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Molecular Endocrinology 17 (7): 1315-1331
Copyright © 2003 by The Endocrine Society

Specific Ubiquitin-Conjugating Enzymes Promote Degradation of Specific Nuclear Receptor Coactivators

Feng Yan, Xiuhua Gao, David M. Lonard and Zafar Nawaz

Department of Surgery, Medical Microbiology, and Immunology and Cancer Center Criss II (F.Y., X.G., Z.N.), Creighton University, Omaha, Nebraska 68178; and Department of Molecular and Cellular Biology (D.M.L.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Zafar Nawaz, Ph.D., Department of Surgery, Medical Microbiology & Immunology and Cancer Center, Criss II, Room 518, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178. E-mail: znawaz{at}creighton.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptor coactivators (NRCoAs) are nuclear hormone receptor-associated regulatory proteins that interact with members of the nuclear receptor superfamily in the presence of their cognate ligand, enhancing their transcriptional activity. The identification of ubiquitin-proteasome pathway proteins as coactivators provides evidence that ubiquitin-proteasome-mediated protein degradation plays an integral role in eukaryotic gene transcription. It has also been observed that nuclear receptors themselves are ubiquitinated and degraded in a hormone-dependent manner and that ubiquitin-proteasome function is essential for most nuclear receptors to function as transactivators. Here, we show that specific ubiquitin-proteasome pathway enzymes target specific NRCoA proteins in vivo and in vitro. First, using a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, we confirmed that NRCoA proteins are targets of the ubiquitin-proteasome pathway. Then using coimmunoprecipitation studies, we also demonstrate that in vivo, NRCoA proteins are ubiquitinated. Finally, we illustrate that in vitro, NRCoA ubiquitination and degradation depend on the ubiquitin-activating enzyme (E1) and on specific ubiquitin-conjugating enzymes (E2) for each of the coactivators.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTOR COACTIVATORS (NRCoAs) are molecules that interact with ligand-bound nuclear receptors and serve to facilitate the efficient transcriptional regulation of target genes (1, 2, 3, 4, 5, 6, 7, 8, 9). Originally, the existence of NRCoA was predicted based upon the fact that different receptors compete for a limiting pool of accessory proteins that are required for maximal gene transcription. Activation of one receptor can trans-repress the activity of another receptor by depleting a common coactivator pool, and overexpression of these limiting coactivators can reverse this trans-repression or squelching phenomenon (10, 11, 12). Coactivators possess enzymatic activities, which are thought to contribute to their ability to promote gene transcription. Histone acetyl transferase (HAT) activity was the first enzymatic activity that was attributed to coactivators (13, 14, 15, 16). Recently, ATPase, methyltransferase, and ubiquitin-conjugation and ubiquitin-ligase activities also have been detected in coactivators (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). It has been proposed that coactivators are able to enhance gene transcription either by acting as a bridge between the activated nuclear receptor and general transcription factors (GTFs) and/or as catalytic enzymes, which may covalently modify histones, GTFs, receptors, coactivators, and other proteins (9, 16, 20, 27).

The steroid receptor coactivator (SRC) family contains three members, SRC-1 (28), TIF-2 (transcriptional intermediary factor 2), GRIP1 (glucocorticoid receptor-interacting protein 1) (29, 30, 31), and RAC-3 (retinoid acid receptor coactivator 3), p/CIP (p300/CBP interacting protein), ACTR (activator of thyroid and retinoid acid receptors), AIB1 (steroid receptor coactivator amplified in breast cancer 1), TRAM-1 (thyroid receptor activator molecule 1) (13, 32, 33, 34, 35). These coactivators have been shown to contain HAT activity that may contribute to their ability to enhance receptor-mediated gene transcription (13, 14, 15, 16). Coactivator-associated arginine methyltransferase 1 is a coactivator that has been shown to contain methyltransferase activity (17, 20). Also, the members of another coactivator family, the switch/sucrose nonfermentation (SWI/SNF) complex, contain ATPase activity (36, 37, 38, 39). It has been proposed that when assembled at the promoter of hormone-responsive genes, the concert of HAT, methyltransferase, ATPase, and bridging activities contributed by coactivators stimulate transcription through nucleosome remodeling and/or covalent modification of other components of the transcriptional complex (6, 9, 16, 20, 36).

Recent identification of the components of the ubiquitin-proteasome pathway as coactivators by our laboratory and others links this pathway to nuclear receptor-mediated gene transcription. These studies demonstrate that the ubiquitin-conjugating enzyme, UBC9, and the E3 ubiquitin-protein ligases, E6-associated protein (E6-AP) and receptor potentiation factor 1/reverse Spt phenotype 5 (RPF1/RSP5), interact with nuclear receptors and modulate their transcriptional activity (19, 23, 24, 25, 40). Similarly, another coactivator protein, yeast suppressor for Gal 1/thyroid receptor interacting protein 1 (SUG1/TRIP1), an ATPase subunit of the 26S-proteasome complex, also modulates nuclear hormone receptor function (18, 21, 22, 41). These ubiquitin-proteasome pathway coactivators lie at different places in the ubiquitin-proteasome protein degradation system, harboring enzymatic activities such as ubiquitin conjugation, ubiquitin-protein ligation, and ATPase activities (18, 19, 21, 22, 23, 24, 25, 40). It is possible that the enzymes of the ubiquitin-proteasome pathway exert a positive effect on transcription by promoting degradation of negative regulators of gene transcription (42). Alternatively, these enzymes may be employed in the obligate turnover of positively acting factors such as receptors, GTFs, and coactivators. Consistent with the latter possibility, it has been shown recently that the estrogen, progesterone, glucocorticoid, retinoid, and thyroid receptors are ubiquitinated and degraded through the ubiquitin-proteasome pathway (40, 43, 44, 45, 46, 47, 48, 49, 50). Concomitant with their ligand-mediated turnover and activation, we also showed that ubiquitin-proteasome activity is required for the transcriptional activities of most members of the nuclear hormone receptor superfamily, but not other nonnuclear receptor transcription factors (51). The glucocorticoid receptor, in contrast to that seen for the other receptors examined so far, is transcriptionally active when the ubiquitin-proteasome system is disrupted, suggesting that the requirement for ubiquitination-mediated turnover is both nuclear receptor and transcription factor specific (51, 52, 53). The ubiquitin-proteasome pathway also has been implicated in the regulation of protein levels of other transcription factors such as signal transducer and activator of transcription 5a (STAT5a) tramtrack, nuclear factor-{kappa}B (NF-{kappa}B), STAT1, and fos/jun (54, 55, 56, 57). In addition, RNA polymerase II itself has also been shown to be ubiquitinated, indicating that protein turnover is an integral part of gene transcription (58). Microscopic analysis has also revealed the presence of proteasome subunits at the loci of hormone-responsive genes (59). These observations all imply that ubiquitin-proteasome-mediated protein degradation is an important component in eukaryotic gene transcription.

