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Molecular Endocrinology, doi:10.1210/me.2003-0308
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Molecular Endocrinology 18 (5): 1131-1143
Copyright © 2004 by The Endocrine Society

Protein Phosphatase 5 Is a Negative Regulator of Estrogen Receptor-Mediated Transcription

Kazuhiro Ikeda, Sumito Ogawa, Tohru Tsukui, Kuniko Horie-Inoue, Yasuyoshi Ouchi, Shigeaki Kato, Masami Muramatsu and Satoshi Inoue

Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine (K.I., T.T., K.H.-I., M.M., S.I.), and Department of Molecular Biology (T.T.), Saitama Medical School, Hidaka-shi, Saitama 350-1241, Japan, Department of Geriatric Medicine (S.O., Y.O., S.I.), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan; and Institute of Molecular and Cellular Biosciences (S.K.), University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

Address all correspondence and requests for reprints to: Satoshi Inoue, Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical School, 1397-1 Yamane, Hidaka-shi, Saitama 350-1241, Japan. E-mail: INOUE-GER{at}h.u-tokyo.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptors (ERs) are transcription factors that can be modulated by both estrogen-dependent and growth factor-dependent phosphorylation. A yeast two-hybrid screening identified a serine/threonine protein phosphatase (PP5) as an interactant of ERß (1–481), a dominant negative ERß mutant. Glutathione S-transferase pull-down assays, mammalian two-hybrid assays, and immunoprecipitation studies showed that PP5 directly binds to both ER{alpha} and ERß via its tetratricopeptide repeat domain. E domains of ER{alpha} and ERß, without containing activation domain core regions in transcription activation function 2, were required for the binding to PP5. In ER{alpha}-positive breast cancer MCF7 cells, estrogen- and epidermal growth factor-dependent phosphorylation of ER{alpha} on serine residue 118, a major phosphorylation site of the receptor, was reduced by expressing PP5 but enhanced by PP5 antisense oligonucleotide. Estrogen-induced transcriptional activities of both ER{alpha} and ERß and mRNA expression of estrogen-responsive genes, including pS2, c-myc, and cyclin D1, were suppressed by PP5 but enhanced by PP5 antisense oligonucleotide. A truncated PP5 mutant consisting only of its tetratricopeptide repeat domain acted as a dominant negative PP5 that enhanced serine residue 118 phosphorylation of ER{alpha} and transactivations by ER{alpha} and ERß. We present the first evidence that PP5 functions as an inhibitory regulator of ER phosphorylation and transcriptional activation in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PHYSIOLOGICAL FUNCTIONS of estrogen are mediated by the two estrogen receptors, ER{alpha} and ERß (1). ERs are transcription factors that regulate expressions of estrogen-targeted genes in response to hormone binding. Like other transcription factors, ERs are phosphoproteins (2). It has been observed that direct phosphorylation of ER{alpha} is induced upon addition of ligands (3) as well as mediated by protein kinases in a ligand-independent manner (4, 5, 6, 7). Serine 118 (S118) is a major phosphorylation site within A/B domain, or N-terminal transcription activation function (AF)-1 of ER{alpha}, the mutation of which reduces transactivation by ER{alpha} (3, 8). Previous literature shows that S118 is phosphorylated by MAPK (4) or by Cdk-activating kinase, a cyclin-dependent kinase that phosphorylates the PolII C-terminal tail domain (7). S118 phosphorylation is also induced by ligand binding to the receptor in a MAPK- or Cdk-activating kinase-independent manner (9).

The molecular mechanism(s) by which the phosphorylation of ERs is regulated remain(s) to be clarified. The reversible phosphorylation of proteins is catalyzed by protein kinases and phosphatases. Among the enzymes, serine/threonine protein phosphatases belong to the PPP family that specially targets phosphorylation of serine/threonine residues (10). The PPP family of phosphatases is comprised of several members including protein phosphatase 1 (PP1), PP2A, PP2B, PP2C, and PP4–7 (11). PP5 (serine/threonine protein phosphatase), another member of the PPP family, has a unique character in that it consists of a single polypeptide chain containing a phosphatase catalytic domain near its C terminus and four tetratricopeptide repeat (TPR) domains as a regulatory region in its N terminus (12). The TPR domain consists of a highly degenerate 34-amino acid repeat initially identified in several cell-cycle gene products and in proteins involved in the regulation of RNA synthesis (13, 14). TPR domains mediate protein-protein interactions (15), and there is evidence that the TPR domain of PP5 targets the phosphatase to other proteins, including heat shock protein 90-glucocorticoid receptor complex (16), apoptosis signal-regulating kinase 1 (17), the atrial natriuretic peptide receptor (18), the anaphase-promoting complex (19), and PP2A (20).

Here we show that PP5 directly binds to ER{alpha} and ERß and inhibits transcriptional activities of the receptors. ER{alpha} and ERß interact with the TPR domain of PP5 through their E domains without including activation domain (AD) core regions of helix 12 within C-terminal transcription AF-2, which was previously shown to be important for binding to coactivators (21, 22, 23). 17ß-Estradiol (E2)-dependent or epidermal growth factor (EGF)-dependent phosphorylation of ER{alpha} on S118 is inhibited by PP5 but enhanced by a truncated mutant of PP5 consisting of only the TPR domain. PP5 suppresses the transactivations by ER{alpha} and ERß but not those by ER{alpha} mutants with one amino acid substitution, S118A (mutation of S118 to alanine) or S118E (substitution of S118 to glutamic acid (3). PP5 inhibits E2-induced mRNA expression of estrogen-targeted genes including pS2, c-myc, and cyclin D1. Furthermore, an antisense (AS) oligonucleotide against PP5 that could reduce endogenous PP5 expression enhanced both E2-dependent and EGF-dependent phosphorylation of ER{alpha}, transactivation by ER{alpha}, and E2-induced mRNA expression of estrogen-targeted genes. Our results may present a novel molecular mechanism that PP5 is a key regulator of the signaling pathways of ER{alpha} and ERß in a negative manner.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of PP5 that Interacts with a Dominant Negative ERß
To identify novel binding partners of ERs, we used the yeast two-hybrid system based on a Lex A-ERß (1–481) fusion protein to screen a cDNA library derived from estrogen-depleted MCF7 breast cancer cells. ERß (1–481) mutant is a dominant negative form of ER that has potential to repress both ER{alpha}- and ERß-mediated transactivation (24). Among positive clones, three independent clones encoding PP5 were obtained. The interactions of the PP5 clones between ERß (1–481) were confirmed by the galactose-dependent growth of yeast strain EGY48, which was cotransformed with pSH18–34 LacZ reporter plasmid, pEG202NLS-ERß (1–481), and the PP5 clones in galactose-inducible pJG4–5 (Fig. 1Go).



