Estrogen receptor-alpha (ER alpha) is downregulated in the presence of its cognate ligand, estradiol (E2), through the ubiquitin proteasome pathway. Here, we show that ubiquitin proteasome function is required for ER alpha to serve as a transcriptional activator. Deletion of the last 61 amino acids of ER alpha, including residues that form helix 12, abolishes ligand-mediated downregulation of the receptor as do point mutations in the ligand binding domain that impair coactivator binding. In addition, coactivators also are subject to degradation by the 26S proteasome, but their intrinsic transcriptional activity is not affected. These data provide evidence that protein interactions with ER alpha coactivator binding surfaces are important for ligand-mediated receptor down-regulation and suggest that receptor and coactivator turnover contributes to ER alpha transcriptional activity.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradioldependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin-proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA͞UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor ␤ proteins.The ubiquitin-proteasome pathway is the major system in the eukaryotic cell for the selective degradation of short-lived regulatory proteins (1, 2). A common feature of proteasomemediated protein degradation is the covalent attachment of ubiquitin, a highly conserved 8.6-kDa protein, to lysine residues of proteins targeted for degradation followed by the formation of polyubiquitin chains attached covalently to the targeted protein. Ubiquitinated proteins are recognized and degraded by the multi-subunit protease complex, the 26S-proteasome (3-6). In addition to the role it plays in protein degradation, ubiquitination may serve regulatory functions such as directing the subcellular localization of proteins (3, 4). The ubiquitin-proteasome pathway also plays an important role in various cellular processes such as cell-cycle regulation, signal transduction, differentiation, antigen processing, and degradation of tumor suppressors (3, 4, 7-11).Protein ubiquitination involves three classes of enzymes, namely the E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzymes (UBCs), and E3 ubiquitin-protein ligases. The UBA first activates ubiquitin in an ATPdependent manner. The activated ubiquitin then forms a thioester bond between the carboxyl-terminal glycine residue of ubiquitin and a cysteine residue of the UBA. Next, ubiquitin is transferred from the E1 to one of the several E2s (UBCs), preserving the high-energy thioester bond (1, 2, 5, 11). In some cases, ubiquitin is transferred directly from the E2 to the target protein through an isopeptide bond between the -amino group of lysine residues of the target protein and the carboxy terminus of ubiquitin. In other instances, the transfer of ubiquitin from UBCs to target proteins proceeds through an E3 ubiquitin-protein ligase intermediate (12,13). It has been proposed th...

In this study, we found that the E6-associated protein (E6-AP/UBE3A) directly interacts with and coactivates the transcriptional activity of the human progesterone receptor (PR) in a hormone-dependent manner. E6-AP also coactivates the hormone-dependent transcriptional activities of the other members of the nuclear hormone receptor superfamily. Previously, it was shown that E6-AP serves the role of a ubiquitin-protein ligase (E3) in the presence of the E6 protein from human papillomavirus types 16 and 18. Our data show that the ubiquitinprotein ligase function of E6-AP is dispensable for its ability to coactivate nuclear hormone receptors, showing that E6-AP possesses two separable independent functions, as both a coactivator and a ubiquitin-protein ligase. Disruption of the maternal copy of E6-AP is correlated with Angelman syndrome (AS), a genetic neurological disorder characterized by severe mental retardation, seizures, speech impairment, and other symptoms. However, the exact mechanism by which the defective E6-AP gene causes AS remains unknown. To correlate the E6-AP coactivator function and ubiquitin-protein ligase functions with the AS phenotype, we expressed mutant forms of E6-AP isolated from AS patients and assessed the ability of each of these mutant proteins to coactivate PR or provide ubiquitin-protein ligase activity. This analysis revealed that in the majority of the AS patients examined, the ubiquitin-protein ligase function of E6-AP was defective whereas the coactivator function was intact. This finding suggests that the AS phenotype results from a defect in the ubiquitin-proteosome protein degradation pathway.Steroids, thyroid hormones, vitamin D, and retinoids regulate diverse biological processes including growth, development, and homeostasis through their cognate nuclear hormone receptors, which make up a superfamily of structurally related intracellular ligand-activated transcription factors (18,34,40,47). Nuclear hormone receptors contain common structural motifs which include a poorly conserved amino-terminal activation function (activation factor 1 [AF-1]) that affects transcription efficiency, a central DNA-binding domain, which mediates receptor binding to specific DNA enhancer sequences and determines target gene specificity, and a carboxy-terminal hormone-binding domain. The latter domain contains AF-2, a region which mediates the hormone-dependent activation function of receptors (40). When bound to hormone, these receptors undergo a conformational change, dissociation from heat shock proteins, receptor dimerization, phosphorylation, DNA binding at an enhancer element of the target gene, interaction with coactivators, and subsequent recruitment of basal transcription factors to form a stable preinitiation complex. These events are followed by either up-regulation or down-regulation of target gene transcription (40).Nuclear hormone receptor coactivators represent a growing class of proteins which interact with receptors in a ligandspecific manner and serve to enhance their transcrip...