The ubiquitin-proteasome pathway is the major system in eukaryotic cells for selective degradation of short-lived regulatory proteins. This pathway modulates the levels of target proteins and/or compositions of multiprotein complexes in cells by targeted protein degradation (60, 61, 62). In the ubiquitin-proteasome pathway, the highly conserved 76-amino acid (aa) ubiquitin protein is covalently attached to target proteins, which are then degraded by the 26S proteasome. The conjugation of ubiquitin to target proteins involves three consecutive steps mediated by activities of the sole E1 ubiquitin-activating enzyme (UBA), multiple E2 ubiquitin-conjugating enzymes (UBCs), and multiple E3 ubiquitin-protein ligases (UBLs), respectively. Initially, the UBA enzyme activates ubiquitin in an ATP-dependent reaction by forming a thioester bond between the active-site cysteine residue of the UBA enzyme and the carboxyl-terminal glycine residue of ubiquitin. Ubiquitin is then transferred from the UBA enzyme to any one of a number of UBC enzymes, maintaining the high-energy thioester linkage. Finally, UBC enzymes transfer ubiquitin covalently to target proteins either directly or in conjunction with a UBL enzyme that defines target specificity (62, 63, 64). The first ubiquitin molecule binds to the {epsilon}-amino group of lysine residues of the target protein via the carboxy terminus of ubiquitin. In succeeding reactions, a polyubiquitin chain is synthesized by transferring activated ubiquitin molecules to lysine (48) of the ubiquitin moiety previously linked to the target protein. Subsequently, the polyubiquitinated target protein is degraded by the 26S proteasome, a large multisubunit protease that resides both in the nucleus and cytoplasm (60, 61, 62).

In this study, we show that the ubiquitin-proteasome pathway regulates the endogenous levels of NRCoA proteins. Furthermore, by using a temperature-sensitive mutant mammalian cell line (ts85) that is defective in UBA enzyme activity, which abolishes protein ubiquitination (65), we also demonstrate that at the restrictive temperature, NRCoA proteins are at higher steady-state levels compared with that observed at the permissive temperature; these results support the conclusion that NRCoAs are degraded by the ubiquitin-proteasome pathway. Moreover, coimmunoprecipitation studies presented here confirm that in vivo, NRCoA proteins are modified by ubiquitin. Finally, we demonstrate that in vitro, NRCoA ubiquitination and degradation depend on UBA and specific UBCs for each of the NRCoA proteins.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Endogenous Protein Levels of Various NRCoA Proteins Are Regulated via the Ubiquitin-Proteasome Pathway
The ubiquitin-proteasome pathway targets a wide variety of short-lived regulatory proteins for degradation (60, 61, 62). Identification of ubiquitin-proteasome pathway enzymes as coactivators of nuclear receptors and observations that a variety of nuclear receptors are degraded through the ubiquitin-proteasome pathway in a hormone-dependent manner have suggested a link between this pathway and receptor-mediated gene transcription (19, 22, 23, 24, 25, 40, 43, 45, 46, 47, 48, 49, 50, 51). Furthermore, it has been suggested recently that transiently overexpressed NRCoA proteins are also degraded through the ubiquitin-proteasome pathway (51, 66).

To determine whether the endogenous protein levels of various NRCoA proteins are targets of the ubiquitin-proteasome pathway, HeLa cells were grown overnight (3 x 105 per well) in DMEM containing 10% serum. Cells were then treated with the proteasome inhibitor MG132, or its vehicle, dimethylsulfoxide (DMSO). Twenty-four hours later, cells were harvested and the endogenous levels of various coactivator proteins were examined by Western blot analyses using coactivator-specific antibodies. In vitro transcribed and translated coactivator proteins were included as positive controls [lanes indicated as SRC-1, TIF-2, RAC-3, CBP, and E6-AP]. As shown in Fig. 1Go, A–E, the steady-state levels of various endogenous coactivator proteins (SRC-1, TIF-2, RAC-3, CBP, and E6-AP) in vehicle (DMSO)-treated HeLa cells are lower than in the presence of MG132. MG132 treatment resulted in an increase in the amount of SRC-1 (5.4-fold), TIF-2 (3.9-fold), RAC-3 (7.6-fold), CBP (4.2-fold), and E6-AP (1.9-fold) protein levels (Fig. 1Go, A–E, lanes marked as MG132). However, MG132 had no significant effect on the expression levels of the ß-actin protein (Fig. 1FGo) or an exogenously expressed luciferase protein (data not shown; Ref. 51). The fact that the steady-state levels of these NRCoAs are higher in the presence of MG132 indicates that they are targets of ubiquitin-proteasome-mediated turnover.



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  		Figure 1. The Proteasome Inhibitor, MG132, Stabilizes the Expression of Endogenous Coactivator Proteins

HeLa cells (3 x 105 per well) were grown in DMEM containing 10% stripped serum overnight. Cells were then treated with either vehicle (DMSO) or proteasome inhibitor (1 µM MG132). Cells were harvested 24 h after treatment, and 100 µg of total proteins were analyzed by Western blot either by using SRC-1-, TIF-2-, RAC-3-, CBP-, or E6-AP-specific antibodies. As positive controls, in vitro transcribed and translated coactivator proteins were also analyzed (lanes indicated as SRC-1, TIF-2, RAC-3, CBP, and E6-AP). Arrows indicate the position of the specific coactivator proteins. A, Endogenous expression of SRC-1 coactivator protein in DMSO and proteasome inhibitor, MG132 treated cells. B, The expression levels of endogenousTIF-2 coactivator protein in DMSO- and MG132-treated cells. C, Expression analysis of RAC-3 coactivator in DMSO- and MG132-treated cells. D, The protein levels of CBP in control (DMSO)- and proteasome inhibitor (MG132)-treated cells. E, Endogenous expression of E6-AP protein in DMSO- and MG132-treated cells. F, The proteasome inhibitor, MG132, has no significant effect on the stability of the ß-actin protein. As a negative control, HeLa cells were treated with either vehicle (DMSO) or proteasome inhibitor (MG132). Cells were harvested 24 h after treatments, and 25 µg of total proteins were analyzed by Western blot using ß-actin-specific antibody. All Western blots were repeated at least three times.

