scholarly journals SPBP Is a Phosphoserine-Specific Repressor of Estrogen Receptor α

2005 ◽  
Vol 25 (9) ◽  
pp. 3421-3430 ◽  
Author(s):  
Valentina Gburcik ◽  
Nathalie Bot ◽  
Marcello Maggiolini ◽  
Didier Picard
Keyword(s):  
Estrogen Receptor ◽  
Activation Function ◽  
Estrogen Receptor Α ◽  
Fine Tuning ◽  
Breast Cancer Cell Lines ◽  
Interaction Domain ◽  
Phosphorylated Form ◽  
Element Binding Protein ◽  
Responsive Element

ABSTRACT Multiple signaling pathways stimulate the activity of estrogen receptor α (ERα) by direct phosphorylation within its N-terminal activation function 1 (AF1). How phosphorylation affects AF1 activity remains poorly understood. We performed a phage display screen for human proteins that are exclusively recruited to the phosphorylated form of AF1 and found the stromelysin-1 platelet-derived growth factor-responsive element-binding protein (SPBP). In a purified system, SPBP bound only the in vitro-phosphorylated form of the ERα AF1 or the phosphoserine mimic S118E, and the interaction domain could be mapped to a 42-amino-acid fragment of SPBP. In cells, SPBP preferentially interacted with liganded and phosphorylated ERα. Functionally, SPBP behaved as a repressor of activated ERα, which extends its previously demonstrated roles as a DNA binding transactivation factor and coactivator of other transcription factors. By targeting the phosphorylated form of AF1, SPBP may contribute to attenuating and fine-tuning ERα activity. A functional consequence is that SPBP inhibits the proliferation of ERα-dependent but not ERα-independent breast cancer cell lines, mirroring a reported negative correlation with the ERα status of breast tumors.

scholarly journals The Human Estrogen Receptor-α Isoform hERα46 Antagonizes the Proliferative Influence of hERα66 in MCF7 Breast Cancer Cells

Endocrinology ◽  
2005 ◽  
Vol 146 (12) ◽  
pp. 5474-5484 ◽  
Author(s):  
Graziella Penot ◽  
Christine Le Péron ◽  
Yohann Mérot ◽  
Eva Grimaud-Fanouillère ◽  
François Ferrière ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Estrogen Receptor ◽  
Cell Growth ◽  
Activation Function ◽  
Estrogen Receptor Α ◽  
Breast Cancer Cell Lines ◽  
Mcf7 Cells ◽  
Exact Function ◽  
Human Estrogen Receptor

The expression of two human estrogen receptor-α (hERα) isoforms has been characterized within estrogen receptor-α-positive breast cancer cell lines such as MCF7: the full-length hERα66 and the N terminally deleted hERα46, which is devoid of activation function (AF)-1. Although hERα66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERα46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERα46 is mainly expressed in the nucleus at relatively low levels, whereas hERα66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERα46 accumulating within the nucleus. Although hERα46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G0/G1 phases. To gain further details on the influence of hERα46 on cell growth, we used PC12 estrogen receptor-α-negative cell line, in which stable transfection of hERα66 but not hERα46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERα46 inhibits the hERα66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERα46 antagonizes the proliferative action of hERα66 in MCF7 cells in part by inhibiting hERα66 AF-1 activity.

scholarly journals pp90rsk1 Regulates Estrogen Receptor-Mediated Transcription through Phosphorylation of Ser-167

1998 ◽  
Vol 18 (4) ◽  
pp. 1978-1984 ◽  
Author(s):  
Peteranne B. Joel ◽  
Jeffrey Smith ◽  
Thomas W. Sturgill ◽  
Tracey L. Fisher ◽  
John Blenis ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcriptional Activation ◽  
Ectopic Expression ◽  
Activation Function ◽  
Estrogen Receptor Α ◽  
Kinase Domain ◽  
Stable Complex ◽  
Wild Type

ABSTRACT The estrogen receptor α (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

scholarly journals The cell-specific activity of the estrogen receptor a may be fine-tuned by phosphorylation-induced structural gymnastics

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04005 ◽  
Author(s):  
Valentina Gburcik ◽  
Didier Picard
Keyword(s):  
Estrogen Receptor ◽  
Posttranslational Modifications ◽  
Target Genes ◽  
Specific Activity ◽  
Estrogen Receptor Α ◽  
Fine Tuning ◽  
Differentiated Cells ◽  
Phosphorylated Form ◽  
A Cell ◽  
Key Residues

The estrogen receptor α (ERα) regulates the transcription of target genes by recruiting coregulator proteins through several domains including the two activation functions AF1 and AF2. The contribution of the N-terminally located AF1 activity is particularly important in differentiated cells, and for ERα to integrate inputs from other signaling pathways. However, how the phosphorylation of key residues influences AF1 activity has long remained mysterious, in part because the naturally disordered AF1 domain has resisted a structural characterization. The recent discovery of two coregulators that are specific for a phosphorylated form of AF1 suggests that phosphorylation, possibly in conjunction with the subsequent binding of these coregulators, may enforce a stable structure. The binding of the “pioneer” coregulators might facilitate the subsequent recruitment of yet other coregulators. Different AF1 folds may be enabled by the combinatorial action of posttranslational modifications and coregulator binding thereby fine-tuning ERα activities in a cell- and promoter-specific fashion.

