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Division of Biochemistry, Faculty of Medicine, Campus Gasthuisberg, University of Leuven, B-3000 Leuven, Belgium
Address all correspondence and requests for reprints to: F. Claessens, Katholieke Universiteit Leuven, Campus Gasthuisberg, O/N, Here-straat 49, 3000 Leuven, Belgium. E-mail: frank.claessens{at}med.kuleuven.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Overexpression of SUMO-1 represses the AR-mediated transcription, and this effect is abolished after mutating both SUMO-1 acceptor sites. On the other hand, the mutation of lysine 385 clearly affects the cooperativity of the receptor on multiple hormone response elements. Lysine 511 is not implicated in this function. Surprisingly, these effects on cooperativity clearly depend on the nature of the response elements. When selective androgen response elements, which are organized as direct repeats of 5'-TGTTCT-3'-like sequences, were tested, the lysine 385 mutation did not increase the androgen response. Point mutations changing the direct-repeat elements into inverted-repeat elements restored the effects of the lysine 385 mutation on cooperativity. In conclusion, SUMO-ylation of the AR might have a differential function in the control of cooperativity, depending on the conformation of the AR dimer bound to DNA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The AR-mediated response involves the recruitment of coactivators of which the group of the p160 or NR-interacting proteins are the best studied (5). Steroid receptor coactivator-1 (SRC-1), human and rat transcription-intermediary factor 2 and its mouse ortholog glucocorticoid receptor-interacting protein 1 (GRIP1), and receptor-associated coactivator 3 belong to this group (6, 7, 8, 9). They interact with the NR-LBDs via highly conserved 
-helical LxxLL motifs, arranged in a centrally located NR-interacting region (5, 10). For the AR, however, a glutamine-rich region of SRC-1 (Qr) is the main interaction site for the AR-NTD (11, 12, 13).
The NTD of the AR is about 530 amino acids (aa) long and contains a strong hormone-dependent transactivation unit 1, called Tau-1, residing between aa 100–370. When the LBD is deleted, this activation domain shifts more C terminally and is called the autonomous transactivation unit (Tau)-5 (aa 360–529) (14, 15). A strong amino/carboxy (N/C)-interaction is necessary for AR-mediated activation on canonical but not selective androgen response elements (16, 17, 18).
The transcriptional activity of the NRs can be controlled, or at least modulated, by posttranslational modifications such as phosphorylation and acetylation (19, 20, 21, 22, 23). Another posttranslational modification is ubiquitination (24, 25). Covalent attachment of at least four ubiquitin molecules targets the substrates to the proteasome where they undergo degradation. Recently, a new posttranslational modification system was discovered, which resembles, but is distinct from, the ubiquitination system. It was called SUMO-1 (small ubiquitin-like modifier-1) modification or SUMO-ylation. The lysine residue where SUMO-ylation can occur resides in a consensus motif 
KxE where is a large hydrophobic residue, K the lysine of SUMO-1 attachment, x any amino acid, and E a glutamic acid (26, 27, 28, 29, 30). The conjugation pathway is mediated by three types of enzymes: an activating enzyme consisting of the Aos1/Uba2 dimer, a conjugation enzyme Ubc9, and ligation enzymes (26, 27, 28, 29, 30). Only one conjugating enzyme for SUMO-1 is known, but several SUMO-1 ligating enzymes have been discovered recently, e.g. the protein inhibitor of activated signal transducer and activator of transcription (PIAS) (31, 32). Moreover, both Ubc9 and PIASx/AR-interacting protein 3 have been demonstrated to interact with the AR (32).
