of inhibitor concentrations tested (0 to 100 μM). In the same experiment, SRC-1 FLIN values did not significantly change. Due to the fact that SRC-3 can also be phosphorylated by p38MAPK (20, 84), we also examined the effects of the stress-activated protein kinase 2/p38 kinase inhibitor SB202190 (20 μM) but observed no change in SRC-3 localization (data not shown).
FIG. 5.
FIG. 5.
Inhibition of SRC-3 phosphorylation impedes its nuclear localization. (A) HeLa cells expressing GFP-SRC-3, GFP-SRC-1, or GFP-SRC-3(A1-6) were cultured for 48 h in hormone-free medium and then incubated for 4 h with the MEK kinase inhibitor U0126 (45 μM) (more ...)
To confirm that the effect of U0126 on SRC-3 localization was due to the inactivation of ERK1/2, we used RNAi to knock down the expression of ERK1/2 in HeLa cells. The reduction of both ERK1 and ERK2 together (Fig. (Fig.5C)5C) had no effect on the SRC-3 level and resulted in a significant shift of the coactivator to the cytoplasm (FLIN value of ~0.78) (Fig. (Fig.5D,5D, bottom panel). Due to functional redundancy, the knockdown of either p42 or p44 alone failed to alter SRC-3 compartmentation (see Fig. S2 in the supplemental material). The effect on SRC-3 relocation was specific since the subcellular distribution of SRC-1 was not changed upon the same siRNA treatment (see Fig. S2 in the supplemental material). Both the U01216 and RNAi studies clearly suggest a role for ERK1/2-directed phosphorylation in regulating the residency of SRC-3 in the nucleus.
Finally, we used an SRC-3 mutant in which six previously identified phosphorylation sites were replaced by alanines [GFP-SRC-3(A1-6)] (84, 90). The results indicate that the average FLIN value for GFP-SRC-3(A1-6) is ~82%, compared to the value of ~95% obtained for wild-type GFP-SRC-3 (Fig. (Fig.5E).5E). Interestingly, when treated with various amounts of U0126 (0 to 100 μM), this mutant was not further shifted in the cytoplasm (Fig. 5A and B). These data suggest that the mutant (nonphosphorylatable at these sites) is more cytoplasmic and that the main phosphorylation sites involved in SRC-3 localization are located within the six mutated sites.
EGF induces phosphorylation of SRC-3 and promotes SRC-3 nuclear localization.
To further investigate how the nuclear localization of SRC-3 is regulated by phosphorylation, we took advantage of the fact that the EGF receptor is an essential link in the G protein-coupled receptor-mediated ERK1/2 activation pathway. Indeed, if our previous conclusion is correct, EGF treatment should induce an enhancement in SRC-3 nuclear localization. To test our hypothesis, we used in vivo real-time monitoring in combination with HTM analysis of HeLa cells transfected with GFP-SRC-3, GFP-SRC- 3(A1-6), or GFP-SRC-1. Compared to control experiments where cells were exposed to vehicle only (Fig. (Fig.6A,6A, lane 1; see Movie S1 in the supplemental material), the addition of EGF stimulated a significant increase of SRC-3 within the nucleus over 1.5 h (Fig. (Fig.6A,6A, lane 2; see Movie S2 in the supplemental material), while it had no effect on SRC-3(A1-6) (lane 4) or SRC-1 (lane 5) (see also Movies S3 and S4 in the supplemental material); pretreatment of cells with U0126 abrogated the nuclear accumulation of SRC-3 (lane 3). These results were supported with HTM analyses in which EGF induced a 38% increase in nuclear intensity with cells expressing SRC-3 but only minimal changes with cells expressing SRC-3(A1-6) or SRC-1 or when cells were preincubated with U0126 (Fig. (Fig.6B).6B). We also showed that an SRC-3-AAA mutant in which the three NR boxes have been mutated (90) similarly accumulated in the nucleus in presence of EGF (see Fig. S3 in the supplemental material), suggesting that the nuclear increase was independent of the NR interaction. The increase in nuclear intensity is, at least in part, due to an increase in the average FLIN value. Indeed, although already high in SRC-3-untreated cells (~0.95), this value increased to ~0.97 after EGF treatment (data not shown). However, we believe that a part of the increased nuclear intensity is also due to the nuclear accumulation of a diffuse nondetectable pool of SRC-3 in the cytoplasm.
