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Endocrinology, doi:10.1210/en.2008-0037
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Endocrinology Vol. 149, No. 9 4336-4345
Copyright © 2008 by The Endocrine Society

Recruitment of Coactivator Glucocorticoid Receptor Interacting Protein 1 to an Estrogen Receptor Transcription Complex Is Regulated by the 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase

Ingvild S. Fenne, Tuyen Hoang, Marianne Hauglid, Jørn Vegard Sagen, Ernst A. Lien and Gunnar Mellgren

Institute of Medicine, Section for Endocrinology, University of Bergen (I.S.F., T.H., M.H., J.V.S., E.A.L., G.M.) and The Hormone Laboratory, Haukeland University Hospital (I.S.F., E.A.L., G.M.), N-5021 Bergen, Norway

Address all correspondence and requests for reprints to: Gunnar Mellgren, M.D., Ph.D., The Hormone Laboratory, Institute of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. E-mail: gunnar.mellgren{at}med.uib.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Steroid receptor coactivators (SRCs), such as glucocorticoid receptor interacting protein 1 (GRIP1) are recruited to the DNA-bound nuclear receptors (NRs) and are also shown to enhance the gene transactivation by other transcription factors. In contrast to the two other members of the SRC family, SRC-1 and SRC-3/amplified in breast cancer 1, SRC-2/GRIP1 is regulated by the cAMP-dependent protein kinase [protein kinase A (PKA)] that stimulates its ubiquitination and degradation. In this report we demonstrate that COS-1 and MCF-7 cells treated with cAMP-elevating agents and 8-para-chlorophenylthio-cAMP for short periods of time showed an increase in GRIP1 coactivator function, whereas prolonged stimulation of the cAMP/PKA pathway led to a decline in GRIP1-mediated activation and protein levels. Furthermore, MCF-7 breast cancer cells were subjected to chromatin immunoprecipitation assays after stimulation of the cAMP/PKA pathway. cAMP/PKA initiated a rapid recruitment of GRIP1 to the endogenous estrogen receptor (ER)-{alpha} target pS2 gene promoter. In contrast to the estradiol-induced recruitment of GRIP1 to pS2, we observed an additional increase in GRIP1 recruitment on inhibition of the proteasome, suggesting that inhibition of GRIP1 degradation leads to accumulation at the pS2. Real-time PCR experiments confirmed that cAMP/PKA enhanced the expression of pS2. Moreover, confocal imaging of COS-1 cells transfected with yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ER{alpha} revealed that PKA led to redistribution and colocalization of yellow fluorescent protein-GRIP1 and cyan fluorescent protein-ER{alpha} in subnuclear foci. In conclusion, these results suggest that activation of the cAMP/PKA pathway stimulates recruitment of GRIP1 to an ER-responsive gene promoter. The initial stimulation of GRIP1 coactivator function is followed by an increased turnover and subsequent degradation of GRIP1 protein.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID RECEPTORS are members of the nuclear receptor (NR) superfamily of transcription factors regulating the expression of target genes in response to steroid hormone binding and through cross talk with other signaling pathways. Activated NRs bind to hormone response elements within the promoter regions of target genes and recruit different complexes of coactivators, RNA polymerase II (Pol II), and members of the basal transcription machinery leading to initiation of transcription. Steroid receptor coactivators (SRCs) consist of three structurally and functionally related proteins: 1) steroid receptor coactivator (SRC)-1 (1); 2) SRC-2/glucocorticoid receptor interacting protein 1 (GRIP1) or its human homolog, transcription intermediary factor 2 (2, 3); and 3) SRC-3/amplified in breast cancer 1 (AIB1), also named activator of thyroid and retinoic acid receptors, thyroid hormone receptor activator molecule 1, cAMP-response element-binding protein/p300-interacting protein, and receptor-associated coactivator 3 (4, 5, 6, 7, 8). The SRCs interact with ligand-activated NRs via their central NR interaction domain composed of three conserved LXXLL motifs and serve as adapters recruiting other coactivator complexes with different enzymatic activities in a sequential manner, including the histone acetyl transferases, cAMP-response element-binding protein/p300, and p300/cAMP-response element-binding protein-associated factor (5, 8); the histone methyl transferases, coactivator-associated arginine methyltransferase 1 and protein methyltransferase-1/2 (9, 10, 11); and the ATP-dependent chromatin remodeling complex Switch/Sucrose Nonfermentable (12). These coactivators contribute to the unfolding of the chromatin structure facilitating binding of additional regulators. Coactivators possessing ubiquitin conjugating activity, such as ubiquitin conjugation enzyme H7 (13), or ubiquitin ligase activity such as E6-assosiated protein (14) are also associated with the NR complex, contributing to activation of transcription (15). Finally, association of the thyroid receptor-associated protein/vitamin D receptor-interacting protein mediator complex facilitates the interaction of NRs with Pol II and general transcription factors (16).

Ligand-stimulated estrogen receptor (ER)-{alpha}-mediated transcription of the pS2 gene has been extensively characterized and is a highly dynamic process involving continual assembly and disassembly of receptors and the various coactivator complexes at the target pS2 promoter region in a cyclic, coordinated manner (17, 18).

The mechanisms controlling the dynamic association are only partly elucidated. It has been proposed that posttranslational modifications and/or proteasomal degradation of complex members that occur in response to ligand-dependent or ligand-independent cellular signaling pathways are involved (15, 19).

Sumoylation, ubiquitination, phosphorylation, and acetylation have been demonstrated to regulate the function of the SRCs, including their subcellular dynamics and localization, their interaction with NRs and other coregulators, and their protein stability (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). These modifications collectively affect the dynamics of NR-mediated transcription by influencing the combinatorial recruitment of coactivators into active transcriptional complexes at distinct promoters. The coactivator GRIP1 is regulated by sumoylation (26, 27), phosphorylation (23, 24, 28), and ubiquitination (20, 25, 34). Sumoylation of GRIP1 modulates its subnuclear localization and its interaction with NRs (26, 27). We and others have shown that GRIP1 colocalizes with proteasome components in specific nuclear foci and that GRIP1 is degraded through ubiquitin/proteasome-mediated degradation (20, 25). Phosphorylation of GRIP1 by the MAPKs p38 and ERK have been shown to potentiate ER-dependent transcription (24, 28). We previously demonstrated that activation of cAMP-dependent protein kinase (PKA) down-regulates GRIP1 through ubiquitination and proteasomal degradation, leading to the inhibition of GRIP1-mediated coactivation of NRs such as glucocorticoid receptor and steroidogenic factor-1 (21, 25).