A wide range of proteins are subject to SUMO-ylation, e.g. promyelocytic leukemia protein, inhibitor of nuclear factor 
B, p53-related p73 protein, PIAS proteins, and RanGap1 (26, 27, 28, 29, 30, 31, 32). Recently, several steroid receptors have also been reported to be conjugated with SUMO-1. The glucocorticoid receptor has three major SUMO-1 attachment sites, two of which are situated in the NTD and one in the LBD (33, 34). The PR can be SUMO-ylated in the NTD, and this modification is thought to regulate its autoinhibition and transrepression (35). The AR-NTD has two SUMO-1 consensus modification sites at positions 385 and 511 (36). In this paper, we analyze the SUMO-1 conjugation of the AR and its impact on AR-mediated transcriptional activity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We mutated one or both lysines of the SUMO-1 attachment sites in the hAR into arginines (K385R and K511R) and compared the SUMO-ylation efficiency to that of the wild-type (wt)AR in the presence of AR agonist methyltrienolone (R1881), pure AR antagonist hydroxyflutamide (OH-F), or partial AR antagonists medroxyprogesterone acetate (MPA) and cyproterone acetate (CPA). Clearly, both lysines are independent SUMO-1 acceptor sites, and the modification status of the AR depends on the nature of the ligand (Fig. 1
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, Flag-tagged hAR and mutant ARs were transiently expressed in COS-7 cells and coexpressed with c-myc-tagged SUMO-1 in the presence or absence of R1881 (10–8 M). When wild-type AR (wtAR) is coexpressed with SUMO-1, two major bands appear on Western blot in the absence of hormone (Fig. 1A, upper panel, lane 1). The fastest migrating band corresponds with the unmodified AR, whereas the slower band is dependent on coexpression with SUMO-1. As described by Poukka et al. (36), stimulation of the cells with R1881 enhances the SUMO-1 modification of the AR, whereby three bands appear (Fig. 1A, upper panel, lane 2). From immunoprecipitation with anti-Flag antibody and subsequent immunoblotting with anti-c-myc antibody, we can conclude that the slower migrating bands are the SUMO-ylated AR forms (Fig. 1A, lower panel). We assume that the middle band is explained by mono-SUMO-ylation, whereas the slowest band corresponds with di-SUMO-ylated AR. It is clear that these lysines are the only SUMO-1 conjugation targets in the wtAR because no modification is observed when both sites are mutated (K385R/K511R, lanes 7 and 8). When lysine 385 is mutated, mono-SUMO-ylation takes place only after stimulation with R1881 (lane 4 compared with lane 3). However, SUMO-ylation of the K511R construct already takes place in the absence of R1881 (lane 5) but is much more pronounced in the presence of the agonist (lane 6).
The SUMO-ylation pattern of the AR and AR mutants in the presence of 10–8 M OH-F is shown in Fig. 1B. The Western blots have been overexposed to detect low SUMO-ylation efficiencies. Clearly, SUMO-1 attachment to the wtAR or the K385R and K511R constructs is only very weakly enhanced by OH-F.
When the cells are treated with the partial antagonists MPA (Fig. 1C) or CPA (data not shown), SUMO-ylation of the AR constructs resembles the pattern obtained with agonist R1881.
Ubc9-Binding Sites in the hAR
Many of the proteins that can be SUMO-ylated interact with Ubc9, the SUMO-1 conjugating enzyme. The hinge region of the AR has been implicated in Ubc9 interaction (38). Therefore, we predicted that the deletion of the hinge region of the AR (AR
H) should affect the SUMO-ylation efficiency. However, the Western blot in Fig. 2A shows that the SUMO-1 pattern for both wtAR and ARH are superimposable. To identify the Ubc9-interacting part of the AR, two-hybrid assays were performed in COS-7 cells (Fig. 2, B and C). Surprisingly, no interaction is observed between Ubc9 and the DBD/H/LBD under conditions in which a good interaction is observed between the NTD and the DBD/H/LBD. We therefore looked for additional Ubc9 interaction sites in the AR. Coexpression of AR-NTD fused to the DBDGal4 domain with Ubc9 fused to the VP16 activation domain clearly shows that Ubc9 binds well to the AR-NTD (Fig. 2C). There is already a high luciferase activity measured in the presence of the AR-NTD alone because of a strong constitutive active activation domain. To verify this interaction and to analyze the interaction of AR with Ubc9 in vitro, we performed glutathione-S-transferase (GST) pull-down experiments. Bacterially expressed GST or GST-Ubc9, immobilized on glutathione-Sepharose beads, was incubated with in vitro translated and [35S]methionine-labeled hAR-NTD1–529 or deletions hAR-NTD1–360 and hAR-NTD360–529 (Fig. 2D). The first deletion construct still contains both SUMO-1 consensus motifs in contrast to the latter deletion construct, which lacks both sites. In this assay the hAR-NTD bound specifically to GST-Ubc9 but not to GST alone. Other than the wtAR-NTD, only the fragment of the NTD encompassing the Tau-5 domain and the SUMO-1 sites (hAR-NTD1–360) showed an interaction with Ubc9, whereas the mutant AR NTD360–529 is not able to interact with the conjugating enzyme.