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  		Fig. 1. Specific Interaction between PP5 and ERß (1–481) in Yeast

Galactose-inducible plasmids pJG4–5 including PP5 clones (nos. 55, 76, and 81) obtained from yeast two-hybrid screening were transformed into yeast EGY48 along with LacZ reporter plasmid and pEG202NLS containing ERß (1–481) or its empty vector. The specific interaction between PP5 clones and ERß (1–481) construct was observed in galactose-containing medium through ß-galactosidase staining.

 
ERs Directly Bind to and Are Substrates for PP5
To characterize whether PP5 directly interacts with ERs in vitro, we performed glutathione S-transferase (GST) pull-down assays using bacterially expressed GST or GST-fused PP5 mutants and in vitro translated ER{alpha} and ERß (Fig. 2Go). Both ER{alpha} and ERß were pulled down by full-length PP5, but not by GST alone. GST-PP5 (28–165), GST-PP5 (2–181), and GST-PP5 (2–312) can bind both ERs, whereas neither GST-PP5 (2–71) nor GST-PP5 (181–499) can bind the receptors (Fig. 2Go, B and C). The data suggest that both ER{alpha} and ERß directly bind to PP5, and PP5 interacts with ERs through its TPR domains.



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  		Fig. 2. ER{alpha} and ERß Proteins Are Pulled Down by GST Fusion Proteins Containing PP5 or Its TPR Domains

A, Schematic diagrams of human PP5 constructs. PP5 cDNAs were inserted into pGEX4T-1 downstream of and in frame with the GST tag and GST or GST fusion proteins were expressed in Escherichia coli. Solid and shaded boxes indicate TPR domains and catalytic domain, respectively. B and C, In vitro translated ER{alpha} (B) and ERß (C) proteins labeled with 35S-methionine were incubated with GST or GST fusion proteins containing full-length PP5 or PP5 mutants. Labeled proteins corresponding to 10% of input and materials bound to glutathione-Sepharose were separated by 10% SDS-PAGE and detected by radioautography. D and E, Estrogen stimulation does not affect the in vitro interaction of full-length PP5 or its TPR domains [PP5 (28–165)] with ER{alpha} (D) and ERß (E). GST pull-down assays were performed as described above except for the addition of 10 nM E2 in the binding solution.

 
We next examined whether the interaction between PP5 and ERs has ligand dependency (Fig. 2Go, D and E). The amounts of pulled-down ER{alpha} or ERß did not change for GST-PP5 or GST-PP5 (28–165) in the presence or absence of E2, indicating that the association of PP5 with ERs does not require ligand activation.

We further investigated which domain(s) of ERs is/are responsible for the association with PP5 in vivo by performing mammalian two-hybrid assays. Cotransfection of expression constructs for the herpes simplex viral protein 16 (VP16) transactivation domain fused to PP5 and the GAL4 DNA binding domain fused to full-length or fragments of ER{alpha}/ERß was performed into 293T cells, and the binding ability of PP5 with ER fragments was assessed by measuring luciferase activity that is derived from a GAL4-driven luciferase reporter (Fig. 3Go). The most significant luciferase activity was observed when PP5 interacted with E domains of ERs without containing the activation domain (AD) core regions within AF-2 [i.e. ER{alpha} (302–530) and ERß (248–481)] (Fig. 3BGo). ER fragments including ABCD domains, ABC domains, and the AD core regions within E/F domains showed no binding activity to PP5.



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  		Fig. 3. Interaction of PP5 with ER{alpha} and ERß in Mammalian Two-Hybrid Assays

A, Structure of full-length and truncated mutants of human ER{alpha} and ERß. The cDNAs encoding wild-type (WT) ER{alpha} and ERß or cDNA fragments encoding structural ABCD domains (amino acids 1–302), ABC domains (amino acids 1–263), E domain (amino acids 302–530), and a part of EF domains (amino acids 530–595) of ER{alpha}, and ABCD domains (amino acids 1–248), ABC domains (amino acids 1–213), E domain (amino acids 248–481), and a part of EF domains (amino acids 481–530) of ERß were subcloned into an expression vector pCMX-GAL4 downstream of and in frame with GAL4 DNA binding domain. B, Mammalian two-hybrid assays were carried out using expression vectors of the pCMX-GAL4 containing full-length or mutants of ER{alpha} and ERß (GAL4-ER), and pCMX-VP16 containing PP5 (VP16-PP5), in which PP5 was inserted into pCMX-VP16 downstream of and in frame to the VP16 transactivation domain in pCMX-VP16. 293T cells were cotransfected with 0.7 µg pRL-CMV vector for internal control, 0.8 µg TK-MH100 x 4Luc, 0.2 µg VP16-PP5, and 0.1 µg each pCMX-GAL4 constructs of ER{alpha} or ERß in a well of 24-well plates. The cells were cultured for 24 h and the luciferase assay was performed. Data are the mean ± SD of three independent experiments performed in triplicate.

 
The finding that TPR domains in PP5 are requisite sites for interaction with ERs was further confirmed by coimmunoprecipitation experiments, using 293T cells cotransfected with green fluorescent protein (GFP)-tagged ER{alpha} along with Flag-tagged full-length PP5 or a PP5 mutant with only its TPR domains (Fig. 4BGo). The in vivo interaction between endogenous PP5 and ER{alpha} in human breast cancer MCF7 cells was also shown by coimmunoprecipitation study (Fig. 4CGo).