In this paper we explored the role of the cAMP/PKA pathway in regulating GRIP1-mediated coactivation of ER{alpha}-dependent transcription. We demonstrate that activation of the cAMP/PKA signaling pathway stimulates GRIP1 coactivation function and the recruitment of GRIP1 to an endogenous ER target gene promoter. Recruitment of GRIP1 can be further enhanced by inhibition of the proteasome and is followed by GRIP1 down-regulation. Moreover, live cell imaging revealed that activation of PKA stimulates the redistribution and colocalization of yellow fluorescent protein (YFP)-GRIP1 and cyan fluorescent protein (CFP)-ER{alpha} into subnuclear foci. These findings suggest that extracellular signals acting through the cAMP/PKA pathway regulate GRIP1 coactivator function through stimulated recruitment to an ER{alpha}-transcription complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The luciferase reporter plasmid ERE-TATA-luc and the expression plasmid pSG5-hER{alpha} were generously supplied by Dr. E. Treuter (Karolinska Institutet, Stockholm, Sweden). The luciferase reporter construct pG5-luc for GAL-4 was obtained from Promega (Madison, WI). The expression plasmid pM-GRIP1 encoding GAL-4 DNA-binding domain fused to GRIP1 and the pSG5-HA-GRIP1 expression plasmid was kindly provided by Dr. M. R. Stallcup (University of Southern California, Los Angeles, CA). The pCMV5-C{alpha} expression plasmid encoding the catalytic subunit of PKA was provided by Dr. G. S. McKnight (University of Washington, Seattle, WA). Fluorescent expression plasmid pECFP-ER{alpha} was generously supplied by Dr. K. Matsuda (Kyoto Prefectural University of Medicine, Kyoto, Japan). pEYFP-GRIP1 expression plasmid was constructed by introducing a SalI and KpnI restriction site at the N- and C-terminal ends of the GRIP1 nucleotide sequence in the pSG5-HA-GRIP1 plasmid by using the QuickChange XL site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) followed by cloning of the GRIP1 fragment into a pEYFP-C1 vector (CLONTECH Laboratories, Inc., Mountain View, CA).

Cell culture and transfection experiments
COS-1 African monkey kidney cells and MCF-7 human breast adenocarcinoma cells were grown at 37 C under 5% CO2, in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% (vol/vol) penicillin/streptomycin solution. The growth medium for the MCF-7 cells was additionally supplemented with 4.5 g/liter glucose and 1 µM insulin. For transfection experiments the cells were seeded in 12- or 24-well plates with phenol red-free DMEM containing charcoaled-stripped fetal bovine serum and the contents described above. The cells were transfected by using SuperFect (QIAGEN, Valencia, CA) and treated with forskolin, 3-isobutyl-1-methylxanthine (IBMX), 8-para-chlorophenylthio-cAMP (8-CPT-cAMP), and 17β-estradiol (E2) as indicated in the figure legends. After incubation for different periods of time, the cells were harvested in a buffer containing 25 mM Tris Acetate-EDTA (pH 7.8), 2 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and 1% Triton X-100. Luciferase assays were performed using the luciferase assay kit (BIOThema AB, Handen, Sweden).

Quantitative (Q) real-time PCR of pS2 mRNA
For Q-real-time PCR assays, MCF-7 cells were harvested in Trizol (Life Technologies, Carlsbad, CA), and RNA was extracted according to the manufacturer’s recommendations. Total RNA was resuspended in PCR-grade water, and the concentration was estimated using Nanodrop (Saveen Werner, Malmö, Sweden). In each sample, 1 µg RNA was reverse transcribed in a total reaction mix of 26 µl containing 2.6 µl reaction buffer, 5.2 mM MgCl2, 2.6 µl deoxynucleotide mix, 2.6 µl random primer, 1.3 µl ribonuclease inhibitor, and 1.04 µl reverse transcriptase using the first-strand cDNA synthesis kit (Roche, Basel, Switzerland). Q-real-time PCR was performed using LightCycler-480 SYBR Green I master (Roche). Primers used were: pS2, 5'-GAGGCCCAGACAGAGACGTG-3' (forward) and 5'-CCCTGCAGAAGTGTCTAAAATTCA-3' (reverse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ACCACAGTCCATGCCATCAC-3' (forward) and 5'-TCCACCACCCTGTTGCTGTA-3' (reverse). Fold change in mRNA pS2 expression in treated cells, compared with untreated cells, were calculated using the crossing point (Cp) for each sample and the efficiency (Eff) of each transcript, using the formula EffpS2 {Delta}Cp/EffGAPDH {Delta}Cp.

Western blotting and coimmunoprecipitation
Procedures for Western blotting are previously described (25). Antibodies used in the immunoblotting experiments were rabbit anti-HA (Zymed Laboratories Inc., South San Francisco, CA) and rabbit anti-ER (Santa Cruz Biotechnology, Santa Cruz, CA) in combination with horseradish peroxidase-conjugated goat antirabbit IgG (Pierce, Rockford, IL), rat anti-hemagglutinin (HA)-peroxidase, high affinity (Roche, Basel, Germany), mouse anti-ER Ab-10 (Neomarkers, Fremont, CA), and mouse anti-GAPDH (Chemicon International, Inc., Temecula, CA) in combination with horseradish peroxidase-conjugated donkey antimouse IgG (Santa Cruz Biotechnology). For coimmunoprecipitation, COS-1 cells were grown in 90 mm petri dishes and transfected with expression plasmids, as indicated in the figure legends. Cells were harvested at 48 h and lysed in immunoprecipitation (IP) buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 µg/ml aprotinin, 5 mM N-ethylmaleimide, 100 nm sodium orthovanadate, 0.2 mM phenylmethylsulfonyl fluoride, and the Complete Mini-EDTA-free protease inhibitor tablets (Roche). Cell lysates were incubated with 2 µg anti-ER (Santa Cruz Biotechnology) or 5 µg anti-HA peroxidase high affinity (Roche) at 4 C overnight. Thereafter 40 µl protein G-Sepharose (Amersham Bioscience, Uppsala, Sweden) were added, and the samples were incubated for another 2 h. Subsequently the beads were washed four times for 5 min in IP buffer. Proteins were eluted from the beads by boiling in SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blotting using anti-HA and anti-ER.

Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed according to the manufacturer’s description (Upstate, New York, NY). In brief, MCF-7 cells were grown in 9- or 14-cm dishes for 3 d in phenol red-free DMEM supplemented with charcoaled-stripped serum. The cells were then treated with E2 (0.1 µM), forskolin (10 µM), IBMX (50 µM), and 8-CPT-cAMP (150 µM) for different periods of time. Thereafter the cells were cross-linked with 1% formaldehyde for 10 min at room temperature, washed twice in ice-cold PBS, and harvested in PBS supplemented with the protease inhibitors pepstatin (1 µg/ml), aprotinin (1 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). The cells were lysed in a buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris (pH 8.1). Chromatin was sheared by sonicating the lysate seven times for 15 sec at 30% power using a Vibra Cell sonicator (Sonic & Materials Inc., Newtown, CT). The sheared chromatin was then diluted in a buffer containing 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, and protease inhibitors, as described above. A portion of the chromatin was saved as input sample. The diluted chromatin was immunocleared with salmon sperm DNA/protein A agarose for 2 h at 4 C followed by overnight immunoprecipitation with specific antibodies, including anti-NCOA2 (Bethyl, Montgomery, TX), anti-Pol II (Santa Cruz Biotechnology), anti-ER (Santa Cruz Biotechnology), and anti-AIB1 (Abcam, Cambridge, UK). The immunoprecipitate was incubated with salmon sperm DNA/protein A agarose for 1 h, and the beads were washed sequentially for 10 min with the following wash buffers: low-salt immune complex wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl]; high-salt immune complex wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl]; LiCl immune complex wash buffer [0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris (pH 8.1)], and Tris/EDTA buffer of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). DNA was eluted twice in 1% SDS and 0.1 M NaHCO3 followed by reverse cross-linking of DNA overnight at 65 C and incubation with proteinase K for 1 h at 45 C. Purification of DNA was performed by using a PCR purification kit (QIAGEN).