 
Disruption of the UBA Stabilizes the Expression of Various NRCoAs
To date, only a single UBA enzyme has been identified for the ubiquitin-proteasome pathway, carrying out the activation of ubiquitin required for all subsequent ubiquitin shuttling and placement on target proteins. Therefore, the mutant mammalian cell line, ts85 (derived from the FM3A mouse mammary carcinoma cell line) that harbors a temperature-sensitive mutation in UBA, which disrupts all protein ubiquitination and degradation, was used to address the role of ubiquitination of NRCoAs in influencing their turnover (63). To ascertain whether NRCoAs are targets of the ubiquitin-proteasome pathway, ts85 cells were transiently transfected with coactivator expression plasmids for SRC-1, TIF-2, RAC-3, CBP, and E6-AP. After transfection, cells were incubated either at a permissive (30 C) or restrictive temperature (37 C) for 24 h. Cells were subsequently harvested and lysates were analyzed by Western blot analysis using coactivator-specific antibodies. Western analyses of the above NRCoAs in the ts85 mutant cell line demonstrated that at the restrictive temperature, the protein levels of all the coactivators are higher than at the permissive temperature (Fig. 2Go, A–E, compare 30 C vs. 37 C). As shown previously (51) and in this study, incubation of ts85 cells at either restrictive or permissive temperatures had no influence on luciferase protein levels produced from a transiently transfected luciferase expression vector (Fig. 2GGo). Conversely, estrogen receptor-{alpha} was not turned over in an estradiol-dependent manner at the restrictive temperature, indicating that the ubiquitin-proteasome system was functionally disrupted at this temperature (51). Furthermore, the steady state level of endogenously expressed ß-actin protein is also identical at both permissive (30 C) and restrictive (37 C) temperatures (Fig. 2FGo, compare 30 C vs. 37 C), indicating that disruption of UBA does not globally alter the expression of either endogenous or exogenous proteins. This demonstrates that not all proteins are targets of the ubiquitin-proteasome pathway. Because the UBA enzyme activity and all downstream ubiquitin-proteasome pathway function is impaired at the restrictive temperature, an increase in the steady-state levels of coactivator proteins resulting from disruption of the ubiquitin-proteasome system provides another piece of evidence indicating that NRCoAs are targets of turnover.



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  		Figure 2. Disruption of the UBA Stabilizes the Epression of Various Nuclear Coactivators

A, The temperature-sensitive UBA mutant ts85 cell line was transiently transfected with 1 µg of SRC-1 expression expression plasmid, pIRES.SRC-1. After transfection half of the cells were shifted from 30 C to a restrictive temperature (37 C) at which UBA enzyme is inactive. Cells were harvested 24 h later, and SRC-1 expression was analyzed by Western blot analysis using flag antibody. Panels B–E are the same as panel A except that the respective expression vector and antibodies were used for each coactivator. To quantify the expression levels of coactivators both in the presence of vehicle DMSO and proteasome inhibitor MG132, the Western blots were scanned by NIH image 1.62 program; data are plotted as fold increased stability, and the levels of coactivators in the presence of vehicle (DMSO) are set as 1-fold. F, Disruption of the ubiquitin-activating enzyme E1 has no significant effect on the stability of the ß-actin protein. As a negative control, ts85 cells were incubated either at 30 C or at 37 C for 24 h and expression levels of ß-actin were analyzed by Western blot using ß-actin-specific antibody. To quantify the expression levels of coactivators at 30 C or at 37 C, Western blots were scanned by the NIH image 1.62 program; data are plotted as fold increased stability, and the levels of coactivators at 30 C are set as 1-fold. Arrows indicate the position of coactivator proteins. Western blots for each coactivator were repeated at least three times.

 
Various NRCoA Proteins Are Modified by Ubiquitination in Vivo
The elevated level of coactivator proteins in MG132-treated HeLa cells and ts85 mutant cells at the restrictive temperature imply that coactivators are targets of the ubiquitin-proteasome pathway. To further explore whether ubiquitin-conjugated coactivator proteins exist in cells, we performed coimmunoprecipitation studies using anticoactivator and antiubiquitin antibodies. HeLa cells were transiently transfected with expression vector for SRC-1, RAC-3, CBP, and E6-AP. After transfection, cell lysates were immunoprecipitated with either a nonspecific antibody ({alpha}-T7) or specific antibodies such as anti-SRC-1 ({alpha}-SRC-1), anti-RAC-3 ({alpha}-RAC-3), anti-CBP ({alpha}-CBP), and anti-E6-AP ({alpha}-E6-AP), followed by Western blotting with the respective anti-coactivator-specific antibody (Fig. 3Go, A–D, left panels) or an antiubiquitin antibody (Fig. 3Go, A–D, right panels). The results presented in Fig. 3Go, A–D (left panels), demonstrate that coactivator precipitates were detected only when cell lysates were incubated with coactivator-specific antibodies ({alpha}-SRC-1, {alpha}-RAC-3, {alpha}-CBP, and {alpha}-E6-AP). However, the nonspecific antibody did not precipitate any coactivator protein ({alpha}-T7). As controls, coactivator proteins were also precipitated from in vitro transcribed and translated coactivator proteins (Fig. 3Go, lanes marked as C). As shown in Fig. 3Go, A–D, right panels, ubiquitination of coactivator proteins was observed only in lysates that were immunoprecipitated with coactivator-specific antibodies, suggesting that coactivators are modified by ubiquitin in vivo. Because ubiquitin usually conjugates to multiple lysine residues of target proteins and also forms polyubiquitin chains, ubiquitin-conjugated proteins are often visualized as a ladder of higher molecular weight species on Western blots. The multiple bands seen in Fig. 3Go, A–D (right panels), should represent the polyubiquitinated forms of coactivator proteins. In contrast to coactivators, we were unable to detect any ubiquitination of the luciferase protein (Fig. 3EGo, right panel), indicating that ubiquitin antibody specifically recognized ubiquitin-conjugated coactivator proteins. These data imply that NRCoAs are modified by ubiquitin and are substrates for the ubiquitin-proteasome pathway in vivo.



