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Regulation of SRC-3 Intercompartmental Dynamics by Estrogen Receptor and Phosphorylation ^,

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. Mancini^1*

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

Received 9 September 2006/ Returned for modification 19 October 2006/ Accepted 23 May 2007

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (ER) 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 ER, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ER, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ER-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.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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-B (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 IB 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 (ER) 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.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids. 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(NES) 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(A_1-6) construct (expression plasmid in which all six identified phosphorylation sites have been mutated to alanine) (90) were generated by subcloning the SRC-3(A_1-6) fragment from pCMV-Flag-SRC3(A_1-6) 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(NLS) 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. 6 to 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).

View larger version (60K): [in this window] [in a new window]   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 with EGF for 1.5 h. In lane 3 (U0126 plus EGF), cells were first preincubated for 3 h with U0126 (45 µg/ml). (B) Automated quantification of SRC-3 subcellular localization upon EGF treatment using high-resolution HTM. HeLa cells were transfected with GFP-SRC-3, GFP-SRC-3(A1-6), or GFP-SRC-1 and treated for 1.5 h with EGF (100 ng/ml). Cells were fixed, DAPI stained, and analyzed by HTM. The histograms show the nuclear fluorescence intensity values obtained for hundreds of cells in the absence or presence of EGF and in cells pretreated with U0126 inhibitor (**, P < 0.01) (error bars represent SD). (C) Real-time monitoring of SRC-3 extractability upon EGF treatment. HeLa cells expressing GFP-SRC-3 or GFP-SRC-3(A1-6) were grown in hormone-free medium during 48 h. Cells were then incubated with 100 ng/ml EGF during 1.5 h and monitored before and 1, 5, and 10 min after perfusion with ice-cold HYPO buffer. Bright-field phase images show that the cell remained in place and became rounded and swollen as a result of exposure to the buffer. All imaging parameters were held constant within each set of images. Note the resistance of SRC-3 to extraction compared to SRC-3(A1-6). (D) Automated HTM quantification of GFP-SRC-3 and GFP-SRC-3(A1-6) after in situ extraction in the presence of EGF. Transfected cells were grown for 48 h in hormone-free medium and treated for 1.5 h with vehicle or EGF (100 ng/ml). Cells were then permeabilized (indicated by "–") and treated with ice-cold HYPO or CSK buffer for 3 min. After fixation, cells were analyzed by HTM: the same numbers of fields (100 fields) were automatically scanned, and the cell population obtained was quantified for the average nuclear fluorescence/cell for each condition. (E) Intranuclear mobility of SRC-3 is reduced in the presence of EGF. Photobleaching experiments were performed on HeLa cells expressing GFP-SRC-3 or GFP-SRC-3(A1-6). Transfected cells were grown for 48 h in hormone-free medium and treated with or without 100 ng/ml EGF for 1.5 h. The graphs represent the averages of normalized intensity (in arbitrary units) (mean ± SEM). In the left panel, the graph representing GFP-SRC-3 alone (Fig. 3B) was inserted for comparison. The inset box indicates the t1/2 of fluorescence recovery for each condition and the number of cells analyzed (n). (F) EGF induces phosphorylation of SRC-3 at specific sites. HEK293 cells grown in phenol red-free DMEM (supplemented with 5% charcoal-dextran-stripped FBS) were cotransfected with an expression plasmid for wild-type Flag-SRC-3. After transfection, cells were grown in medium containing 0.5% FBS for 36 h and stimulated with EGF (100 ng/ml) for 1 h before harvest. Flag-SRC-3 was immunoprecipitated by anti-Flag antibodies and separated on an 8% sodium dodecyl sulfate-polyacrylamide gel. Immunoblotting was performed using the indicated phosphorylation state-specific antibodies. All membranes used for experiments were stripped and reprobed with anti-Flag antibodies to control for protein loading (total f-SRC-3). (G) U0126 inhibits EGF-induced phosphorylation of SRC-3 at specific sites. Cells were treated as described above (F), except for the right lane, where the cells were preincubated with U0126 (45 µg/ml) for 1 h. Immunoblotting was performed using the indicated phosphorylation state-specific antibodies.