The DNA samples were analyzed either by semiquantitative PCR and subsequent analysis of the product by agarose gel electrophoresis or Q-real-time PCR using SYBR Green and a LightCycler rapid thermal cycler system (Roche). The primers used were specific for the pS2 gene promoter region: 5'-TATGAATCACTTCTGCAGTGAG-3' (forward) and 5'-GCAAATGTTATCTAACGCTC-3' (reverse).

Confocal microscopy
For confocal imaging, COS-1 cells were seeded in phenol red-free DMEM in 35-mm glass bottom dishes (MatTek Corp., Ashland, MA) and transfected with the expression plasmids encoding YFP-GRIP1 and CFP-ER{alpha}. To stimulate the cAMP/PKA pathway, the cells were cotransfected with the pCMV5-C{alpha} expression plasmid. The cells were either treated with 0.1 µM E2 or left untreated. Imaging was performed 24 h after transfection by using a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems, Wetzar, Germany). CFP was monitored using a blue diode laser excitating with a 405-nm laser line, and emission was viewed through a 410- to 494-nm filter. Detection of YFP was performed by using an argon/argon krypton laser excitating with a 514-nm laser line and emission through a 525- to 612-nm filter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of the cAMP/PKA-pathway regulates GRIP1-mediated coactivator function in a time-dependent manner
We have previously reported that stimulation of PKA down-regulates GRIP1 function and protein level (25). To investigate the time-dependent effects of PKA-activation on GRIP1 coactivator function, COS-1 cells transfected with expression plasmids encoding GAL-4-GRIP1 together with the GAL-4-responsive reporter plasmid pG5-luc were treated with a cAMP analog (8-CPT-cAMP) for different periods of time (1, 2, 4, and 24 h). 8-CPT-cAMP was subsequently removed from the media by extensive washing of the cells. In contrast to long-term cAMP-elevation, treatment of the cells with the cAMP analog for short periods of time (1–2 h) stimulated GRIP1 coactivator function (Fig. 1AGo). Long-term PKA activation was also induced by treatment with a combination of 8-CPT-cAMP, forskolin, and IBMX for 24 h or cotransfecting the cells with an expression plasmid encoding the catalytic subunit of PKA (PKA-C{alpha}). To examine the expression levels of GRIP1 at different time lags of cAMP treatment, lysates from COS-1 cells transfected with HA-GRIP1 plasmid and treated with cAMP analog as described above were analyzed by Western blotting. GAPDH was used as a loading control (Fig. 1BGo). Whereas short-term treatment with cAMP analog had no significant effect on the GRIP1 levels, long-term PKA activation leads to a down-regulation of the GRIP1. Based on these results, we hypothesized that cAMP/PKA initially stimulates GRIP1 function and subsequently leads to its degradation.


Figure 1
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  		FIG. 1. The cAMP/PKA signaling pathway regulates GRIP1 activity in a time-dependent manner. A, COS-1 cells were transfected with the expression plasmids encoding GAL-4-GRIP1 (1.5 µg) and PKA-C{alpha} (0.1 µg), and the GAL-4-responsive reporter plasmid, pG5-luc (1.1 µg). Twenty-four hours after transfection, the cells were treated with 8-CPT-cAMP (300 µM) for 1, 2, 4, and 24 h or a combination of 8-CPT-cAMP, forskolin (10 µM), and IBMX (50 µM) for 24 h. 8-CPT-cAMP was removed from the cells at different periods of time by extensive washing of the cells with PBS. The cells were lysed 48 h after transfection and luciferase assays were performed. The figure shows the mean ± SD of triplicate transfections. The results are representative of three independent experiments. B, COS-1 cells were transfected with pSG5-HA-GRIP1 (2.0 µg) and treated with 8-CPT-cAMP (300 µM) or 8-CPT-cAMP, forskolin (10 µM), and IBMX (50 µM) for different periods of time and subjected to Western blotting using anti-HA and anti-GAPDH antibodies. Expression of GAPDH was used as a control. The Western blot presented is representative of three independent experiments.

 
To further explore the time-dependent regulation of GRIP1 by cAMP/PKA in another cell line, MCF-7 breast cancer cells were transfected with GAL-4-GRIP1 together with the GAL-4-responsive reporter plasmid pG5-luc and treated with forskolin, IBMX, and 8-CPT-cAMP for 1, 2, 4, and 24 h. As observed in COS-1 cells, luciferase assays demonstrated that short-term stimulation of cAMP/PKA (1–4 h) led to an evident increase in GRIP1 coactivator function. However, after 4 h of treatment, the activity of GRIP1 gradually abated (Fig. 2AGo). To confirm the regulation of GRIP1 in MCF-7 cells, an expression plasmid encoding GRIP1 was cotransfected with the ER-responsive luciferase reporter plasmid ERE-TATA-luc. The cells were treated as described above, and stimulation of cAMP/PKA for 2–4 h led to enhanced GRIP1 coactivation of ER{alpha}, whereas longer times of stimulation led to a clear reduction in GRIP1-mediated coactivation of ER (Fig. 2BGo). To investigate whether the diminished GRIP1 coactivation of ER{alpha} was accompanied with reduced levels of GRIP1 protein, Western blotting of cell lysates from transfected MCF-7 cells was performed using anti-ER and anti-HA antibodies to detect ER{alpha} and GRIP1, respectively. The level of ER{alpha} increased on cAMP/PKA stimulation, whereas the GRIP1 protein level was markedly reduced (Fig. 2CGo). Cells overexpressing ER{alpha} displayed similar enhancement in ER{alpha} levels on cAMP/PKA activation as cells expressing only endogenous ER{alpha}. The observed cAMP/PKA-mediated increase of ER{alpha} was in accordance with previous findings by others demonstrating that the activation of PKA leads to stabilization of ER{alpha} (36, 37). Our results suggest that persistent activation of PKA inhibits transcriptional activation of ER{alpha} in MCF-7 cells and reduces the level of GRIP1 protein.