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  		Fig. 4. Interaction and Phosphorylation Inhibition of ER{alpha} with PP5

A, Schematic representation of the full-length and TPR domain of PP5. B, Coimmunoprecipitation experiments of PP5 with ER{alpha} in 293T cells. Flag-tagged full-length and TPR domain of PP5 or control pcDNA3 vector containing Flag tag (Vec) and GFP-tagged ER{alpha} or control pEGFP-C2 vector (Vec) were transfected for 24 h in 293T cells. Cell extracts were obtained after a chemical cross-linking with 200 µg/ml of dithiobis[succinimidyl propionate] (DSP). Extract (1 mg) was incubated with an anti-Flag antibody M2 for 3 h at 4 C and then incubated with protein G-Sepharose beads for 1.5 h at 4 C. Proteins in the immune complexes or 20 µg of the cell extracts (corresponding to 2% of input) were resolved by SDS-PAGE, and the Western blot (WB) was probed with an antibody against GFP. Signals were visualized by an enhanced chemiluminescence system. C, Coimmunoprecipitaion of PP5 with ER{alpha} in MCF7 cells. Cell extract (1 mg) from MCF7 cells that were chemically cross-linked with DSP was incubated with either anti-PP5 antibody (PP5/PPT) or nonimmune serum for 3 h at 4 C and then incubated with protein G-Sepharose beads for 1.5 h at 4 C. Proteins in the immune complexes or 10 µg of the cell extracts (corresponding to 1% of input) were resolved by SDS-PAGE, and the Western blot was probed with an antibody against ER{alpha} (H-184). IP, Immunoprecipitation.

 
PP5 Inhibits Phosphorylation of ER{alpha} on S118
To assess the phosphatase activity of PP5 on ERs, we next examined whether PP5 modulates the phosphorylation of ER{alpha} on S118, which is a major phosphorylation site for ER{alpha}. In MCF7 cells, maximal phosphorylation levels of ER{alpha} were observed 60 min and 5 min after E2 stimulation and EGF stimulation, respectively, analyzed by immunoblotting using a specific antibody against ER{alpha} phosphorylated at S118 (ER{alpha}-P-S118). In cells transfected with PP5, the phosphorylation level of ER{alpha} 60 min after E2 stimulation was 29% reduced compared with control cells transfected with a control vector (Fig. 5AGo), and the phosphorylation level 5 min after EGF stimulation was 29% reduced relative to control cells (Fig. 5BGo). The maximal S118 phosphorylation levels induced by either E2 (Fig. 5AGo) or EGF (Fig. 5BGo) were approximately 65% and 80% enhanced, respectively, in MCF7 cells transfected with the TPR domains of PP5 compared with control cells. To confirm the inhibitory activity of PP5 on ER{alpha} phosphorylation at S118, we investigated the effect of PP5 AS oligonucleotide on the phosphorylation levels of ER{alpha} in MCF7 cells. Either E2-dependent (Fig. 5CGo) or EGF-dependent (Fig. 5DGo) phosphorylation of ER{alpha} was enhanced by treatment with PP5 AS compared with control mismatch scrambled (Scr) or PP5 sense (Sense) oligonucleotides (~150% increase by 60-min E2 incubation in AS vs. Scr, ~80% increase by 5-min EGF stimulation in AS vs. Scr). The results indicate that PP5 has an inhibitory activity on ER{alpha} phosphorylation at S118, and the disruption of PP5 activity either by TPR alone or PP5 AS oligonucleotide causes an enhancement of S118 phosphorylation levels.



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  		Fig. 5. PP5 Inhibits Phosphorylation of ER{alpha} on S118 in MCF7 Cells

MCF7 cells were plated at a density of 1.4 x 105 cells per well on six-well plates overnight, transfected with 1 µg expression plasmids (A and B) or with 1 µg oligonucleotides (C and D) for 12 h, serum starved for 24 h, and subsequently treated with E2 (10 nM)(A and C) or EGF (100 ng/ml)(B and D) for indicated times. Cell extracts were collected using 80 µl of sample buffer for SDS-PAGE. Extracts (25 µl) were separated by SDS-PAGE and detected by antibody against phosphorylated human ER{alpha} on S118 (ER{alpha}-P), antibody against whole ER{alpha} (H-184), and anti-PP5 antibody (PP5/PPT). A–D, Upper panels show representative data from three independent experiments of Western blots after chemiluminescent detection. A–D, Lower panels show phosphorylation levels at ER{alpha} S118 normalized by total protein amounts of ER{alpha}. Quantification of signal intensities was performed using LAS 1000 image analyzer, and data are expressed as the mean ± SD of three independent experiments.

 
Suppression of ER Transcription Activity by PP5
To determine whether PP5 affects estrogen-regulated transcription, we next examined transcriptional activity of ERs and ER mutants using an estrogen response element (ERE)-driven luciferase reporter cotransfected with full-length PP5 or truncated PP5 mutants (Fig. 6Go). PP5 attenuated the ER transcriptional activity in a concentration-dependent manner, as E2-dependent transactivations by both ER{alpha} and ERß in 293T cells transfected with 50 ng of PP5 were approximately 50% decreased compared with control cells without PP5 transfection (Fig. 6AGo). Transfection with equal amounts of TPR domain cDNA (5 ng) relative to ER expression vector did not affect E2-dependent transactivation, whereas 10-fold excess dose of TPR cDNA (50 ng) relative to ER cDNAs elicited a 30–40% increase in E2-dependent transactivations by both ER{alpha} and ERß compared with control cells. Another PP5 mutant with its catalytic domain alone did not affect ER{alpha} and ERß transactivations. Because levels of S118 phosphorylation of ER{alpha} were modulated by PP5 expression in Fig. 5Go, we next investigated whether PP5 affects transactivations by ER{alpha} mutants on S118. Mutation of S118 to alanine (S118A) causes a reduction in E2-dependent transcriptional activation in a number of cell types, whereas substitution of S118 to glutamic acid (S118E) resulting in a constitutive negative charge leads to transactivation at levels higher than wild-type ER{alpha} (3). E2-dependent transactivation by S118A was 30% reduced compared with wild-type ER{alpha}, whereas that by S118E was approximately 30% enhanced compared with wild-type ER{alpha} (Fig. 6BGo). PP5 expression did not further alter E2-dependent transactivation by either S118A or S118E, suggesting that PP5 exerts its phosphatase activity on S118. To confirm the inhibitory activity of PP5 on ER-dependent transactivation, we examined the effect of PP5 AS oligonucleotide on the transcriptional activity by ER{alpha} (Fig. 6CGo). The E2-stimulated ER{alpha} transactivation was 2-fold increased by PP5 AS transfection compared with Scr or PP5 sense oligonucleotides transfection, indicating that PP5 negatively regulate ligand-dependent transactivation by ER{alpha}.