Consistent with these results, in situ extraction using both HYPO and CSK buffers showed that EGF dramatically increased the nuclear affinity of SRC-3 but not SRC-3(A1-6) (Fig. 6C and D). Finally, FRAP analysis showed a clear reduction of SRC-3 mobility with EGF treatment (t1/2 increased by ~3.4 s), an effect that was not significant with SRC-3(A1-6) (t1/2 increased by ~1.4 s) (Fig. (Fig.6E).6E). Taken together, these results indicate that increased nuclear accumulation of SRC-3 is concomitant with decreased mobility and resistance to real-time detergent extraction and suggest that EGF induces the phosphorylation of the coactivator, leading to an increased residency time of SRC-3 in the nuclear compartment.
To definitely demonstrate the link between the EGF-induced nuclear localization of SRC-3 and its phosphorylation code, we investigated the effect of EGF on SRC-3 phosphorylation using phosphorylation site-specific antibodies in HEK293 cells transfected with Flag-tagged SRC-3. As shown in Fig. Fig.6F,6F, the phosphorylation of SRC-3 at threonine 24, and at serines 857 and 860, was enhanced in the presence of EGF. With preincubation using U0126, both serines 857 and 860 showed a reduced EGF-induced phosphorylation (Fig. (Fig.6G).6G). This result suggests that ERK1/2 is able to phosphorylate serines 857 and 860 upon EGF treatment. Interestingly, when these two sites were mutated in combination, but not independently, the coactivator localized slightly in the cytoplasm (FLIN value of 0.87) (see Fig. S4 in the supplemental material). This result indicates that serines 857 and 860 are important for the nuclear localization of SRC-3 and that each phosphorylation is not individually sufficient to regulate subcellular localization.
Phosphorylation events govern SRC-3 and ER colocalization/interaction.
To determine the role of SRC-3 phosphorylation in the context of ER interactions at the single-cell level, we next examined the effect of U0126 inhibition on cells coexpressing ER and SRC-3. HeLa cells were cotransfected with GFP-SRC-3 and HcRed-ER and incubated with 10 nM E2 for 2 h with or without a 1-h preincubation with U0126. In E2-treated cells, we observed a co-reorganization of SRC-3 and ER into a hyperspeckled colocalization pattern (Fig. (Fig.7A).7A). Strikingly, when the cells were pretreated with U0126, nuclear SRC-3 and ER showed much less colocalization (Fig. (Fig.7A,7A, bottom). In addition, we observed that the presence of ER prevented the cytoplasmic shift of SRC-3 in the presence of UO126, which might be due to the previously observed increase of SRC-3 nuclear affinity (Fig. (Fig.2C).2C). To accurately quantify this phenomenon, we used HTM and measured the variance of the fluorescent signal in the nucleus (CN_VAR) of GFP-SRC-3 and HcRed-ER under the different hormone and inhibitor treatment regimens. The CN_VAR values are directly linked to the presence or absence of subnuclear hyperspeckling; in the absence of hormone, the average nuclear fluorescence variation of both proteins was very low (0.24 and 0.14 for SRC-3 and ER, respectively) (Fig. (Fig.7B).7B). In the presence of E2, the average fluorescence variation of both SRC-3 and ERα increased significantly to 0.9 and 1.3, which correspond to increases of about four and nine times, respectively (Fig. (Fig.7B).7B). However, when the cells were pretreated with U0126, this increase was abolished. We also found that with SRC-3(A1-6), E2 did not induce the co-reorganization of SRC-3(A1-6) and ER into a hyperspeckled colocalization pattern (Fig. (Fig.7C),7C), and accordingly, the corresponding CN_VAR values were not significantly modified (Fig. (Fig.7D7D).
The E2-induced phosphorylation by ERK1/2 at serine 118 is still controversial (8, 32, 55). However, to test whether the inhibitory effects of U0126 on SRC-3/ER colocalization is due to a decrease in ERK1/2 phosphorylation of SRC-3 alone not involving ER phosphorylation, we abrogated the direct regulation of ER by ERK1/2 through the use of the S118A mutation (37). The result shows that in the presence of E2, this mutant could still colocalize with SRC-3 (Fig. (Fig.7E)7E) at a level comparable to that of wild-type ER (Fig. (Fig.7F),7F), indicating that the phosphorylation of ER at serine 118 is not a prerequisite for promoting ER/SRC-3 colocalization and complex formation.