Figure 2
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  		FIG. 2. cAMP/PKA regulates GRIP1-mediated coactivation of ER{alpha} in MCF-7 cells. A, MCF-7 cells were transfected with the expression plasmids encoding GAL-4-GRIP1 (1.5 µg) and the GAL-4-responsive reporter plasmid, pG5-luc (1.1 µg). B, MCF-7 cells were transfected with an expression plasmid encoding HA-GRIP1 (1.5 µg) along with the luciferase reporter construct ERE-TATA-luc (1.1 µg). E2 (0.1 µM) was added after 24 h. The cells in A and B were treated with forskolin (10 µM), IBMX (50 µM), and 8-CPT-cAMP (150 µM) (cAMP). Twenty-four hours after transfection, cAMP elevating agents and analog were removed from the cells at different periods of time by extensive washing of the cells with PBS. Forty-eight hours after transfection the cells were lysed and subjected to luciferase assays. The figures show the mean ± SD of triplicate transfections of three representative experiments. C, MCF-7 cells were transfected with expression plasmids encoding HA-GRIP1 (1.5 µg), pSG-ER{alpha} (0.1 µg), treated with forskolin, IBMX, and 8-CPT-cAMP for 24 h and subjected to Western blotting using anti-HA-, anti-ER-, and anti-GAPDH-specific antibodies. The Western blot presented is representative of three independent experiments.

 
cAMP/PKA stimulates GRIP1 recruitment to the pS2 gene promoter in MCF-7 cells
Ligand-regulated recruitment of ER{alpha} and coactivators, including GRIP1, to the ER-responsive pS2 gene promoter has been examined previously (17, 18). However, knowledge of the intracellular signaling pathways regulating coactivator recruitment is limited. To investigate whether cAMP/PKA modulates the recruitment of GRIP1 to the pS2 gene promoter in MCF-7 cells, ChIP assays were used. MCF-7 cells were cultured in steroid hormone-free media for 3 d followed by treatment with vehicle or forskolin, IBMX, 8-CPT-cAMP, and/or E2 for the indicated periods of time. Immunoprecipitation of GRIP1-bound chromatin was performed using an anti-NCOA2 antibody. The levels of GRIP1 protein associated with the pS2 gene promoter were analyzed by Q-real-time PCR using pS2 promoter specific primers. In accordance with other reports (18, 38), stimulation of the cells with E2 for 30 and 60 min promoted recruitment of GRIP1 to the pS2 gene promoter (Fig. 3Go, A and B). Treatment of the cells with cAMP analog and elevating agents also stimulated recruitment of GRIP1, and E2 combined with cAMP resulted in the highest occupancy by GRIP1 at the pS2 promoter, showing an approximately 3.5-fold increase in GRIP1 association, compared with the control after 60 min of stimulation (Fig. 3BGo).


Figure 3
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  		FIG. 3. Stimulation of the cAMP/PKA signaling pathway recruits GRIP1 to the pS2 gene promoter. MCF-7 cells were treated with forskolin (10 µM), IBMX (50 µM), and 8-CPT-cAMP (150 µM) (cAMP), E2 (0.1 µM), or a combination of E2 and cAMP. ChIP was performed, and the quantity of purified DNA bound to the endogenous pS2 gene promoter was analyzed by PCR using primer pairs amplifying a region of the pS2 promoter. A, MCF-7 cells were treated with E2 and cAMP agents as described above and incubated for 60 min before cross-linking of the cells. ChIP was preformed by using anti-NCOA2 antibody and the level of endogenous GRIP1 bound to the pS2 promoter was amplified by PCR and analyzed on a 2% agarose gel. B, The levels of GRIP1 protein recruited to the pS2 gene promoter after 30 and 60 min of treatment with cAMP and/or E2 were analyzed by Q-real-time PCR using primers spanning the pS2 gene promoter region. The quantitative values from the PCR were normalized to the input samples and presented as fold recruitment, compared with the control (vehicle) defined as 1. The graphs represent the mean ± SE from seven to nine independent ChIP experiments. C, MCF-7 cells were treated with cAMP and/or E2 for 45, 90, 120, and 180 min, and the cells were analyzed for GRIP1 recruitment to the pS2 promoter by Q-real-time PCR. The figure illustrates the cyclical recruitment of GRIP1 to the promoter region in response to the different treatments. The results presented in the figure show the fold recruitment, compared with the control, at each of the given time points and are representative of three to six independent experiments. D and E, MCF-7 cells treated with cAMP and/or E2 for 60 min were subjected to ChIP with antibodies to Pol II and ER{alpha}, respectively. The levels of Pol II and ER{alpha} associated with the pS2 promoter were quantified by real-time PCR. The graphs represent the mean ± SE from three independent ChIP experiments. F, MCF-7 cells were incubated with the proteasome inactivating agent, MG132 (10 µM), for 5 h followed by treatment with forskolin, IBMX, and 8-CPT-cAMP or E2 for 45 min. ChIP assays were performed by using anti-NCOA2 antibody, and the level of GRIP1 recruited to the pS2 promoter in response to the different treatments was determined by Q-real-time PCR using pS2 promoter-specific primers. The results present mean values ± SE obtained from three individual Q-real-time PCRs.

 
ChIP analyses of ER{alpha} and its coregulators have shown that they cycle on and off target promoters in response to ligand (17, 39). To investigate whether the cAMP/PKA pathway elicits a cyclic, dynamic pattern of GRIP1 recruitment similar to that observed for ligand-dependent ER{alpha} transcription, MCF-7 cells prepared for ChIP assays were treated with cAMP and/or E2 for 45, 90, 120, and 180 min (Fig. 3CGo). ChIP analyses of treated cells were compared with vehicle-treated cells (control) at each of the given time points and presented as fold of the control. Interestingly, two distinct periods of maximal GRIP1 recruitment were seen, at approximately 45 and at 150 min after exposure to cAMP. These data suggest that the transient activation of the cAMP/PKA signaling pathway induces a cyclic recruitment of GRIP1 to the pS2 gene promoter region.

To confirm that stimulation of the cAMP/PKA actually activated transcription at the pS2 promoter region, we performed ChIP assays using antibodies against Pol II and ER{alpha}. MCF-7 cells were treated with cAMP and/or E2 for 60 min as described above. Activation of the cAMP/PKA pathway clearly recruited Pol II to the pS2 promoter. cAMP induced higher levels of Pol II at the promoter after 60 min than observed on treatment with E2 (Fig. 3DGo). However, it should be noted that treatment with E2 has been reported to stimulate the recruitment of Pol II to the pS2 gene promoter at later time points (18, 40). At 60 min after treatment, cAMP induced a small increase in ER{alpha} recruitment to the promoter, compared with vehicle, whereas treatment with E2 led to a 20-fold increase in the recruitment of ER{alpha} (Fig. 3EGo).

Based on these results and our previous findings demonstrating that long-term stimulation of the cAMP/PKA pathway leads to ubiquitination and degradation of GRIP1 (25), we hypothesized that inhibition of the proteasome could lead to an increase in cAMP-mediated GRIP1 recruitment to the pS2 promoter. As shown in Fig. 3FGo, treatment of MCF-7 cells with MG132 led to marked stimulation of GRIP1 recruitment after cAMP treatment, whereas no such stimulation was observed after treatment with E2.