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  		Figure 3. NRCoAs Are Modified by Ubiquitination in Vivo

A, The SRC-1 expression plasmid, pIRESneo.SRC-1, was transiently transfected into HeLa cells. Cell lysates were immunoprecipitated (IP) 24 h after transfection with either nonspecific antibody ({alpha}-T7) or SRC-1-specific antibody ({alpha}-SRC-1), and precipitated proteins were immunobloted with either anti-SRC-1 antibody (left panel, WB: {alpha}-SRC-1) or antiubiquitin antibody (right panel, WB: {alpha}-Ub). As a positive control, in vitro transcribed and translated SRC-1 protein was also immunoprecipitated (lane marked as C). Panels B–D are the same as for panel A except that expression vectors and antibodies for the respective proteins were used as indicated. E, Luciferase (Luc) protein is not ubiquitinated in vitro. The experiment was performed as indicated above. Each experiment was repeated at least three times.

 
To verify that NRCoA proteins are indeed modified by ubiquitin and are targets of the ubiquitin-proteasome pathway, we also transiently transfected HeLa cells with coactivator expression plasmids for TIF-2, RAC-3, and CBP together with hemagglutinin (HA)-tagged ubiquitin (HA-UB) or an empty plasmid (control). After transfection, cell lysates were immunoprecipitated with either a nonspecific antibody ({alpha}-T7) or HA-tagged specific antibody ({alpha}-HA), followed by Western blotting with either anti-HA-tagged specific antibody (Fig. 4Go, A–C, left panels) or anti-TIF-2 ({alpha}-TIF-2), anti-RAC-3 ({alpha}-RAC-3), and anti-CBP ({alpha}-CBP) (Fig. 4Go, A–C, right panels). The results in Fig. 4Go, A–C (left panels), demonstrate that immunoprecipitated proteins were detected only when cell lysates were incubated with HA-tagged specific antibody ({alpha}-HA). Furthermore, HA-tagged antibody ({alpha}-HA) was able to immunoprecipitate proteins only from cells that were transfected with HA-tagged ubiquitin expression plasmid (HA-UB). In contrast, no immunoprecipitates were observed in cells that were transfected with empty plasmid (control). Similarly, the nonspecific antibody was unable to immunoprecipitate any of the coactivators ({alpha}-T7). To demonstrate that HA-tagged ubiquitin conjugated to coactivator proteins, precipitated proteins were also examined by Western blotting using either anti-TIF-2 ({alpha}-TIF-2), anti-RAC-3 ({alpha}-RAC-3), or anti-CBP ({alpha}-CBP). As shown in Fig. 4Go, A–C, right panels, coactivator proteins were detected as multiple bands, confirming that coactivators are modified by ubiquitin in vivo. Because the luciferase protein is not a target of the proteasome, we examined the luciferase protein for ubiquitination as a negative control. As shown in Fig. 4DGo, right panel, we were unable to detect any ubiquitination of the luciferase protein. These results demonstrate that NRCoAs are targets of the ubiquitin-proteasome pathway.



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  		Figure 4. Ubiquitination of Nuclear Receptor Coactivators

A, To confirm whether TIF-2 is a target of the ubiquitin pathway, the TIF-2 expression plasmid, pCR3.1.TIF-2 was transiently transfected into HeLa cells either along with an empty vector (control) or HA-tagged ubiquitin expression plasmid (HA-UB). cell lysates were immunoprecipitated (IP) 24 h after transfection with either nonspecific antibody ({alpha}-T7) or HA tag-specific antibody ({alpha}-HA) and precipitated proteins were immunobloted with either anti-HA antibody (left panel, WB: {alpha}-HA) or anti-TIF-2 antibody (right panel, WB: {alpha}-TIF-2). B–D, Performed as above except using the appropriate expression vectors and indicated antibodies. Brackets indicate the ubiquitinated forms of NRCoA. Experiments were repeated at least three times.

 
Coactivator Ubiquitination and Degradation Depend on UBA and UBCs in Vitro
To further confirm that NRCoAs are targets of the ubiquitin-proteasome pathway and to determine the specificity of individual UBCs for specific coactivator target proteins, we performed in vitro protein degradation and ubiquitination assays. 35S-Labeled coactivator proteins were synthesized in vitro using TNT-coupled rabbit reticulocyte extracts in the presence of radiolabeled methionine. Reticulocyte lysates contain components of the endogenous ubiquitin-proteasome machinery that are required for the degradation of the NRCoA studied here, which could potentially result in the degradation of proteins in the absence of introduced exogenous UBA/UBCs. The radiolabeled coactivator proteins were incubated with ATP and ubiquitin either in the absence of UBA/UBCs/UBL or in the presence of bacterially expressed UBA, specific UBCs, and UBL and incubated at 30 C. To study the in vitro ubiquitination and degradation of SRC-1 and to identify the UBC that promotes SRC-1 ubiquitination, radiolabeled SRC-1 protein was incubated either without UBA/UBC/UBL or with UBA and various UBCs in the presence of ATP and ubiquitin. As shown in Fig. 5AGo, UBA and UBC2 (E214K) enzymes promote the degradation of SRC-1 compared with a control that lacks UBA and UBC2 (lane 1). In addition, Fig. 5AGo also suggests that the UBC3 (UbcH3) enzyme is less effective in promoting SRC-1 degradation. Furthermore, other UBC enzymes such as UBC4 (rat UBC4-1), UBC5 (UbcH5), UBC6 (UbcH6), UBC7 (UbcH7), and UBC8 (UbcH8) were unable to promote SRC-1 degradation in our in vitro assay (Table 1Go), suggesting that the ubiquitin-proteasome pathway promotes degradation of SRC-1 primarily via the UBC2 enzyme.



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  		Figure 5. In Vitro Coactivator Ubiquitination and Degradation Depends on UBA and Specific Sets of UBCs

A, To study the in vitro ubiquitination and degradation of SRC-1, 35S-labeled SRC-1 protein was synthesized in vitro with TNT-coupled rabbit reticulocyte extracts. The labeled SRC-1 protein was incubated with ATP and ubiquitin either in the absence of UBA/UBCs/UBL or presence of bacterially expressed UBA/UBC2 (E214K) and UBA/UBC3 (UbcH3) (for 4 h). Reactions were terminated by adding SDS-loading buffer and analyzed by SDS-PAGE and autoradiography. The arrows indicate the position of intact protein. B–F, Experiments were performed as above, using the indicated in vitro transcribed/translated proteins and UBA/UBC proteins. Protein levels were quantitated using NIH image 1.62 and normalized so that -UBA/UBC/UBL lanes have a value of 1. Experiments were repeated at least three times.