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  		Fig. 6. Inhibition of ER-Mediated Transcription by PP5

A, 293T cells were plated at a density of 1 x 104 cells per well on 24-well plates and transfected with 0.8 µg ERE-tk-Luc, 0.7 µg pRL-CMV, 0–50 ng expression vectors for full-length (PP5), TPR domain (TPR), or catalytic domain (C) of PP5, and 5 ng expression vectors for ER{alpha} and ERß. Cells were treated with or without E2 (10 nM) for 24 h, and luciferase assasy were performed. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. B, 293T cells were transfected with 0.8 µg ERE-tk-Luc, 0.7 µg pRL-CMV, 0–50 ng expression vectors for full-length PP5, TPR domain, or catalytic domain (C), and 5 ng expression vestors for wild-type ER{alpha} or ER{alpha} mutants S118A and S118E. Data are expressed as the mean ± SD of three independent experiments performed in triplicate. C, Effect of PP5 AS oligonucleotide on ER-mediated transcription. Luciferase assays were performed as in panel B except transfection with 0.8 µg ERE-tk-Luc, 0.7 µg pRL-CMV, and 200 ng oligonucleotides into MCF7 cells. Upper panel shows the data of three independent experiments performed in triplicate. Lower panels show representative data from three independent experiments of Western blots (WB) using the identical cell extracts for luciferase assays. Signals were detected by antibodies against ER{alpha} and PP5.

 
PP5 Attenuates Induction of Estrogen-Responsive Genes
The ligand-induced ER activation leads to transcriptional induction of estrogen-responsive genes. We next investigated whether PP5 affects E2-induced expression of the estrogen-responsive genes including pS2 (25), c-myc (26), and cyclin D1 (27, 28) by Northern blot analysis (Fig. 7AGo). Adenoviral expression of PP5 in MCF7 cells markedly suppressed the E2 (10 nM)-induced mRNA expression of pS2, c-myc, and cyclin D1 under the serum-depleted condition, compared with the results of a control adenovirus (Ad-GFP) or vehicle alone. Each mRNA level was normalized by that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the same sample (Fig. 7Go, B–D). Approximately 40% reduction of pS2 expression was observed in adenoviral PP5-transduced MCF7 cells 24 h after E2 stimulation, compared with the gene expression at the same period in control cells including adenoviral GFP-transduced cells and vehicle-transfected cells (Fig. 7BGo). Regarding c-myc, the maximal mRNA expression in Ad-PP5 cells was observed at 2 h after E2 treatment, and it was only 30% of the maximal expression levels in the control cells (Fig. 7CGo). The maximal expression of cyclin D1 mRNA in Ad-PP5 cells was delayed compared with that in control cells: the former was at 12 h whereas the latter was at 2 h after E2 treatment (Fig. 7DGo). The maximal level of cyclin D1 in Ad-PP5 cells was approximately half of that in vehicle-transfected cells.



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  		Fig. 7. PP5 Attenuates E2-Induced Transcription of Estrogen-Responsive Genes in MCF7 Cells

MCF7 cells were plated at a density of 1 x 106 cells per well on 10-cm plates and infected with recombinant adenoviruses expressing PP5 (Ad-PP5) and GFP (Ad-GFP), or transfected with vehicle (Vehicle) for 12 h at a multiplicity of infection of 10. Cells were serum starved for 24 h after the infection and treated with E2 (10 nM) for indicated times. Northern blot analysis was carried out using 20 µg total RNA and hybridized with 32P-labeled cDNAs for pS2, c-myc, cyclin D1 (CycD1), and GAPDH. A, The representative result of three independent experiments of Northern blotting. B, C, and D, Quantification of mRNA levels of estrogen-responsive genes including pS2 (B), c-myc (C), and CycD1 (D) after normalization to GAPDH mRNA levels in MCF7 cells after E2 treatment. Data are expressed as the mean ± SD values of fold change over control from three independent experiments. Data for cells treated with vehicle and harvested at the starting point of E2 treatments are used as control mRNA levels.

 
Because S118 phosphorylation levels and ER{alpha} transactivation were augmented by treatment with PP5 AS oligonucleotide, we further investigated whether PP5 AS enhances mRNA expression of estrogen-responsive genes (Fig. 8Go). Transfection with PP5 AS increased basal levels of pS2 (Fig. 8BGo), c-myc (Fig. 8CGo), and cyclin D1 (Fig. 8DGo) mRNAs in serum-starved condition (~170%, ~100%, and ~80% increase for pS2, c-Myc, and CycD1, respectively; AS vs. Scr). E2 (10 nM)-induced mRNA expression of those three genes was 25–50% enhanced by PP5 AS compared with PP5 sense or Scr oligonucleotides, without affecting the time course of each gene. The maximal mRNA expression of pS2, c-myc, and cyclin D1 detected in 24 h after E2 stimulation was 25%, 48%, and 36% increased by treatment with PP5 AS, respectively, when compared with Scr oligonucleotide (at 24 h, 2 h, and 6 h after E2 stimulation for pS2, c-Myc, and CycD1, respectively. P < 0.05 for pS2 at 24 h, Scr vs. AS; P < 0.001 for c-Myc at 2 h, Scr vs. AS; P < 0.05 for CycD1 at 6 h, Scr vs. AS).



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  		Fig. 8. PP5 AS Oligonucleotide Enhances E2-Induced Transcription of Estrogen-Responsive Genes in MCF7 Cells

MCF7 cells were plated at a density of 1 x 106 cells per well on 10-cm plates and transfected with 5 µg control mismatch Scr, PP5 sense (Sense), or PP5 AS oligonucleotides for 12 h. Cells were serum starved for 24 h after the transfection and treated with E2 (10 nM) for indicated times. Northern blot analysis was performed as indicated in Fig. 7Go. A, The representative result of three independent experiments of Northern blotting. B, C, and D, Quantification of mRNA levels of estrogen-responsive genes including pS2 (B), c-myc (C), and CycD1 (D) after normalization to GAPDH mRNA levels in MCF7 cells after E2 treatment. Data are expressed as the mean ± SD values of fold change over control from three independent experiments. Data for cells transfected with Scr oligonucleotide and harvested at the starting point of E2 treatments are used as control mRNA levels.

 
Taken together, the data demonstrate that PP5 negatively regulates ER phosphorylation levels, transcriptional activity, and transcriptional induction of estrogen-responsive genes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we isolated a serine/threonine phosphatase PP5 as a direct interactant with ERß through yeast two-hybrid studies. GST pull-down assays, mammalian two-hybrid assays, and immunoprecipitation studies show that PP5 interacts with both ER{alpha} and ERß through its TPR domains. We demonstrated that PP5 negatively regulates the degree of both estrogen-dependent and EGF-dependent ER{alpha} phosphorylation on S118, leading to the suppression of ER transcriptional activity and the reduction of E2-induced expression of estrogen-responsive genes including pS2, c-myc, and cyclin D1. We thus present the first evidence that PP5 acts as an inhibitory regulator of ER signaling pathway in a negative direction.

The status of ER phosphorylation can be regulated by phosphatases, as okadaic acid treatment enhances ER{alpha} phosphorylation and ER{alpha} transactivation (29). While preparing this manuscript, another member of the PPP family PP2A has been found to interact with ER{alpha} and dephosphorylate S118 of the receptor (30). Here we have shown the evidence that ER phosphorylation and function can be modulated by PP5. There are several differences between PP5 and PP2A regarding the interactions with ERs. In our results, the TPR domains of PP5 are the sites responsible for binding to ER{alpha} and ERß. The TPR domains of PP5 may also function as an autoinhibitory region against phosphatase activity as removal of the domain produces a marked increase in activity (31). In contrast, PP2A acts in a form of heterotrimer like other members of the PPP family, except PP5, and it has been reported that the catalytic subunit of the phosphatase is required for the interaction with ER{alpha}. In regard to interacting functional domains of ERs, we have found E domains not containing an AD core region within AF-2 bind to PP5, whereas the A/B domain within AF-1 of ER{alpha} was reported to associate with PP2A. Thus it seems that ERs can be dephosphorylated by several kinds of protein phosphatases at different functional domains, leading to the more complex and subtle regulatory mechanisms for the receptors. Yet it is also noted that PP5 can exist in a native complex in vivo with the A subunit of PP2A via its TPR domains (20). It may be possible that PP5 directly interacts and cooperates with PP2A when the phosphatases bind to ERs and regulate the phosphorylation status of the receptors.

Notably, we showed that the truncated PP5 mutant consisting of only its TPR domains acts as a dominant negative PP5 because it increased the levels of ER{alpha} phosphorylation on S118 and transcriptional activation by ER{alpha} and ERß. Our data suggest a physiological role for PP5 in ER signaling in vivo. It has been shown that the TPR domains of PP5 also have dominant negative effects on glucocorticoid receptor (GR)-mediated transactivation (16). PP5 could regulate the phosphorylation state of steroid receptors or associated phosphoproteins. We speculate that the TPR domains of PP5 may form complexes with endogenous PP5, leading to inhibition of phosphatase activity of endogenous PP5. Another possibility is that the TPR domains of PP5 displace other interacting proteins of steroid receptors, which are crucial for the regulation of the receptors. Indeed, some of the TPR-containing proteins appear to compete with each other for binding to heat shock protein 90, a major molecular chaperone that forms heterocomplexes with steroid receptors and participates in the signaling pathways of the receptors including glucocorticoid receptor (GR) and ER (32, 33).

To confirm endogenous PP5 activity toward ER{alpha} function, we used an AS strategy to decrease PP5 expression in MCF7 cells. The phosphorothioate AS oligonucleotide was designed to target the region overlapping the translation start site. Treatment with PP5 AS oligonucleotide, but not with control Scr or PP5 sense oligonucleotides, led to a significant reduction of PP5 expression (Fig. 6CGo) and a proportional increase in S118 phosphorylation of ER{alpha} (Fig. 5Go, C and D), transcriptional activity by ER{alpha} (Fig. 6CGo), and expression of estrogen-targeted gene mRNAs (Fig. 8Go, A–D) in MCF cells. In a report using PP5 AS oligonucleotide targeting the 3'-untranslated region of PP5 for MCF7 cells, Urban et al. (34) concluded that treatment with up to 500 nM of PP5 AS had no apparent effect on estrogen-induced expression of c-myc and cyclin D1 mRNA. In contrast to their Northern blot result, we investigated the time-dependent mRNA expression of estrogen-targeted genes and confirmed that PP5 AS oligonucleotide at a concentration less than 70 nM significantly suppressed E2-dependent mRNA expression of pS2, c-myc, and cyclin D1. Thus, we consider that PP5 is a critical regulator of ER function that could modulate phosphorylation states and transcriptional activities of the receptors.

On the basis of its interactions with other proteins and of studies in which PP5 activity was inhibited using recombinant DNA approaches and antisense oligonucleotide treatment, potential biological roles of PP5 have begun to be elucidated. PP5 has been shown to modulate GR signaling (16, 35), to promote cell growth (34, 36), and to terminate responses to oxidative stress (17). In regard to oxidative stress, PP5 is a physiological inhibitor of apoptosis signal-regulating kinase 1-c-Jun N-terminal kinase/p38 pathway, which plays a pivotal role in stress-induced apoptosis (17). PP5 also interacts with the anaphase-promoting complex and preserves the dephosphorylated or inactivated state of the complex before the activation occurs (19). The growth-promoting effect of PP5 on cell proliferation appears to be exerted by inhibiting both glucocorticoid- and p53-mediated signaling pathways leading to p21WAF1/Cip1-mediated growth arrest (35, 36). Constitutive overexpression of PP5 in MCF7 cells converted the E2-dependent phenotype of the cells into an E2-independent one (34). Indeed, we also observed that adenoviral delivery of PP5 into MCF7 cells increases the number of proliferating cells by cell cycle analysis, as it decreases the percentage of cells at G1 phase and accumulates the cells at S phase (data not shown). Although it seems paradoxical, we speculate that the proliferative function of overexpressed PP5 observed in MCF7 cells may result from a hyperactivity of the enzyme to inhibit growth-arresting factors including GR and p53, which overcomes PP5-mediated inhibition of ER function at physiological concentrations of the phosphatase.

In summary, we have demonstrated that PP5 directly binds to ERs and regulates ER phosphorylation and transcriptional activity in a negative manner. The inhibitory action of PP5 against ER phosphorylation and function may contribute to a regulatory system of ER-mediated signaling at physiological and pathophysiological status. Further study will be required to understand the distinct activity of PP5 in estrogen-dependent cell proliferation, which may be responsible for developing hormone-dependent tumors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast Two-Hybrid Screening
To analyze the regulatory mechanism of ERs, we employed the yeast two-hybrid system to search for proteins that bind to ERs by using ERß (1–481), a truncated human ERß cDNA fragment containing 1–481 amino acids (24) as a bait. The bait plasmid was constructed in-frame with the LexA DNA-binding domain of the pEG202NLS plasmid (Origene Technologies Inc., Rockville, MD). A cDNA library derived from estrogen-depleted MCF7 cells in the pJG4–5 prey plasmid was screened for proteins that interact with ERß (1–481) using the EGY48 yeast reporter strain and the pSH18–34 LacZ reporter plasmid. Plasmids of positive clones were recovered, and the cDNA inserts were sequenced. To examine the interaction between ERß (1–481) and PP5, ERß (1–481) and PP5 constructs were cotransformed along with the pSH18–34 reporter plasmid into the EGY48 yeast strain. Transcriptional activation of LacZ gene was examined in X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside)-containing medium. As PP5 cDNA was included in a galactose-inducible vector, pJG4–5, the interaction between ERß (1–481) and PP5 was observed when the reporter yeast was transformed with a ERß (1–481) construct pEG202NLS-ERß (1–481) and cultured in galactose-containing medium.

GST Pull-Down Assay
GST constructs for full-length PP5 (GST-PP5) and truncated PP5 mutants GST-PP5 (2–71), GST-PP5 (28–165), GST-PP5 (2–181), GST-PP5 (2–132), and GST-PP5 (181–499), were prepared in pGEX4T-1 (Amersham Biosciences, Inc., Piscataway, NJ). With regard to structure of the truncated PP5 mutants, GST-PP5 (2–71) includes only one TPR domain. GST-PP5 (28–165) and GST-PP5 (2–181) contain four TPR domains. GST-PP5 (2–312) consists of four TPR domains plus the N-terminal region of catalytic domain. GST-PP5 (181–499) includes the whole catalytic domain but not TPR domains. GST fusion proteins were induced, solubilized in solution A (20 mM Tris-HCl, pH 7.9; 10% glycerol; 80 mM KCl; 1 mM MgCl2; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.5 mM phenylmethylsulfonyl fluoride; and 1% Triton X-100), and bound to glutathione-Sepharose 4B beads following the manufacturer’s instruction (Amersham Biosciences, Inc.). GST fusion proteins bound to glutathione beads were incubated at 4 C for 1.5 h with 35S-labeled ER{alpha} or ERß, which was synthesized in vitro using the TnT-coupled reticulocyte lysate system (Promega Corp., Madison, WI). After the incubation, the beads were washed three times with solution A, and the complexes were separated by SDS-PAGE. The results were visualized using a Fuji FLA 3000 phosphoimaging analyzer (Fuji Film, Tokyo, Japan).

Mammalian Two-Hybrid Assay
The luciferase reporter plasmid TK-MH100 x 4Luc and the expression plasmids pCMX-GAL4 and pCMX-VP16 were kindly provided by K. Umesono (Kyoto University, Kyoto, Japan). pCMX-VP16-PP5 was constructed by an in-frame ligation of human PP5 cDNA to the VP16 transactivation domain in pCMX-VP16. pCMX-GAL4 constructs were generated by in-frame ligations of various ER{alpha} and ERß cDNA fragments to the GAL4 DNA binding domain in pCMX-GAL4. The receptor domains encoded by ER{alpha} and ERß cDNA fragments were as follows: for ER{alpha}, ABCD (amino acids 1–302), ABC (amino acids 1–263), E (amino acids 302–530), and a part of EF (amino acids 530–595) domains; for ERß, ABCD (amino acids 1–248), ABC (amino acids 1–213), E (amino acids 248–481), and a part of EF (amino acids 481–530) domains. 293T cells were plated at a density of 6 x 104 cells per well of 24-well plates and incubated overnight. Cells were then cotransfected with plasmids containing 0.8 µg TK-MH100 x 4Luc, 0.7 µg pRL-TK vector (Promega), 0.2 µg VP16-PP5, and 0.1 µg GAL4 fusion constructs of ER{alpha} or ERß using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN). Cells were cultured for 24 h and luciferase assays were performed using a Dual-Luciferase Assay System (Promega). Data are expressed as the mean ± SD of three independent experiments performed in triplicate.

Coimmunoprecipitation Assay
The GFP-tagged ER{alpha} construct was generated by an in-frame ligation of human ER{alpha} cDNA to downstream of GFP in pEGFP-C2 (BD Biosciences CLONTECH, Palo Alto, CA). The Flag-tagged pcDNA3 (Invitrogen, San Diego, CA) constructs pcDNA3-Flag-PP5 and pcDNA3-Flag-TPR were prepared by insertions of full-length PP5 and TPR domains (amino acids 28–165) into pcDNA3 containing Flag tag. 293T cells were plated at a density of 1 x 106 cells per dish in 10-cm dishes and cotransfected with 7.5 µg GFP-ER{alpha} and 7.5 µg Flag-PP5 or Flag-TPR by the calcium phosphate method. After 24 h, cells were incubated with PBS containing 5 mM hybrophobic lysine-specific cross-linker dithiobis[succinimidyl propionate] (Pierce Biotechnology, Inc., Rockford, IL) at 4 C for 30 min. The reaction was stopped by addition of 100 mM Tris-HCl, pH 7.5, at room temperature for 10 min. Cells were washed with PBS and lysed in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5 mM aprotinin, 0.5 mM phenylmethylsulfonylfluoride). Lysates were cleared by centrifugation, and protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA). For immunoprecipitation, 1 mg of lysates were incubated with 10 µg of anti-Flag antibody M2 (Sigma Chemical Co., St. Louis, MO) for 3 h at 4 C, and then incubated with 20 µl of protein G-Sepharose beads (50% vol/vol slurry) (Amersham Biosciences, Inc.) for 90 min at 4 C. The beads were washed three times with immunoprecipitation buffer and resuspended in 20 µl of sample buffer for SDS-PAGE. Eluted proteins were subjected to SDS-PAGE, followed by electroblotting onto polyvinylidene difluoride membrane, and probed with antibodies against GFP (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan). The antibody-antigen complexes were detected using the enhanced chemiluminescence system (Amersham Biosciences, Inc.) according to the manufacturer’s instruction. In experiments for examining the interaction between endogenous PP5 and ER{alpha}, the extracts from MCF7 cells were immunoprecipitated with an anti-PP5 antibody PP5/PPT (BD Biosciences, San Jose, CA) or nonimmune serum and probed by an anti-ER{alpha} antibody H-184 (Santa Cruz Biotechnology, Santa Cruz, CA).

Adenoviral Gene Expression
Adenoviral constructs of Flag-tagged human PP5 (Ad-PP5) and GFP-fusion histone H2B (Ad-GFP) were prepared in an adenovirus vector using Adenovirus Expression Vector Kit (Takara Bio Inc., Shiga, Japan) (37, 38). MCF7 cells were infected with the recombinant adenoviruses at a multiplicity of infection (MOI) of 10 for 12 h. The infected cells were serum starved for 24 h in phenol red-free DMEM and treated with 10 nM E2 in DMEM containing 10% dextran-coated charcoal-treated fetal calf serum (dccFCS) for the indicated times.

Oligonucleotide Treatment
Twenty-three-base phosphorothioate oligonucleotides were prepared by Invitrogen. Sequences for PP5 AS, PP5 sense, and control Scr oligonucleotides were 5'-CTCTCGCCCTCCGCCATCGCCAT-3', 5'-ATGGCGATGGCGGAGGGCGAGAG-3', and 5'-GCAGTGGCGAGCTGAGAGAGGGG-3', respectively. MCF7 cells were incubated with phenol red-free DMEM containing 10% dccFCS before experiments. Cells were transfected with oligonucleotides using GeneSilencer reagent (GeneTherapy Systems, Inc., San Diego, CA) according to the procedure recommended by the manufacturer. Twelve hours after transfection, cells were fed with serum-starved DMEM, or phenol red-free DMEM containing 10% dccFCS supplemented with or without E2. Cells were used for experiments involving ER phosphorylation, transactivation, and expression of estrogen target genes.

Transcription Assay of ER
Expression vectors of S118A (HE457) and S118E (HE458) mutants of ER{alpha} were the gifts from Dr. P. Chambon (3). N-terminal Flag-tagged pcDNA3 constructs including full-length PP5, TPR domains, and catalytic domain were generated. 293T cells at a density of 1 x 104 cells per well on 24-well plates were transfected with 0.8 µg ERE-tk-Luc, 0.7 µg pRL-CMV (Promega), 5 ng of expression vectors for full-length ER{alpha}/ERß or ER{alpha} mutants, and 0–50 ng of expression vectors for full-length or truncated PP5 in phenol red-free DMEM containing 10% dccFCS using FuGENE 6 transfection reagent (Roche Diagnostics). Twelve hours after transfection, cells were treated with or without 10 nM E2 for 24 h and luciferase assays were performed. Data were represented as the mean ± SD of three independent experiments performed in triplicate.

Northern Blot Analysis and Probes
The cDNAs encoding full-length human pS2, human c-myc, human cyclin D1, and human GAPDH were cloned by RT-PCR and verified by sequencing. Probes for Northern blot analysis were prepared by labeling the cloned cDNAs with [{alpha}-32P]dCTP using the Random Primer Labeling Kit (Takara Bio Inc.). Total RNAs (20 µg) were separated in 1% formaldehyde denaturing agarose gels and transferred to Hybond-NX membranes (Amersham Biosciences, Inc.). Blotted membranes were hybridized with the 32P-labeled probes in a hybridization buffer (0.1% sodium dodecyl sulfate (SDS), 50% formamide, 5x sodium saline citrate (SSC), 50 mM NaPO4 (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt’s solution, and 50 µg/ml salmon sperm DNA] at 42 C overnight. Membranes were then washed with 2x SSC, 0.1% SDS at 42 C for 30 min and 0.2x SSC, 0.1% SDS at 42 C for 30 min. Radioactivities of the signals were quantified using a Fuji FLA 3000 phosphoimaging analyzer (Fuji Photo Film, Tokyo, Japan). The mRNA levels for estrogen-targeted genes at each time point were determined by the signal intensities normalized by that of GAPDH mRNA level in an identical sample. Data for cells treated with vehicle and harvested at the starting point of E2 treatments are used as control mRNA levels. Data are expressed as the mean ± SD values of fold change over control from three independent experiments.

Analysis for Phosphorylation State of ER{alpha} at S118
MCF7 cells were plated at a density of 1.4 x 105 cells on six-well plates and transfected with 1 µg of expression plasmids for full-length PP5, TPR domains, or an empty vector for 12 h using FuGENE 6 transfection reagent (Roche Diagnostics). In experiments using oligonucleotides, cells were transfected with PP5 sense, AS, or control mismatch Scr oligonucleotides for 12 h using GeneSilencer reagent. In a preliminary experiment, we confirmed that the transfection efficiency of pEGFP-C1 vector (BD Biosciences CLONTECH) into MCF7 cells was 32% using FuGENE 6 transfection reagent. Transfected cells were serum starved for 24 h and treated with E2 (10 nM), EGF(100 ng/ml), or vehicles. Cell extracts were subjected to immunoblotting using a specific antibody against phosphorylated ER{alpha} at S118 (16J4)(Cell Signaling Technology, Beverly, MA). Quantification of signal intensities was performed using LAS 1000 image analyzer (Fuji Photo Film). Three independent experiments were performed, and phosphorylation levels at ER{alpha} S118 were normalized by total protein amounts of ER{alpha}.

Cell Cycle Analysis
For flow cytometry analysis, MCF7 cells were transduced with recombinant adenoviruses Ad-PP5 or Ad-GFP. Twelve hours after infection, cells were cultured in serum-deleted medium for 24 h and treated with E2 (10 nM). Cells were trypsinized, fixed with 70% ethanol, treated with RNase A (100 µg/ml), and then stained with propidium iodide (10 µg/ml). Cells were analyzed by FACS Calibur flow cytometer (Becton Dickinson and Co., Mountain View, CA) and the cell-cycle profile was determined using ModFit LT software (Becton Dickinson).


   ACKNOWLEDGMENTS
 
We thank Dr. P. Chambon for kindly providing HE457 and HE458 plasmids, and T. Hishinuma, T. Ichikawa, and A. Harashima for their technical assistance.


   FOOTNOTES
 
This work was supported in part by grants-in-aid from the Ministry of Health, Labor and Welfare; from the Ministry of Education, Culture, Sports, Science and Technology of Japan; from Kanzawa Medical Research Foundation; and from Novartis Foundation (Japan) for the Promotion of Science.

Abbreviations: AD core, activation domain core; AF, activation function; AS, antisense; E2, 17ß-estradiol; dccFCS, dextran-coated charcoal-treated fetal calf serum; EGF, epidermal growth factor; ER, estrogen receptor; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GR, glucocorticoid receptor; GST, glutathione S-transferase; PP5, serine/threonine protein phosphatase type 5; S118, serine 118; S118A, mutation of S118 to alanine; S118E, substitution of S118 to glutamic acid; Scr, scrambled; SDS, sodium dodecyl sulfate; SSC, sodium saline citrate; TPR, tetratricopeptide repeat; VP16, herpes simplex viral protein 16; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside.

Received for publication August 9, 2003. Accepted for publication January 27, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Muramatsu M, Inoue S 2000 Estrogen receptors: how do they control reproductive and nonreproductive functions? Biochem Biophys Res Commun 270:1–10[CrossRef][Medline]
  2. Shao D, Lazar MA 1999 Modulating nuclear receptor function: may the phos be with you. J Clin Invest 103:1617–1618[Medline]
  3. Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region. EMBO J 12:1153–1160[Medline]
  4. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  5. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  6. Chen D, Pace PE, Coombes RC, Ali S 1999 Phosphorylation of human estrogen receptor {alpha} by protein kinase A regulates dimerization. Mol Cell Biol 19:1002–1015[Abstract/Free Full Text]
  7. Chen D, Riedl T, Washbrook E, Pace PE, Coombes RC, Egly JM, Ali S 2000 Activation of estrogen receptor {alpha} by S118 phosphorylation involves a ligand-dependent interaction with TFIIH and participation of CDK7. Mol Cell 6:127–137[CrossRef][Medline]
  8. 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]
  9. Joel PB, Traish AM, Lannigan DA 1998 Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J Biol Chem 273:13317–13323[Abstract/Free Full Text]
  10. Barford D, Das AK, Egloff MP 1998 The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164[CrossRef][Medline]
  11. Cohen PT 1997 Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem Sci 22:245–251[CrossRef][Medline]
  12. Chen MX, McPartlin AE, Brown L, Chen YH, Barker HM, Cohen PT 1994 A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J 13:4278–4290[Medline]
  13. Hirano T, Kinoshita N, Morikawa K, Yanagida M 1990 Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+. Cell 60:319–328[CrossRef][Medline]
  14. Sikorski RS, Boguski MS, Goebl M, Hieter P 1990 A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 60:307–317[CrossRef][Medline]
  15. Blatch GL, Lassle M 1999 The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21:932–939[CrossRef][Medline]
  16. Chen MS, Silverstein AM, Pratt WB, Chinkers M 1996 The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant. J Biol Chem 271:32315–32320[Abstract/Free Full Text]
  17. Morita K, Saitoh M, Tobiume K, Matsuura H, Enomoto S, Nishitoh H, Ichijo H 2001 Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress. EMBO J 20:6028–6036[CrossRef][Medline]
  18. Chinkers M 1994 Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor. Proc Natl Acad Sci USA 91:11075–11079[Abstract/Free Full Text]
  19. Ollendorff V, Donoghue DJ 1997 The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex. J Biol Chem 272:32011–32018[Abstract/Free Full Text]
  20. Lubert EJ, Hong Y, Sarge KD 2001 Interaction between protein phosphatase 5 and the A subunit of protein phosphatase 2A: evidence for a heterotrimeric form of protein phosphatase 5. J Biol Chem 276:38582–38587[Abstract/Free Full Text]
  21. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  22. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
  23. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljunggren J, Gustafsson JA, Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full antagonist. EMBO J 18:4608–4618[CrossRef][Medline]
  24. Ogawa S, Inoue S, Orimo A, Hosoi T, Ouchi Y, Muramatsu M 1998 Cross-inhibition of both estrogen receptor {alpha} and ß pathways by each dominant negative mutant. FEBS Lett 423:129–132[CrossRef][Medline]
  25. Brown AM, Jeltsch JM, Roberts M, Chambon P 1984 Activation of pS2 gene transcription is a primary response to estrogen in the human breast cancer cell line MCF-7. Proc Natl Acad Sci USA 81:6344–6348[Abstract/Free Full Text]
  26. Dubik D, Shiu RP 1992 Mechanism of estrogen activation of c-myc oncogene expression. Oncogene 7:1587–1594[Medline]
  27. Sabbah M, Courilleau D, Mester J, Redeuilh G 1999 Estrogen induction of the cyclin D1 promoter: involvement of a cAMP response-like element. Proc Natl Acad Sci USA 96:11217–11222[Abstract/Free Full Text]
  28. Castro-Rivera E, Samudio I, Safe S 2001 Estrogen regulation of cyclin D1 gene expression in ZR-75 breast cancer cells involves multiple enhancer elements. J Biol Chem 276:30853–30861[Abstract/Free Full Text]
  29. Smith CL 1998 Cross-talk between peptide growth factor and estrogen receptor signaling pathways. Biol Reprod 58:627–632[Abstract/Free Full Text]
  30. Lu Q, Surks HK, Ebling H, Baur WE, Brown D, Pallas DC, Karas RH 2003 Regulation of estrogen receptor {alpha}-mediated transcription by a direct interaction with protein phosphatase 2A. J Biol Chem 278:4639–4645[Abstract/Free Full Text]
  31. Sinclair C, Borchers C, Parker C, Tomer K, Charbonneau H, Rossie S 1999 The tetratricopeptide repeat domain and a C-terminal region control the activity of Ser/Thr protein phosphatase 5. J Biol Chem 274:23666–23672[Abstract/Free Full Text]
  32. Owens-Grillo JK, Hoffmann K, Hutchison KA, Yem AW, Deibel Jr MR, Handschumacher RE, Pratt WB 1995 The cyclosporin A-binding immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor. J Biol Chem 270:20479–20484[Abstract/Free Full Text]
  33. Ratajczak T, Carrello A 1996 Cyclophilin 40 (CyP-40), mapping of its hsp90 binding domain and evidence that FKBP52 competes with CyP-40 for hsp90 binding. J Biol Chem 271:2961–2965[Abstract/Free Full Text]
  34. Urban G, Golden T, Aragon IV, Scammell JG, Dean NM, Honkanen RE 2001 Identification of an estrogen-inducible phosphatase (PP5) that converts MCF-7 human breast carcinoma cells into an estrogen-independent phenotype when expressed constitutively. J Biol Chem 276:27638–27646[Abstract/Free Full Text]
  35. Zuo Z, Urban G, Scammell JG, Dean NM, McLean TK, Aragon I, Honkanen RE 1999 Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest. Biochemistry 38:8849–8857[CrossRef][Medline]
  36. Zuo Z, Dean NM, Honkanen RE 1998 Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate growth arrest. J Biol Chem 273:12250–12258[Abstract/Free Full Text]
  37. Niwa H, Yamamura K, Miyazaki J 1991 Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199[CrossRef][Medline]
  38. Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I 1996 Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA 93:1320–1324[Abstract/Free Full Text]

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