Because cAMP stimulated GRIP1 recruitment to the pS2 promoter, we decided to examine whether activation of the cAMP/PKA pathway also enhanced pS2 expression in MCF-7 cells. mRNA from MCF-7 cells treated with vehicle or forskolin, IBMX, 8-CPT-cAMP, and/or E2 for different periods of time were extracted. Q-real-time PCR of pS2 mRNA relative to GAPDH mRNA demonstrated that 1 h of cAMP treatment led to an approximately 1.4-fold increase in expression of pS2 after 1 h and 2.0-fold increase in pS2 after 4 h (Fig. 4Go). The rapid effect of cAMP on pS2 expression was in line with the increase in cAMP-mediated recruitment of Pol II observed after 1 h (Fig. 3DGo). The expression level of pS2 increased with time and reached a top at 24 h after treatment with cAMP. As expected, E2 also stimulated pS2 expression. However, compared with cAMP-treated cells, E2-mediated pS2 expression was delayed and less pronounced, and we also observed a lower level of Pol II recruited to the promoter 1 h after E2 treatment. The combined treatment with cAMP and E2 led to the highest expression levels of pS2 (Fig. 4Go). The levels of pS2 appeared to abate after 24 h of treatment with cAMP and cAMP/E2. Taken together, these data indicate that cAMP stimulates the recruitment of GRIP1 to the pS2 promoter in MCF-7 cells. This is accompanied by an increased recruitment of Pol II and an increase in the expression of pS2. In contrast to E2-mediated recruitment of GRIP1, the cAMP-induced increase in GRIP1 occupancy at the pS2 promoter is further stimulated by the proteasome inhibitor MG132.


Figure 4
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  		FIG. 4. cAMP/PKA stimulation leads to enhanced pS2 gene expression. MCF-7 cells were treated with forskolin (10 µM), IBMX (50 µM), and 8-CPT-cAMP (150 µM) (cAMP) and/or E2 (0.1 µM) for 1, 4, 24, and 30 h. The cells were harvested in Trizol and total RNA was isolated. Q-real-time PCR of pS2 mRNA relative to GAPDH mRNA was performed using pS2- and GAPDH-specific primers. Fold change in mRNA pS2 expression in treated cells, compared with untreated cells, were calculated using the Cp for each sample and the Eff of each transcript, using the formula EffpS2{Delta}Cp/EffGAPDH{Delta}Cp. The figure shows mean fold change in relative pS2 expression, compared with untreated cells, at each time point ± SD.

 
Activation of PKA leads to colocalization of GRIP1 and ER{alpha} in subnuclear foci
Posttranslational modifications of NRs and their coactivators are known to affect their mobility and subcellular localization (26, 32, 35, 41). We have shown that PKA activation changes the subnuclear localization of GRIP1 (25). To study the effects of PKA on the nuclear distribution of both GRIP1 and ER{alpha}, COS-1 cells were transfected with expression plasmids encoding YFP-GRIP1 and CFP-ER{alpha} and examined by scanning confocal microscopy after 24 h. In addition, some cell cultures were cotransfected with PKA-C{alpha} expression plasmid and/or treated with E2. In the absence of E2 and PKA-C{alpha} overexpression, YFP-GRIP1 and CFP-ER{alpha} were distributed in a diffuse, uniform pattern in the nuclei in most of the cells (Fig. 5AGo). Treatment with E2 led to a marked change in the distribution of YFP-GRIP1 and CFP-ER{alpha}, resulting in the formation of subnuclear foci containing YFP-GRIP1 and CFP-ER{alpha}. This observation was in accordance with others showing that GRIP1 is recruited to ER{alpha} foci in response to ligand (42, 43). Interestingly, cotransfection with PKA-C{alpha} also altered the distribution of GRIP1 and ER{alpha} in a similar manner as observed on treatment with E2, leading to reorganization and colocalization of YFP-GRIP1 and CFP-ER{alpha} in subnuclear foci. Furthermore, a combination of E2 stimulation and PKA activation led to an even larger number of cells with focal distribution of colocalized YFP-GRIP1 and CFP-ER{alpha}, suggesting a combined effect of PKA and E2 in the recruitment of GRIP1 to ER{alpha} (Fig. 5AGo). The number of cells containing YFP-GRIP1 foci was recorded after treatment with E2 and/or PKA-C{alpha} overexpression (Fig. 5BGo). In accordance with observations by others (20), approximately 16% of the cells displayed YFP-GRIP1 organized in subnuclear foci in the absence of E2. Treatment with E2 or overexpression of PKA-C{alpha} led to a 2-fold increase in the number of cells containing the nuclear YFP-GRIP1 foci, and a combination of E2 and PKA-C{alpha} induced an almost 4-fold increase in the number of GRIP1 foci-containing cells. The results show that stimulation of PKA leads to an subnuclear redistribution of YFP-GRIP and that YFP-GRIP1 is colocalized with CFP-ER{alpha} in transfected COS-1 cells.


Figure 5
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  		FIG. 5. Subcellular distribution of YFP-GRIP1 and CFP-ER{alpha} and coimmunoprecipitation of GRIP1 and ER{alpha} after PKA activation. A, COS-1 cells seeded in glass-bottom dishes were transfected with plasmids expressing YFP-GRIP1 (0.4 µg) and CFP-ER{alpha} (0.4 µg) and with or without PKA-C{alpha} (0.1 µg). The cells were treated with E2 (0.1 µM) or left untreated. Confocal scanning microscopy was performed 24 h after transfection. The different cell treatments or transfections are indicated at the left and above the confocal images, and the overlay images are shown in the right column. The figures are representative of three to six independent experiments. B, The number of nuclei containing YFP-GRIP1 foci in A was determined by manually counting the foci-positive and foci-negative cells within a restricted area of each cell culture. The graphs show the average number of cells containing YFP-GRIP1 foci ± SE from three to six independent experiments. C, COS-1 cells were transfected with pSG5-HA-GRIP1 (5.0 µg), pSG5-ER{alpha} (0.33 µg), and pCMV5-C{alpha} (0.33 µg) expression plasmids, treated with E2 (0.1 µM) after 24 h and incubated for additional 24 h. Cell lysates were IP with anti-ER{alpha} antibody and immunoblotted (IB) with anti-HA antibody (upper panel) or anti-HA (IP) and anti-ER{alpha} (IB) antibodies (lower panel). Two percent of the original cell lysate were saved as input control and visualized by anti-HA (upper panel), whereas 1% input control was analyzed by anti-ER{alpha} antibody (lower panel). The results are representative of two independent experiments.

 
To further explore the in vivo association of ER{alpha} and GRIP1 after PKA stimulation, coimmunoprecipitation of ER{alpha} and GRIP1 was performed from COS-1 cells transfected with ER{alpha}, HA-GRIP1, and PKA expression plasmids. The cells were also treated with E2. Identical lysates were subjected to separate assays using anti-ER or anti-HA antibodies for the immunoprecipitation. As expected, ER{alpha} and GRIP1 were associated in the same in vivo complex after E2 treatment (Fig. 5CGo). Using anti-ER antibody for immunoprecipitation and anti-HA antibody for immunoblotting, we observed that activation of PKA reduced the GRIP1 level (input samples) and that the amount of GRIP1 associated with ER{alpha} was reduced accordingly. Using anti-HA antibody for immunoprecipitation and anti-ER antibody for immunoblotting, it was shown that the amount of ER{alpha} was not reduced by PKA (input samples), whereas the amount of HA-GRIP1 complexed with ER{alpha} decreased after PKA activation (Fig. 5CGo). These results demonstrate that the down-regulation of GRIP1 induced by long-term PKA activation is accompanied by a decrease in GRIP1 associated with ER{alpha}. Thus, even though we observed an increased subnuclear colocalization of YFP-GRIP1 and CFP-ER{alpha} after long-term PKA activation, the amounts of GRIP1 that interacted with ER{alpha} was reduced in accordance with the total reduction of GRIP1 protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NRs modulate gene expression through dynamic interactions with their coregulators. Proteasomal degradation is important for the coordinated cyclic association of NRs and coregulators on the target promoters (15). We previously reported that PKA leads to ubiquitination and proteasomal degradation of GRIP1 (25). In the present study, we show that activation of the cAMP pathway initially stimulates GRIP1 coactivator function and recruitment to the ER{alpha} target gene pS2, whereas long-term PKA activation leads to GRIP1 degradation. Because the effect on GRIP1 was mediated by overexpression of PKA-C{alpha} as well as cAMP-elevating agents and cAMP-analog, our data indicate that cAMP regulates GRIP1 through the classical PKA pathway and not primarily by dependent activation of the novel exchange nucleotide protein directly activated by cAMP and PKA-independent cAMP-pathway (44).