 

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  		Table 1. Specific Sets of Ubiquitin-Conjugating Enzymes E2 Promote the Degradation of Specific NRCoA

 
Next, we asked which UBC enzymes promote the degradation of TIF-2 coactivator. Like SRC-1, radiolabeled TIF-2 protein was incubated either without UBA/UBC/UBL or with UBA and several UBC enzymes. Our data demonstrate that UBC5, UBC7, and UBC8 enzymes were able to promote TIF-2 degradation (Fig. 5BGo and Table 1Go), whereas UBC2, UBC3, UBC4, and UBC6 had no significant effect on in vitro TIF-2 degradation (Table 1Go).

To identify the UBC enzymes that are responsible for RAC-3 ubiquitination and degradation, 35S-labeled RAC-3 protein was incubated with ATP and ubiquitin either in the absence of UBA/UBC/UBL or in the presence of UBA and different UBC enzymes. As shown in Fig. 5CGo, UBC3 was able to promote degradation of RAC-3, whereas UBC8 promoted degradation to a lesser extent. Like UBC2, UBC3, UBC4, UBC5, and, to a lesser extent, UBC8 enzymes were also effective in promoting the degradation of the RAC-3. However, UBC6 and UBC7 were unable to promote RAC-3 degradation (Table 1Go). These data suggest that RAC-3 degradation by the ubiquitin-proteasome pathway proceeds predominantly through UBC2, UBC3, UBC4, or UBC5.

CBP, which is not a part of the SRC family, was initially characterized as a coactivator of the CREB (67). CBP also acts as a coactivator for a wide variety of transcription factors such as p53 (68), NF-{kappa}B (69), and nuclear hormone receptors (70, 71, 72). CBP has been referred to as a cointegrator, influencing the cross-talk between different groups of transcription factors, e.g. between the glucocorticoid receptor and NF-{kappa}B (73). In addition to its interactions with nuclear receptors, CBP interacts with SRC-1 and RAC-3, forming ternary complexes with SRC family members and nuclear hormone receptors (35, 72, 74). Because CBP is also a target of the ubiquitin-proteasome pathway (51), we examined which UBC enzymes promote the degradation of CBP as well. The 35S-labeled CBP protein was synthesized in vitro using rabbit reticulocyte extracts and then incubated with ATP and ubiquitin either in the absence or presence of bacterially expressed UBA and different UBC enzymes. As shown in Fig. 5DGo, the ubiquitin pathway enzyme UBC3 promotes the degradation of CBP in vitro, whereas UBC2 has no significant effect. In addition, the ubiquitin pathway enzymes UBC2, UBC4, UBC5, UBC6, UBC7, and UBC8 have no effect on CBP protein levels (Table 1Go). These data suggest that the ubiquitin-proteasome pathway promotes the degradation of CBP via UBC3.

E6-AP is a ubiquitin-protein ligase E3 enzyme that plays a role in defining the substrate specificity of the ubiquitin-proteasome pathway (60, 61, 62). In addition to its originally characterized role, we have characterized E6-AP as a coactivator of nuclear receptors (23). In this study we ask which UBCs are important for E6-AP ubiquitination and degradation. E6-AP autoubiquitinates itself, promoting its own degradation (75). To examine ubiquitination and degradation of E6-AP in vitro, reticulocyte-transcribed/translated radiolabeled E6-AP protein was incubated in the presence of ATP and ubiquitin without or with UBA and various UBCs (shown in Fig. 5EGo)/UBL (E6-AP). As shown in Fig. 5EGo, UBC5A (UbcH5A), UBC7, and UBC8 were able to promote the ubiquitination and degradation of E6-AP whereas UBC2, UBC3, UBC4, UBC5B (UbcH5B), and UBC6 had no significant effect on the protein levels of E6-AP. These data confirm that the E6-AP protein is a substrate of UBC5A, UBC7, and UBC8 enzymes as shown previously (75).

To confirm that ubiquitin pathway enzymes specifically promote the degradation of NRCoA proteins, we also incubated UBA and UBCs enzymes with the luciferase protein in our in vitro protein degradation and ubiquitination assays. 35S-labeled luciferase protein was incubated with ATP and ubiquitin either in the absence or presence of UBA and different UBC enzymes. Results shown in Fig. 5EGo suggest that all UBCs assayed were unable to promote luciferase degradation, demonstrating that UBCs can specifically promote the degradation of NRCoA proteins and that these enzymes have no significant effect on a protein that is not a target of the ubiquitin-proteasome pathway. In vitro degradation of SRC-1, TIF-2, RAC-3, and CBP could be inhibited by MG132, indicating that their turnover was mediated through the proteasome (Fig. 6Go). To determine whether coactivator degradation in our in vitro system could be due to UBCs that are already present in the reticulocyte lysate, we performed an immunodepletion experiment. Antibodies specific to the preferred UBC for each coactivator tested above were added to the reticulocyte lysate before the transcription/translation of each coactivator and subsequently incubated with the respective UBC and other reaction components as described above. Coactivator degradation was abolished in all of the immunodepleted reactions, indicating that coactivator turnover is specific to the exogenously supplied UBC (data not shown). Interestingly, we were unable to observe higher molecular weight products in the presence of MG132 as might be expected when proteins can be ubiquitinated but the proteasome is unable to promote their turnover. However, our results could be due to these coactivators being efficient targets of deubiquitinating enzymes. Also, it has recently been shown that the ubiquitination machinery (E3 ligases) is directly associated with the proteasome (76). Because of this, it may be impossible to observe the ubiquitinated coactivator because its entry into the proteasome and degradation would be simultaneous with its ubiquitination.



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  		Figure 6. The Proteasome Inhibitor, MG132, Blocks in Vitro Coactivator Degradation

To determine whether in vitro degradation of coactivators is blocked by proteasome inhibitor, MG132, 35S-labeled coactivator proteins were synthesized in vitro in the presence of either vehicle only or 33 µM MG132 with TNT-coupled rabbit reticulocyte extracts. The labeled proteins were incubated with ATP and ubiquitin either in the absence of UBA/UBCs/UBL or presence of bacterially expressed UBA/UBCs (for 2 h). Reactions were terminated by adding SDS-loading buffer and analyzed by SDS-PAGE and autoradiography. The arrows indicate the position of intact protein. A–D, Experiments were repeated at least three times.

