scholarly journals Ligand-Independent Interactions of p160/Steroid Receptor Coactivators and CREB-Binding Protein (CBP) with Estrogen Receptor-α: Regulation by Phosphorylation Sites in the A/B Region Depends on Other Receptor Domains

2003 ◽  
Vol 17 (7) ◽  
pp. 1296-1314 ◽  
Author(s):  
Martin Dutertre ◽  
Carolyn L. Smith
Keyword(s):  
Estrogen Receptor ◽  
Binding Protein ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Phosphorylation Sites ◽  
Creb Binding Protein ◽  
Steroid Receptor Coactivators

scholarly journals Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

2001 ◽  
Vol 21 (13) ◽  
pp. 4404-4412 ◽  
Author(s):  
David L. Stenoien ◽  
Anne C. Nye ◽  
Maureen G. Mancini ◽  
Kavita Patel ◽  
Martin Dutertre ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Real Time ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Living Cells ◽  
Live Cells ◽  
Creb Binding Protein ◽  
High Accumulation

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

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.

scholarly journals Analysis of the Steroid Receptor Coactivator 1 (SRC1)-CREB Binding Protein Interaction Interface and Its Importance for the Function of SRC1

2001 ◽  
Vol 21 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Hilary M. Sheppard ◽  
Janet C. Harries ◽  
Sagair Hussain ◽  
Charlotte Bevan ◽  
David M. Heery
Keyword(s):  
Binding Protein ◽  
Steroid Receptor ◽  
Creb Binding Protein ◽  
Interaction Domain ◽  
Steroid Receptor Coactivator ◽  
Helix Loop Helix ◽  
Rich Domain ◽  
Interaction Interface ◽  
Acetyltransferase Domain

ABSTRACT The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300. SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300. In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two α-helices. We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID). We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300. In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region. In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligand-induced activity. Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays.

scholarly journals Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

2003 ◽  
Vol 23 (1) ◽  
pp. 335-348 ◽  
Author(s):  
Mari Luz Acevedo ◽  
W. Lee Kraus
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Preinitiation Complex ◽  
Transcription System ◽  
Steroid Receptor Coactivator ◽  
Transcription Reinitiation

ABSTRACT Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor α (ERα) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERα-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERα. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERα-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERα-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERα interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERα-dependent transcription with chromatin templates.

scholarly journals Identification of Regions within the F Domain of the Human Estrogen Receptor α that Are Important for Modulating Transactivation and Protein-Protein Interactions

2007 ◽  
Vol 21 (4) ◽  
pp. 829-842 ◽  
Author(s):  
Akiko Koide ◽  
Changqing Zhao ◽  
Misuzu Naganuma ◽  
Judith Abrams ◽  
Sarah Deighton-Collins ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Ligand Binding ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Steroid Receptor Coactivator ◽  
Human Estrogen Receptor

Abstract The estrogen receptor (ER)α is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERα modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ERα (hERα) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins (“monobodies”) that specifically bind to ERα/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579–584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a β-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERα ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERα selectively modulate its activity and its interactions with other proteins.

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