The present results suggest that PKA stimulates both the activity and turnover of GRIP1. A similar mechanism has recently been reported for SRC-3/AIB1 (45, 46). Phosphorylation of SRC-3/AIB1 by pMAPK38 and glycogen synthase kinase-3 was found to exert a biphasic effect on SRC-3/AIB1 activity, first by stimulating its coactivator function and subsequently by stimulation of its proteasomal degradation. Thus, intracellular signals may stimulate both the activity and turnover of different SRCs and subsequently NR-dependent transcriptional activity. cAMP is known to modulate MAPKs ERK and p38, and activation of MAPKs have been reported to lead to phosphorylation of GRIP1 and modulation of its activity (23, 24, 28). However, inhibition of ERK and p38 by the specific inhibitors PD98058 and SB203580 did not affect the PKA-stimulated degradation of GRIP1 (data not shown).

cAMP/PKA is also known to regulate the activity of ER{alpha} (36, 37, 47, 48, 49, 50), but the exact mechanisms are unclear. PKA activation leads to phosphorylation of ER at Ser-236 and Ser-305 (49), and PKA-induced phosphorylation of ER{alpha} has recently been shown to increase its interaction with SRC-3/AIB1 in the presence of E2 (50). Thus, SRCs may play an important role for the PKA-mediated regulation of ER{alpha} activation. Recently it has also been shown that PKA decreases estradiol-mediated activation of ER{alpha}-dependent reporter genes through changes in ligand binding, promoter interaction, and ER{alpha} phosphorylation (47). However, PKA has also been reported to enhance ER{alpha}-dependent transcription, in both the presence and absence of E2 (37, 47, 51, 52), and the mechanisms by which PKA regulates ER{alpha} activity appears to depend on cell type and/or promoter-specific factors.

Both GRIP1 and ER{alpha} are targets of the ubiquitin-proteasome degradation system. GRIP1 is degraded on prolonged PKA activation (25), whereas ER{alpha} is stabilized by cAMP/PKA, protecting it from ligand-induced proteasomal degradation (37). Thus, it appears that PKA regulates GRIP1 and ER{alpha} through opposite mechanisms. Ligand-dependent proteasome-mediated protein degradation serves as a mechanism to allow temporal recruitment and exchange of coregulators required for ER{alpha}-mediated transcription (39). However, little is known about the ligand-independent regulation of coregulator recruitment to target promoters. Phosphorylation of NRs and coactivators by protein kinases is known to regulate transcription through modulating their affinities for other proteins (33, 53, 54). Several reports also suggest that phosphorylation of NRs and coactivators target them for degradation by the ubiquitin-proteasome pathway (45, 55, 56, 57, 58). However, we were not able to observe any PKA-induced phosphorylation of GRIP1 (data not shown).

In this paper, we report that inhibition of the proteasome led to increased recruitment of GRIP1 to pS2 as well as an enhancement of the PKA-induced recruitment of GRIP1. In contrast, proteasome inhibition on E2 treatment led to reduced recruitment of GRIP1. This suggests that PKA and E2 regulate coactivator function through distinct mechanisms. It has been shown that inhibition of the proteasome activity in the presence of ligand leads to immobilization and polyubiquitination of ER{alpha} in the nuclear matrix (39). This may result in reduced promoter-associated GRIP1 levels.

Transcriptional activation by ER{alpha} is a complex process requiring multiple coactivators, and different promoters show different sequential orders of coactivator recruitment (17). Because stimulation of the cAMP/PKA pathway specifically targets GRIP1 and not SRC-1 or SRC-3/AIB1 (25), signals that lead to PKA activation may alter the pattern of SRC recruitment to the gene promoter.

Steroid receptors may change their subnuclear localization upon ligand binding (42, 59, 60, 61). Many of these changes involve reorganization from a diffuse, homogenous distribution into highly concentrated subnuclear foci that may colocalize with coactivators, such as the SRCs. Ligand-independent regulation of the subcellular localization of NR and coregulators is less characterized. Here we demonstrate that PKA, similar to E2, triggered the accumulation and colocalization of YFP-GRIP1 and CFP-ER{alpha} into nuclear foci. Activation of PKA in the presence of E2 further increased the number of foci containing cells, suggesting a combined effect of PKA and E2 in recruiting YFP-GRIP1 and CFP-ER{alpha} to specific subnuclear sites. However, the functional role of foci formation is not clear. It has been suggested that they may represent active forms of NRs and coactivators or sites for recruitment of transcription partners required for transcriptional activation (62, 63, 64). These foci may also represent areas of protein degradation. We and others have previously demonstrated that GRIP1 is colocalized with components of the proteasome in the foci (20, 25). Here we observed an increase in the accumulation of YFP-GRIP1 foci after long-term PKA activation that also leads to a decrease in GRIP1-ER{alpha} complex formation in vivo. PKA triggers the recruitment of GRIP1 into specific foci, but it remains to be determined whether these foci are sites for ubiquitin-proteasomal degradation of GRIP1.

In this paper, we focused on GRIP1 in the regulation of ER{alpha}-dependent transcription, and we showed that GRIP1 recruitment to the ER{alpha}-target gene pS2 is stimulated by cAMP/PKA. The role of cAMP/PKA-mediated GRIP1 recruitment to and regulation of other target genes remains to be investigated.


   Acknowledgments
 
The confocal imaging was performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen. We thank M. R. Stallcup, K. Matsuda, G. S. McKnight, and E. Treuter for the supplied plasmids. Expert technical assistance from C. Cook, A. M. Sellevold, B. Skjellstad, and A. Folloso is highly appreciated.


   Footnotes
 
This work was supported by grants from The Research Council of Norway, The Norwegian Cancer Society, Margareth Solbergs legat, Det regionale samarbeidsorganet, Helse Vest, and Meltzerfondet.

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online May 22, 2008

Abbreviations: AIB1, Amplified in breast cancer-1; CFP, cyan fluorescent protein; ChIP, chromatin immunoprecipitation; Cp, crossing point; 8-CPT-cAMP, 8-parachlorophenylthio-cAMP; E2, 17β-estradiol; Eff, efficiency; ER, estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRIP1, glucocorticoid receptor interacting protein 1; HA, hemagglutinin; IBMX, 3-isobutyl-1-methylxanthine; IP, immunoprecipitation; NR, nuclear receptor; PKA, protein kinase A; Pol II, RNA polymerase II; Q-real-time PCR, quantitative real-time PCR; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; YFP, yellow fluorescent protein.