Since the CN_VAR values are directly linked to the presence or absence of subnuclear hyperspeckling, these results indicate that the ERK1/2 phosphorylation of SRC-3 is necessary for both its nuclear interaction with ER and its concomitant intranuclear reorganization. To determine the SRC-3 and ER interaction, steroid induction was carried out in MCF-7 cells that express both endogenous SRC-3 and ER. Following immunoprecipitation with an anti-ER antibody, the coprecipitated SRC-3 was detected by Western blotting with an anti-SRC-3 antibody. As expected, in the absence of hormone, wild-type SRC-3 interacted minimally with ERα, and this interaction was greatly enhanced by E2 (Fig. (Fig.7G).7G). However, when the cells were preincubated with the U0126 inhibitor, a marked reduction in the interaction between ER and SRC-3 was observed (Fig. (Fig.7G).7G). This reduction was not due to reduced protein expression since the total amounts of SRC-3 and ER were constant (Fig. (Fig.7G,7G, Total SRC-3).
Since U0126 affected both the ability of SRC-3 to interact with ER and its subnuclear localization, we next tested whether the presence of the inhibitor could impact the transcriptional activity of the coactivator; we did this by employing an ER response element/luciferase reporter. SRC-3 efficiently coactivated ER upon the addition of E2, while the presence of U0126 diminished coactivation (Fig. (Fig.7H).7H). This result is not due to a defect in SRC-3 intrinsic to its transcriptional activation domain function because U0126 treatment did not decrease transactivation when a Gal4-SRC-3 construct was transfected together with a 5× UAS-TATA-luciferase reporter (Fig. (Fig.7I7I).
Promoter interaction of SRC-3 and ER requires phosphorylation.
The results described above indicate that a sizable fraction of the nuclear pools of SRC-3 and ER can interact in a ERK1/2 phosphorylation-dependent manner. However, those studies do not allow us to discriminate the minute fraction (e.g., a small percentage of the total nuclear volume) involved in the transcriptional activation of genes from the whole pool of nuclear SRC-3 and ER. Therefore, we next visually evaluated the amount of receptor and coactivator involved in transcriptional activation using a chromosomally integrated reporter gene array, allowing us to visualize the recruitment of both ER and SRC-3 at a PRL promoter/enhancer reporter array (63). In this system (clone 19) (see Materials and Methods), under hormone-free conditions for 24 h, the accumulation of ER, SRC-3 or SRC-1 (control), and polymerase II at the multicopy gene locus does not rise above nucleoplasmic levels (Fig. (Fig.8A;8A; see Fig. S5A in the supplemental material). At the transcriptional level, the integrated reporter gene function measurement by mRNA fluorescent in situ hybridization indicated negligible activity (see Fig. S5B in the supplemental material). When cells were treated with E2, the transgene array became visible within a few minutes, as revealed by GFP-ER and endogenous SRC-3 and SRC-1 colocalization (Fig. 8A and B). This accumulation was also accompanied by a clear accumulation of RNA polymerase II (see Fig. S5A in the supplemental material) and mRNA from the reporter (>10-fold over vehicle) (see Fig. S5B in the supplemental material). However, when the cells were pretreated with U0126, whereas ER still accumulated at the promoter, SRC-3 recruitment decreased (Fig. (Fig.8A),8A), and a marked reduction of the colocalization pattern with ER was observed (Fig. (Fig.8B).8B). Interestingly, ER was still able to recruit other coregulators such as SRC-1 at levels comparable to those of cells treated only with E2.