 
Identification of Potential PEST Sites within Coactivator Proteins
Many unstable proteins contain stretches of amino acids enriched in proline, glutamic acid, serine and threonine, termed PEST domains, which have been implicated in promoting protein turnover (61, 77, 78). Because NRCoA proteins are targets of the ubiquitin-proteasome pathway, we analyzed NRCoA proteins for potential PEST sites using the PEST-FIND program (http://www.at.embnet.org/embnet/tools/bio/PESTfind/). This analysis revealed that SRC-1, TIF-2, RAC-3, CBP, and E6-AP proteins contain PEST sequences. The location of potential PEST sites and PEST scores for SRC-1, TIF-2, RAC-3, CBP, and E6-AP proteins are shown in Table 2Go. SRC-1 contains two potential PEST sites located between amino acids (aa) 1–17 and aa 202–217. In our in vitro degradation assay, the SRC-1B isoform of SRC-1, which lacks the two PEST motifs, was only minimally degraded, suggesting that its turnover is mediated through a PEST-dependent mechanism. The TIF-2 coactivator protein contains four potential PEST sites located between aa 610–625, aa 648–666, aa 713–731, and aa 788–826. RAC-3 analysis revealed that it contains only one potential PEST site encompassing aa 647–674. The CBP protein has two PEST sites located between aa 592–603 and aa 640–667. Finally, E6-AP has only one potential PEST site. In contrast to NRCoA proteins, the luciferase protein, which is not degraded via the ubiquitin-proteasome pathway, has no PEST sequences.


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  		Table 2. The Potential PEST Sites within Coactivator Proteins

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In eukaryotic cells, the ubiquitin-proteasome pathway mediates selective degradation of many key regulatory proteins (60, 61, 62). These regulatory proteins include transcription factors such as NF-{kappa}B (57), STAT1 (54), SMADs (Sma- and Mad-related protein) (79, 81), MyoD (myogenic determination) (81), and nuclear hormone receptors (40); cell cycle-regulatory proteins such as cyclins, inhibitors of cyclin-dependent kinases, and anaphase inhibitors (60, 61, 62); tumor suppressors such as p53 (60, 61, 62); nuclear oncoproteins (56); mutated and abnormal proteins in the endoplasmic reticulum, cell surface receptors (60), and RNA polymerase II (58). Degradation of these regulatory proteins has linked the ubiquitin-proteasome pathway to various diverse processes such as gene transcription, cell cycle regulation, DNA repair, cell differentiation, signal transduction, antigen processing, development, and apoptosis (60, 61, 62).

The identification of a number of ubiquitin-proteasome pathway enzymes such as E6-AP, receptor potentiation factor 1/reverse Spt phenotype 5 (RPF1/RSP5), and UBC9 as coactivators has suggested a link between this pathway and nuclear receptor-mediated gene transcription (19, 23, 24, 25). Similarly, another coactivator protein, yeast suppressor for Gal 1/thyroid receptor interacting protein 1 (SUG1/TRIP1), an ATPase subunit of the 26S-proteasome complex, also modulates nuclear receptor function (18, 21, 22, 41). It is possible that this class of coactivators exerts an effect on gene transcription by promoting protein degradation. In contrast, the SRC coactivator family is thought to modulate transcription by promoting the initiation of transcription through the acetylation of histones in the proximity of the hormone-responsive promoter. Paradoxically, numerous nuclear receptors are degraded coincident with their activation by ligand (40, 43, 45, 46, 47, 48, 49, 50, 51). Inhibition of the ubiquitin-proteasome pathway abrogates the transcriptional activities of the estrogen and progesterone receptors, suggesting that turnover of the receptor contributes to transcription (51). However, this is contrasted with that observed for the glucocorticoid receptor which, like the estrogen and progesterone receptors, is turned over in the presence of its agonist ligand and is not transcriptionally impaired in the presence of proteasome inhibitors (51, 52, 53). Conversely, androgen and vitamin D receptors are stabilized in the presence of their agonist ligands, indicating that the stability of each nuclear receptor and its requirement for proteasome function vary. Furthermore, the degradation of coactivators and their colocalization with components of the proteasome at sites of hormone-responsive promoter elements (59, 66) provides further evidence that ubiquitin-proteasome-mediated protein degradation plays a central role in nuclear receptor-mediated gene transcription. Interestingly, estrogen receptor-{alpha} and thyroid hormone receptors have both been shown to be ubiquitinated even in the absence of hormone (82, 83), suggesting that hormone binding must occur in addition to receptor ubiquitination for accelerated turnover to occur.

It has been recently reported that proteasome inhibition has deleterious effects on the enzymatic activity of the luciferase reporter protein used to assess the transcriptional activity of a variety of nuclear receptors in the presence of MG132 (84). However, we have not observed a decrease in luciferase enzymatic activity (expressed from a cytomegalovirus-based expression vector) in HeLa cells treated with MG132 or in ts85 cells incubated at the restrictive temperature regimen (Ref. 51 ; data not shown). Furthermore, we observe a similar reduction in the transcriptional activity of the estrogen and progesterone receptor in the presence of MG132 when using chloramphenicol acetyltransferase-based estrogen- or progesterone-responsive reporters, respectively (data not shown). It is possible that the different susceptibility of luciferase to MG132 could be cell type or promoter specific.

In this manuscript, we show that the proteasome inhibitor MG132 stabilizes the steady-state levels of SRC-1, TIF-2, RAC-3, CBP, and E6-AP coactivator proteins, suggesting that NRCoAs are targets of the ubiquitin-proteasome pathway. These observations were substantiated by utilizing a temperature-sensitive mutant cell line that contains a thermolabile UBA enzyme (65). We demonstrate that at a restrictive temperature where UBA enzyme activity is disrupted, which subsequently abolishes ubiquitin-targeted protein degradation, the protein levels of NRCoA are higher compared with the temperature at which the pathway is active (permissive temperature), substantiating the notion that NRCoA proteins are targeted and degraded by the ubiquitin-proteasome pathway. These data were confirmed by coimmunoprecipitation experiments, which demonstrated that NRCoA proteins are ubiquitinated in cells. It has been shown that, after the attachment of the first ubiquitin molecule to the substrate protein, a polyubiquitin chain is usually formed, in which the carboxy terminus of each ubiquitin is attached to a specific lysine residue of the previous ubiquitin. The 26S-proteasome complex, in an ATP-dependent manner usually, degrades only polyubiquitinated target proteins (60, 61, 62). Data presented here also indicate that NRCoA proteins are polyubiquitinated in vivo, consistent with them being targets for degradation by the 26S-proteasome pathway.