View larger version (44K): [in this window] [in a new window]   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 ER. (A) PRL-HeLa cells transiently expressing GFP-ER were treated with or without E2 (10 nM) (– or E2, respectively) for 30 min, fixed, and immunolabeled. Where indicated, cells were pretreated with U0126 (45 µM) for 2 h and then further incubated with E2 for 30 min (U0126/E2). Endogenous SRC-3 and SRC-1 were immunodetected with their respective antibodies (p160). A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images. Arrows indicate the accumulation of ER or SRC-3/SRC-1 at the ER element integration site. Note that in contrast to SRC-1, the ligand-dependent recruitment of SRC-3 is inhibited by the presence of U0126. Bar, 2.5 µm. (B) Quantification of the colocalization pattern between p160 coactivators (SRC-3 or SRC-1) and ER at the PRL-promoter using the Pearson's colocalization coefficient [R(r)] (see Materials and Methods). The inset box shows examples of areas (i.e., transgene array) that were selected for calculation and the corresponding R(r) values.

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 x 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(NLS) was cotransfected with 450 ng the plasmid encoding Flag-tagged SRC3 or Flag-tagged SRC3(NES) 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 5x 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.

View larger version (46K): [in this window] [in a new window]   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) (40 nM) or vehicle (dimethyl sulfoxide) for 4 h. Cells were fixed, immunolabeled for endogenous SRC-3, and then analyzed by HTM. FLIN values were determined for each cell, and the corresponding filled-area histogram was plotted for each condition. The average FLIN values are also indicated in addition to the number of cells analyzed. Note the shift of the histogram to the right in the presence of leptomycin B, which is indicative of an increased nuclear localization of SRC-3 in the cell population. (B) Transfer of SRC-3 from human to mouse nuclei in interspecific heterokaryons. Human (HeLa) and mouse (MEF) (arrows) cells are distinguished by the DAPI staining showing specific heterochromatic DNA in MEF cells. HeLa cells containing endogenous SRC-3 were fused to mouse SRC-3–/– MEFs (devoid of SRC-3). One hour before fusion, cycloheximide (50 µg/ml) was administered to prevent new synthesis of the coactivator. One hour after fusion, cells were fixed and immunolabeled for SRC-3. Note the absence of SRC-3 in the MEF–/– cells when the cells were cocultured together but not fused (bar, 5 µm). (C) Kinetics of SRC-3 nucleocytoplasmic shuttling. HeLa cells with a cell-wide distribution of GFP-SRC-3 were analyzed by FLIP. Cells were photobleached in the cytoplasm, and fluorescence was subsequently measured (every 5 s for 60 s) in both the nucleus and cytoplasm. Following cytoplasmic bleaching, nuclear GFP-SRC-3 fluorescence decreased, while cytoplasmic fluorescence increased, reaching new steady-state levels within 60 s. (D) Hormone-dependent interaction between SRC-3 and ER(NLS) takes place and results in the cotransportation of coactivator/receptor into the nucleus. A GFP-ER mutant lacking amino acids 250 to 303 [ER(NLS)] was cotransfected with empty vector (–), Flag-SRC-3, or a Flag-SRC-3 mutant lacking amino acids 1031 to 1130 [Flag-SRC-3(NES)]. Forty hours after transfection, cells were incubated with E2 (10 nM) during 8 h in the presence of 50 µg/ml cycloheximide and then fixed and immunolabeled with anti-Flag antibodies. Where indicated, cells were pretreated with leptomycin B for 30 min and then further incubated with E2/leptomycin B during 8 h. A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate a high-resolution picture. DAPI staining delineates the nuclei (bar, 2.5 µm). Note that the E2-induced shift of ER(NLS) in the nucleus does not occurs in the presence of leptomycin B or when coexpressed with SRC-3(NES) (bar, 5 µm). (E) Automated quantification of ER(NLS) subcellular localization using high-resolution HTM. The histograms show the average FLIN values obtained for hundreds of cells scanned from the experiment described for panel D. Error bars represent SE.