scholarly journals Phosphorylation at serines 104 and 106 by Erk1/2 MAPK is important for estrogen receptor-α activity

2008 ◽  
Vol 40 (4) ◽  
pp. 173-184 ◽  
Author(s):  
Ross S Thomas ◽  
Naveed Sarwar ◽  
Fladia Phoenix ◽  
R Charles Coombes ◽  
Simak Ali
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Activation Function ◽  
Estrogen Receptor Α ◽  
Mitogen Activated Protein Kinase ◽  
Breast Cancer Patients ◽  
Independent Manner ◽  
Stimulation Of

Phosphorylation of estrogen receptor-α (ERα) at specific residues in transcription activation function 1 (AF-1) can stimulate ERα activity in a ligand-independent manner. This has led to the proposal that AF-1 phosphorylation and the consequent increase in ERα activity could contribute to resistance to endocrine therapies in breast cancer patients. Previous studies have shown that serine 118 (S118) in AF-1 is phosphorylated by extracellular signal-regulated kinases 1 and 2 (Erk1/2) mitogen-activated protein kinase (MAPK) in a ligand-independent manner. Here, we show that serines 104 (S104) and 106 (S106) are also phosphorylated by MAPK in vitro and upon stimulation of MAPK activity in vivo. Phosphorylation of S104 and S106 can be inhibited by the MAP-erk kinase (MEK)1/2 inhibitor U0126 and by expression of kinase-dead Raf1. Further, we show that, although S118 is important for the stimulation of ERα activity by the selective ER modulator 4-hydroxytamoxifen (OHT), S104 and S106 are also required for the agonist activity of OHT. Acidic amino acid substitution of S104 or S106 stimulates ERα activity to a greater extent than the equivalent substitution at S118, suggesting that phosphorylation at S104 and S106 is important for ERα activity. Collectively, these data indicate that the MAPK stimulation of ERα activity involves the phosphorylation not only of S118 but also of S104 and S106, and that MAPK-mediated hyperphosphorylation of ERα at these sites may contribute to resistance to tamoxifen in breast cancer.

Identification of two independent nucleosome-binding domains in the transcriptional co-activator SPBP

2012 ◽  
Vol 442 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Sagar Darvekar ◽  
Sylvia Sagen Johnsen ◽  
Agnete Bratsberg Eriksen ◽  
Terje Johansen ◽  
Eva Sjøttem
Keyword(s):  
Transcription Factors ◽  
Dependent Manner ◽  
Chromatin Binding ◽  
Binding Region ◽  
Interaction Domain ◽  
Binding Domains ◽  
Element Binding Protein ◽  
Inducible Protein ◽  
Responsive Element ◽  
Nucleosome Binding

Transcriptional regulation requires co-ordinated action of transcription factors, co-activator complexes and general transcription factors to access specific loci in the dense chromatin structure. In the present study we demonstrate that the transcriptional co-regulator SPBP [stromelysin-1 PDGF (platelet-derived growth factor)-responsive element binding protein] contains two independent chromatin-binding domains, the SPBP-(1551–1666) region and the C-terminal extended PHD [ePHD/ADD (extended plant homeodomain/ATRX-DNMT3-DNMT3L)] domain. The region 1551–1666 is a novel core nucleosome-interaction domain located adjacent to the AT-hook motif in the DNA-binding domain. This novel nucleosome-binding region is critically important for proper localization of SPBP in the cell nucleus. The ePHD/ADD domain associates with nucleosomes in a histone tail-dependent manner, and has significant impact on the dynamic interaction between SPBP and chromatin. Furthermore, SPBP and its homologue RAI1 (retinoic-acid-inducible protein 1), are strongly enriched on chromatin in interphase HeLa cells, and both proteins display low nuclear mobility. RAI1 contains a region with homology to the novel nucleosome-binding region SPBP-(1551–1666) and an ePHD/ADD domain with ability to bind nucleosomes. These results indicate that the transcriptional co-regulator SPBP and its homologue RAI1 implicated in Smith–Magenis syndrome and Potocki–Lupski syndrome both belong to the expanding family of chromatin-binding proteins containing several domains involved in specific chromatin interactions.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

scholarly journals Rapid Estrogen-Induced Phosphorylation of the SRC-3 Coactivator Occurs in an Extranuclear Complex Containing Estrogen Receptor

2005 ◽  
Vol 25 (18) ◽  
pp. 8273-8284 ◽  
Author(s):  
Fuzhong F. Zheng ◽  
Ray-Chang Wu ◽  
Carolyn L. Smith ◽  
Bert W. O'Malley
Keyword(s):  
Estrogen Receptor ◽  
Ligand Binding ◽  
Direct Interaction ◽  
Activation Function ◽  
Estrogen Receptor Α ◽  
Binding Domain ◽  
Phosphorylation Sites ◽  
Binding Domains ◽  
Nongenomic Action ◽  
Binding Groove

ABSTRACT SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor α (ERα) or activated ERβ is present. Both the activation function 1 and the ligand binding domains of ERα are required for maximal induction. Mutations in the coactivator binding groove of the ERα ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERα must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERα mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERα DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERα and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.

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