Terminal Domain of Miner

3 downloads 0 Views 480KB Size Report
... M.L.) and INSERM U409 (M.-C.L.), Institut Fe´de´ratif de Recherche Claude Bernard, Faculte´ ...... Kumar R, Thompson EB 2003 Transactivation functions.

0888-8809/03/$15.00/0 Printed in U.S.A.

Molecular Endocrinology 17(12):2529–2542 Copyright © 2003 by The Endocrine Society doi: 10.1210/me.2003-0299

Protein Inhibitor of Activated Signal Transducer and Activator of Transcription 1 Interacts with the NTerminal Domain of Mineralocorticoid Receptor and Represses Its Transcriptional Activity: Implication of Small Ubiquitin-Related Modifier 1 Modification LAURENT PASCUAL-LE TALLEC, OLIVIER KIRSH, MARIE-CHRISTINE LECOMTE, SAY VIENGCHAREUN, MARIA-CHRISTINA ZENNARO, ANNE DEJEAN, AND MARC LOMBE`S Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) Unite´ (U) 478 (L.P.-L.T., S.V., M.-C.Z., M.L.) and INSERM U409 (M.-C.L.), Institut Fe´de´ratif de Recherche Claude Bernard, Faculte´ de Me´decine Xavier Bichat, 75870 Paris cedex 18, France; and INSERM U579 (O.K., A.D.), Institut Pasteur, 75724 Paris cedex 15, France Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators. To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASx␤, described as SUMO (small ubiquitin-related modifier) E3-ligases. Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated. Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation. The agonist or antagonist nature of the

bound ligand also determines PIAS1 corepressive action. We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo. Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties. Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status. Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications. (Molecular Endocrinology 17: 2529–2542, 2003)

B

cular (4) systems are also responsive to aldosterone. MR binds both aldosterone and glucocorticoids with approximately the same affinity. Because cortisol has a 1000-fold higher concentration in plasma than that of aldosterone, the mineralocorticoid selectivity depends mainly on the enzymatic activity of 11␤-hydroxysteroid dehydrogenase type 2 (5, 6), which converts cortisol into inactive cortisone at a cellular level, resulting in the preferential action of aldosterone on MR. Furthermore, this selectivity is also conferred at a receptor level by ligand-induced conformational changes, which differ between gluco- and mineralocorticoids (7, 8), leading to different transactivation properties. The general structure of steroid receptors [type I nuclear receptor (NR) family] is highly conserved among all members of the family and is defined by separate domains possessing specific functions (9). They include a ligand-binding domain (LBD or D/EF), located in the C-terminal part of the receptor, a central

IOLOGICAL ACTIONS OF aldosterone are mediated through the mineralocorticoid receptor (MR), a member of the steroid receptor family (1). Polarized epithelial tissues, such as the distal nephron or colon, are considered to be the main targets of aldosterone, which regulates ion homeostasis mainly through modulation of ion channel expression (2). Nonepithelial tissues such as the central nervous (3) and cardiovasAbbreviations: AF, Activating function; 3-AT, 3-amino1,2,4 triazole; DBD, DNA-binding domain; DO-WLH, drop out mix without tryptophan, leucine, and histidine; DTT, dithiothreitol; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; GST, glutathione S-transferase; h, human; ID, inhibition domain; LBD, ligand-binding domain; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; NR, nuclear receptor; N-CoR, nuclear receptor corepressor; NTD, N-terminal domain; PIAS, protein inhibitor of activated STAT (signal transducer and activator of transcription); PMSF, phenylmethylsulfonylfluoride; SMRT, silencing mediator of retinoid and thyroid-hormone receptors; SUMO, small ubiquitin-related modifier; TIF, transcriptional intermediary factor.

2529

2530 Mol Endocrinol, December 2003, 17(12):2529–2542

DNA-binding domain (DBD or C), and a N-terminal domain (NTD or A/B). The DBD shares more than 90% homology between all steroid receptors whereas the LBD is usually less conserved (50–60%) even between closely related receptors, thus conferring hormone specificity. In contrast, the NTD is highly variable in length and amino acid sequence. Indeed, MR possesses the longest NTD described for all NRs with its approximately 600 amino acids encoded by a unique exon (10). This domain is very distinct from those of other steroid receptors NTDs, sharing less than 15% homology with that of the glucocorticoid receptor (GR), MR’s closest related receptor, but is highly conserved more than 85% among MRs of all known mammalian species. Thus, this MR NTD sequence may have important and specific functions conferring, in part, specificity for aldosterone action. In addition to the agonist-dependent activating function (AF2) domain located in the LBD, NRs have a constitutive AF1 transactivation domain located in the NTD. The relative contributions of AF1 and AF2 in mediating transcriptional activity vary among receptors. Maximal activity requires the synergistic activities of both AF1 and AF2 through their interaction with transcriptional coregulators (11). Coactivators act as adapter molecules of NRs that spatially recruit CREBbinding protein/p300 complexes and the general transcription machinery to promote transcription (12). They predominantly interact with NRs through LXXLL motifs (13) which are present in nearly all coactivators. Whereas numerous ligand-dependent or -independent GR coactivators or corepressors are now characterized (14–19), very little is known about MR coregulators. GR-interacting protein 1 (15), transcriptional intermediary factor 1␣ (TIF1␣), and steroid receptor coactivator-1 (20) have been implicated in the AF2 aldosterone-dependent transactivation. With respect to AF1 constitutive activity, we showed the functional interaction of steroid receptor coactivator-1e and TIF1␣ coactivators on a naturally occurring LBDdeleted hMR mutant (21). Other reports indicate that the rat MR AF1 domain indirectly interacts with TIF2 (22) and is able to recruit the RNA helicase A CREBbinding protein complex in a ligand-specific manner (23). To date, no MR corepressor has been clearly identified even though SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) are likely to participate in its repression. All these coregulators influence different steroid receptor functions, but no specific MR partner has been identified thus far. Given the unique MR NTD characteristics, it was tempting to postulate that it could play a key role in mineralocorticoid signaling by recruiting specific coactivators or corepressors. In the present paper, we used hMR NTD as bait in a yeast two-hybrid approach to identify specific molecular partners. PIAS family proteins, recently identified as SUMO (Small Ubiquitin related Modifier) E3-ligases (24), were isolated as hMR NTD interacting proteins. In our cellular models, under

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

specific experimental conditions, PIAS1 acts as a specific repressor of hMR-mediated transactivation in a ligand-dependent manner. We further explored the molecular mechanisms of this repression by assessing the implication of hMR sumoylation modifications.

RESULTS Characterization of AF Domains in hMR NTD Recent papers reported that the MR N-terminal region encompasses one or two activation domains (AFs) responsible for ligand-independent transactivation function (22, 25). However, the presence and exact location of these domains are still controversial. Before two-hybrid screening and bait elaboration, we investigated N-terminal hMR secondary structures by computer predictive studies [Network Protein Sequence Analysis (pbil.ibcp.fr/NPSA)]. Analysis revealed a very well organized structure in the aminoterminal end with multiple repeats of ␣-helices and ␤-strands, a central unfolded domain, and a less organized structure in the carboxy-terminal end (Fig. 1, top of panel A), all of which is in accordance with the initial description of the structure proposed for the rat MR NTD (22). We then decided to use the domains presented in Fig. 1A. These constructs were cloned into yeast (pLexA) and mammalian (pcDNA3.1) expression vectors and tested for correct expression (Fig. 1B). We investigated the ability of N-terminal subdomains to autonomously transactivate HIS and LacZ reporter genes by a one-hybrid approach. Figure 1C shows that the 1–167 and 445–602 fragments fused to LexA-DBD permitted yeast growth on selective medium [drop out mix without tryptophan, leucine, histidine (DO-WLH)], which was completely inhibited by 60 mM 3-amino-1,2,4 triazole (3-AT) (data not shown). The 1–602 and particularly the 163–602 fragments supported only moderate growth which was inhibited by 20 mM 3-AT. In contrast, the 163–437 fragment was completely unable to promote yeast cell growth. These results were quantitatively assessed by ␤-galactosidase assays (Fig. 1D). Of note, the 163–602 fragment retained only 20% of ␤-galactosidase activity induced by the 445–602 fragment, suggesting that the 163–437 part of the 163–602 fragment could negatively modulate transactivation mediated by the 445–602 fragment. Taken together, these results indicate that the NTD of hMR is composed of both a distal (1–167) and a proximal (445– 602) activation domain (AF) and probably a central (163– 437) inhibition domain (ID). Identification of PIAS Proteins as hMR N-Terminal Molecular Partners The full-length NTD of hMR spanning from amino acid 1–602 was used as bait for two-hybrid screening of a human kidney library. Because this fragment possesses strong intrinsic activation properties, 60 mM

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Mol Endocrinol, December 2003, 17(12):2529–2542

2531

Fig. 1. Characterization of hMR N-Terminal Subdomains A, A schematic representation of hMR domains. The top of the panel represents the predictive secondary structure of hMR NTD (tall bar, ␣-helix; short bar, ␤-strand) obtained by Network Protein Sequence Analysis (pbil.ibcp.fr/NPSA). B, An in vitro translation of the NTD fragments separated onto 10% SDS-PAGE. C, NTD fragments of hMR fused to the LexA DBD protein were evaluated for their autonomous transcriptional activity using yeast-mating tests between a L40 strain expressing the NTD constructs-LexA DBD or empty LexA DBD fusion proteins, and a AMR70 strain expressing empty GAL4 activation domain protein performed on Trp⫺, Leu⫺, His⫺ medium (DO-WLH). Two different protein domains known to interact together were used as a positive control (⫹). D, Liquid ␤-galactosidase assays of the same L40 yeast strain grown in liquid Trp⫺ medium (drop out mix without leucine). Results are expressed as means ⫾ SD of triplicate assays.