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).

Immunocytochemistry. 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).

HTM. Cells were imaged using the Cell Lab IC 100 image cytometer from Beckman Coulter, Inc., with a Nikon 40x Plan S fluor 0.90-numerical-aperture (NA) objective, except for the experiment shown in Fig. 7B, D, and F, where a Nikon 63x 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.

View larger version (61K): [in this window] [in a new window]   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 indicated, U0126/E2 cells were pretreated for 2 h with U0126 (45 µM) and further treated with E2 for 2 h. A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images. The inset shows a magnification of the selected subnuclear region (white square). (B) Automated HTM quantification of the subnuclear pattern variation. HeLa cells transiently expressing GFP-SRC-3 and HcRed-ER were treated as described above (A) and analyzed by high-resolution HTM with a 60x objective. The histograms show the subnuclear variation measurement (CN_VAR x 10–2), which is the statistical variation in pixel brightness for each channel (SRC-3 and ER) in the nuclear compartment. Note that the increase in CN_VAR for both SRC-3 and ER in the presence of hormone is inhibited by U0126 (error bars represent SE). (C) HeLa cells expressing GFP-SRC-3(A1-6) and HcRed-ER were treated and analyzed as described above (A). (D) Automated HTM quantification of the variation in the subnuclear pattern of GFP-SRC-3(A1-6) and ER. CN_VAR quantifications were processed as described above (B). (E) HeLa cells expressing Flag-tagged SRC-3 (Flag-SRC-3) (in red) and GFP-ER(S118A) (in green) were treated and analyzed as described above (A) except that after fixation, SRC-3 was immunodetected with anti-Flag antibody. (F) Automated HTM quantification of the variation in the subnuclear pattern of Flag-SRC-3 and GFP-ER(S118A). CN_VAR quantifications were processed as described above (B). (G) MCF-7 cells were incubated for 3 days in 5% stripped-dialyzed serum. E2 (10–8 M) and U0126 (45 µM) (preincubation of 2 h) were added for 1 h (indicated by a +). A coimmunoprecipitation assay was performed using anti-ER antibodies (top), and coprecipitated SRC-3 was detected by Western blotting using anti-SRC-3 antibodies (middle). Note that the ligand-induced SRC-3/ER interaction is dramatically reduced in the presence of U0126. (H) HeLa cells were cotransfected with an ER response element/luciferase reporter gene (150 ng/well) and expression plasmids for ER (5 ng/well) and SRC-3 (150 ng/well). E2 (10–8 M) with or without U0126 (45 µM) was added to the transfected cells 24 h posttransfection and incubated for an additional 8 h. Cells were harvested, and luciferase activities were measured and normalized to protein amounts. Note that the ligand-induced SRC-3 transcriptional coactivation effect is diminished in the presence of U0126. Error bars represent SD. RLU, relative light units. (I) Inhibition of MAPK does not affect SRC-3 intrinsic transcriptional activation domains. HeLa cells were cotransfected with a UAS-TK luciferase reporter gene (150 ng/well) with or without an expression plasmid for Gal4-SRC-3 (50 ng/well). Cells were incubated for 24 h with or without U0126 (45 µM) and harvested, and luciferase activities were then measured and normalized against the total protein amount. Note that SRC-3 intrinsic transcriptional activation is not affected by the presence of U0126. Error bars represent SD.