Received January 9, 2008.

Accepted for publication May 12, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Onate SA, Tsai SY, Tsai MJ, O'Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  2. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  3. Voegel JJ, Heine MJ, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675[Medline]
  4. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  5. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[CrossRef][Medline]
  6. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2. Proc Natl Acad Sci USA 94:8479–8484[Abstract/Free Full Text]
  7. Takeshita A, Cardona GR, Koibuchi N, Suen CS, Chin WW 1997 TRAM-1, A novel 160-kDa thyroid hormone receptor activator molecule, exhibits distinct properties from steroid receptor coactivator-1. J Biol Chem 272:27629–27634[Abstract/Free Full Text]
  8. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677–684[CrossRef][Medline]
  9. Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, Aswad DW, Stallcup MR 1999 Regulation of transcription by a protein methyltransferase. Science 284:2174–2177[Abstract/Free Full Text]
  10. Koh SS, Chen D, Lee YH, Stallcup MR 2001 Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J Biol Chem 276:1089–1098[Abstract/Free Full Text]
  11. Qi C, Chang J, Zhu Y, Yeldandi AV, Rao SM, Zhu YJ 2002 Identification of protein arginine methyltransferase 2 as a coactivator for estrogen receptor {alpha}. J Biol Chem 277:28624–28630[Abstract/Free Full Text]
  12. Chiba H, Muramatsu M, Nomoto A, Kato H 1994 Two human homologues of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic Acids Res 22:1815–1820[Abstract/Free Full Text]
  13. Verma S, Ismail A, Gao X, Fu G, Li X, O'Malley BW, Nawaz Z 2004 The ubiquitin-conjugating enzyme UBCH7 acts as a coactivator for steroid hormone receptors. Mol Cell Biol 24:8716–8726[Abstract/Free Full Text]
  14. Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, O'Malley BW 1999 The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19:1182–1189[Abstract/Free Full Text]
  15. Rochette-Egly C 2005 Dynamic combinatorial networks in nuclear receptor-mediated transcription. J Biol Chem 280:32565–32568[Free Full Text]
  16. Lewis BA, Reinberg D 2003 The mediator coactivator complex: functional and physical roles in transcriptional regulation. J Cell Sci 116:3667–3675[Abstract/Free Full Text]
  17. Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F 2003 Estrogen receptor-{alpha} directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763[CrossRef][Medline]
  18. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843–852[CrossRef][Medline]
  19. Baek SH, Rosenfeld MG 2004 Nuclear receptor coregulators: their modification codes and regulatory mechanism by translocation. Biochem Biophys Res Commun 319:707–714[CrossRef][Medline]
  20. Baumann CT, Ma H, Wolford R, Reyes JC, Maruvada P, Lim C, Yen PM, Stallcup MR, Hager GL 2001 The glucocorticoid receptor interacting protein 1 (GRIP1) localizes in discrete nuclear foci that associate with ND10 bodies and are enriched in components of the 26S proteasome. Mol Endocrinol 15:485–500[Abstract/Free Full Text]
  21. Borud B, Hoang T, Bakke M, Jacob AL, Lund J, Mellgren G 2002 The nuclear receptor coactivators p300/CBP/cointegrator-associated protein (p/CIP) and transcription intermediary factor 2 (TIF2) differentially regulate PKA-stimulated transcriptional activity of steroidogenic factor 1. Mol Endocrinol 16: 757–773
  22. Chauchereau A, Amazit L, Quesne M, Guiochon-Mantel A, Milgrom E 2003 Sumoylation of the progesterone receptor and of the steroid receptor coactivator SRC-1. J Biol Chem 278:12335–12343[Abstract/Free Full Text]
  23. Frigo DE, Basu A, Nierth-Simpson EN, Weldon CB, Dugan CM, Elliott S, Collins-Burow BM, Salvo VA, Zhu Y, Melnik LI, Lopez GN, Kushner PJ, Curiel TJ, Rowan BG, McLachlan JA, Burow ME 2006 p38 mitogen-activated protein kinase stimulates estrogen-mediated transcription and proliferation through the phosphorylation and potentiation of the p160 coactivator glucocorticoid receptor-interacting protein 1. Mol Endocrinol 20:971–983[Abstract/Free Full Text]
  24. Gregory CW, Fei X, Ponguta LA, He B, Bill HM, French FS, Wilson EM 2004 Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer. J Biol Chem 279:7119–7130[Abstract/Free Full Text]
  25. Hoang T, Fenne IS, Cook C, Borud B, Bakke M, Lien EA, Mellgren G 2004 cAMP-dependent protein kinase regulates ubiquitin-proteasome-mediated degradation and subcellular localization of the nuclear receptor coactivator GRIP1. J Biol Chem 279:49120–49130[Abstract/Free Full Text]
  26. Kotaja N, Karvonen U, Janne OA, Palvimo JJ 2002 The nuclear receptor interaction domain of GRIP1 is modulated by covalent attachment of SUMO-1. J Biol Chem 277:30283–30288[Abstract/Free Full Text]
  27. Kotaja N, Vihinen M, Palvimo JJ, Janne OA 2002 Androgen receptor-interacting protein 3 and other PIAS proteins cooperate with glucocorticoid receptor-interacting protein 1 in steroid receptor-dependent signaling. J Biol Chem 277:17781–17788[Abstract/Free Full Text]
  28. Lopez GN, Turck CW, Schaufele F, Stallcup MR, Kushner PJ 2001 Growth factors signal to steroid receptors through mitogen-activated protein kinase regulation of p160 coactivator activity. J Biol Chem 276:22177–22182[Abstract/Free Full Text]
  29. Rowan BG, Garrison N, Weigel NL, O'Malley BW 2000 8-Bromo-cyclic AMP induces phosphorylation of two sites in SRC-1 that facilitate ligand-independent activation of the chicken progesterone receptor and are critical for functional cooperation between SRC-1 and CREB binding protein. Mol Cell Biol 20:8720–8730[Abstract/Free Full Text]
  30. Rowan BG, Weigel NL, O'Malley BW 2000 Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway. J Biol Chem 275:4475–4483[Abstract/Free Full Text]
  31. Wu H, Sun L, Zhang Y, Chen Y, Shi B, Li R, Wang Y, Liang J, Fan D, Wu G, Wang D, Li S, Shang Y 2006 Coordinated regulation of AIB1 transcriptional activity by sumoylation and phosphorylation. J Biol Chem 281:21848–21856[Abstract/Free Full Text]
  32. Wu RC, Qin J, Hashimoto Y, Wong J, Xu J, Tsai SY, Tsai MJ, O'Malley BW 2002 Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I{kappa}B kinase. Mol Cell Biol 22:3549–3561[Abstract/Free Full Text]
  33. Wu RC, Smith CL, O'Malley BW 2005 Transcriptional regulation by steroid receptor coactivator phosphorylation. Endocr Rev 26:393–399[Abstract/Free Full Text]