Because NRCoA proteins are important modulators of gene transcription and these proteins amplify the magnitude of the biological responses to hormones in target tissues, it is probably significant that an appropriate amount of NRCoA proteins are present within the cell. This possibility is consistent with previously published data mentioned in the introduction, which suggests that the protein levels of NRCoA are regulated by the ubiquitin-proteasome pathway (51, 59). Furthermore, it has been shown that coactivator proteins exist in cells as large preformed distinct subcomplexes (5, 74). It is possible that the composition and/or concentration of these multiprotein complexes in cells are regulated by targeted protein degradation via the ubiquitin-proteasome pathway. Additionally, the degradation of NRCoA via the ubiquitin-proteasome pathway could be a way to disassemble and reassemble coactivator complexes. For instance, it has been reported that distinct NRCoA complexes interact with nuclear receptors in a competitive and exclusive manner. It has been postulated that initially, an SRC-1-containing NRCoA complex associates with nuclear receptors, which is then followed by a TRAP220-containing complex (85). It is conceivable that ubiquitin-proteasome-mediated degradation of coactivators may facilitate such an exchange of NRCoA complexes, thereby promoting increased transcription.

Previously, it also has been suggested that ubiquitination of proteins often requires a PEST sequence, a stretch of amino acids enriched in proline, glutamic acid, serine, and threonine (77, 78). Sequence analysis of NRCoA proteins by the PEST-FIND program revealed that NRCoA proteins contain potential PEST sequences (Table 2Go). It is possible that these PEST sequences might act as degradation signals in NRCoA as well. Additionally, the destruction box motif, which is necessary for the ubiquitination and degradation of mitotic cyclins and certain other cell-cycle regulators, could also play a role (86). Proteins that are not known to be involved in cell-cycle regulation, such as uracil permease, can also be degraded in a destruction box-dependent manner, suggesting that such a motif may also by involved in the degradation of NRCoA as well (61, 87). Sequence analysis of NRCoA (SRC-1, TIF-2, RAC-3, CBP, and E6-AP) reveals that these NRCoAs contain destruction box/destruction box-related sequences, pointing to a diversity in the pathways that may impinge upon NRCoA protein turnover.

The UBC enzyme family represents a large number of closely related proteins. In Saccharomyces cerevisiae alone, 13 genes have been identified encoding different UBC enzymes (60, 61, 62). Gene inactivation studies in yeast have demonstrated that different UBC enzymes play a role in distinct cellular processes. For instance, the UBC2 enzyme is required for DNA repair and degradation of N-end rule substrates, the UBC3 enzyme is required for G1 to S phase transition in the cell cycle, UBC4 and UBC5 enzymes are required for the degradation of abnormal and short lived proteins, the UBC6 enzyme is involved in the degradation of yeast transcriptional repressor MAT{alpha}2, and UBC7 is responsible for resistance to cadmium toxicity and degradation of MAT{alpha}2 protein (61, 88). Our data demonstrate that specific UBC enzymes promote the degradation of specific target proteins in mammalian cells, impinging upon distinct cofactors that modulate nuclear receptor-mediated gene transcription (Table 1Go).

In addition to the other enzymatic roles of NRCoAs that are thought to contribute to transcription, ubiquitin conjugation is also likely to contribute. Protein degradation could possibly contribute to a positive transcriptional response by promoting the disruption of the preinitiation complex, allowing for transcriptional elongation to proceed. RNA polymerase II is also a target of the ubiquitin-proteasome pathway, and it is tempting to think that degradation of the elongated form of RNA polymerase II may allow for subsequent reinitiation of multiple rounds of transcription. Finally, it is plausible that targeted degradation of nuclear receptors and NRCoA proteins may enable efficient remodeling of components of receptor-NRCoA complexes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid Constructions
The mammalian expression plasmids pCR3.1.E6-AP, pCR3.1.SRC-1, and pCR3.1.Luc have been described previously (23, 51). To construct the mammalian expression plasmid pIRESneo.SRC1, a pSp64-based plasmid containing SRC-1 was digested with SalI-BglII, and then the SalI-BglII fragment containing the SRC1 cDNA was cloned into the corresponding sites of the plasmid pIRESneo (CLONTECH Laboratories, Inc., Palo Alto, CA). To incorporate a Flag tag at the amino terminus of the SRC1 protein, the following oligos containing Flag tag sequences with NotI and SalI ends were inserted into the corresponding sites of the pIRES.SRC1 vector: 5'-GGCCGCCATGGACTACAAGGACGACGATGACAAGG-3' (sense strand) and 5'-TCGACCTTGTCATCGTCGTCCTTGTAGTCCATGGC-3' (antisense strand). The pCR3.1.TIF-2 vector was generated by inserting the BglII fragment of pSG5.TIF-2, containing the TIF-2 cDNA, into the BamHI site of pCR3.1 (CLONTECH Laboratories, Inc.). To construct pCR3.1.RAC-3, the NheI fragment of pCMX.RAC-3 was subcloned into the XbaI site of pCR3.1. The pCR3.1.CBP plasmid was created by ligating the HindIII-NotI fragment of pRc/RSV.mCBP into the corresponding sites of pCR3.1.

Cell Growth and Transfections
HeLa and ts85 (containing a temperature-sensitive mutation in UBA) cell lines were maintained in DMEM supplemented with 10% fetal bovine serum. Twenty-four hours before transfection, HeLa (3 x 105 per well) or ts85 (9 x 105 per well) cells were plated in Falcon six-well dishes in DMEM containing 10% dextran-coated charcoal-stripped serum. Cells were transfected with the indicated constructs using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s recommendations. Cells were washed and fed with DMEM containing 10% stripped serum. Where indicated, HeLa cells were treated with either DMSO or 1 µM proteasome inhibitor, MG132 (Sigma, St. Louis, MO), and ts85 cells were incubated either at a permissive temperature (30 C) or at a restrictive temperature (37 C) (63). Cells were harvested 24 h thereafter. Cell extracts were analyzed by Western blot analysis.