View larger version (58K): [in this window] [in a new window]   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 washout (CSK-W), and resuspended CSK pellet (CSK-P) were analyzed. (B) Real-time monitoring of a cell expressing YFP-SRC-3 before and 1, 5, and 10 min after perfusion with ice-cold HYPO buffer. Bright-field phase images show that the cell remained in place and became rounded and swollen as a result of exposure to the buffer. All imaging parameters were held constant within each set of images. For comparison, the same protocol was used to extract YFP-SRC-1 expressed in HeLa cells (right two panels). (C) Real-time monitoring of a cell coexpressing YFP-SRC-3 and CFP-ER before and 1, 5, and 10 min after perfusion with ice-cold HYPO buffer. The coexpression of CFP-ER with YFP-SRC-3, either with or without 10 nM E2, leads to a significant increase in the retention of nuclear SRC-3. (D) Automated HTM quantification of GFP-SRC-3 fluorescence. In situ extraction of SRC-3 was performed in HeLa cells transiently expressing GFP-SRC-3 alone (left histogram) or with GFP-SRC-3 coexpressed with HcRed-ER (middle and right histograms). Cells were grown for 48 h in hormone-free medium and treated for 1 h with vehicle or hormone. Cells were then permeabilized (indicated by "–") and treated with HYPO or CSK buffer for 3 min. Fixed cells were analyzed by HTM: the same numbers of fields (100 fields) were automatically scanned, and the cell population obtained was quantified for the total average nuclear fluorescence/cell for each condition.

FRAP. 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 100x/1.4-NA objective with pinhole set at 4 airy units. Scanning was bidirectional at the highest possible rate using a 3.5x 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 (I_t) and the mean nucleus (N_t) signals as follows:

(1)

(2)

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 (t_1/2) 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. 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.

View larger version (48K): [in this window] [in a new window]   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. In the case of SRC-3, note the dramatic decrease of all the fluorescence of the nuclear pool within the nucleus, which demonstrates the extremely rapid movement of the coactivator. In the case of GFP-SRC-1, only a clearly bleached region is apparent, indicating the relatively slower movement of SRC-1. (B) Recovery of fluorescence over time for both SRC-3 and SRC-1. The data plots are the averages of normalized intensity (in arbitrary units) (mean ± SEM). In the inset, n indicates the number of cells analyzed for each coactivator. (C) Fluorescence microscopy was performed on cells expressing CFP-ER (pseudocolored in green) and YFP-SRC-3 (pseudocolored in red). Forty-eight hours after transfection, cells were treated with 10 nM E2 for 2 h or with vehicle (–) and then fixed and counterstained with DAPI. A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images. In the absence of hormone (top row), CFP-ER and YFP-SRC-3 were diffusely distributed in the nucleus. The addition of ligand (bottom row) resulted in a redistribution of both proteins into the same foci, some of which are indicated by arrows (bar, 5 µm). (D) Intranuclear mobility of SRC-3 is reduced in the presence of ER. SRC-3 photobleaching experiments were performed on cells cotransfected with CFP-ER and YFP-SRC-3. The histogram shows the recovery t1/2 values of YFP-SRC-3 alone and in the presence of ligand-free and ligand-bound CFP-ER (n = 42, n = 14, and n = 17, respectively). In the presence of ER, SRC-3 mobility decreases, as indicated by the increased t1/2, suggesting that basal interactions may occur even in the absence of ligand. SRC-3 mobility is further reduced in the presence of E2, indicating that ER-SRC-3 complexes are stabilized by ligand.

Quantitative colocalization analyses (see Fig. 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 (1x 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.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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. 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).

View larger version (29K): [in this window] [in a new window]   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 cell population scanned. FLIN values for endogenous SRC-3 (A) and transiently expressed GFP-SRC-3 (B) were measured. The numbers of cells analyzed and the FLIN averages are indicated. (Inset) Representative cell objects which belong to the extracted subpopulation are shown as examples. (C) Immunodetection of SRC-3 with an antibody to HA in HeLa cells stably expressing HA-SRC-3 under the control of a Tet promoter. The expression of exogenous SRC-3 was repressed in the presence of doxycycline (+ DOX) (200 ng/ml) in hormone-free medium. To express HA-SRC-3, cells were grown in DOX-free medium (– DOX) during 48 h. HA-SRC-3 was expressed in the nucleus as shown by immunodetection using anti-HA antibodies. DAPI staining delineates the nuclei (bar, 10 µm). (D) FLIN area-filled histograms of the doxycycline-free cell population analyzed in C.

