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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analysis of mRNA prepared from a variety of estrogen-responsive cell lines, breast tumor specimens, and normal breast tissue have established that estrogen receptor-alpha (ER alpha) mRNA is typically expressed as a mixture of transcripts. Using PCR amplification, this heterogeneity has been shown to result largely from an imprecise pattern of mRNA splicing that gives rise to a family of correctly processed and exon-skipped ER alpha transcripts. We have reconstructed ER alpha cDNAs representing the single exon-skipped variants ERdeltaE2 through ERdeltaE7 to enable their functional characterization in a well defined cell transfection system. All six of the ER alpha splicing variants support the efficient expression of stable proteins in Cos7 cells, and each shows a characteristic pattern of subcellular distribution. Each of the variants displays a dramatic reduction in DNA-binding activity with a consensus estrogen response element (ERE) in an in vitro gel mobility shift assay. While this DNA-binding defect appears to be complete for ERdeltaE2, ERdeltaE3, ERdeltaE4, and ERdeltaE6, weak DNA binding is observed for ERdeltaE5 and ERdeltaE7. Scatchard analysis of hormone binding demonstrates that among the variants, only ERdeltaE3 binds 17beta-estradiol (E2) and does so with an affinity similar to wild-type ER alpha (wt ER alpha). Individual variants cotransfected with the pERE-TK-CAT reporter plasmid [a consensus ERE-driven chloramphenicol acetyltransferase (CAT) reporter gene that is highly responsive to E2-liganded wt ER alpha] were ineffective at inducing CAT expression in ER-negative HeLa cells. Only ERdeltaE5 showed indications of positive transcriptional activity on the pERE-TK-CAT reporter, but this activity was limited to approximately 5% of the activity of wt ER alpha. When variants were expressed simultaneously with wt ER alpha, ERdeltaE3 and ERdeltaE5 were observed to have a dominant negative effect on wt ER alpha transcriptional activity. Like the wild-type receptor, both ERdeltaE3 and ERdeltaE5 interact with steroid receptor coactivator-1e (SRC-1e) in vitro; however, only ERdeltaE3 retained the ability to dimerize with wt ER alpha. Transcription from a region of the ovalbumin promoter, which contains an ERE half-site and an AP-1 motif, is positively regulated by liganded wt ER alpha and ERdeltaE3 in phorbol ester-treated, transiently transfected HeLa cells. In both cases, this activity was enhanced by cotransfected cJun. These observations suggest that selected ER alpha splicing variants are likely to exert important transcriptional effects, especially on genes that are regulated by nonconsensus EREs and subject to complex hormonal control.
Mol Endocrinol 2000 May
PMID:An estrogen receptor-alpha splicing variant mediates both positive and negative effects on gene transcription. 1080 28

We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTalpha increases the magnitude of ERalpha transcriptional activity three- to fourfold. It shows lesser enhancement of ERbeta transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTalpha or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTalpha (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTalpha or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTalpha, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTalpha to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain its ability to selectively enhance ER transcriptional activity. These findings highlight a new role for PTalpha as a coregulator activity-modulating protein that confers receptor specificity. Proteins such as PTalpha represent an additional regulatory component that defines a novel paradigm enabling receptor-selective enhancement of transcriptional activity by coactivators.
Mol Cell Biol 2000 Sep
PMID:Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity. 1093 99

The glucocorticoid receptor interacting protein-1 (GRIP1) is a member of the steroid receptor coactivator (SRC) family of transcriptional regulators. Green fluorescent protein (GFP) fusions were made to full-length GRIP1, and a series of GRIP1 mutants lacking the defined regulatory regions and the intracellular distribution of these proteins was studied in HeLa cells. The distribution of GRIP1 was complex, ranging from diffuse nucleoplasmic to discrete intranuclear foci. Formation of these foci was dependent on the C-terminal region of GRIP1, which contains the two characterized transcriptional activation domains, AD1 and AD2. A subpopulation of GRIP1 foci associate with ND10s, small nuclear bodies that contain several proteins including PML, SP100, DAXX, and CREB-binding protein (CBP). Association with the ND10s is dependent on the AD1 of GRIP1, a region of the protein previously described as a CBP-interacting domain. The GRIP1 foci are enriched in components of the 26S proteasome, including the core 20S proteasome, PA28alpha, and ubiquitin. In addition, the irreversible proteasome inhibitor lactacystin induced an increase in the total fluorescence intensity of the GFP-GRIP1 expressing cells, demonstrating that GRIP1 is degraded by the proteasome. These findings suggest the intriguing possibility that degradation of GRIP1 by the 26S proteasome may be a key component of its regulation.
Mol Endocrinol 2001 Apr
PMID: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. 1126 2

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-2 belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/AIB-1. In this study we demonstrate that SRC-2 is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-2 to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-2, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-2 and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.
Mol Endocrinol 2001 May
PMID:Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells. 1132 58

Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.
Mol Endocrinol 2001 Jul
PMID:Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1. 1143 8

The transcriptional activity of ERalpha (or NR3A1) after binding of ligand is mediated through synergistic action between activation functions (AFs) AF-1 and AF-2 and the transcriptional machinery. This is functionally achieved by bridging coactivators such as CEBP binding protein/p300 and members of the p160 subfamily such as steroid receptor coactivator protein-1 (SRC-1). We previously identified a conserved potential alpha-helical structure within the AF-1 functional core, and by evaluating point mutants of human ERalpha (hERalpha) within this region, we show that in transfection experiments this structure is required for synergism between SRC-1 and hERalpha. We report that the transcriptional synergism between AF-1 mutants and SRC-1 was abolished in AF-1-sensitive cells such as HepG2, whereas it was reduced by 50% in CHO-K1 cells, which have a mixed context that is sensitive to both the AF-1 and AF-2 regions of hERalpha. Glutathione-S-transferase pulldown assays demonstrate that the AF-1 core is able and sufficient for the hERalpha N-terminal region to interact with SRC-1. Interestingly, an enhancement of this recruitment in the presence of the hERalpha ligand-binding domain was observed, which was found to be dependent on a direct interaction between the N-terminal B domain and the ligand-binding domain. Another functional consequence of this physical interaction, which is promoted by both partial and full agonists of hERalpha, was an increase in the phosphorylation state of the N-terminal domain. Binding of 4-hydroxytamoxifen (OHT) to the hERalpha C-terminal region induced a functional AF-1 conformation in vitro through this N- and C-terminal interaction. The involvement of an SRC-1-mediated pathway in transactivation mediated by hERalpha AF-1 was further substantiated by transfection experiments using the OHTresponsive human C3 promoter, which showed that OHT-induced hERalpha AF-1 activity was enhanced by SRC-1 and required the AF-1 alpha-helical structure. In conclusion, we demonstrate that the synergism between AF-1 and AF-2 is mediated in part by a cooperative recruitment of SRC-1 by both the AF-1 alpha-helical core and AF-2 regions and that it is stabilized by a direct interaction between the B and C-terminal domains. This interaction of SRC-1 with the AF-1 alpha-helical core is essential for both E2- and OHT-induced ERalpha activity.
Mol Endocrinol 2001 Nov
PMID:Synergism between ERalpha transactivation function 1 (AF-1) and AF-2 mediated by steroid receptor coactivator protein-1: requirement for the AF-1 alpha-helical core and for a direct interaction between the N- and C-terminal domains. 1168 26

We have previously demonstrated that overexpression of Cdc25B in transgenic mice resulted in mammary gland hyperplasia and increased steroid hormone responsiveness. To address how Cdc25B enhances the hormone responsiveness in mammary glands, we showed that Cdc25B stimulates steroid receptor-dependent transcription in transient transfection assays and in a cell-free assay with chromatin templates. Surprisingly, the effect of Cdc25B on steroid receptors is independent of its protein phosphatase activity in vitro. The direct interactions of Cdc25B with steroid receptors, on the other hand, were evidenced in in vivo and in vitro assays, suggesting the potential direct contribution of Cdc25B on the steroid receptor-mediated transcription. In addition, p300/CBP-associated factor and CREB binding protein were shown to interact and synergize with Cdc25B and further enhance its coactivation activity. Thus, we have uncovered a novel function of Cdc25B that serves as a steroid receptor coactivator in addition to its role as a regulator for cell cycle progression. This dual function might likely contribute to its oncogenic action in breast cancer.
Mol Cell Biol 2001 Dec
PMID:Cdc25B functions as a novel coactivator for the steroid receptors. 1168 96

The E6-associated protein (E6-AP), although originally identified as a ubiquitin ligase, has recently been shown to function as a coactivator of steroid receptor-dependent gene expression in in vitro assays. In order to determine whether E6-AP acts as a coactivator in vivo, physiological parameters associated with male and female sex steroid action were assessed in the E6-AP null mouse. Gonadal size was reduced in E6-AP null male and female mice in comparison to wild-type controls in conjunction with reduced fertility in both genders. Consistent with this observation, defects in sperm production and function, as well as ovulation were observed. In comparison to wild-type controls, induction of prostate gland growth induced by testosterone and uterine growth by estradiol were significantly reduced. In contrast, estrogen and progesterone-stimulated growth of virgin mammary gland was not compromised by E6-AP ablation despite E6-AP expression in this tissue. This latter finding contrasts with the impaired estrogen and progesterone-induced mammary gland development observed previously for steroid receptor coactivator type 1 (SRC-1) and SRC-3 female knockout mice. Taken together, these results are consistent with a role for E6-AP in mediating a subset of steroid hormone actions in vivo. Nevertheless, differences observed between SRC and E6-AP knockout phenotypes indicate that these two families of steroid receptor coactivators are not functionally equivalent and supports the hypothesis that coactivators contribute to tissue-specific steroid hormone action.
Mol Cell Biol 2002 Jan
PMID:Genetic ablation of the steroid receptor coactivator-ubiquitin ligase, E6-AP, results in tissue-selective steroid hormone resistance and defects in reproduction. 1175 48

Steroid receptor RNA activator (SRA) is a novel coactivator for steroid receptors that acts as an RNA molecule, whereas steroid receptor coactivator (SRC) family members, such as steroid receptor coactivator-1 (SRC-1) and transcriptional intermediary factor 2 (TIF2) exert their biological effects as proteins. Individual overexpression of each of these coactivators, which can form multimeric complexes in vivo, results in stimulated ERalpha transcriptional activity in transient transfection assays. However there is no information on the consequences of reducing SRC-1, TIF2, or SRA expression, singly or in combination, on ERalpha transcriptional activity. We therefore developed antisense oligodeoxynucleotides (asODNs) to SRA, SRC-1, and TIF2 mRNAs, which rapidly and specifically reduced the expression of each of these coactivators. ERalpha-dependent gene expression was reduced in a dose-dependent fashion by up to 80% in cells transfected with these oligonucleotides. Furthermore, treatment of cells with combinations of SRA, SRC-1, and TIF2 asODNs reduced ERalpha transcriptional activity to an extent greater than individual asODN treatment alone, suggesting that these coactivators cooperate, in at least an additive fashion, to activate ERalpha-dependent target gene expression. Finally, treatment of MCF-7 cells with asODN against SRC-1 and TIF2 revealed a requirement of these coactivators, but not SRA, for hormone-dependent DNA synthesis and induction of estrogen-dependent pS2 gene expression, indicating that SRA and SRC family coactivators can fulfill specific functional roles. Taken together, we have developed a rapid method to reduce endogenous coactivator expression that enables an assessment of the in vivo role of specific coactivators on ERalpha biological action and avoids potential artifacts arising from overexpression of coactivators in transient transfection assays.
Mol Endocrinol 2002 Feb
PMID:Reduction of coactivator expression by antisense oligodeoxynucleotides inhibits ERalpha transcriptional activity and MCF-7 proliferation. 1181 99

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.
Mol Cell Biol 2002 May
PMID:Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I kappa B kinase. 1197 85


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