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), we have analyzed the SUMO-ylation of the separated NTD of the AR (Fig. 3). Flag-tagged hAR-NTD or its mutants (NTDK385R, NTDK511R and NTDK385R/K511R) are transiently expressed in COS-7 cells and coexpressed with either c-myc-tagged SUMO-1 or SUMO-1mut. SUMO-1mut lacks the two carboxy-terminal glycines so that SUMO-1 modification does not occur. The extracts were immunoprecipitated with anti-Flag antibody and subsequently immunoblotted with anti-Flag (Fig. 3, upper panel) or anti-c-myc antibody (Fig. 3, lower panel). After cotransfection of the AR-NTD with SUMO-1, a slower migrating band appears, indicating that the AR-NTD, when tested in isolation, is also SUMO-ylated. We can only detect mono-SUMO-ylation at lysine 385 (Fig. 3, lane 6). However, when both Ubc9 and SUMO-1 are overexpressed with wtAR, we could detect di-SUMO-ylation (data not shown).
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FQNLF and ARG21E (Fig. 4), two mutants for which we know the N/C interaction is abolished (18). Immunoblotting transfected COS-7 cells with a monoclonal M2 anti-Flag antibody shows that the SUMO-1 pattern for ARFQNLF and ARG21E is the same as for wtAR and hence, SUMO-ylation of the AR is independent of the N/C-interaction.
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). COS-7 cells were transfected with expression vectors for wtAR or ARK385R/K511R in the presence of pSG5SUMO-1 or pSG5SUMO-1mut. As DNA probe, we used oligonucleotides covering the rat tyrosine aminotransferase (rTAT)-GRE sequence. The band shift assays showed no decreased binding of wtAR when cotransfected with SUMO-1. Also no difference in DNA binding is observed for the mutant ARs. The SUMO-ylation status of the AR in the extracts was verified by Western blot analysis (data not shown).
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). The extracts were obtained from COS-7 cells, transfected with expression vectors for non-Flag-tagged wtAR in the presence of Flag-tagged SUMO-1 or SUMO-1mut. Western blot confirmed the presence of SUMO-ylated wtAR (data not shown). Anti-Flag antibody induced a partial supershift of the retarded rTAT-GRE probe, indicating that SUMO-ylated AR indeed binds DNA (Fig. 5B, lane 5 compared with lane 3).
SUMO-1-Effect on the Transcriptional Activity of the AR
It has already been suggested that SUMO-1 modification negatively regulates the AR transactivation capacity (36). We analyzed this by cotransfecting wtAR or its mutants (K385R, K511R, and K385R/K511R) with an expression vector for either SUMO-1 or SUMO-1mut and the reporter construct 2xrTAT-GRE(E1b)-Luc (Fig. 6A). Indeed, the transcriptional activity of wtAR or the single mutants decreases with approximately 50% when SUMO-1 is coexpressed. The repressive effect of SUMO-1 for the double mutant (ARK385R/K511R), however, is much smaller. We further investigated this using several other hormone response elements (HREs) (Fig. 6B). As selective AREs, we used slp-HRE2 and sc-ARE1.2 (Table 1). As canonical AREs, we introduced mutations in slp-HRE2 (slp-HRE2 mut-4T-A; +2A-T) and sc-ARE1.2 (sc-ARE1.2 mut-4T-A; –2T-A), leading to a loss of selectivity of these elements (39). Here too, SUMO-1 overexpression leads to a decrease in AR activity.
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. Clearly, mutating lysine 511 does not affect the androgen responses.
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). For four copies of slp-HRE2, there is a more than 10-fold increase in wtAR activity compared with two copies (upper panel). Also a 5-fold higher androgen induction is observed for 4xsc-ARE1.2 compared with 2xsc-ARE1.2 (lower panel). However, in contrast to multiple copies of canonical AREs, mutating the SUMO-1 acceptor sites separately or together did not affect this synergistic effect.
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, the loss of specificity after mutation of the AR-specific HREs (slp-HRE2 mut-4T-A; +2A-T and sc-ARE1.2 mut-4T-A; –2T-A) indeed correlates with an increased transactivation by ARK385R and K385R/K511R in comparison with wtAR activity. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). SUMO-ylation of lysine 511 is agonist dependent. SUMO-ylation of lysine 385, although partly hormone independent, certainly is also a ligand-responsive event. Lysine 385 is the main site, but both lysines 385 and 511 can be SUMO-ylated independently from each other. In the presence of the pure antagonist OH-F, there is no SUMO-1 conjugation at lysine 511, nor enhanced SUMO-1 conjugation at lysine 385. Interestingly, we observed that the SUMO-ylation of the AR mutant T877A, seen in LNCaP cells, is comparable to that of wtAR (data not shown).
Most of the SUMO-1 protein targets interact with Ubc9, and it is likely that substrate recognition is achieved by Ubc9 (41, 42). For the AR, it has been suggested that the hinge region is implicated in Ubc9 interaction because it was isolated in double-hybrid screening with this region as a bait (38). Our assays, however, did not reveal clear interaction between Ubc9 and AR-DBD/H/LBD. Moreover, an AR in which the hinge region has been deleted is as efficiently SUMO-ylated as the wild-type receptor (Fig. 2A). In addition, SUMO-ylation assay of the AR-NTD in Fig. 3 confirms that SUMO-1 attachment at lysine 385 can happen in the absence of the hinge region. We concluded that Ubc9 must interact with the AR-NTD. It has been reported that SUMO-1 consensus motifs are not only necessary for the covalent binding of SUMO-1, but they can also serve as the site of interaction with Ubc9 (43). Indeed, from mammalian double-hybrid assays as well as GST-pull downs, we deduce that the major interaction site for Ubc9 in the AR is the Tau-5 constitutive active activation domain (Fig. 2). Remarkably, the two consensus motifs for SUMO-ylation that lie in Tau-5 are predicted to form a loop structure, which might fit in the catalytic cleft of Ubc9, as demonstrated in the RanGAP1-Ubc9 complex (44).
Ubc9 has been reported to be a potent coactivator of the AR (38), whereas in other studies Ubc9 was shown to enhance AR activity modestly on some reporter constructs but not on others (45). In our hands, cotransfection of low amounts of Ubc9 increased the AR activity only moderately on all constructs tested, but increasing amounts of Ubc9 lead to a repressive effect. Whether this correlates directly with the intrinsic transcription repressing functions of Ubc9 when fused to Gal4DBD is not clear (data not shown).
PIASx has been shown to function as a E3-type SUMO-1 protein ligase and enhances SUMO-ylation of the AR in vitro (31, 32). In COS-7 cells, we could not show enhanced AR-SUMO-ylation after cotransfecting the AR with PIASx, although a clear interaction of PIASx with ARDBD/H/LBD is seen in a two-hybrid assay (data not shown). This may be explained by the fact that SUMO-ylation of the AR is already optimal in COS-7 cells even in the absence of overexpressed PIASx. In functional assays, PIASx represses or activates AR activity on the canonical TAT-GRE and slp-HRE2 mut-4T-A;-2A-T respectively, whereas on the selective slp-HRE2, no effect is observed (data not shown). We therefore agree with literature that overexpressing the SUMO-ylation ligase PIASx can affect AR activity to different extents depending on the response elements tested (45, 46). We postulate that the PIASx-mediated effects are indirect because we could not see a correlation with the SUMO-ylation status of the AR.
We then examined the effect of SUMO-ylation on the transcriptional activity of the AR by coexpression of SUMO-1. The observed effect was dependent on the presence of one or both SUMO-1 acceptor sites (Fig. 6). Similar to Poukka et al. (36), SUMO-1, but not SUMO-1mut, has a negative effect on AR activity.
For the PR, the repression of the transcriptional activity by SUMO-ylation of its NTD requires the liganded LBD, suggesting that the N/C interaction is involved (35). For the AR, the ligand-dependent interaction of the LBD with the NTD is strongly agonist dependent, whereas OH-F, MPA, and CPA fail to induce N/C interaction (47). The SUMO-ylation pattern of the AR after stimulation with MPA and CPA resembles that after stimulation with agonist (R1881) and not after stimulation of antagonist OH-F (Fig. 1). It is therefore not surprising that in contrast to the PR, SUMO-ylation efficiency of the AR is not influenced by N/C interaction nor by the enhanced recruitment of the p160s, induced by the G21E mutation (Fig. 4) (18). Whether the ligand responsiveness of the SUMO-ylation is indirectly a result of a conformational change of the AR-NTD, induced by a ligand-occupied LBD, or whether other modulating proteins are recruited by the latter is still an open question.
One possible explanation for the observed reduction in AR transactivation by SUMO-ylation would be that SUMO-1 modification alters its DNA-binding ability. This has been demonstrated for heat shock transcription factor 2, a transcription factor that regulates heat shock protein gene expression. SUMO-1 attachment to heat shock transcription factor 2 converts this factor to the active DNA-binding form (48). However, the DNA-binding assays in Fig. 5 show that the reduced AR activity seen when SUMO-1 is coexpressed does not reduce the DNA binding. Indeed, the amount of retarded probe is even slightly higher when the AR is SUMO-ylated.
More recently, the p160 coactivators GRIP1 and SRC-1 have been shown to be SUMO-ylated at a site in the nuclear receptor interaction domain (49, 50). The group of Kotaja et al. (49) has shown that mutation of the SUMO-1 attachment sites in this domain of GRIP1 is correlated with a decreased colocalization of GRIP1 with the AR, a diminished coactivator capacity, and a diminished AR-LBD/GRIP1 interaction. It seems unlikely that such SUMO-ylation of GRIP1 or SRC-1 could be responsible for the decreased AR activity seen in our experiments, because the disruption of the SUMO-1 attachment sites (K385R/K511R) in the AR leads to reversal of the negative effects. It could, however, provide an explanation for the residual repression of the AR double mutant by overexpressed SUMO-1 on all AREs tested (Fig. 6, A and B).
The possibility that AR stability, and thus the outcome of these transfection experiments, is affected by SUMO-ylation was contradicted by the immunoblotting results, which revealed no increased proteolysis of the SUMO-1-modified AR and no change in steady-state levels (Fig. 1).
The SUMO-1 consensus modification sites of the GR overlap with the synergy control motifs (33, 40). Disrupting these motifs increases the transcriptional activity of the GR on promotors containing more that one hormone response element. Also the substitutions in the SUMO-1 acceptor sites affect AR activity on reporter constructs with multiple HREs (36). In our experiments, mutation of lysine 385 and the double mutation indeed lead to an increased activity on the reporter construct containing two copies of the rTAT-GRE, and this is even more pronounced when four copies are present (Fig. 7). It seems that lysine 385 plays an important role in this synergy control, whereas lysine 511 is not implicated in synergy.
We further characterized this synergy control in AR transactivation. From earlier experiments, we concluded that the AR transactivation mechanisms on canonical AREs differ from these on selective AREs, because the disruption of the N/C interaction or deletion of the glutamine repeat has a negative or positive effect on AR activity on canonical AREs whereas no change is seen on selective elements (18, 51). Here, we observed cooperativity of the AR on reporter constructs containing multiple selective motifs (Fig. 8A), but when the SUMO-ylation sites in the AR were mutated, no increase in transactivation was seen. It is difficult to compare the experimental data obtained after overexpression of SUMO-1 (Fig. 6), which will affect a multitude of factors, with those obtained when single SUMO-ylation sites are mutated (Fig. 8).
Clearly, lysine 385 is not acting as a synergy control element on selective AREs (Fig. 8A). However, when the selective AREs are mutated into canonical AREs, mutation of the SUMO-ylation sites again resulted in an increased synergy in the androgen response (Fig. 8B). This indicates that the underlying mechanism for cooperation and/or transcription activation and the role of SUMO-ylation in it on selective AREs might be different from that on canonical response elements.
In conclusion, we provide evidence that SUMO-ylation of lysines 385 and 511 is noncooperative, and independent from N/C interactions and the hinge region. We give evidence that Tau-5 of the AR-NTD is the main interaction site for Ubc9 rather than the AR hinge region. This is important because the hinge region is also involved in the recognition of selective AREs (2), and we report differences in the role of SUMO-ylation of lysine 385 in cooperativity on AR-selective vs. canonical elements. These observations must be taken into account in future experiments, e.g. on coactivators and corepressors. It has recently been suggested that both AR SUMO-ylation sites are involved in the binding of silencing mediator of retinoid and thyroid hormone receptor (52, 53) and SRC-1 to the AR-NTD (12, 13). The cell-specific levels of SUMO-1, Ubc9 and PIASx, corepressors, and coactivators, as well as the nature of the response elements, will determine the extent of the androgen responses. Future experiments will also have to direct the issues of the chronological order of events and the regulatory role of SUMO-ylation at the level of AREs integrated into chromatin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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FQNLF, pSG5AR-DBD/H/LBD538–919 and the fusion constructs NTD with VP16 or DBDGal4 are described elsewhere (18, 54). The point mutations K385R, K511R, and K385R/K511R were made by site-directed mutagenesis using the PCR-based method. The generated fragments were cloned into the pSG5(Flag)3 (expression of the full-size AR or AR-NTDs) or pABGal4 (generating AR-DBDGal4NTD fusions) vector. The expression vector for SUMO-1 and Ubc9 was a kind gift of Dr. A. Dejean (Unité de Recombinaison et Expression Génétique, Institut Pasteur, Paris, France). A c-myc-tagged or Flag-tagged SUMO-1 and SUMO-1mut (lacking the two C-terminal glycines) and the expression vector for flag-tagged ARH (hAR lacking the first 56 nucleotides of exon 4) were made by a PCR-cloning method. Similarly, Ubc9 was cloned into the GST expression vector, pGEX-5X-1 (Amersham Pharmacia Biotech, Arlington Heights, IL) and the VP16 expression vector pSNATCHII (15).
Restriction and modifying enzymes were obtained from MBI Fermentas GmbH (St. Leon-Rot, Germany). The luciferase reporter constructs containing the isolated elements TAT-GRE, slp-HRE2, sc-ARE1.2, slp-HRE2 mut-4T-A; +2A-T and sc-ARE1.2 mut-4T-A;–2A-T (Table 1) are driven by the TK minimal promotor or the E1b promotor and have been described elsewhere (Ref.41 and references herein). The pCMV-ßGal vector was obtained from Stratagene (La Jolla, CA).
Transfections
All transfections were performed in COS-7 African green monkey kidney cells, obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA). The cells were seeded in 96-well culture plates and transfected as described elsewhere (18). The amount of luciferase reporter construct was fixed at 100 ng per well, and the amount of pCMV-ß-Gal was fixed at 5 ng per well. After transfection, the cells were incubated for 24 h with medium containing 5% dextran-coated charcoal and supplemented or not with 10–8 M of the synthetic androgen R1881 (methyltrienolone) (PerkinElmer, Boston, MA), the antagonist OH-F (a kind gift of Dr. Neri, Schering Plough, Kenilworth, NJ), or the partial antagonist MPA (Sigma-Aldrich Corp., St. Louis, MO). After 24 h, the cells were lysed in 25 µl of passive lysis buffer (Promega Corp., Madison, WI). The luciferase and ß-galactosidase activities were measured in 2.5 µl of the extracts using the assay systems from Promega and Tropix (Westburg, The Netherlands), respectively. The luciferase activity in cell extracts was corrected for transfection efficiency by normalizing it according to the corresponding ß-galactosidase activity. The values shown are the averages of at least three independent experiments performed in triplicate. Error bars indicate the SEM values.
Preparation of COS-7 Whole-Cell Extracts
COS-7 cells were plated in six-well culture plates (6-cm Petri dishes for immunoprecipitation experiments) and were transiently transfected with 0.5 µg of Flag-tagged AR or AR mutants (full-size AR or AR-NTDs) and 1.0 µg of c-myc-tagged SUMO-1 or SUMO-1mut. At 24 h after transfection, cells were stimulated for 24 h with or without hormone. The cells were treated and lysed as described earlier (51).
Immunoprecipitation and Western Blots
For immunoprecipitation, each protein extract was incubated with anti-Flag M2 agarose beads (10 µl) for 2 h at 4 C. After centrifugation (1 min, 5000 rpm), the supernatant was removed, and the cells were washed three times with Tris-buffered saline (10 mM Tris-HCl, pH 8.0; 150 mM NaCl). The bound proteins were released from the beads in 2 x sodium dodecyl sulfate (SDS) sample buffer. For Western blotting, equal amounts of protein extracts were separated on a 6% or a 8% SDS-PAGE gel (for full-size AR or AR-NTDs, respectively) and blotted onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). The membranes were probed with a monoclonal M2 anti-Flag antibody (Stratagene) or with c-Myc antibody 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactive proteins were visualized with the chemiluminescence reagent plus (PerkinElmer) or with the chromogenic reagent for horseradish peroxidase detection (4CN reagent, PerkinElmer).
DNA-Binding Assays and Supershift Assays
Synthetic complementary oligonucleotides were hybridized, radioactively labeled, and used in band-shift assays as described previously (52). In brief, 15 µg of total cell extract were preincubated with 1 µl poly(dI:dC) (1 µg/µl), 10 µl D100 (20 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, 17% glycerol, 100 mM NaCl), 1 µl dithiothreitol (20 mM), 1 µl Triton X-100 (1%), and 1 µl of water. Subsequently, the probe is added and incubated for 20 min on ice. Bound probe was separated from the free by nondenaturing electrophoresis for 2 h at 120 V in a 5% polyacrylamide gel. To obtain supershifts, a rabbit antiserum against hAR (55) or the monoclonal M2 anti-Flag antibody was added before the probe.
Protein Expression and in Vitro Binding Assay
In vitro transcription and translation of full-size AR or AR fragments were performed in rabbit reticulocyte lysate in the presence of [35S]methionine in a total volume of 25 µl as described by the manufacturer (Promega Corp.). The in vitro translated proteins were diluted to 500 µl with binding buffer (20 mM Tris, pH 7.5; 150 mM NaCl; and 0.1% Tween 20). GST or GSTUbc9 was expressed in the BL21 bacterial strain and bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech). Nonspecific protein-binding sites were blocked by incubation with 2% BSA for 1 h at 4 C, and 50 µl of each in vitro translated protein were incubated with the beads in 250 µl of binding buffer for 30 min at room temperature. Beads were washed three times with binding buffer. Bound proteins were eluted with 2x SDS sample buffer. After SDS-PAGE electrophoresis, the gel was fixed in 10% acetic acid-25% isopropanol for 30 min, incubated in Amplify NAMP 100 (Amersham Pharmacia Biotech) for another 30 min, and dried; finally, labeled proteins were visualized by exposure to autoradiographic film (Hyperfilm ECL, Amersham Pharmacia Biotech).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: aa, Amino acids; AR, Androgen receptor; ARE, androgen response element; CMV, cytomegalovirus; CPA, cyproterone acetate; DBD, DNA-binding domain; GR, glucocorticoid receptor; GRIP, glucocorticoid receptor-interacting protein; GST, glutathione-S-transferase; hAR, human AR; HRE, hormone response element; LBD, ligand-binding domain; MPA, medroxyprogesterone acetate; NR, nuclear receptor; NTD, amino-terminal domain; PR, progesterone receptor; N/C, amino/carboxy; OH-F, hydroxyflutamide; PIAS, protein inhibitor of activated signal transducer and activator of transcription; SDS, sodium dodecyl sulfate; SRC-1, steroid receptor coactivator-1; Tau, transcription activation unit; SUMO-1, small ubiquitin-like modifier-1; TK, thymidine kinase; rTAT, rat tyrosine aminotransferase; wtAR, wild-type AR.