To further confirm the importance of phosphorylation in SRC-3 recruitment at the promoter level, SRC-3 was compared with its mutant, SRC-3(A1-6). Both were cotransfected with HcRed-ER in clone 19 cells and were incubated for 30 min with or without E2. As expected, the results show that GFP-SRC-3 was successfully corecruited at the promoter with ER (Fig. (Fig.9A)9A) in 97.8% of the cells expressing both ER and SRC-3 (Fig. (Fig.9B).9B). In contrast, when SRC-3(A1-6) was coexpressed with ER, E2 treatment led to a marked reduction (i.e., 62.3%) of cells with ER and SRC-3(A1-6) recruited at the array (Fig. (Fig.9A,9A, bottom, and B). Interestingly, in the cell population where GFP-SRC-3(A1-6) was recruited (~38%) (Fig. (Fig.9B),9B), we found that transcriptional activation was markedly reduced compared to that of cells expressing GFP-SRC-3 (see the decreased mRNA fluorescent in situ hybridization signal in Fig. S6 in the supplemental material), probably due to a defect in recruiting other coactivators (e.g., CBP) (84). Taken together, these results indicate that ERK1/2 differentially affects the recruitment of SRC-1 versus SRC-3 to a visible ER-regulated PRL promoter array. Also, these results underscore the functional redundancy of ERK1/2-dependent signaling within the p160 coactivator family, as the ER-SRC-1 interaction and promoter function were retained in the presence of U0126, unlike the ER-SRC-3 interaction. In addition, the SRC-3(A1-6) results (Fig. (Fig.9)9) clearly highlight the importance of the phosphorylation of SRC-3 as a condition for an optimal recruitment at the promoter level and are consistent with previous observations showing that phosphorylation sites are required for the coactivation of ERs (84).
FIG. 9.
FIG. 9.
SRC-3 phosphorylation is essential for recruitment at the promoter. (A) PRL-HeLa cells transiently coexpressing HcRed-ER and GFP-SRC-3 or GFP-SRC-3(A1-6) were treated with or without E2 (10 nM) (− or E2, respectively) for 30 min and then fixed (more ...)
We have shown that SRC-3 is capable of trafficking from the nucleus to the cytoplasm at the steady state and without any stimuli and that this property confers SRC-3 the capability to interact with cytoplasmic partners. Phosphorylation plays a role in the cellular location of SRC-3 by enhancing its nuclear localization. Furthermore, we found that EGF modulates the SRC-3 phosphocode at specific serine residues and consequently enhances its nuclear localization and residency time. Finally, we have established that SRC-3 phosphorylation is important not only for colocalization with ER in the bulk nucleus but also for functional ER/SRC-3 complexes located at a model promoter (PRL array). SRC-3 phosphorylation can be integrated into a comprehensive cellular model (Fig. (Fig.10)10) where it coordinates coactivation function by enhancing its (i) nuclear localization, (ii) interaction with nucleoplasmic NR, and (iii) promoter occupancy.
FIG. 10.
FIG. 10.
Phosphorylation life cycle of SRC-3 and probabilistic interactions. (A) The vast majority of SRC-3 is nuclear resident, but both the nuclear and cytoplasmic pools are dynamic. SRC-3 contains an NLS and an NES that regulate its import and export, respectively. (more ...)
SRC-3 dynamics are influenced by nuclear interactions.
The tools to study cellular compartmentalization and dynamics have become increasingly sophisticated and are now being directed at refining and extending previous models of NRs and coregulators function. The compartmentalization analyses used in this study have been designed and applied to emphasize single-cell measurements and minimize the disadvantages of bulk population averaging. Collectively, these studies emphasize a cell-wide, multimodal, phosphorylation-based approach to the regulation of SRC-3 intracellular trafficking, thereby refining previously described models of where and how SRC-3 functions in the context of NR coactivation. Furthermore, these cellular responses for SRC-3 are clearly distinct from those of other p160 coactivators, thus implying differential mechanisms of regulation.
The results presented combined standard biochemical fractionation techniques with real-time imaging to show the rapid (sometimes instant) and complete extraction of SRC-3 from the nucleus upon exposure to ice-cold hypotonic buffer. This event occurs because, unlike SRC-1, unstimulated SRC-3 is not tightly bound to the relatively immobile nuclear proteins (e.g., chromatin), or an insoluble substructure, and readily exits the nucleus. Interestingly, the presence of coexpressed ER (even without ligand) led to a significant retention of SRC-3 in the nucleus. These data support the concept that SRC proteins can interact with unliganded receptors (66). Expressed alone, SRC-3 is fully eluted from a cell with low-salt buffer (without detergent), while SRC-1 (Fig. (Fig.2B)2B) and unliganded ER are not (65).
The use of live-cell FRAP analysis allowed us to probe the relationships between extractability and protein dynamics. The current SRC-3 mobility studies differ from our previous single-cell analyses of ER and SRC-1 (67), although some parallels exist. SRC-3 nuclear mobility is even more dynamic relative to ER or SRC-1 (66, 67). Once in the nucleus, SRC-3 has decreased mobility in the presence of unliganded, and especially agonist-treated, ERα, presumably owing to interactions with less-mobile nuclear components. The reduced mobility may be associated with a prelude to, or concomitant with, probabilistic interactions with other transcription factors involved in transient complex formation. The fraction of interactions and transient complexes associated with transcription per se, however, is likely small, given the number of molecules and nuclear volume, yet can still be a process required for bona fide transcription initiation. This consideration highlights the issue of functioning versus functional protein pools, with an increasing number of studies suggesting that molecular dynamics and/or stochastics play an important role in gene regulation (57, 78). The increased ER-SRC-3 interactions indicated by FRAP analysis (t1/2 SRC-3 < t1/2 SRC-3 + apo-ER < t1/2 SRC-3 + holo-ER) likely result in the increased nuclear retention seen when cells are challenged with hypotonic buffer. Using a bulk biochemical fractionation approach, this process was previously interpreted in our laboratory as being a form of translocation upon coactivator activation.
SRC-3 subcellular localization.
We established SRC-3 localization to be primarily nuclear in all cell lines tested. These findings are consistent with (i) various immunohistochemical observations (7, 42, 70), (ii) the fact that SRC-3 harbors a nuclear localization signal (39, 56), and (iii) its role as a coactivator of NRs (40). However, using a biochemical approach, other groups reported previously that SRC-3 was localized mainly in the cytoplasm (56, 83). Those results may be explained by the observation that >50% of nuclear proteins are lost from the nucleus within 10 min of cell disruption (54). More specifically, Myc, Myb, DNA ligase 1, and the nucleolar protein Nopp140 can be recovered in the cytosol after cell homogenization (15, 41, 48). Therefore, in studies where the subcellular localization of SRC-3 was analyzed by cell fractionation and concluded to be cytoplasmic, we explain this discrepancy as resulting from condition-dependent SRC-3 extractability, a phenomenon which has also been described previously for some steroid receptors (23, 25). However, some reports indicate increased cytoplasmic SRC-3 directly on tissue sections (35, 42, 56). To reconcile these observations, we should consider several issues. First, SRC-3 can shuttle between the nucleus and the cytoplasm over the course of an hour. In addition, by comparing the expression of SRC-3 in normal breast tissue to that in malignant breast tissue, List and colleagues found that there is an increased nuclear retention with a concomitant decrease of cytoplasmic SRC-3 as normal epithelium progresses to a more tumorigenic phenotype (42). Furthermore, Kuang and colleagues (35) previously examined the cellular and subcellular localizations of SRC-3 in the mammary gland and suggested that the cellular concentrations and subcellular localization of the coactivator in this organ are both developmental stage and malignant state specific. Consistent with these findings, using a transgenic mouse model of breast epithelium, Avivar et al. showed that SRC-3 is cytoplasmic in nonproliferative cells versus nuclear in dividing cells (4). The differential localization of SRC-3 is of interest, since changes in subcellular compartmentalization have also been described for other transcription or replication factors relevant to breast cancer, such as BRCA1 and p53 (11, 51). SRC-3 contains both an NLS and NES (39, 56), and developmental or oncogenic functions of SRC-3 might be activated or repressed by cellular events favoring one of these two types of signal pathways. Our results showing the effect of a MEK1/2 inhibitor on SRC-3 subcellular localization strongly suggest that the activation of cytoplasmic kinases such as ERK1/2, perhaps through membrane-initiated pathways, could be a key to modulating the cellular location and function of SRC-3 observed in the nucleus of normal versus malignant cells.
Physiological relevance of SRC-3 shuttling.
The nucleocytoplasmic shuttling properties of proteins have been ascribed to many NRs (27, 36). These proteins have exclusively nuclear functions, and therefore, it has been difficult to explain the physiological relevance of the phenomenon. In contrast, our SRC-3 observations appear to be critical for a better understanding of its biological functions. In fact, an increasing number of observations reveals that SRC-3/AIB1 has a role in extranuclear events or is modified in the cytoplasmic compartments. For example, in a recent study, SRC-3 has been shown to play an unexpected role in the repression of mRNA cytokine production at the translational level (88). This translational repression is dependent on the cytoplasmic association of SRC-3 with the known translational repressors T-cell intracellular antigen 1 and T-cell intracellular antigen 1-related protein. It has also been shown that SRC-3 is involved in cell migration and promotes metastatis in breast tumor cells, perhaps by promoting the proper localization of focal adhesion kinase to cellular extensions and membrane protrusions (87). Those observations may imply that, at some point, a fraction of the coactivator has to be cytoplasmic to achieve these functions. Furthermore, nucleocytoplasmic shuttling properties may also provide a nexus for cross talk between SRC-3 and cytoplasmic kinase pathways. Indeed, Wang et al. and Wu et al. have independently shown that SRC-3 interacts with three members of the IKK complex (79, 84). Two of these kinases (IKKα and NEMO) have been to shown be localized exclusively in the cytoplasm (30, 31), implying that a fraction of the coactivator is necessarily cytoplasmic. Finally, the nucleocytoplasmic shuttling capabilities of SRC-3 might also serve as a regulatory process for the cell to modulate SRC-3 function. For example, SRC-3 may be associated with different proteins at the steady-state level depending on its localization. Consequently, this phenomenon might lead to a compartment-specific activity of the coactivator, as it has been reported that SRC-3 histone acetyltransferase activity was very robust when nuclear but was dramatically decreased when it was cytoplasmic (56).
Our observations suggest that SRC-3 will have a nuclear coactivation role as a main function but may also have a biological function in the cytoplasm. In this hypothesis, SRC-3 nuclear function can be regulated by membrane-initiated pathways and posttranslational modification occurring in the cytoplasm (e.g., phosphorylation). In the same way, SRC-3 cytoplasmic function can be “activated” or regulated by posttranslational modifications and/or interactions with specifically cytoplasm-localized proteins.
Cellular compartmentalization of SRC-3 is regulated by phosphorylation and the ERK signaling pathway.
Our results indicate that ERK1/2 kinases regulate the cellular localization of SRC-3 at several levels (nuclear retention, subnuclear speckling, and promoter occupancy) (see below). Consistent with these findings, we found that an SRC-3 mutant with alanine substitutions at six identified phosphorylation sites showed a greater tendency to be cytoplasmic. ERKs are the terminal effectors of the classical MAPK cascade, which consists of signal transduction from the MAPK kinase kinases (A-, B-, and C-Raf) to MAPK kinases (MEK1/2) and finally to the MAPKs (ERK1/2). ERK1 and ERK2 have 83% amino acid identity and are variably expressed in all tissues (reviewed in reference 60). They are strongly activated by growth factors like EGF (12). Indeed, in our studies, EGF stimulated SRC-3 phosphorylation at threonine 24, serine 857, and serine 860 and increased its nuclear localization. Interestingly, threonine 24 is located within the NLS of SRC-3 (residues 16 to 38) (39) and is also conserved in the NLS of SRC-1, which also can shuttle between the nucleus and cytoplasm (1). Our cell-based results indicate that SRC-3 localization is regulated by ERK1/2, which is consistent with previous biochemical studies showing that SRC-3 is a substrate of the two kinases (18).
We cannot exclude the possibility that other kinases may also be involved in SRC-3 subcellular localization. The inhibitory effect of U0126 was observed at a low concentration (~15 μM); higher concentrations (100 μM) increased the cytoplasmic localization of SRC-3, but other kinases could be inhibited at this level of inhibitor (16, 33), further reducing SRC-3 phosphorylation. This might explain why the specific knockdown of ERK1/2 did not reduce FLIN values as effectively as 100 μm U0126.
Strikingly, in the presence of ER, U0126 lead to a marked reduction in the colocalization of ER and SRC-3 in nucleoplasmic speckles but not a cytoplasmic shift as seen in the absence of ER. This effect could be due to an increase in the nuclear affinity of SRC-3 in the presence of nonliganded or E2-activated ER, as shown in Fig. Fig.2.2. Consistent with this hypothesis, we showed that E2 promotes a subnuclear repositioning of ER-SRC-3 complexes, as reflected in a dynamic (as shown by FRAP) hyperspeckled pattern where both proteins colocalize. As expected, coimmunoprecipitation assays confirmed that both proteins can physically interact. This ERK1/2-sensitive redistribution of SRC-3 and liganded ER to discrete (albeit transient) subnuclear compartments suggests a role for phosphorylation in cellular reorganization and transient interactions of NR and coactivator. These results using the single-cell approach are consistent with a previous report showing that PD98059 (another MEK1/2 inhibitor) reduced in vitro interactions between ER and SRC-3 (84).
To complete our understanding of the phosphorylation states of SRC-3 in terms of functional complexes, we used a single-cell model to visualize and quantify the transcriptional processes based upon ER regulation of a prolactin promoter/enhancer array (63). Our results demonstrated that ERK phosphorylation is a key posttranslational event not only for regulating interactions between SRC-3 and other coactivators, nuclear targeting, and subnuclear colocalization with ER but also for promoting interactions with ER at the promoter level (Fig. (Fig.10).10). These findings establish a new cellular context for SRC-3 function mediated by distinct spatiotemporal phosphorylation events. At one level, the specific phosphorylation of SRC-3 causes a shift in the equilibrium of probabilistic interactions toward transiently stable (functional) complexes at a promoter. Alternatively, dephosphorylated SRC-3 leads to complex dissociation and the return of coactivator and receptor to stochastic cellular pools (see the model diagram in Fig. Fig.1010).
ERK signaling pathway and SRC-3 links to cancer.
It is interesting to consider our observations in the light of compelling evidence implicating SRC-3 as a causal factor in the genesis of human cancers. The role of SRC-3 phosphorylation in oncogenic stimulation was established previously by Wu et al. (84), who studied the oncogenic potential of SRC-3 and found that wild-type SRC-3 collaborated with Ras to transform SRC-3−/− MEFs, whereas nonphosphorylatable SRC-3 mutants (including T24A) caused only minimal transformation. Thus, it is plausible in normal tissues that a basal, constant stimulation of SRC-3 leads to a neoplastic phenotype as a consequence of misregulation at any one of the multiple regulatory steps involved in its cellular compartmentalization. For example, both normal mammary development and breast cancer growth are under the influence of ER, which regulates cell proliferation (14). In addition to estrogens, a host of polypeptide growth factors, like EGF, may also play an important role in regulating the growth of breast cancer cells by autocrine, paracrine, or endocrine mechanisms (13, 64). As breast cancer cells have membrane receptors for the several growth factors that they secrete, it has been proposed that these growth factors activate the ERK signaling pathway (50). ERKs, in turn, may induce SRC-3 phosphorylation, its translocation to the nucleus, and stimulation of coactivator activity (18, 82). Interestingly, a clinical study of breast cancer patients showed that SRC-3 and HER2/neu (EGF receptor family member) expression levels are closely associated with a decidedly worse prognosis, suggesting that a critical link between HER2/neu and SRC-3 might exist in breast cancer (53). Our current study furthers this notion by suggesting that constitutively active HER2 signaling would result in phosphorylated SRC-3 with a longer nuclear dwell time.
Supplementary Material
[Supplemental material]
We gratefully acknowledge J. Zhong, K. M. Antalis, C. Haueter, and D. Townley for excellent technical support and C. Foulds, Y. K. Kang, J. F. Louet, A. Siddiqui, and A. Immaneni for critical and thorough reading of the manuscript. We thank C. L. Smith and Ping Yi for kindly providing F-SRC-3AAA and F-SRC-3(ΔNES) expression constructs, respectively.
We thank La Fondation pour la Recherche Médicale for their initial financial support to L. Amazit. This work was funded by DK-55622 (M. Mielke), Cain Cancer 1080001204 (B. W. O'Malley), HD08818 (B. W. O'Malley), and DK61589 (M. G. Mancini) and a postdoctoral fellowship to R.-C. Wu (U.S. Department of Defense Breast Cancer Research Program [DAMD17-01-1-0147]). The Integrated Microscopy Core used throughout this study was supported in part by U54 HD-96008 (B. W. O'Malley) and P01 CA-06425 (D. Medina) and the Dan L. Duncan Cancer Center of Baylor College of Medicine.
[down-pointing small open triangle]Published ahead of print on 23 July 2007.
Supplemental material for this article may be found at
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