In a little more than 10 years, nuclear receptor (NR) coregulators (coactivators and corepressors) have contributed to our present realization that a great level of sophistication exists in transcriptional regulation. Here, we discuss the implications of coregulators as versatile regulatory agents, influencing not only transcriptional initiation but also elongation, splicing, and translation. In addition to this, there is an increasing recognition that they also regulate a variety of biological processes outside of the nucleus. An important concept that we wish to emphasize is that coregulators are both targets and propagators of posttranslational modification (PTM) codes. This underlies a sophisticated epigenetic regulatory scheme from which a complex and dynamic mammalian phenotype emanates.

We previously demonstrated that the proteasome activator REGgamma directs degradation of the steroid receptor coactivator SRC-3 by the 20S proteasome in an ATP- and ubiquitin-independent manner. Our efforts to identify additional endogenous direct targets of the REGgamma proteasome revealed that p21(Waf/Cip1), a central cyclin-dependent kinase inhibitor, is another endogenous target. Gain-of-function analysis, RNAi knockdown, REGgamma-deficient MEF analysis, and pulse-chase experiments substantiate that REGgamma promotes degradation of unbound p21. Cell-free proteasome proteolysis assays using purified REGgamma, p21, and the 20S proteasome confirm that REGgamma directly mediates degradation of free p21 in an ATP- and ubiquitin-independent manner. Depletion of REGgamma in a thyroid carcinoma cell line results in cell-cycle and proliferative alterations. Our study reveals that, in addition to degrading the SRC-3 growth coactivator, REGgamma also has a role in the regulation of the cell cycle through its ability to influence the level of a cell-cycle regulator(s).

Steroid receptor coactivator-3 (SRC-3/AIB1) is an oncogene frequently amplified and overexpressed in breast cancers. Here we report that SRC-3 interacts with REGgamma, a proteasome activator known to stimulate the trypsin-like activity of the 20S proteasome. RNAi knockdown and gain-of-function experiments suggest that REGgamma promotes SRC-3 protein degradation. Cellular levels of REGgamma expression affect estrogen-receptor target-gene expression and cell growth as a result of its ability to promote degradation of the SRC-3 protein. In vitro proteasome proteolysis assays using purified REGgamma, SRC-3, and the 20S proteasome reinforce these conclusions and demonstrate that REGgamma promotes the degradation of SRC-3 in a ubiquitin- and ATP-independent manner. This work demonstrates the first example of a physiologically relevant endogenous cellular target for the REGgamma-proteasome complex. It also highlights the fact that an alternative mode of proteasome-mediated protein degradation, independent of the 19S proteasome regulatory cap, targets the SRC-3 protein for degradation.

About 200 coactivators play a central role in promoting gene expression mediated by nuclear receptors. This diverse group of proteins are key integrators of signals from steroid hormones and have been implicated in cancer and other diseases.

The p160 steroid receptor coactivators (SRCs) SRC-1, SRC-2 [nuclear receptor coactivator (NCOA)2], and SRC-3 [amplified in breast cancer 1 (AIB1)/NCOA3] are key pleiotropic "master regulators" of transcription factor activity necessary for cancer cell proliferation, survival, metabolism, and metastasis. SRC overexpression and overactivation occur in numerous human cancers and are associated with poor clinical outcomes and resistance to therapy. In prostate cancer (PC), the p160 SRCs play critical roles in androgen receptor transcriptional activity, cell proliferation, and resistance to androgen deprivation therapy. We recently demonstrated that the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger (POZ) domain protein (SPOP) interacts directly with SRC-3 and promotes its cullin 3-dependent ubiquitination and proteolysis in breast cancer, thus functioning as a potential tumor suppressor. Interestingly, somatic heterozygous missense mutations in the SPOP substrate-binding cleft recently were identified in up to 15% of human PCs (making SPOP the gene most commonly affected by nonsynonymous point mutations in PC), but their contribution to PC pathophysiology remains unknown. We now report that PC-associated SPOP mutants cannot interact with SRC-3 protein or promote its ubiquitination and degradation. Our data suggest that wild-type SPOP plays a critical tumor suppressor role in PC cells, promoting the turnover of SRC-3 protein and suppressing androgen receptor transcriptional activity. This tumor suppressor effect is abrogated by the PC-associated SPOP mutations. These studies provide a possible explanation for the role of SPOP mutations in PC, and highlight the potential of SRC-3 as a therapeutic target in PC.proteasome | MATH domain | BTB domain