Received for publication August 19, 2003. Accepted for publication March 10, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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hinge region by p300 regulates transactivation and hormone sensitivity. J Biol Chem 276:18375–19383 function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription. J Biol Chem 277:41311–41317NURSA Molecule Pages Link:
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L. Davies, N. Karthikeyan, J. T. Lynch, E.-A. Sial, A. Gkourtsa, C. Demonacos, and M. Krstic-Demonacos Cross Talk of Signaling Pathways in the Regulation of the Glucocorticoid Receptor Function Mol. Endocrinol., June 1, 2008; 22(6): 1331 - 1344. [Abstract] [Full Text] [PDF]
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T. Bawa-Khalfe, J. Cheng, Z. Wang, and E. T. H. Yeh Induction of the SUMO-specific Protease 1 Transcription by the Androgen Receptor in Prostate Cancer Cells J. Biol. Chem., December 28, 2007; 282(52): 37341 - 37349. [Abstract] [Full Text] [PDF]
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 M. Geletu, M. Y. Balkhi, A. A. Peer Zada, M. Christopeit, J. A. Pulikkan, A. K. Trivedi, D. G. Tenen, and G. Behre Target proteins of C/EBP{alpha}p30 in AML: C/EBP{alpha}p30 enhances sumoylation of C/EBP{alpha}p42 via up-regulation of Ubc9 Blood, November 1, 2007; 110(9): 3301 - 3309. [Abstract] [Full Text] [PDF]
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 B. Ma, Y. Pan, J. Zheng, A. J. Levine, and R. Nussinov Sequence analysis of p53 response-elements suggests multiple binding modes of the p53 tetramer to DNA targets Nucleic Acids Res., May 14, 2007; 35(9): 2986 - 3001. [Abstract] [Full Text] [PDF]
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U. Moehren, M. Papaioannou, C. A. Reeb, W. Hong, and A. Baniahmad Alien Interacts with the Human Androgen Receptor and Inhibits Prostate Cancer Cell Growth Mol. Endocrinol., May 1, 2007; 21(5): 1039 - 1048. [Abstract] [Full Text] [PDF]
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 K. Schauwaers, K. De Gendt, P. T. K. Saunders, N. Atanassova, A. Haelens, L. Callewaert, U. Moehren, J. V. Swinnen, G. Verhoeven, G. Verrijdt, et al. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model PNAS, March 20, 2007; 104(12): 4961 - 4966. [Abstract] [Full Text] [PDF]
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 M. Vigodner, T. Ishikawa, P. N. Schlegel, and P. L. Morris SUMO-1, human male germ cell development, and the androgen receptor in the testis of men with normal and abnormal spermatogenesis Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E1022 - E1033. [Abstract] [Full Text] [PDF]
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 L. Callewaert, N. Van Tilborgh, and F. Claessens Interplay between Two Hormone-Independent Activation Domains in the Androgen Receptor Cancer Res., January 1, 2006; 66(1): 543 - 553. [Abstract] [Full Text] [PDF]
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S. Sentis, M. Le Romancer, C. Bianchin, M.-C. Rostan, and L. Corbo Sumoylation of the Estrogen Receptor {alpha} Hinge Region Regulates Its Transcriptional Activity Mol. Endocrinol., November 1, 2005; 19(11): 2671 - 2684. [Abstract] [Full Text] [PDF]
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C. Degerny, D. Monte, C. Beaudoin, E. Jaffray, L. Portois, R. T. Hay, Y. de Launoit, and J.-L. Baert SUMO Modification of the Ets-related Transcription Factor ERM Inhibits Its Transcriptional Activity J. Biol. Chem., July 1, 2005; 280(26): 24330 - 24338. [Abstract] [Full Text] [PDF]
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A. J. Miller, C. Levy, I. J. Davis, E. Razin, and D. E. Fisher Sumoylation of MITF and Its Related Family Members TFE3 and TFEB J. Biol. Chem., January 7, 2005; 280(1): 146 - 155. [Abstract] [Full Text] [PDF]
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 D.-Y. Lin, H.-I Fang, A.-H. Ma, Y.-S. Huang, Y.-S. Pu, G. Jenster, H.-J. Kung, and H.-M. Shih Negative Modulation of Androgen Receptor Transcriptional Activity by Daxx Mol. Cell. Biol., December 15, 2004; 24(24): 10529 - 10541. [Abstract] [Full Text] [PDF]
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