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. 2A).

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. 2B, left). In contrast, 10-fold more (55%) nuclear SRC-1 remained under the same conditions (Fig. 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. 2C).

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. 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. 2D, middle). With E2 present, extractability was reduced further and not significantly different from that of unextracted controls (Fig. 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. 3A, bottom) and a short recovery time (t_1/2 = 0.7 ± 0.27 s) (Fig. 3B). Photobleaching of GFP-SRC-1 revealed a more distinct bleach area (Fig. 3A, top) immediately after bleaching and a significantly longer recovery time (t_1/2 = 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. 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. 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 t_1/2 of SRC-3 is significantly higher in the presence of ER (t_1/2 = 0.72 ± 0.12 s) (Fig. 3D). Upon the addition of 10 nM E2 (1 h), SRC-3 mobility in the presence of ER was further reduced (t_1/2 = 1.58 ± 0.14 s) (Fig. 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. 4A, upper panel) than HeLa cells (Fig. 1A). When T47D cells were incubated with leptomycin B for 4 h, average FLIN levels increased to 97% (Fig. 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. 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. 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. 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 [ER(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(NLS) appeared to be cytoplasmic in the absence of hormone (Fig. 4D), with an average FLIN value of 0.46 (Fig. 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(NLS) 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(NLS) in the nucleus (FLIN value 0.59), indicating that the export of the coactivator was necessary for the interaction with ERNLS. Consequently, an SRC-3 mutant containing a deletion encompassing the NES of the coactivator [SRC-3(NES)] failed to carry ER(NLS) 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. 5A, top), while SRC-1 was not affected by the inhibitor (Fig. 5A, middle). To further quantify the redistribution of the coregulator in response to U01216, HTM was used to establish a dose-response curve. Figure 5B 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 p38^MAPK (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).

View larger version (47K): [in this window] [in a new window]   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) or vehicle as a control in the presence of cycloheximide (50 µg/ml). DAPI staining delineated the nucleus. GFP-SRC-3 was partially shifted to the cytoplasm in the presence of the inhibitor. Bar, 5 µm. (B) Dose-response curve showing the effect of U0126 on SRC-3 subcellular localization using high-resolution HTM. HeLa cells were transfected with the indicated expression vector in a 96-well format. Cells were incubated in triplicate with U0126 (0 to 100 µM) 48 h after transfection. Cycloheximide (50 µg/ml) was added to the medium at the same time as the inhibitor. In each well, FLIN values were collected for each cell. Data shown are the average FLIN values of three wells for each condition. Error bars represent standard deviations (SD). (C) Knockdown of both kinases by siRNAs was demonstrated by immunoblotting with anti-ERK1/2 antibodies. Cells were transfected with siRNA ERK1, siRNA ERK2, both together (siRNA ERK1+2), or a control siRNA. Seventy-two hours after transfection, cells were collected, and proteins were extracted for Western blot analysis. For protein loading controls, levels of tubulin and SRC-3 were determined after stripping and reprobing of the membranes with corresponding antibodies. (D) HTM automated quantification of SRC-3 subcellular localization. Coverslips corresponding to lanes 1 (control siRNA) and 4 (siRNA ERK1+2) of the experiment described above (C) were treated for immunohistochemistry and then scanned randomly. The percentage of cells with the corresponding FLIN values were determined, and the corresponding filled-area histogram was plotted for each condition. The average FLIN values are also indicated. Note that RNAi of ERK1 and ERK2 resulted in a decreased FLIN level of SRC-3 compared to control siRNA. (E) A nonphosphorylatable SRC-3 mutant shows altered subcellular localization. HeLa cells were transfected in triplicate with the expression vector for GFP-SRC-3 (wild type) and the nonphosphorylatable mutant form in which the six serine/threonine phosphorylation sites were replaced by alanine [GFP-SRC-3(A1-6)]. Cells were cultured for 48 h in hormone-free medium, fixed, immunolabeled, and then analyzed by high-resolution HTM. The histograms show the average FLIN values collected in triplicate for each condition (**, P < 0.01) (error bars represent SD).

Finally, we used an SRC-3 mutant in which six previously identified phosphorylation sites were replaced by alanines [GFP-SRC-3(A_1-6)] (84, 90). The results indicate that the average FLIN value for GFP-SRC-3(A_1-6) is 82%, compared to the value of 95% obtained for wild-type GFP-SRC-3 (Fig. 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. 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. 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(A_1-6) or SRC-1 or when cells were preincubated with U0126 (Fig. 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 (t_1/2 increased by 3.4 s), an effect that was not significant with SRC-3_(A1-6) (t_1/2 increased by 1.4 s) (Fig. 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. 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. 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. 7A). Strikingly, when the cells were pretreated with U0126, nuclear SRC-3 and ER showed much less colocalization (Fig. 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. 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. 7B). In the presence of E2, the average fluorescence variation of both SRC-3 and ER increased significantly to 0.9 and 1.3, which correspond to increases of about four and nine times, respectively (Fig. 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. 7C), and accordingly, the corresponding CN_VAR values were not significantly modified (Fig. 7D).

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. 7E) at a level comparable to that of wild-type ER (Fig. 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 ER, and this interaction was greatly enhanced by E2 (Fig. 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. 7G). This reduction was not due to reduced protein expression since the total amounts of SRC-3 and ER were constant (Fig. 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. 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 5x UAS-TATA-luciferase reporter (Fig. 7I).

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. 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. 8A), and a marked reduction of the colocalization pattern with ER was observed (Fig. 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. 9A) in 97.8% of the cells expressing both ER and SRC-3 (Fig. 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. 9A, bottom, and B). Interestingly, in the cell population where GFP-SRC-3_(A1-6) was recruited (38%) (Fig. 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. 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).

View larger version (28K): [in this window] [in a new window]   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 and DAPI stained. A z series of focal planes was digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images. Arrows indicate the accumulation of ER or wild-type (WT) SRC-3 at the ER element integration site. Note that in contrast to SRC-3, the nonphosphorylatable mutant SRC-3(A1-6) is not corecruited with ER. (B) Quantitated graph of A. The histogram shows the proportion of the cotransfected cell population that has visible accumulation of both ER and SRC-3 [or SRC-3(A1-6)] at the array in response to E2 treatment (n > 100).

	DISCUSSION

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View larger version (27K): [in this window] [in a new window]   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. External stimuli activate the cytoplasmic ERK signaling pathway, which in turn promotes SRC-3 phosphorylation. This results in an increase in the kinetics of import and the nuclear concentration of SRC-3 where it exhibits rapid molecular dynamics. (B) Phosphorylated SRC-3 has a high-affinity interaction with ER and other coregulators (e.g., CBP, CARM1, and P/CAF) in the nucleoplasm. These interactions lead to a reduced mobility and the formation of random and transient foci, which are generally not associated with transcription sites based strictly upon three-dimensional probabilities. (C) When dynamically bound to chromatin (63), ER can specifically interact with phosphorylated SRC-3, which recruits other members of the chromatin remodeling machinery to initiate transcriptional activation. Phosphorylation-driven equilibria increase the probability of forming transient complexes at a target gene and are critical to ER-regulated gene expression. ERE, ER element; kOFF, constant of dissociation of the ER at the promoter.

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