Western Blot Analysis
To analyze the expression levels of various coactivators, transiently transfected HeLa and ts85 cells were lysed in a buffer containing 400 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% Nonidet P-40 (NP40), 10 mg/ml phenylmethylsulfonyl fluoride (PMSF) (10 µl/ml), aprotinin (30 µl/ml), and 100 nM sodium orthovanadate (10 µl/ml) by vortexing, followed by incubation on ice for 15 min. Subsequently, lysates were centrifuged for 15 min at 4 C (21,000 x g), and equal amounts of protein (50–100 µg) were loaded and resolved by 7.5% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were then incubated in a blocking buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.05% Tween 20, and 5% dried nonfat milk) overnight at 4 C. The membrane was then incubated with appropriate antibodies, recognizing specific coactivators in incubation buffer (20 mM Tris-HCl, pH 7.5; 137 mM NaCl; 0.05% Tween 20; and 2% dried nonfat milk) for 2 h. After extensive washing the membrane was incubated with secondary antibody for 1 h. Detection of specifically bound proteins was carried out using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). To quantify the expression levels of coactivators both in the presence of vehicle (DMSO) and proteasome inhibitor MG132, the Western blots were scanned by NIH image 1.62 program; data are plotted as fold increased stability and the levels of coactivators in the presence of vehicle (DMSO) are set as 1-fold.

Coimmunoprecipitation
Twenty-four hours after transfections, cells were washed in TEN buffer (40 mM Tris-HCl, pH 7.5; 1 mM EDTA; 150 mM NaCl) and lysed in ice-cold RIPA buffer containing salt [400 mM NaCl, 1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mg/ml PMSF (10 µl/ml), aprotinin (30 µl/ml), and 100 nM sodium orthovanadate (10 µl/ml)] by pipeting up and down. Thereafter, cell lysates were placed on ice for 30 min. To bring the salt concentration of cell lysates to 150 mM NaCl, 150 µl of NaCl-free RIPA buffer [1x PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF (10 µl/ml), aprotinin (30 µl/ml), and 100 nM sodium orthovanadate (10 µl/ml)] were added to the lysates. After centrifugation at 4 C (21,000 x g), lysates were incubated with 20 µl of protein A Sepharose and rocked at 4 C for 30 min. After centrifugation, supernatants were transferred to fresh tubes and lysates were mixed either with nonspecific (anti-T7) or specific antibody (anti-SRC-1, anti-RAC-3, anti-CBP, anti-E6-AP, anti-luciferase, and anti-HA) at 4 C for 2 h on rocker. Afterward, 20 µl of protein A Sepharose beads were added, and lysates were incubated for an additional hour at 4 C on a rocker. Finally, after extensive washing with NaCl-free RIPA buffer, immunoprecipitates were subjected to SDS-PAGE and analyzed by Western blotting using either an antiubiquitin, anti-SRC-1, anti-TIF-2, anti-RAC-3, anti-CBP, anti-E6-AP, antiluciferase, or anti-HA antibody.

Bacterial Expression of Ubiquitin Pathway Enzymes
Arabidopsis thaliana UBA, various ubiquitin-conjugating enzymes E2 [UBC2 (E214K, UBC3 (UbcH3), UBC4 (rat UBC4-1), UBC5 (UbcH5), UBC6 (UbcH6), UBC7 (UbcH7), and UBC8 (UbcH8)] and ubiquitin-protein ligase E3 (UBL, E6-associated protein, E6-AP) were expressed in Escherichia coli BL21 ({lambda}DE3) using the pET expression system (Novagen, Madison, WI) (89). Bacterial cells containing the appropriate expression plasmids were grown overnight in 400-ml cultures at 25 C. The next morning, expression of proteins was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 3–4 h. Subsequently, cells were lysed in sonication buffer [10 mM Tris-HCl (pH 7.9), 10% glycerol, 0.5 M NaCl, 0.1% NP40, 5 mM ß- mercaptoethanol, and protease inhibitors (100 µg/ml PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin)]. Coomassie blue staining of an aliquot of each lysate separated by 4–20% SDS-PAGE was used to determine the relative amounts of each protein.

In Vitro Expression of Various Coactivator and Luciferase Proteins
In vitro expression of various radiolabeled coactivator proteins and luciferase were performed using TNT-coupled rabbit reticulocyte extracts in the presence of [35S]methionine according to manufacturer’s recommended conditions (Promega Corp., Madison, WI).

Protein Degradation and Ubiquitination Assays
Various 35S-labeled NRCoA and luciferase proteins were incubated either without or with UBA (~5–10 ng), indicated UBCs (~100 ng), and UBL (~50–100 ng) in reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM ATP, 10 mM MgCl2, 0.2 mM dithiothreitol, and 4 µg of ubiquitin (Sigma) for 2–4 h at 30 C. Reactions were terminated by boiling samples in the presence of SDS-loading buffer [100 mM Tris-HCl (pH 8.0), 200 mM dithiothreitol, 4% SDS, 20% glycerol, and 0.2% bromophenol blue]. The reaction mixtures were resolved by 7.5% SDS-PAGE, and radiolabeled bands were visualized by autoradiography.


   ACKNOWLEDGMENTS
 
We thank Peter Howley and Sushant Kumar for UbcH7 and UbcH8 expression plasmids; Allan Weissman for A. thaliana UBA1 and UbcH5B expression plasmids; Martin Scheffner for UbcH3, UbcH5, and UbcH6 expression plasmids; Simon S. Wing for UBC2 and UBC4 expression plasmids; Jiemin Wong for antibodies against SRC-1, TIF-2, RAC-3, and CBP; N. Maitland for E6-AP antibody; and Sue Fox for the ts85 cell line; we also thank Neil McKeena, Fred Pereira, and Andrew P. Dennis for critical reading of the manuscript. The coactivator proteins were analyzed using the PEST-FIND program for PEST sequences available at the following web site: http://www.at.embnet.org/embnet/tools/bio/PESTfind/.


   FOOTNOTES
 
This work was supported by NIH Grant DK-56833 (to Z.N.).

Abbreviations: aa, Amino acids; CBP, CREB binding protein; CREB, cAMP response element binding protein; DMSO, dimethylsulfoxide; E6-AP, E6-associated protein; GTF, general transcription factor; HA, hemagglutinin; HAT, histone acetyl transferase; NF-{kappa}B, nuclear factor {kappa}B; NP40, Nonidet P-40; NRCoA, nuclear receptor coactivator; PMSF, phenylmethylsulfonyl fluoride; RAC-3, retinoid acid receptor coactivator 3; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; TIF-2, transcriptional intermediary factor 2; UBA, ubiquitin-activating enzyme; UBC, ubiquitin-conjugating enzyme; UBL, ubiquitin-protein ligase.

Received for publication June 6, 2002. Accepted for publication March 18, 2003.


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 DISCUSSION
 MATERIALS AND METHODS
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Coregulators:   E6AP  |  UBCH7  |  CBP  |  SRC-1  |  GRIP1  |  AIB1



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