  34. Yan F, Gao X, Lonard DM, Nawaz Z 2003 Specific ubiquitin-conjugating enzymes promote degradation of specific nuclear receptor coactivators. Mol Endocrinol 17:1315–1331[Abstract/Free Full Text]
  35. Zheng FF, Wu RC, Smith CL, O'Malley BW 2005 Rapid estrogen-induced phosphorylation of the SRC-3 coactivator occurs in an extranuclear complex containing estrogen receptor. Mol Cell Biol 25:8273–8284[Abstract/Free Full Text]
  36. Schreihofer DA, Resnick EM, Lin VY, Shupnik MA 2001 Ligand-independent activation of pituitary ER: dependence on PKA-stimulated pathways. Endocrinology 142:3361–3368[Abstract/Free Full Text]
  37. Tsai HW, Katzenellenbogen JA, Katzenellenbogen BS, Shupnik MA 2004 Protein kinase A activation of estrogen receptor {alpha} transcription does not require proteasome activity and protects the receptor from ligand-mediated degradation. Endocrinology 145:2730–2738[Abstract/Free Full Text]
  38. Kim JH, Li H, Stallcup MR 2003 CoCoA, a nuclear receptor coactivator which acts through an N-terminal activation domain of p160 coactivators. Mol Cell 12:1537–1549[CrossRef][Medline]
  39. Reid G, Hubner MR, Metivier R, Brand H, Denger S, Manu D, Beaudouin J, Ellenberg J, Gannon F 2003 Cyclic, proteasome-mediated turnover of unliganded and liganded ER{alpha} on responsive promoters is an integral feature of estrogen signaling. Mol Cell 11:695–707[CrossRef][Medline]
  40. Shao W, Keeton EK, McDonnell DP, Brown M 2004 Coactivator AIB1 links estrogen receptor transcriptional activity and stability. Proc Natl Acad Sci USA 101:11599–11604[Abstract/Free Full Text]
  41. Gioeli D, Black BE, Gordon V, Spencer A, Kesler CT, Eblen ST, Paschal BM, Weber MJ 2006 Stress kinase signaling regulates androgen receptor phosphorylation, transcription, and localization. Mol Endocrinol 20:503–515[Abstract/Free Full Text]
  42. Htun H, Holth LT, Walker D, Davie JR, Hager GL 1999 Direct visualization of the human estrogen receptor {alpha} reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471–486[Abstract/Free Full Text]
  43. Schaufele F, Chang CY, Liu W, Baxter JD, Nordeen SK, Wan Y, Day RN, McDonnell DP 2000 Temporally distinct and ligand-specific recruitment of nuclear receptor-interacting peptides and cofactors to subnuclear domains containing the estrogen receptor. Mol Endocrinol 14:2024–2039[Abstract/Free Full Text]
  44. de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL 1998 Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396:474–477[CrossRef][Medline]
  45. Gianni M, Parrella E, Raska Jr I, Gaillard E, Nigro EA, Gaudon C, Garattini E, Rochette-Egly C 2006 P38MAPK-dependent phosphorylation and degradation of SRC-3/AIB1 and RAR{alpha}-mediated transcription. EMBO J 25:739–751[CrossRef][Medline]
  46. Wu RC, Feng Q, Lonard DM, O'Malley BW 2007 SRC-3 coactivator functional lifetime is regulated by a phospho-dependent ubiquitin time clock. Cell 129:1125–1140[CrossRef][Medline]
  47. Al-Dhaheri MH, Rowan BG 2007 PKA exhibits selective modulation of estradiol dependent transcription in breast cancer cells that is associated with decreased ligand binding, altered ER{alpha} promoter interaction and changes in receptor phosphorylation. Mol Endocrinol 21:439–456[Abstract/Free Full Text]
  48. El-Tanani MK, Green CD 1997 Two separate mechanisms for ligand-independent activation of the estrogen receptor. Mol Endocrinol 11:928–937[Abstract/Free Full Text]
  49. Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 269:4458–4466[Abstract/Free Full Text]
  50. Likhite VS, Stossi F, Kim K, Katzenellenbogen BS, Katzenellenbogen JA 2006 Kinase-specific phosphorylation of the estrogen receptor changes receptor interactions with ligand, deoxyribonucleic acid, and coregulators associated with alterations in estrogen and tamoxifen activity. Mol Endocrinol 20:3120–3132[Abstract/Free Full Text]
  51. Cho H, Katzenellenbogen BS 1993 Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol Endocrinol 7:441–452[Abstract/Free Full Text]
  52. Lazennec G, Thomas JA, Katzenellenbogen BS 2001 Involvement of cyclic AMP response element binding protein (CREB) and estrogen receptor phosphorylation in the synergistic activation of the estrogen receptor by estradiol and protein kinase activators. J Steroid Biochem Mol Biol 77:193–203[CrossRef][Medline]
  53. Lannigan DA 2003 Estrogen receptor phosphorylation. Steroids 68:1–9[CrossRef][Medline]
  54. Rochette-Egly C 2003 Nuclear receptors: integration of multiple signalling pathways through phosphorylation. Cell Signal 15:355–366[CrossRef][Medline]
  55. Floyd ZE, Stephens JM 2002 Interferon-{gamma}-mediated activation and ubiquitin-proteasome-dependent degradation of PPAR{gamma} in adipocytes. J Biol Chem 277:4062–4068[Abstract/Free Full Text]
  56. Gianni M, Bauer A, Garattini E, Chambon P, Rochette-Egly C 2002 Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RAR{gamma} degradation and transactivation. EMBO J 21:3760–3769[CrossRef][Medline]
  57. Lange CA, Shen T, Horwitz KB 2000 Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome. Proc Natl Acad Sci USA 97:1032–1037[Abstract/Free Full Text]
  58. Lin HK, Wang L, Hu YC, Altuwaijri S, Chang C 2002 Phosphorylation-dependent ubiquitylation and degradation of androgen receptor by Akt require Mdm2 E3 ligase. EMBO J 21:4037–4048[CrossRef][Medline]
  59. Fejes-Toth G, Pearce D, Naray-Fejes-Toth A 1998 Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc Natl Acad Sci USA 95:2973–2978[Abstract/Free Full Text]
  60. Georget V, Lobaccaro JM, Terouanne B, Mangeat P, Nicolas JC, Sultan C 1997 Trafficking of the androgen receptor in living cells with fused green fluorescent protein-androgen receptor. Mol Cell Endocrinol 129:17–26[CrossRef][Medline]
  61. Htun H, Barsony J, Renyi I, Gould DL, Hager GL 1996 Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc Natl Acad Sci USA93:4845–4850
  62. Chen WY, Juan LJ, Chung BC 2005 SF-1 (nuclear receptor 5A1) activity is activated by cyclic AMP via p300-mediated recruitment to active foci, acetylation, and increased DNA binding. Mol Cell Biol 25:10442–10453[Abstract/Free Full Text]
  63. Elbi C, Misteli T, Hager GL 2002 Recruitment of dioxin receptor to active transcription sites. Mol Biol Cell 13:2001–2015[Abstract/Free Full Text]
  64. Ruas JL, Poellinger L, Pereira T 2005 Role of CBP in regulating HIF-1-mediated activation of transcription. J Cell Sci 118:301–311[Abstract/Free Full Text]



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals