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Originally published In Press as doi:10.1074/jbc.M313703200 on February 2, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17524-17534, April 23, 2004
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TSG101 Interacts with Apoptosis-antagonizing Transcription Factor and Enhances Androgen Receptor-mediated Transcription by Promoting Its Monoubiquitination*

Sven Burgdorf{ddagger}, Peter Leister, and Karl Heinz Scheidtmann§

From the Institute of Genetics, University of Bonn, D-53117 Bonn, Germany

Received for publication, December 15, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis-antagonizing transcription factor (AATF), also termed Che-1, was identified as interacting protein of Dlk/ZIP kinase and RNA polymerase II, respectively. Che-1 has additionally been shown to bind Rb, thereby activating transcription factor E2F and promoting cell cycle progression. Moreover, AATF enhances steroid receptor-mediated transactivation in a hormone- and dose-dependent manner (Leister, P., Burgdorf, S., and Scheidtmann, K. H., (2003) Signal Transduction 3, 18–25). These data suggest that AATF exerts its functions through interaction with different transcription factors. In search of novel interaction partners of AATF, we identified the tumor susceptibility gene product TSG101, which had also been recognized as a co-regulator of nuclear hormone receptors. Interestingly, TSG101 and AATF functioned as cooperative coactivators in androgen receptor-mediated transcription. Because TSG101 was also shown to play a role in regulation of ubiquitin conjugation, we asked whether its coactivating function might be linked to ubiquitination. Indeed, TSG101 enhanced monoubiquitination of the androgen receptor in a ligand-dependent manner, and this correlated with enhanced transactivating capacity. Furthermore, a dominant-negative mutant of ubiquitin preventing polyubiquitination also stimulated androgen receptor-mediated transcription, which in this case could not be enhanced by TSG101. We propose that TSG101 activates androgen receptor-induced transcription by transient stabilization of the monoubiquitinated state, thus revealing a novel regulatory mechanism for nuclear receptors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis-antagonizing transcription factor (AATF)1 was first identified as a protein interaction partner of Dlk/ZIP kinase (1). Dlk/ZIP kinase, in turn, is a member of the death-associated protein kinase family that plays a role in different processes like apoptosis (reviewed in Ref. 2) and mitosis (3). AATF was shown to interfere with Dlk-induced apoptosis, thus explaining its name (1). AATF is conserved from yeast to man, suggesting that it fulfills an important function. Indeed, the mouse ortholog (termed traube) has been shown to play an essential role in early embryogenesis (4). Moreover, the human ortholog of AATF (termed Che-1) was independently identified as an interacting protein of the RNA polymerase II, subunit 11, and the tumor-suppressor protein Rb (5). This latter interaction releases the repression function of Rb on E2F-mediated transcription, presumably by displacing histone deacetylase-1 from the Rb·E2F complex, thereby promoting cell cycle progression (6). Thus, AATF/Che-1 appears to play a role in cell cycle control. Consistent with this assumption is its down-regulation upon TGF-{beta}-induced differentiation (7).

The AATF protein has a modular structure. At its N terminus it contains an acidic region that is characteristic of several transcription factors, such as VP16 or BRCA-1. In its C-terminal half, it contains three highly conserved regions, the significance of which is not known. Furthermore, AATF contains a leucine zipper and three LXXLL motifs, the latter of which are characteristic interaction motifs of coactivators for nuclear hormone receptors (8).

Nuclear hormone receptors (NR) comprise a large family of ligand-dependent transcription factors that play a role in diverse physiological processes like cell cycle control, differentiation, and apoptosis. They are classified into three classes. Class I includes the classical steroid hormone receptors (androgen, estrogen, glucocorticoid, progesterone, and mineralocorticoid receptor), class II receptors comprise non-steroid hormone receptors (thyroid, vitamin D, and retinoic acid receptor), and class III are the so-called orphan receptors for which the ligands or functions have yet to be identified. One of the best studied examples is the androgen receptor (AR). Like other members of the NR superfamily, AR contains four conserved functional domains: an N-terminal ligand-independent transactivation domain AF-1 (activation function 1), a central DNA-binding domain (DBD), a C-terminal ligand binding domain (LBD) containing the ligand-dependent transactivation domain (AF-2), and a hinge region connecting the LBD and the DBD. In the absence of hormone, AR is kept inactive in the cytoplasm by complex formation with HSP90. After activation by testosterone, AR is translocated to the promoter or enhancer regions of target genes where it can activate transcription (for reviews, see Refs. 9 and 10).

Recent studies revealed that NRs mediate their function by the recruitment of coregulators (coactivators or corepressors). These coregulators are involved in modification and remodeling of chromatin, and they facilitate the interaction between transcriptional activators and the general transcription machinery. Thus, several histone acetyl transferases and methyl transferases like SRC-1/p160, p300/cAMP-response element-binding protein-binding protein, or CARM-1, respectively, have been identified as coactivators of NR, whereas corepressors exhibit or recruit histone deacetylase activity (reviewed in Refs. 11–13). Many coactivators can interact directly with the LBD of activated receptors via an LXXLL signature motif (8, 14). AATF contains three LXXLL motifs. A recent investigation on their significance revealed that AATF can indeed directly interact with NRs and enhance NR-mediated transactivation in a hormone-dependent manner (15). Thus, AATF behaved like a bona fide coactivator of NR. Together with the data on Rb-E2F, these findings suggest that AATF exerts its functions through interaction with different transcription factors and may, in fact, be a more general coactivator of transcription.

In search of further interaction partners of AATF, we identified the TSG101 (tumor susceptibility gene 101) protein as a putative complex partner. TSG101 was originally identified by random gene inactivation to isolate putative tumor suppressor proteins (16). Inactivation of tsg101 led to malignant transformation of NIH3T3 cells. However, tsg101 might also have a growth-promoting function as suggested from different knock-out models. tsg101 knock-out mice die by day 6.5 and fail to induce mesoderm upon gastrulation (17). Cells derived from knock-out embryos exhibit a dramatic decrease in proliferation, presumably due to accumulation of p53 and p21 (17) and inhibition of CDK2 activity (18). These data can be explained by recent findings showing that TSG101 is included in the MDM2-p53 regulatory circuit. The levels of MDM2 and p53 are tightly balanced: MDM2 is a target gene of p53, which in turn is targeted by MDM2 for degradation through its ubiquitin ligase activity (reviewed in Ref. 19). TSG101 stabilizes MDM2, which then leads to down-regulation of p53 (20). Consequently, loss of TSG101 would result in up-regulation of p53.

Stabilization of MDM2 by TSG101 seems to be due to inhibition of ubiquitination (20), consistent with the notion that TSG101 might play a regulatory role in the ubiquitin system (21, 22). Initial studies on the biochemical function(s) of TSG101 revealed an interaction with nuclear hormone receptors and down-modulation of their transactivation activities (23–25). More recent investigations, however, point to a role in ubiquitination-dependent processes. TSG101 contains at its N terminus a region with high homology to ubiquitin conjugases. However, the cysteine residue in the active center, which normally mediates the covalent linkage to ubiquitin, is replaced by tyrosine. Therefore, TSG101 was proposed to be a dominant negative regulator of ubiquitination (21, 22). The findings that TSG101 stabilizes proteins like MDM2 and p21 are consistent with this idea (20, 26). Moreover, TSG101 has been implicated in protein sorting and virus budding, both of which include ubiquitination events (27–29).

In the present investigation, we identified TSG101 as an interaction partner of AATF. Because both TSG101 and AATF have individually been characterized as coregulators of nuclear hormone receptors, we investigated a possible functional relationship of these proteins. Both AATF and TSG101 functioned as synergistic coactivators of the AR. Furthermore, we investigated a possible link between ubiquitin-related functions of TSG101 and transcription. Interestingly, TSG101 enhanced monoubiquitination of the AR, and this modification correlated with transcriptional activation. We propose that TSG101 stimulates androgen receptor-induced transcription by promoting its monoubiquitination, thus revealing a novel regulatory mechanism for nuclear receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—For the yeast two-hybrid screen, fragments of AATF cDNA (encoding amino acids 396–523 or 447–523) were cloned into pGBT9 (Clontech, Heidelberg, Germany). For expression of fusion proteins bearing the GAL4 transactivation domain, inserts were cloned into pGAD424 (Clontech). For the expression and purification of GST fusion proteins, TSG101 was cloned into pGEX-6T (Amersham Biosciences). For expression studies, TSG101 was cloned into pEGFP-C1 (Clontech). A plasmid encoding the AR under the control of the SV40 promoter (SV-AR0) was kindly provided by A. Brinkmann (Erasmus University, Rotterdam, The Netherlands). An expression vector for human TSG101 was kindly provided by S. Cohen (Stanford University). For transient expression assays, AATF and rat TSG101 were cloned into pCMV-Tag2 (Stratagene, La Jolla, CA), thereby adding a FLAG-tag to the N terminus. Expression plasmids for His6-tagged ubiquitin (H6M-Ub) and the dominant negative ubiquitin mutant (H6M-UbK48R) were a kind gift from R. Kopito (Stanford University). All cloning experiments were performed using standard methods. Expression plasmid encoding E2F and E2F-responsive cdc25 promoter-luciferase reporter construct were a kind gift from K. Helin, Milan, Italy. Expression plasmids encoding rat p53 and p53-responsive mdm2 promoter-luciferase reporter were described previously (30).

Yeast Two-hybrid Screen—The Matchmaker GAL4 two-hybrid system (Clontech) with the yeast strain AH109 was used in this study. A total of 8.6 x 105 colonies was screened as described before (31). The cDNA insert of the positive clones was amplified by PCR using the whole cell yeast PCR kit (Qbiogene, Grünberg, Germany) and 5'-CCAAACCCAAAAATGGAGATCGAATTC-3' and 5'-CTAGAGTCGACCCGGGCTCGA-3' as primers and analyzed by sequence analysis (AG-OWA, Berlin, Germany).

Isolation of Full-length Rat TSG101—Total RNA was isolated from rat fibroblast cell line REF52.2 using the Qiagen RNeasy mini-kit (Qiagen, Hilden, Germany) and reverse-transcribed using the Super-script II RT (Invitrogen). Rat TSG101 was then amplified by PCR from the cDNA using 5'-GCACTAGAATTCATGGCGGTGTCCGAGAG-3' and 5'-GAGCTTGTCGACTCAGTAGAGGTCACTAAGGCC-3' as primers. The resulting PCR product was verified by sequence analysis.

In Vitro Binding Assays—GST pull-down assays were performed using full-length TSG101 fused to GST as binding matrix and in vitro translated [35S]methionine-labeled AATF. GST·TSG101 was expressed in Escherichia coli M15 and immobilized on glutathione-Sepharose beads. In vitro translation of AATF was performed in the TNT-T7/T3-coupled reticulocyte translation system (Promega). Binding of AATF was carried out in phosphate lysis buffer (20 mM sodium phosphate, pH 8.0, 140 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P40, 1 mM dithiothreitol, 50 µM leupeptin,) at 4 °C for 3 h. Thereafter, the beads were washed twice with phosphate lysis buffer, bound proteins were eluted with 2 x SDS sample buffer, separated by SDS-PAGE, and visualized by fluorography.

Mammalian Cell Culture and Transfection—REF52.2, CV1 and Rat-1 cells were maintained in Dulbecco's minimal essential medium (Sigma) supplemented with 10% fetal bovine serum (Biochrom Seromed, Berlin, Germany). MCF-7 (human breast carcinoma) and PC3 (human prostate carcinoma) cells were grown in RPMI medium containing 10% fetal calf serum. Transfections were carried out either by calcium phosphate coprecipitation, as for Rat-1, or by lipofection with jetPEI (QBiogene) (MCF-7 and PC3) or LipofectAMINE (Invitrogen) (REF52.2 and CV1) according to the manufacturers' instructions.

Immunofluorescence Analysis—Transfected cells were washed twice with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS for 15 min, washed again, and permeabilized and blocked in 0.1% Triton X-100, 5% dry milk, and 1% goat serum in PBS for 1 h. Cells were incubated with a mouse monoclonal antibody against TSG101 (sc-7964; Santa Cruz Biotechnology) (1:500) for 1 h, washed again, and incubated with goat anti-mouse Cy3-conjugated secondary antibody (Dianova, Hamburg, Germany) (1:500) for 30 min. 4',6-diamidino-2-phenylindole staining was performed with 1 µg/ml 4,6-diamidino-2-phenylindole for 15 min. The subcellular distribution of expressed proteins was analyzed by fluorescence microscopy with an Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany).

Transactivation Assays—For transactivation assays, 2 x 105 cells were plated on 35-mm Petri dishes and transfected with the indicated amounts of reporter construct, bearing the luciferase gene driven by the mouse mammary tumor virus (MMTV) promoter, and expression plasmids. The total amount of transfected DNA was balanced by supplementing with pCMV-Tag2B empty expression vector. After transfection, cells were treated with 10 nM dihydrotestosterone for another 16 h. Induced cells were washed twice in phosphate-buffered saline and harvested in lysis buffer (100 mM KHPO4, pH 7.8, 0.2% Triton X-100, 1 mM dithiothreitol). Luciferase activity was determined in a reaction mixture containing 20 mM Tricine, 1 mM MgCO3, 2.6 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, 470 µM D-luciferin, and 530 µM ATP and measured in a Lumat LB9501 luminometer (Berthold, Bad Wildbad, Germany). All transactivation experiments were performed in triplicate and repeated at least twice.

In Vivo Ubiquitination Assay—Ubiquitinated proteins are rapidly degraded by the proteasome pathway and are therefore difficult to analyze. To circumvent this problem, His-tagged recombinant ubiquitin has been employed. This allows isolation and purification of ubiquitinated proteins on Ni columns under denaturing conditions, that is, upon inactivation of the proteolytic system (32). Plasmids H6M-Ub encoding His-tagged ubiquitin or H6M-UbK48R, a dominant negative mutant of ubiquitin that cannot be further ubiquitinated (33), were kindly provided by R. Kopito (Stanford University). Plasmids were introduced into MCF-7 cells by cotransfection with plasmids expressing AR and TSG101. 16 h after transfection, cells were induced with 10 nM dihydrotestosterone. After another 8 h, 2 µM MG132 (Calbiochem) was added for an additional 16 h. Cells were harvested in denaturing lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2PO4, 20 mM imidazole, 10 mM Tris-HCl at pH 8), sonicated on ice for 1 min, and incubated with Ni-NTA-agarose (Qiagen) for 1 h. Thereafter, the beads were washed four times with washing buffer (8 M urea, 100 mM NaH2PO4, 20 mM imidazole, 10 mM Tris-HCl at pH 6.2), eluted with 2 x SDS sample buffer, separated by SDS-PAGE, and analyzed by Western blotting using anti-AR antibody (sc-13062; Santa Cruz Biotechnology).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of TSG101 as an Interaction Partner of AATF—To identify protein interaction partners of AATF, a yeast two-hybrid screen was performed using a cDNA library derived from SV40-transformed rat fibroblasts. Because the acidic domain at the N terminus of AATF displays transactivation activity in a Gal4-linked reporter assay in yeast, we employed a C-terminal fragment spanning a region from amino acid 396 to 523 as bait. This fragment contains two of three highly conserved sequence stretches, CR2 and CR3, and includes one of three LXXLL NR interaction motifs (7), thus providing a suitable bait for two-hybrid screening. One of the putative interaction partners isolated was TSG101. TSG101 was originally identified by random gene targeting as a gene whose inactivation leads to malignant transformation (16). Isolation of the plasmid encoding the GAL4 AD·TSG101 fusion protein and its retransformation together with the bait plasmid into yeast cells confirmed the interaction. The portion of rat TSG101 isolated from the cDNA library comprised the C-terminal part from amino acids 191 to 391 as deduced from sequence comparison with human TSG101. The full-length clone of the rat ortholog was obtained by PCR. Elucidation of the complete coding sequence predicted a 391-amino acid protein with 97% sequence identity with mouse and 92.6% identity with human TSG101 at the amino acid level, indicating that this protein is highly conserved. Even the yeast homolog Vps23p still displays a similarity of 34%. The complete sequence of rat TSG101 cDNA is available under GenBankTM accession number AY293306 [GenBank] . Rat TSG101 contained all structural motifs that were elucidated for human TSG101, an N-terminal region with homology to ubiquitin conjugases (termed Ubc region), a proline-rich region, and a coiled coil motif, including a leucine zipper in the central part, and a C-terminal {alpha}-helical domain, including a so-called steadiness box (34) (see scheme in Fig. 1A).



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  		FIG. 1.
Rat TSG101 binds AATF in vitro. A, structure of rat TSG101 containing an (inactive) ubiquitin-conjugating enzyme domain (Ubc), a proline-rich region (Pro), a coiled coil (CC2) region including a leucine zipper (LZ), and a C-terminal {alpha}-helical domain ({alpha}-helix) with the so-called steadiness box (SB). B, TSG101 was expressed as GST fusion protein, immobilized on glutathione-Sepharose, and used for binding of full-length in vitro translated and [35S]methionine-labeled AATF. Binding to GST served as control. Bound proteins were eluted with SDS sample buffer and analyzed by SDS-PAGE and fluorography. The input lane represents one tenth of the input employed in the binding experiment.

 
The interaction of AATF and TSG101 was confirmed in vitro by a GST pull-down experiment with full-length TSG101 expressed as GST fusion protein in E. coli and radiolabeled in vitro translated AATF. GST was used as negative control. AATF bound to the fusion protein, but not to GST, as shown in Fig. 1B.

Interaction between AATF and TSG101 Is Mediated by Their C-terminal Regions—To determine the interaction regions between the two proteins, deletion mutants of both AATF and TSG101 were constructed as shown in Fig. 2A and tested for interaction in the two-hybrid system. The AATF construct originally used as bait implied that the interaction domain resided in the C-terminal region of AATF (residues 396–523). This conclusion was verified by employing this C-terminal fragment of AATF with constructs of TSG101 in the interaction assay (Fig. 2B). Further deletion of AATF to residue 446 still allowed interaction of the two proteins (data not shown), thus narrowing the interaction domain to the C-terminal 80 residues. Likewise, the portion of TSG101 isolated as two-hybrid clone, comprising residues 191–391, indicated that the interaction with AATF was mediated with the C-terminal half. Accordingly, we created additional mutants in which the N-terminal 235 or 317 residues were deleted. All these constructs, including full-length wild-type TSG101, were able to bind AATF (Fig. 2B), indicating that TSG101 also interacts with its C-terminal part (the last 74 residues) comprising the {alpha}-helical domain. To verify this conclusion, deletion mutants lacking the putative interaction domains were employed in the two-hybrid interaction assay. As expected, these truncated proteins did not interact, neither AATF (1–390) with full-length TSG101 nor TSG101 (1–326) with full-length AATF (Fig. 2C). Thus, although both proteins contain a leucine zipper, their interaction is mediated by their C termini.



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  		FIG. 2.
Deletion mapping of the interaction domains between AATF and TSG101 using the yeast two-hybrid system. A, deletion mutants of AATF (upper part) and TSG101 (lower part) were constructed as shown. In the full-length proteins, the structural motifs are indicated. For AATF, LX represents LXXLL motifs; TA, transactivation domain; CR, conserved region; LZ, leucine zipper. For TSG101, see Fig. 1. The deletion constructs were fused to Gal4-DB or Gal4-AD as outlined under "Experimental Procedures." B and C, yeast cells were transformed with various combinations of full-length or deletion constructs of AATF or TSG101, as indicated at the margins of the culture dishes, to determine the interaction domains of both proteins. AATF mutants AATF 386–523 and AATF 447–523 (not shown) were still able to interact with full-length TSG101; on the other hand, TSG101 236–391 and TSG101 317–391 retained the ability to bind wild type AATF (B). Deletion of the C termini of either protein abolished interaction (C).

 
AATF Is Able to Recruit TSG101 to the Nucleus—Next we investigated whether both proteins would colocalize in intact cells. AATF is a nuclear protein (1), whereas TSG101 has been localized to different compartments. It was predominantly localized in the cytoplasm, partially associated with Golgi (35) and endosomes (28), but showed also some nuclear accumulation during S and G2 phases (35, 36). TSG101 was expressed as GFP-tagged fusion protein, and its subcellular localization was analyzed by fluorescence microscopy. As shown in Fig. 3, GFP·TSG101 displayed a punctuate staining in the cytoplasm (Fig. 3, left panels). Similar patterns were seen by others (28, 36). This staining pattern was reminiscent of endosomes. To verify this assumption, we performed an endosome uptake experiment in which cells were incubated with Texas Red-labeled ovalbumin. Upon pinocytosis, ovalbumin is translocated to the endosomal compartment and can be visualized by fluorescence microscopy (37). Indeed, Texas Red-labeled ovalbumin and GFP·TSG101 colocalized to a large extent (Fig. 3A, right panel). A similar distribution was obtained in other cell lines such as REF52.2, HeLa, or Saos-2 cells (data not shown). This subcellular localization of TSG101 is in agreement with a role of TSG101 in endosomal protein sorting (27, 28). To examine whether endogenous TSG101 shared the same distribution, cells were stained for immunofluorescence with a monoclonal antibody against TSG101. Because of low expression levels, this antibody detected TSG101 only in transfected but not in untransfected cells (Fig. 3B). Thus, endogenous TSG101 could not be localized in this way, although it could be detected by Western blotting. However, in the GFP·TSG101-expressing cells, the antibody revealed a diffuse staining of both cytoplasm and nucleus in addition to the punctuate endosome-associated staining (Fig. 3B). This diffuse staining was otherwise obscured by the bright GFP fluorescence in the endosomes. Thus, a subpopulation of TSG101 appears to be localized to the nucleus, in agreement with previous data (35, 36).



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  		FIG. 3.
Subcellular localization of AATF and TSG101. A, TSG101 colocalizes with endosomes. Cells were transfected with GFP·TSG101; 4 h prior to fixation, Texas Red-conjugated ovalbumin (10 µg/ml) (Molecular Probes, Leiden, The Netherlands) was added. The merged image shows that TSG101 colocalized with most endosomes that were labeled with Texas Red-ovalbumin. B, anti-TSG101 staining detects TSG101 in the nucleus. GFP·TSG101-transfected cells were stained with anti-TSG101 at 24 h post-transfection, revealing diffuse cytoplasmic and nuclear staining in addition to the dot-like endosomal staining. C, coexpression of TSG101 and AATF results in accumulation of TSG101 in the nucleus. Cells were cotransfected with GFP·AATF and FLAG-TSG101 and stained with a monoclonal anti-TSG antibody and Cy3-conjugated secondary antibodies. D, deletion mutant AATF 1–390 lacking the interaction domain for TSG101 fails to recruit TSG101 to the nucleus. The AATF mutant was expressed as GFP fusion protein and TSG101 as the FLAG-tagged version, as before.

 
We next investigated whether co-expression of TSG101 with AATF might lead to relocation of one of the partners. Under these conditions, TSG101 accumulated in the nucleus, as shown in Fig. 3C. This accumulation was not observed when the C-terminal truncation mutant AATF (1–390) lacking the interaction domain was expressed with full-length TSG101 or vice versa (shown for AATF (1–390) in Fig. 3D), thus confirming the two-hybrid interaction data (see above). This partial relocation of TSG101 suggests that AATF is able to recruit TSG101 to the nucleus.

AATF and TSG101 Are Both Coactivators of the AR— Interestingly, both TSG101 and AATF are coregulators of nuclear hormone receptors. TSG101 has been shown to interact via its coiled coil region with the activation function-1 transactivation domain of glucocorticoid receptor (24) and to repress NR-mediated transcription (23–25). AATF, on the other hand, was recently recognized as coactivator of several steroid hormone receptors (15). Thus, these proteins seemed to affect NRs in opposite ways. To investigate whether TSG101 and AATF might act as antagonistic coregulators of NRs, we performed transient transactivation assays with a luciferase reporter construct driven by the MMTV promoter. This promoter is responsive to androgen and glucocorticoid receptors in a ligand-dependent manner (38). In a setup experiment, we confirmed that the reporter construct is responsive to AR and dependent on testosterone (Fig. 4A; see also Ref. 15). Surprisingly, including TSG101 in our transactivation experiments resulted in a dose-dependent activation of AR-mediated transcription rather than repression (Fig. 4B). Again, this co-activation was strictly hormone-dependent. Because these observations are at variance to previously reported data, we asked whether the differences might be due to cell type-specific effects of recipient cells used for transfection or to species-specific differences between rat and human TSG101. When we employed CV-1 cells instead of REF52.2 cells or human TSG101 instead of rat TSG101, AR-mediated transcription was still enhanced by TSG101 as shown in Fig. 4D (compare columns 1 and 3). The reasons for these discrepancies are not known.



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  		FIG. 4.
AATF and TSG101 cooperate in the activation of AR-mediated transcription. A, rat-1 cells were transfected with 350 ng of the AR-responsive MMTV promoter luciferase construct (MMTV-luc) and increasing amounts of AR expression plasmid as indicated. Activation of the reporter gene was determined in the absence or presence of dihydrotestosterone (DHT) as outlined under "Experimental Procedures." Hormone treatment was 16–32 h post-transfection. The MMTV promoter response is AR- and hormone-dependent. Therefore, the samples without hormone treatment are not shown for the other experiments. In the other transactivation experiments, cells were transfected with constant amounts of MMTV-luc (350 ng) and AR expression plasmid (100 ng) and varying amounts or combinations of FLAG-AATF or FLAG-TSG101 or GFP·TSG101. B, increasing amounts of TSG101 activate AR-mediated transcription. C, cooperative effect of AATF and TSG101 on reporter gene transcription. AATF and TSG101 expression plasmids were administered at 125 and 250 ng, respectively. D, same experiment as in panel C but in CV-1 cells and with human TSG101. E, effect of deleting the C-terminal domain (mutant TSG1–326) or the N-terminal Ubc region of TSG101 (mutant TSG142–391) on its coactivation function. Rat-1 cells were transfected with 350 ng of MMTV-luc, 100 ng of FLAG-AR, 150 ng of Flag-AATF, and 500 ng of GFP-TSG101 wild type (column 2) or GFP·TSG101 mutants TSG1-326 (column 3), or TSG 142-391 (column 4). F, Western blot analysis demonstrating that the GFP·TSG101 constructs employed in panel E were expressed at similar levels. The blot was probed with anti-GFP antibodies.

 
We then included AATF in this assay. AATF can stimulate transactivation by AR, estrogen receptor, and glucocorticoid receptor up to 5-fold in a hormone-dependent manner (15). Interestingly, coexpression of both TSG101 and AATF resulted in further enhancement of transcription. This effect was more than additive, suggesting a functional cooperation between the two proteins (Fig. 4C). Again, these results applied for CV1 cells as well as for rat fibroblasts (Fig. 4D). In CV1 cells, coexpression of AATF with AR caused a 2-fold and that of TSG101 a 4-fold induction of AR-mediated transactivation. Strikingly, coexpression of both proteins caused a more than 50-fold induction.

To investigate which domains of TSG101 were responsible for this cooperative effect, we generated deletion mutants of TSG101 lacking the N-terminal Ubc region (TSG101 142–391) or the C-terminal {alpha}-helical region containing the interaction domain for AATF (TSG101 1–326) and tested them for coactivating activity. Although both mutants still contained the region required for the interaction with NR (24), they were both impaired in their capacity to enhance AR-mediated transcription (Fig. 4E). That both mutants were expressed at similar levels as wild type TSG101 was verified by Western blot analysis (Fig. 4F). These findings suggest that both the Ubc domain and the interaction region for AATF are essential for efficient coactivation by TSG101.

TSG101 Activates AR-mediated Transcription by Affecting Its Ubiquitination State—The necessity of the Ubc region of TSG101 for its coactivating function led us to investigate a possible involvement of ubiquitination in AR-induced transcription. The rationale for this was 2-fold. First, TSG101 has been shown to be involved in several ubiquitin-dependent processes such as endosomal protein sorting and trafficking (27, 28) and protein stabilization (20, 26). Second, recent studies suggest a tight connection between ubiquitination/proteasome-dependent degradation and transactivation of several transcription factors, including NRs (39–42).

We first investigated whether AR is ubiquitinated upon testosterone-mediated activation. Because ubiquitination of target proteins is difficult to analyze, due to their rapid degradation, we included an expression plasmid for His-tagged ubiquitin (H6M-Ub) in our transactivation assays. Incorporation of His-ubiquitin into target proteins facilitates their isolation and purification by affinity chromatography on Ni-NTA-agarose under denaturing conditions (32, 33). That overexpressed H6M-Ub was indeed incorporated into AR is demonstrated in Fig. 5A. When total cell extracts were probed on Western blots with antibodies against AR, we observed two forms of AR, the regular form of 90 kDa and a second form with increased apparent molecular mass of about 100 kDa, consistent with monoubiquitination (Fig. 5A, upper part, middle lane). This upper form accumulated as a result of ubiquitin overexpression and inhibition of proteasomes and was not seen without ectopic expression of H6M-Ub (left lane). Upon purification of His-ubiquitin-tagged proteins on Ni-NTA agarose (see "Experimental Procedures"), only the ubiquitinated form was retained and eluted (Fig. 5A, lower part, middle lane). Polyubiquinated forms were not detected, presumably because of degradation. AR was not retained on the Ni-NTA resin without overexpression of His-tagged ubiquitin (Fig. 5A, lower panel, left lane). Significantly, ubiquitination of the AR was ligand-dependent, because in the absence of testosterone the upper band was almost absent from the lysate and only marginal amounts of ubiquitinated AR were retained by the Ni-NTA matrix (right lanes).



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  		FIG. 5.
Ubiquitination affects AR-mediated transcription. A, MCF-7 cells were transfected with 3 µg of FLAG-AR, 3 µg of H6M-Ub, 3 µg of H6M-UbK48R as indicated. Ubiquitinated proteins were purified as described under "Experimental Procedures." AR was visualized by Western blotting using anti-AR antibodies. Expression of ubiquitin generates a second form of AR with a higher molecular mass (upper part, middle lane). This band represents ubiquitinated AR as shown by Ni-NTA affinity chromatography (lower part, middle lane). Ubiquitination of the AR is ligand-dependent because in the absence of testosterone little ubiquitinated AR could be detected (right lanes). B, differential effects of wild type ubiquitin and ubiquitin K48R mutant on AR-mediated transcription. Rat-1 cells were transfected with 500 ng of MMTV-luc, 450 ng of FLAG-AR, 400 ng of H6M-Ub, or 400 ng of H6M-UbK48R as indicated. Luciferase activity was measured as before. Similar results were obtained with MCF-7 cells. C and D, the effects of ubiquitin on reporter gene expression are AR-dependent and specific. In panel C, PC3 cells lacking endogenous AR were transfected with MMTV-luc, H6M-Ub, H6M-UbK48R, and/or AR expression plasmids as indicated. Ubiquitin revealed an enhancing effect only in the presence of AR. In panel D, MCF-7 cells were transfected either with CMV promoter-luciferase (CMV-LUC) without activator, or mdm2 promoter-luciferase plus p53 expression plasmid (Mdm2-LUC/p53), or cdc25 promoter-luciferase plus E2F expression plasmid (Cdc25-LUC/E2F), and the effect of ubiquitin on luciferase expression was determined as before.

 
We then investigated whether overexpression of His-tagged ubiquitin would influence AR-mediated transactivation. Indeed, coexpression of AR with H6M-Ub (in the absence of TSG101) resulted in an ~2-fold enhancement of luciferase expression (Fig. 5B).

Because polyubiquitination of AR might lead to its rapid degradation, we considered that the monoubiquitinated form seen in Fig. 5A might represent the transcriptionally active form. To address this issue and to distinguish whether the transcriptional enhancement by ubiquitination was due to the modification as such or rather linked to proteasome-mediated degradation (39), we included a dominant negative ubiquitin mutant (H6M-UbK48R) (33). Extension of ubiquitin chains occurs at Lys-48; thus the mutation of Lys-48 to Arg precludes polyubiquitination and, consequently, degradation, which requires proteins to be at least tetraubiquitinated (43). Indeed, expression of the ubiquitin mutant enhanced AR-mediated transcription even higher (up to 3.5-fold; see Fig. 5B), suggesting that monoubiquitination of the AR might be critical for efficient activation.

To investigate whether the observed enhancement of transactivation upon overexpression of ubiquitin was specific for AR or due to more general effects on transcription, we performed a set of control experiments. First, the experiment was performed in PC3 cells lacking endogenous AR. Without ectopic expression of AR there was no effect of ubiquitin or of the UbK48 mutant on MMTV promoter-driven transactivation, either in the absence or the presence of hormone (Fig. 5C). Second, we included other promoter-reporter constructs containing either the viral CMV-promoter or the p53-responsive mdm2-promoter or the E2F-responsive cdc25 promoter. Of these, the CMV promoter was only marginally affected by overexpression of ubiquitin, whereas the mdm2 and cdc25 promoters were even inhibited (Fig. 5D), presumably reflecting enhanced degradation of p53 or E2F, respectively. The inhibitory effects were less pronounced with the ubiquitin mutant, presumably because it interferes with polyubiquitination and, therefore, with degradation. These data argue against ubiquitin having some generalized effects on transcription.

We then asked whether ubiquitination of AR would be influenced by TSG101, as suggested from the considerations and results discussed above. Indeed, including TSG101 in the in vivo ubiquitination assay lead to a 5-fold increase of the monoubiquitinated form of the AR as shown in Fig. 6A. It should be mentioned that we did not observe significant changes in the half-life of AR by TSG101, as determined by pulse-chase experiments (data not shown). The observed accumulation of monoubiquitinated AR could be interpreted in two ways, either that TSG101 stimulated monoubiquitination or it prevented polyubiquitination. When we included the ubiquitin mutant in this assay, TSG101 had only a minor effect (1.6-fold increase) (Fig. 6B), suggesting that TSG101 prevents polyubiquitination rather than stimulating monoubiquitination. Including H6M-Ub and TSG101 in the transactivation assay resulted in more than 4-fold enhancement of AR-mediated transactivation compared with TSG101 alone (Fig. 6C). As expected, this enhancement was not further increased with the ubiquitin mutant, suggesting that TSG101 and the ubiquitin mutant influenced AR-mediated transcription in the same way, by preventing polyubiquitination.



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  		FIG. 6.
TSG101 enhances monoubiquitination. A, Western blot showing that TSG101 promotes accumulation of the monoubiquitinated form of AR. B, same experiment as in panel A, using the ubiquitination mutant UbK48R that precludes polyubiquitination. In this case, the higher level of monoubiquitination is only marginally enhanced by TSG101. For normalization of transfection efficiency, cells were cotransfected with GFP expression plasmid and GFP was detected by Western blotting, using anti-GFP monoclonal antibody. C, TSG101 further enhances ubiquitin-mediated activation of AR. This effect is not exceeded by the ubiquitin mutant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we identified rat TSG101 as an interacting protein of AATF. Sequence analysis of the rat TSG101 revealed a protein with 93 and 97% sequence identity with human and mouse TSG101, respectively. In particular, all structural features, including the Ubc region, the coiled coil domain along with the leucine zipper, and the C-terminal "steadiness box" were preserved. The interaction between AATF and TSG101 was verified by GST pull-down experiments, and their interaction domains were mapped to their C termini. More importantly, both proteins cooperatively enhanced AR-mediated transcription, thus demonstrating a functional relationship.

Interestingly, both TSG101 and AATF had been independently recognized as coregulators of nuclear hormone receptors. However, human TSG101 was reported to have a repressing effect (23–25), whereas AATF was shown to be a coactivator (15). Therefore, we expected these proteins to behave as antagonists with respect to AR-mediated transcription. Surprisingly, in our hands, TSG101 behaved as a coactivator as well. Coactivation was dose- and hormone-dependent, irrespective of the cellular background or whether rat or human TSG101 was employed in the transactivation assays. Moreover, coexpression of TSG101 and AATF revealed a cooperative rather than antagonistic effect on AR-induced transcription. Finally, the coactivation function of TSG101 and the cooperativity with AATF were lost upon deletion of the interaction domain for AATF from the C terminus of TSG101. Thus, the reasons for the discrepancies to published data are not known.

How do AATF and TSG101 exert their coactivation function? In a simplified view, coactivators may serve in chromatin modification or remodeling or they may act as scaffolds facilitating recruitment of and interaction with the basal transcription apparatus (reviewed in Refs. 12, 13, 44). AATF contains an extremely acidic region at its N terminus that in other proteins, e.g. Pho4, VP16, or BRCA1, was shown to possess chromatin remodeling activity (45–47). Indeed, the acidic domain of AATF exhibited transactivating activity in a GAL4-based reporter gene assay (1).2 Furthermore, AATF was shown to interact with RNA polymerase II subunit 11 (5), suggesting that AATF might act as a mediator or scaffold that provides contacts with the general transcription machinery. The present investigation revealed a further possibility, recruitment of TSG101, as suggested from the relocation of TSG101 to the nucleus in AATF-expressing cells. TSG101, on the other hand, seems to exert its coactivating function via its Ubc domain, which on its own exhibited transactivation activity in a Gal4-based reporter assay (23).

TSG101 has been postulated to be a (negative) regulator in ubiquitination-dependent processes because of its inactive Ubc domain (21, 22) (see the Introduction). Indeed, recent reports confirmed this proposal showing that TSG101 plays a role in protein sorting, trafficking, and stabilization, all of which are linked to ubiquitination. At the late endosomes, proteins destined for degradation are internalized by invagination of the endosomal membrane. For efficient invagination these proteins need to be monoubiquitinated (reviewed in Ref. 48). TSG101 is thought to recognize these monoubiquitinated proteins and to mediate their internalization (27, 28, 49, 50). Furthermore, TSG101 is involved in virus budding. In this case, TSG101 binds the L-domain of the retroviral gag protein and promotes its monoubiquitination (29, 51), which is a prerequisite for viral release (52). Finally, TSG101 is able to stabilize proteins like MDM2 and p21, presumably by preventing their polyubiquitination (20, 26). Our data extend these findings to nuclear TSG101 and its role in transcription. As we showed in this study, TSG101 stimulated monoubiquitination of AR in a hormone-dependent manner and this correlated with enhanced transcriptional activity of AR. A similar effect could be achieved by expressing a ubiquitin mutant, K48R, that can be linked to target proteins but prevents their polyubiquitination. The effect of overexpression of ubiquitin on transactivation of the MMTV promoter was clearly not due to general effects on transcription, because it had no or even an inhibitory effect on activation of the CMV promoter or on E2F- or p53-mediated transactivation, respectively. These latter findings are well in agreement with published data showing that proteasome inhibitors impair transactivation by some nuclear receptors (estrogen receptor, progesterone receptor, and thyroid hormone receptor) but not of the CMV promoter or of E2F-, p53-, or SP1-responsive promoters (39).

There is increasing evidence that ubiquitination and proteasome-mediated degradation of transcription factors are closely linked to their transactivating function. First, components of the ubiquitin/proteasome system, in particular ubiquitin ligase E6-AP, have been recognized as interaction partners or even coactivators of NRs and other transcription factors (39, 53, 54 and further references therein). Second, inhibition of proteasome-mediated degradation resulted in loss of transactivation activity of estrogen receptor (39, 55), AR (42), and glucocorticoid receptor (41). In fact, hormone-dependent transcriptional activation and ubiquitin/proteasome-mediated degradation seem to be interdependent in that blocking the proteasome system inhibits transactivation and inhibiting transcription prevents ubiquitination and proteolytic degradation (55). Coupling of activation and degradation might be a means to label those receptor molecules that actively participated in transcription or to facilitate promoter clearance after initiation or to limit hormone-induced gene activation.

It must be assumed, however, that it is not the polyubiquitinated form, destined for degradation, that actively participates in transcription. Rather, there might be a transient state of low, i.e. monoubiquitination, that mediates transcription initiation before it is targeted for degradation. In line with this assumption, expression of the transcriptional activator VP16 in a ubiquitination-deficient yeast strain resulted in loss of its transactivating activity, but this activity could be recovered by covalent linkage of a single ubiquitin residue to the transcription factor (40). In this case, degradation of the protein remained unaffected. These data indicated that it is ubiquitination, and not ubiquitin-dependent proteolysis, that is crucial for the activation of transcription. Moreover, coupling of a single ubiquitin residue appeared to be necessary and sufficient for activation of the transcription factor.

In our investigation TSG101 caused accumulation of the monoubiquitinated form of AR. This could be because of stimulation of monoubiquitination or prevention of subsequent polyubiquitination. Because TSG101 had little additional effect on monoubiquitination when the ubiquitination mutant was employed, we conclude that TSG101 acts to prevent polyubiquitination.Thisisinagreementwithdatafromtheotherubiquitin-dependent processes in which TSG101 is involved (see above). Because overexpression of the ubiquitin mutant stimulated AR-induced transcription to a similar extent as coexpression of TSG101 and wild type ubiquitin, we propose (i) that the monoubiquitinated form represents the transcriptionally active form and (ii) that TSG101 can stimulate transcription by NRs by transiently locking them in a monoubiquitinated active state.

From our and recent data from the literature, we propose the following model (see Fig. 7). Upon activation by its ligand, the AR is translocated to the nucleus and binds to its response elements where it recruits coactivators such as SRC-1, cAMP-response element-binding protein-binding protein /p300, CARM-1, and, additionally, AATF, TSG101, and ubiquitin ligase, e.g. E6-AP. Interestingly, TSG101 also binds to p300 (23) and p300 has recently been shown to possess ubiquitin ligase activity (56). Upon monoubiquitination of the receptor, TSG101 binds to this ubiquitin moiety and locks the receptor in this state until transcription commences. Alternatively, ubiquitination and binding of TSG101 could as well take place in the cytoplasm to protect AR from premature degradation. In any case, during transcriptional initiation additional interactions or post-translational modifications may induce conformational changes, thereby releasing the lock by TSG101 and allowing polyubiquitination and degradation. This facilitates promoter clearance and allows for a new cycle of activation (55). Our data extend the functions of TSG101 in ubiquitination-dependent processes to a role in transcription and contribute to our understanding of the complex sequence of events leading to transcriptional activation by nuclear hormone receptors.



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  		FIG. 7.
Model of cooperative coactivation by AATF and TSG101. Based on data from this and previously published papers, we propose that upon activation by hormone, AR binds to its response elements and sequentially recruits various coactivators such as SRC-1, CARM-1 (omitted for clarity), AATF, ubiquitin ligase, TSG101, p300, etc. In the model, TSG101 is recruited by AATF and concomitantly binds to the monoubiquitinated receptor, thereby protecting it from polyubiquitination until transcription starts. Upon completion of initiation or transition to elongation, which involve additional interactions or post-translational modifications, TSG101 is released and the receptor is targeted for polyubiquitination and degradation.

 

   FOOTNOTES
 
* This study was supported by Deutsche Forschungsgemeinschaft Grant Sche246/14–1 and by the Fond der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Institute of Molecular Medicine and Experimental Immunology, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Back

§ To whom correspondence should be addressed. Tel.: 49-228-734-258; Fax: 49-228-734-263; E-mail: kh.scheidtmann{at}uni-bonn.de.

1 The abbreviations used are: AATF, apoptosis-antagonizing transcription factor; NR, nuclear hormone receptor; AR, androgen receptor; TSG, tumor susceptibility gene; Ni-NTA, nickel-nitrilotriacetic acid; GST, glutathione S-transferase; Ubc, ubiquitin conjugase; GFP, green fluorescent protein; MMTV, mouse mammary tumor virus; CMV, cytomegalovirus. Back

2 I. Lödige and K. H. Scheidtmann, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. A. Brinkman, Erasmus University Rotterdam, The Netherlands, for AR expression plasmid, Dr. S. Cohen, Stanford University, for human TSG101 expression plasmid, Dr. R. Kopito, Stanford University, for His-ubiquitin expression constructs, and Dr. K. Helin, European Institute of Oncology, Milan, Italy, for E2F expression plasmid and cdc25-promoter construct. We also thank Dr. A. Haas, Institute of Cell Biology, University of Bonn, Germany, for helpful hints and discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Page, G., Lödige, I., Kögel, D., and Scheidtmann, K. H. (1999) FEBS Lett. 462, 187-191[CrossRef][Medline] [Order article via Infotrieve]
  2. Kögel, D., Prehn, J. H., and Scheidtmann, K. H. (2001) BioEssays 23, 352-358[CrossRef][Medline] [Order article via Infotrieve]
  3. Preuss, U., Landsberg, G., and Scheidtmann, K. H. (2003) Nucleic Acids Res. 31, 878-885[Abstract/Free Full Text]
  4. Thomas, T., Voss, A. K., Petrou, P., and Gruss, P. (2000) Dev. Biol. 227, 324-342[CrossRef][Medline] [Order article via Infotrieve]
  5. Fanciulli, M., Bruno, T., Di Padova, M., De Angelis, R., Iezzi, S., Iacobini, C., Floridi, A., and Passananti, C. (2000) FASEB J. 14, 904-912[Abstract/Free Full Text]
  6. Bruno, T., De Angelis, R., De Nicola, F., Barbato, C., Di Padova, M., Corbi, N., Libri, V., Benassi, B., Mattei, E., Chersi, A., Soddu, S., Floridi, A., Passananti, C., and Fanciulli, M. (2002) Cancer Cell 2, 387-399[CrossRef][Medline] [Order article via Infotrieve]
  7. Lindfors, K., Halttunen, T., Huotari, P., Nupponen, N., Vihinen, M., Visakorpi, T., Maki, M., and Kainulainen, H. (2000) Biochem. Biophys. Res. Commun. 276, 660-666[CrossRef][Medline] [Order article via Infotrieve]
  8. Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997) Nature 387, 733-736[CrossRef][Medline] [Order article via Infotrieve]
  9. Weigel, N. L. (1996) Biochem. J. 319 (Pt. 3), 657-667[Medline] [Order article via Infotrieve]
  10. Beato, M., and Klug, J. (2000) Hum. Reprod. Update 6, 225-236[Abstract/Free Full Text]
  11. Robyr, D., Wolffe, A. P., and Wahli, W. (2000) Mol. Endocrinol. 14, 329-347[Free Full Text]
  12. Rosenfeld, M. G., and Glass, C. K. (2001) J. Biol. Chem. 276, 36865-36868[Free Full Text]
  13. McKenna, N. J., and O'Malley, B. W. (2002) Cell 108, 465-474[CrossRef][Medline] [Order article via Infotrieve]
  14. Heery, D. M., Hoare, S., Hussain, S., Parker, M. G., and Sheppard, H. (2001) J. Biol. Chem. 276, 6695-6702[Abstract/Free Full Text]
  15. Leister, P., Burgdorf, S., and Scheidtmann, K. H. (2003) Signal Transduction 3, 17-25[CrossRef]
  16. Li, L., and Cohen, S. N. (1996) Cell 85, 319-329[CrossRef][Medline] [Order article via Infotrieve]
  17. Ruland, J., Sirard, C., Elia, A., MacPherson, D., Wakeham, A., Li, L., de la Pompa, J. L., Cohen, S. N., and Mak, T. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1859-1864[Abstract/Free Full Text]
  18. Krempler, A., Henry, M. D., Triplett, A. A., and Wagner, K. U. (2002) J. Biol. Chem. 277, 43216-43223[Abstract/Free Full Text]
  19. Lohrum, M. A., and Vousden, K. H. (2000) Trends Cell Biol. 10, 197-202[CrossRef][Medline] [Order article via Infotrieve]
  20. Li, L., Liao, J., Ruland, J., Mak, T. W., and Cohen, S. N. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 1619-1624[Abstract/Free Full Text]
  21. Koonin, E. V., and Abagyan, R. A. (1997) Nat. Genet. 16, 330-331[CrossRef][Medline] [Order article via Infotrieve]
  22. Ponting, C. P., Cai, Y. D., and Bork, P. (1997) J. Mol. Med. 75, 467-469[CrossRef][Medline] [Order article via Infotrieve]
  23. Sun, Z., Pan, J., Hope, W. X., Cohen, S. N., and Balk, S. P. (1999) Cancer 86, 689-696[CrossRef][Medline] [Order article via Infotrieve]
  24. Hittelman, A. B., Burakov, D., Iniguez-Lluhi, J. A., Freedman, L. P., and Garabedian, M. J. (1999) EMBO J. 18, 5380-5388[CrossRef][Medline] [Order article via Infotrieve]
  25. Watanabe, M., Yanagi, Y., Masuhiro, Y., Yano, T., Yoshikawa, H., Yanagisawa, J., and Kato, S. (1998) Biochem. Biophys. Res. Commun. 245, 900-905[CrossRef][Medline] [Order article via Infotrieve]
  26. Oh, H., Mammucari, C., Nenci, A., Cabodi, S., Cohen, S. N., and Dotto, G. P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5430-5435[Abstract/Free Full Text]
  27. Babst, M., Odorizzi, G., Estepa, E. J., and Emr, S. D. (2000) Traffic 1, 248-258[CrossRef][Medline] [Order article via Infotrieve]
  28. Bishop, N., Horman, A., and Woodman, P. (2002) J. Cell Biol. 157, 91-101[Abstract/Free Full Text]
  29. Myers, E. L., and Allen, J. F. (2002) J. Virol. 76, 11226-11235[Abstract/Free Full Text]
  30. Lohrum, M., and Scheidtmann, K. H. (1996) Oncogene 13, 2527-2539[Medline] [Order article via Infotrieve]
  31. Fields, S., and Sternglanz, R. (1994) Trends Genet. 10, 286-292[CrossRef][Medline] [Order article via Infotrieve]
  32. Treier, M., Staszewski, L. M., and Bohmann, D. (1994) Cell 78, 787-798[CrossRef][Medline] [Order article via Infotrieve]
  33. Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127[CrossRef][Medline] [Order article via Infotrieve]
  34. Feng, G. H., Lih, C. J., and Cohen, S. N. (2000) Cancer Res. 60, 1736-1741[Abstract/Free Full Text]
  35. Xie, W., Li, L., and Cohen, S. N. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1595-1600[Abstract/Free Full Text]
  36. Zhong, Q., Chen, Y., Jones, D., and Lee, W. H. (1998) Cancer Res. 58, 2699-2702[Abstract/Free Full Text]
  37. Refolo, L. M., Sambamurti, K., Efthimiopoulos, S., Pappolla, M. A., and Robakis, N. K. (1995) J. Neurosci. Res. 40, 694-706[CrossRef][Medline] [Order article via Infotrieve]
  38. Otten, A. D., Sanders, M. M., and McKnight, G. S. (1988) Mol. Endocrinol. 2, 143-147[Abstract]
  39. Lonard, D. M., Nawaz, Z., Smith, C. L., and O'Malley, B. W. (2000) Mol. Cell 5, 939-948[CrossRef][Medline] [Order article via Infotrieve]
  40. Salghetti, S. E., Caudy, A. A., Chenoweth, J. G., and Tansey, W. P. (2001) Science 293, 1651-1653[Abstract/Free Full Text]
  41. Wallace, A. D., and Cidlowski, J. A. (2001) J. Biol. Chem. 276, 42714-42721[Abstract/Free Full Text]
  42. Kang, Z., Pirskanen, A., Janne, O. A., and Palvimo, J. J. (2002) J. Biol. Chem. 277, 48366-48371[Abstract/Free Full Text]
  43. Thrower, J. S., Hoffman, L., Rechsteiner, M., and Pickart, C. M. (2000) EMBO J. 19, 94-102[CrossRef][Medline] [Order article via Infotrieve]
  44. Dilworth, F. J., and Chambon, P. (2001) Oncogene 20, 3047-3054[CrossRef][Medline] [Order article via Infotrieve]
  45. Svaren, J., Schmitz, J., and Horz, W. (1994) EMBO J. 13, 4856-4862[Medline] [Order article via Infotrieve]
  46. Tumbar, T., Sudlow, G., and Belmont, A. S. (1999) J. Cell Biol. 145, 1341-1354[Abstract/Free Full Text]
  47. Hu, Y. F., Hao, Z. L., and Li, R. (1999) Genes Dev. 13, 637-642[Abstract/Free Full Text]
  48. Hicke, L. (2001) Cell 106, 527-530[CrossRef][Medline] [Order article via Infotrieve]
  49. Dupre, S., Volland, C., and Haguenauer-Tsapis, R. (2001) Curr. Biol. 11, R932-R934[CrossRef][Medline] [Order article via Infotrieve]
  50. Katzmann, D. J., Babst, M., and Emr, S. D. (2001) Cell 106, 145-155[CrossRef][Medline] [Order article via Infotrieve]
  51. VerPlank, L., Bouamr, F., LaGrassa, T. J., Agresta, B., Kikonyogo, A., Leis, J., and Carter, C. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 7724-7729[Abstract/Free Full Text]
  52. Garrus, J. E., von Schwedler, U. K., Pornillos, O. W., Morham, S. G., Zavitz, K. H., Wang, H. E., Wettstein, D. A., Stray, K. M., Cote, M., Rich, R. L., Myszka, D. G., and Sundquist, W. I. (2001) Cell 107, 55-65[CrossRef][Medline] [Order article via Infotrieve]
  53. Nawaz, Z., Lonard, D. M., Smith, C. L., Lev-Lehman, E., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1999) Mol. Cell. Biol. 19, 1182-1189[Abstract/Free Full Text]
  54. Saji, S., Okumura, N., Eguchi, H., Nakashima, S., Suzuki, A., Toi, M., Nozawa, Y., Saji, S., and Hayashi, S. (2001) Biochem. Biophys. Res. Commun. 281, 259-265[CrossRef][Medline] [Order article via Infotrieve]
  55. Reid, G., Hubner, M. R., Metivier, R., Brand, H., Denger, S., Manu, D., Beaudouin, J., Ellenberg, J., and Gannon, F. (2003) Mol. Cell 11, 695-707[CrossRef][Medline] [Order article via Infotrieve]
  56. Grossman, S. R., Deato, M. E., Brignone, C., Chan, H. M., Kung, A. L., Tagami, H., Nakatani, Y., and Livingston, D. M. (2003) Science 300, 342-344[Abstract/Free Full Text]

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