3-AT was added to reduce background. One hundred clones displaying evident growth advantage over the background on 3-AT medium during 5 d were isolated. ␤-Galactosidase assays on filters revealed 100% positive clones as expected. To eliminate false-positive clones, specificity tests were performed between yeast L40 strain containing the different N-terminal fragments and AMR70 strain containing different purified plasmids. At this stage, five clones were retained and further characterized. Two of them encoded for a 2.2-kb mRNA perfectly homologous to hPIAS1 mRNA except for the first four amino acids, which were deleted. Because the 163–437 fragment gave no detectable background, a second screening was performed in the same library using amino acid 163–437 as bait. Once again, this repetitively allowed us to isolate hPIAS1, hPIASx␤, and Ubc 9, providing additional support for the fact that PIAS family proteins interact with hMR NTD. Characterization of the PIAS1-hMR Protein Interactions To delineate the hMR N-terminal subdomains responsible for the interaction with PIAS1, we used a yeastmating strategy to test different fragments. Figure 2A

shows comparative growths of yeast expressing different fragments of the hMR NTD alone (pACT2-Gal4-AD) or with PIAS1 (pACT2-Gal4-AD-PIAS1) on highly selective medium (DO-WLH with 60 mM 3-AT). The 1–602 and 163–437 fragments alone were unable to promote yeast growth, whereas the two other fragments, previously demonstrated to harbor AFs, did yield basal growth. In contrast, all N-terminal subdomains were able to interact with PIAS1, promoting massive cell growth over basal. This was quantitatively confirmed by ␤-galactosidase assays (Fig. 2B) performed in drop out mix without leucine medium, suggesting that PIAS1 physically interacts with different parts of the NTD of hMR. Glutathione-S-transferase (GST) pull-down experiments were performed to confirm the physical interaction between the NTD of hMR and PIAS1 biochemically. GST-fused proteins were expressed in bacteria, purified with glutathione Sepharose, and incubated with in vitro translated [35S]Met-labeled putative protein partners. Figure 2C shows that the GST 1–602 fused protein interacts with PIAS1 (middle panel), whereas the nonrelevant luciferase protein used as control was unable to bind the hMR NTD (left panel). Reciprocal pull-down experiments performed with GST-PIAS1 fused protein and in vitro translated NTDs further confirmed the hMR-

2532

Mol Endocrinol, December 2003, 17(12):2529–2542

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Fig. 2. PIAS1 Interacts with hMR N-Terminal Subdomains A, Semiquantitative representation of yeast growth. L40 yeast strain expressing the different NTD-LexA fusion proteins was mated with AMR70 yeast strain expressing empty GAL4 AD or GAL4-PIAS1 fusion protein and grown on selective medium. B, The same yeast strains either expressing (black bars) or not expressing (white bars) PIAS1 were grown in a selective liquid medium and used for ␤-galactosidase assays. Results are expressed as means ⫾ SD of triplicate assays. Numbers indicate the fold increase over basal in the absence of PIAS1. Panels C and D illustrate biochemical hMR-PIAS1 interactions by GST pull-down experiments. PIAS1 (C, middle panel) and 1–602 hMR (C, right panel) were labeled with [35S]methionine by in vitro translation and incubated with glutathione sepharose-bound GST-1–602 and GST-PIAS1, respectively. After extensive washes, bound proteins were eluted in sample buffer and separated onto 10% SDS-PAGE. In vitro translated luciferase protein was used as a control (C, left panel). D, Determination of the hMR N-terminal subdomains responsible for PIAS1 interaction using GST-NTD fusion proteins and in vitro translated PIAS1 or luciferase as a control.

PIAS1 interaction (right panel). In addition, different fragments were tested to determine which N-terminal subdomains were involved in the physical interaction with PIAS1. To this end, GST-fused N-terminal fragments were incubated with in vitro translated [35S]Met-labeled PIAS1 or luciferase as a control (Fig. 2D). All five Nterminal subdomains were equally able to specifically interact with PIAS1. In conclusion, both yeast-mating strategy and GST pull-down experiments indicated that multiple contacts exist between PIAS1 and hMR NTD. PIAS1 Corepression of hMR-Mediated Transactivation To examine the functional significance of the PIAS1hMR interaction, the ability of PIAS1 to modulate hMR-

mediated transactivation of a murine mammary tumor virus (MMTV) promoter driving luciferase gene expression was tested by transient cotransfections in a rabbit renal RC.SV3 cell line. Figure 3A shows that PIAS1 was able to inhibit, by approximately 50%, aldosterone-induced transactivation, whereas it had no effect on basal hMR activity in the absence of hormone. Figure 3B (left panel) demonstrates that PIAS1 significantly repressed hMR-mediated transactivation in a dose-dependent manner (from 1:1 to 1:5 hMR-PIAS1 expressing vectors ratio). In parallel experiments (Fig. 3B, right panel), Western blot analysis with an antiFlag antibody shows that PIAS1 cotransfection (from 1:1 to 1:5 hMR-PIAS1 plasmid ratio) did not modify the total Flag-hMR protein levels. Corepressive activity of

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Mol Endocrinol, December 2003, 17(12):2529–2542

2533

PIAS1 was further evaluated on various corticosteroid receptors (Fig. 3C). As compared with the 50% inhibition of the full-length hMR activity, PIAS1 similarly repressed the activity of hMR-deleted mutant N454 (lacking the first 453 N-terminal amino acids), suggesting that PIAS1-repressive effects require neither the AF1a nor the ID of hMR even though it interacts with these domains. In contrast, PIAS1 had only a minor inhibitory effect on the activity of hMR mutant ⌬5,6 lacking the entire LBD. The effects of PIAS1 were hMR specific because dexamethasone-stimulated hGR transactivation properties remained unchanged under the same experimental conditions. Similar results were observed in the HK human renal cell line (data not shown). Given that PIAS1 impaired hMR transcriptional properties in a ligand-dependent manner, we evaluated whether the nature of the bound ligand affected its repressive activity. Dose-response experiments showed that PIAS1 did not modify the EC50 of hMR for aldosterone (5.10⫺11 M), with an approximately constant 50% inhibition on hMR transactivation from 10⫺11 to 10⫺7 M aldosterone (Fig. 4A). Similarly, PIAS1 inhibits the response of hMR to cortisol by 50% from 10⫺10 to 10⫺6 M with an unchanged EC50 (5.10⫺10 M) (Fig. 4B). In contrast, PIAS1 had no effect on the partial agonist activity of spironolactone, a mineralocorticoid antagonist (Fig. 4C). These results suggest that an appropriate and specific ligand-dependent conformational change of the LBD is required for PIAS1 to repress hMR activity. Fig. 3. PIAS1 Acts as a hMR-Specific Corepressor The functional effects of PIAS1 were evaluated by transient cotransfections in the rabbit kidney RC.SV3 cell line. A, Basal and aldosterone-induced hMR (0.1 ␮g pcDNA3.hMR) transactivation with (black bars) or without (white bars) PIAS1 (0.5 ␮g pcDNA3.1-PIAS1). Results are expressed as relative transcriptional activity (Luc/␤-gal activity). B (Left panel), Cells were cotransfected with hMR (0.1 ␮g pcDNA3.hMR) and increasing amounts of pcDNA3.1-PIAS1 plasmid (0.1, 0.2, and 0.5 ␮g), and then treated with 10⫺8 M aldosterone for 18 h. Relative transcriptional activities were expressed as a percentage of the control (same amount of pcDNA3 plasmid). Right panel, HeLa cells were transiently cotransfected with FlaghMR plasmid (0.1 ␮g) in the absence or the presence of increasing amount of PIAS1 plasmid, (0.1, 0.2, and 0.5 ␮g). Cells were recovered 24 h after transfection, and total proteins were separated onto SDS-PAGE. Western blot was performed with mouse anti-Flag antibody (M2, Sigma) and anti-␣-tubulin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for control of protein loading. C, Transient cotransfections were carried out with 0.1 ␮g pcDNA3.hMR, 0.5 ␮g pcDNA3.N454-hMR, 0.33 ␮g pcDNA3.⌬5,6-hMR, or 0.1 ␮g pRS.hGR, in the presence of pcDNA3 (white bar) or pcDNA3.1-PIAS1 (black bar) plasmids in a 1:5 ratio. All transfections were performed along with 0.33 ␮g pSV.␤-Gal and 1 ␮g pFC31-MMTV-Luc plasmids. Efficiencies were normalized with ␤-galactosidase activities, and results were expressed as a percentage of the control. All results represent the mean ⫾ SD of three independent experiments performed in triplicate. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. RLU, Relative light units.

PIAS1 Activity and CoRNR Motifs Although PIAS1 was initially described as a steroid receptor coactivator, we have established its ability to repress hMR transcription. We thus sought to determine which mechanisms mediate this repression. Based on N-CoR and SMRT corepressor structure analysis, a NR/repressor interface, named CoRNR box, has been characterized in corepressor amino acid sequences (26). Interestingly, consensus motifs (I/L)XX(I/V)I were found in the C-terminal end of some (but not all) members of the PIAS protein family. Figure 5A shows C-terminal end alignments of PIAS proteins. Both human and mouse PIAS1 encompass two overlapping CoRNR boxes, whereas human PIASx␤ and its mouse homologous miz1 contain only one. PIAS3, however, lacks the motif in which an isoleucine to arginine amino acid substitution occurs. PIASx␣ and PIASy completely diverge in their C-terminal end and do not possess any CoRNR motifs. Therefore, we addressed whether CoRNR boxes could be involved in PIAS1 repressive action. A PIAS1 mutant deprived of the two C-terminal CoRNR boxes (the last 641–651 amino acids were deleted as represented in the bottom of Fig. 5A and named hPIAS1-ECoRNR) was generated and its effects tested on hMR transactivation of a MMTV promoter. The ECoRNR mutant was equally able to inhibit hMR transactivation to the same extent

2534

Mol Endocrinol, December 2003, 17(12):2529–2542

Fig. 4. PIAS1 Represses hMR Transactivation Activity in a Ligand-Dependent Manner Transient cotransfections were carried out in RC.SV3 kidney rabbit cell line as described in legend of Fig. 3. Cells were treated for 24 h with increasing concentrations of aldosterone (A), cortisol (B), or spironolactone (C) in a steroid-free medium. Transfection efficiencies were normalized with ␤-galactosidase activities, and results were expressed as percentage of the maximal activity. Results represent the mean ⫾ SD of three independent determinations.

as the full-length PIAS1 (Fig. 5B) excluding CoRNR motifs as a primary explanation for PIAS1 repressive action. Modification of hMR by SUMO-1: The Role of PIAS Proteins It has been recently described that the PIAS family proteins exhibit SUMO ligase activities. To explore the molecular mechanisms of PIAS action on hMR, sumoylation processes were investigated. Figure 6A shows that hMR displays several ⌿KXE consensus sumoylation sites, previously described as synergy control motifs by Iniguez-Lluhi et al. (27). Five lysine acceptor sites are present in the hMR sequence: four

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Fig. 5. CoRNR Motifs in PIAS Family Proteins and Their Role in PIAS1 Corepressive Action A, Human PIAS1 protein (651 amino acids) possesses three LXXLL motifs (two are inverted) or NR boxes (large bars) in its NTD. Two CoRNR motifs (stars) are present in the extreme C-terminal end. PIAS1 also has a zinc finger domain (MizZnF), a RING domain responsible for SUMO-ligase activity, a SID domain involved in STAT interaction, and a SAP domain. The bottom of the panel represents the alignment between amino acids in the C-terminal ends of different proteins within the PIAS family. The hooks or brackets represent CoRNR motifs, which are defined as (I/L)XX(I/V)I amino acid sequences (bold). B, Corepressive action of PIAS1 and ECoRNR-PIAS (0.5 ␮g) on hMR (0.1 ␮g)-mediated transactivation (aldosterone 10⫺8 M) of MMTV and GRE2 promoters. Results are expressed as a percentage of the control (0.5 ␮g pcDNA3) and represent the mean ⫾ SD of two independent experiments performed in triplicate. ***, P ⬍ 0.001. NS, Not significant (P ⱖ 0.05).

in the NTD at positions K89, K399, K428, and K494 (named K1, K2, K3, and K4, respectively) and one in the LBD at position K953 (named K5). The ability of hMR to undergo SUMO-1 modifications was analyzed by an in vitro sumoylation assay previously described (28). Figure 6B (left panel) shows that in vitro translated [35S]Met-labeled hMR, incubated with HeLa fraction providing SUMO-1 activating E1 activity and 50 ng recombinant Ubc9, is modified in the presence of SUMO-1, as demonstrated by the apparition of several conjugated forms. It could not be excluded that multiple/poly-SUMO modifications take place in hMR. As would be expected given the location of the consensus SUMO-1 binding motifs, each N-terminal subdomain tested was able to undergo SUMO-1 modifica-

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Fig. 6. PIAS Family Proteins Are Responsible for in Vitro SUMO-1 Modification of hMR A, Localization and sequence of SUMO consensus sites in hMR. B, In vitro translated [35S]-labeled full-length and NTD fragments of hMR were incubated for 1 h at 37 C in a sumoylation mix containing a fraction of HeLa cells providing the E1 activity together with recombinant Ubc9 and ATP, either in the presence (⫹) or in the absence (⫺) of recombinant His-SUMO-1GG. Asterisks show conjugated forms. C, SUMO E3-ligase activity of PIAS proteins was tested on in vitro translated [35S]-labeled hMR incubated for 1 h at 37 C with bacterially expressed E1 (His Aos1 and Uba2) and E2 (Ubc9) enzymes, with or without recombinant His-SUMO-1GG and PIAS proteins. Samples were separated by SDS-PAGE and analyzed by autoradiography.

tion in vitro (right panel), as evidenced by the appearance of higher molecular mass hMR bands. We next evaluated the involvement of different PIAS proteins as specific SUMO E3-ligases for hMR. Figure 6C shows an in vitro sumoylation assay using recombinant E1 (100 ng), E2 (50 ng), SUMO-1 (1 ␮g), and various recombinant PIAS proteins as SUMO E3ligases. hMR sumoylated forms appeared using 180 ng hPIAS1, hPIASx␣, hPIASx␤, and hPIASy but not with mPIAS3. Other experiments focusing on PIAS protein sumoylation capacity in the presence of HeLa fraction showed that hPIAS1 and hPIASy acted as the most potent SUMO E3-ligases for hMR (data not

Mol Endocrinol, December 2003, 17(12):2529–2542

2535

Fig. 7. In Vivo Sumoylation of hMR A, Detection by immunofluorescence of Flag-MR and FlagK12345 in HeLa cells. B, In vivo sumoylation of wild-type and K12345 Flag-MRs in HeLa cells. Cells were recovered 24 h after transfection and aldosterone treatment, and total proteins were separated onto SDS-PAGE. Western blot was performed with mouse anti-Flag antibody. C, Anti-Flag Western blot after nickel purification of His-SUMO proteins from HeLa cell extracts transfected with the indicated plasmids.

shown). PIAS1 and PIASx␤ behave as SUMO E3ligases responsible for SUMO modification of hMR in vitro in accordance with their identification as hMR interacting proteins in the yeast two-hybrid system. In vivo sumoylation of the receptor was then evaluated in HeLa cells transiently cotransfected with plasmids encoding for Flag-hMR or Flag-K12345 (hMR mutated for the five lysine acceptor sumoylation sites, see below) together with SUMO-1 expression vectors. Both wild-type and K12345 hMRs were correctly localized in the nucleus upon aldosterone exposure as demonstrated by immunofluorescence detection (Fig. 7A) and expressed to a similar extent as evidenced by Western blotting of total cell extracts with an anti-Flag antibody (Fig. 7B). Of interest, high-molecular weight forms of hMR were observed when SUMO-1 was coexpressed whereas these modified forms were barely detected with the Flag K12345 hMR mutant (Fig. 7B). To confirm the in vivo sumoylation of hMR, His-SUMO proteins were purified on nickel agarose beads and analyzed by Western blot. As presented in Fig. 7C,

2536

Mol Endocrinol, December 2003, 17(12):2529–2542

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Fig. 8. Influence of Lysine Substitutions on hMR Sumoylation and Transcriptional Activity Transient transfections were performed as described in Fig. 3. Upper panel shows wild-type and mutant hMR transactivation of 1 ␮g pFC31-MMTV-Luc; lower panel shows that of 0.15 ␮g pGL3-GRE2-Luc. All results are expressed as a percentage of maximal transcriptional activity obtained with wild-type hMR (10⫺8 M aldosterone) and represent the mean ⫾ SD of three independent experiments performed in triplicate. The bottom of the figure shows in vitro sumoylation of hMR (wild type and lysine mutants) by HeLa extracts as described in Fig. 6B.

His-SUMO-conjugated forms of hMR migrating at approximately 175 kDa were observed (lane 1); however, no hMR was detected when SUMO-1 expression vector was cotransfected (lane 2). Interestingly, when PIAS1 was overexpressed, a strong increase in HisSUMO conjugated forms of hMR was noted (lane 3) consistent with the SUMO E3-ligase activity of PIAS1 previously demonstrated in vitro. Finally, as expected, no in vivo sumoylation occurred with the Flag-K12345 hMR (lanes 4–6). Taken together, our results demonstrate that hMR undergoes both in vitro and in vivo SUMO modifications by PIAS1. Functional Role of hMR Sumoylation Acceptor Sites To decipher the regulation of hMR activity by sumoylation, the respective contributions of the five lysine acceptor sites of MR were evaluated. For this purpose, the SUMO acceptor sites were turned off by mutating lysine (K) into arginine (R). Single or multiple additive K to R mutations of hMR were analyzed both biochem-

ically and functionally. As shown in Fig. 8 (lower panel), single mutations had no significant effect on hMR SUMO modifications in vitro compared with the wild type, whereas hMR mutants progressively lost their ability to be SUMO-1 modified as a function of additional lysine mutations from K12 to K12345. Indeed, the K1234 and K12345 mutants were not modified by sumoylation (lower panel) in accordance to the in vivo data (Fig. 7C). The transactivation properties of hMR mutants were then tested on the MMTV-Luc promoter. All hMR mutants displayed the same aldosteroneinduced transactivation ability compared with the wild type regardless of the number, position, or combination of K mutations (Fig. 8, upper panel). This unexpected result led us to investigate the influence of the promoter context on MR transactivation functions. In contrast, when another promoter [glucocorticoid response element 2 (GRE2)-Luc] was used, hMR mutants exhibited enhanced transcriptional activities compared with the wild type. No single mutations altered transactivation except for K2, which significantly increased it by 1.8-fold as compared with the

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

Fig. 9. Repressive Action of PIAS1 on Transcriptional Activity of Lysine Mutant hMRs PIAS1 action on wild-type or lysine mutants hMR was evaluated in a MMTV or GRE2 promoter context. Results are expressed as a percentage of aldosterone-induced (10⫺8 M) hMR transactivation in the absence of PIAS1 set at 100%. Results represent the mean ⫾ SD of three independent experiments performed in triplicate. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001 (Student’s t test). NS, Not significant (P ⱖ 0.05).

wild type. Additive mutations, however, resulted in 1.8-, 2.0-, 2.5-, and 3.2-fold increases in transactivation for K12, K123, K1234, and K12345, respectively (Fig. 8, middle panel). Thus, a dual sumoylation impact on hMR transcriptional function with GRE2 and MMTV promoters clearly appeared. Along this line, we compared the effects of PIAS1 on hMR mutants in both MMTV and GRE2 promoter contexts (Fig. 9). Of interest, PIAS1 retained its repressive activity on wild-type and K mutated hMR transactivation with the MMTV promoter. In contrast, in the context of GRE2, PIAS1 repressive action, although weaker than that observed in MMTV, was progressively impaired with addition of K mutations to be completely abolished with the K12345 mutant. Taken together, our data of hMR sumoylation mutants and the ability of PIAS1 to repress their transcriptional activity illustrate the crucial importance played by the promoter context.

DISCUSSION All steroid receptors contain two separated domains involved in transactivation functions: a ligand-dependent activation function (AF2) located in the C-terminal domain and a constitutive activation function (AF1) in the NTD. We have recently emphasized the importance of the hMR NTD with the identification of an hMR spliced variant that lacks the complete LBD but acts as a receptor with AF1 constitutive transcriptional activity (21). Initial description of the MR NTD reported two divergent AF1 organizations. Govindan et al. (25)

Mol Endocrinol, December 2003, 17(12):2529–2542

2537

defined the presence of a unique AF1 domain localized between amino-acids 328–382 in the central part of the hMR NTD (25) resembling the ␶1 domain of hGR (29). Recently, Fuse et al. (22) described a different organization for the rat MR NTD, with two separated AF1 regions similar to that described for hPR-B (30) and h/rAR (31, 32). In accordance with the findings obtained in the mammalian system (22), our onehybrid results clearly demonstrate that hMR possesses two distinct functional regions mapping to fragments 1–167 and 445–602, both of which are able to transactivate HIS and LacZ reporter gene expression and should thus be considered as AF1-a and AF1-b hMR subdomains. Therefore, the proposition of an AF1 located in 328–382 amino acids (25) is very unlikely because the central 163–437 fragment completely lacks transactivation properties in our experimental conditions. Further investigations are required to precisely delineate the residues implicated in transactivation functions. We also propose that the 163– 437 region possesses an intrinsic inhibitory function because it strongly reduces transcriptional activity directed by the 445–602 fragment as suggested by previous studies (22). Taken together, our results indicate that the structural organization of the hMR NTD is more closely related to that of the hPR-B (30) than that of the hGR (29). Two-hybrid screenings of a human kidney library with the full-length hMR NTD allowed us to identify PIAS1 as a protein that interacts with this fragment. The corepressive effects observed for PIAS1, together with our delineation of an ID within the NTD, prompted us to screen the same library again with the 163–437 fragment. PIAS1 and PIASx␤ were then repetitively isolated, highlighting the importance of PIAS family proteins as hMR NTD molecular partners. Additional experiments demonstrated that other PIAS family members (PIASx␣ and PIASy) were also fully competent to inhibit hMR transcriptional activity (data not shown). Subsequent GST pull-down experiments showed that PIAS1 physically interacts with the hMR NTD through multiple contact sites. This could be related to the four SUMO attachment consensus sequences within the NTD. The fact that each NTD fragment tested for physical interaction contains at least one functional site (see Fig. 6B) suggests that sumoylation catalyzed by PIAS1 occurs within the close vicinity of the PIAS1-hMR interfaces. PIAS1, originally cloned as an inhibitor of STAT-1 activated protein (33), has been recently described as a coactivator for androgen receptor (34). Since then, its role has been extended to other steroid receptors including GR and progesterone receptor (34–36). Indeed, PIAS1 can act as a coactivator for androgen receptor and GR, but in other contexts, PIAS could also exert partial corepressive effects on steroid receptors. The balance between coactivation and corepression seems to be extremely variable according to NR and its level of expression, the promoter, and the cellular contexts (36, 37). This is the first description of

2538 Mol Endocrinol, December 2003, 17(12):2529–2542

an MR corepressor that further discriminates the inhibition of MR-mediated transactivation from that mediated by GR. This specific action of PIAS1 on hMR may be considered as an additional molecular mechanism for mineralocorticoid selectivity by down-regulating the MR pathway without affecting GR-dependent signaling. A number of lines of evidence strongly suggest that the full corepressive activity of PIAS1 requires an appropriate LBD conformation: 1) PIAS1 represses hMR transactivation properties in a ligand-dependent manner; 2) PIAS1 action on MR is influenced by the nature of the bound ligand which is known to induce specific agonist/antagonist compaction of the LBD necessary for interaction with the transcription initiation complex and/or its stabilization (8, 38); 3) PIAS1 has very little effect on hMR-⌬5,6 transactivation whereas it inhibits hMR-N454 transcriptional activity to the same extent as that of the wild-type hMR. In the light of these data and because PIAS proteins interact with the NTD of the receptor, functional interactions may exist between the amino- and carboxyl-terminal domains of hMR for its full activation/repression as has been described for steroid receptors (39, 40). Unlike in AR, in which NTD (W/F)XXLF motifs are involved in NTD-LBD interaction (41), the MR NTD possesses no such motifs. It could be PIAS1, therefore, that plays a role by bridging these functional domains together. Disruption of N/C-terminal interaction has been proposed as a potential molecular defect in androgen insensitivity syndromes (42). Thus, dysregulation of PIAS protein expression and/or action could be implicated in the pathogenesis of some mineralocorticoidsignaling disorders. Consensus motifs, which are interfaces between corepressors and NRs (26), have been described for N-CoR and SMRT. These motifs, named CoRNRs, have very similar sequences, (I/L)XX(I/V)I to LXXLL, of NR boxes described for coactivators that are based on a hydrophobic core able to bind the hydrophobic cleft of NR. It has been shown that the substitution of an I for a L in CoRNR motifs prevents a corepressor binding to unliganded thyroid hormone receptor, and, conversely, an L to I substitution in NR boxes was unable to recruit any corepressors to the liganded thyroid hormone receptor (43). Furthermore, repeat of CoRNR motifs (43), as well as the amino acid context surrounding CoRNR boxes, seems to be involved in the preferential binding of SMRT or N-CoR to distinct NR (44) and be responsible for specific action (45). Therefore, PIAS1 specificity over other members of the PIAS family may rely on its two overlapping CoRNR motifs, which are present at the extreme C-terminal end of the protein (26). Deletion of these CoRNR motifs, however, did not alter PIAS1-repressive effects, suggesting that other functional domains, including its RING-carrying SUMO E3 ligase ability, are involved in repressive action on MR. PIAS proteins are coregulators not only of various transcription factors such as p53 (46), c-Jun (47), lymphoid enhancer factor (48), and almost all steroid re-

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

ceptors, but also of various NR coactivators including TIF1␣ (49), TIF2 (50), and GR-interacting protein 1 (51). To date, all PIAS protein action on NRs or their partners has been shown to rely upon a common regulatory pathway involving sumoylation modification. Our results show that hMR is modified by sumoylation both in vitro and in vivo, and that PIAS1 (and other PIAS family proteins) acts as a SUMO E3-ligase. The MR possesses five consensus sumoylation sites that are perfectly conserved across MR from various species including Xenopus. Such a high number of SUMO sites is unique in the steroid receptor family (52) and may be responsible for fine regulation through sumoylation. We were unable to identify any major SUMO acceptor sites in MR regardless of the lysine substitution combination used. Rather, the impact of sumoylation seems to be remarkably proportional to additive SUMO attachment sites in a GRE2-TATA promoter context, but, surprisingly, hMR lysine mutations were totally ineffective in a MMTV context. This intriguing result strongly suggests that SUMO modification could play a prominent role in MR dimerization and/or its stability. Indeed, in a GRE2 context, MR dimers bind cooperatively to responsive elements in a synergistic manner (53) whereas MMTV contains three halfsites and one distal responsive element on which MR binds (54). Previous elegant studies have already reported the importance of GRE features (the number of response elements, their distance to the TATA box, and their half-site spacing) and the direct implications of SUMO motifs in GR-mediated transactivation (27). More importantly, the negative regulatory functions of SUMO motifs have been suggested in GRE cooperativity and GR dimerization. Thus, sumoylation achieved by PIAS1 SUMO-ligase activity could destabilize the MR dimer formation, leading to transactivation repression. Alternatively, sumoylation could also interfere with the differential recruitment of cofactors by MR, depending on the promoter and cellular contexts. The finding of a sumoylation-independent corepressive activity of PIAS1, as demonstrated by the results obtained with the MMTV promoter, is somehow surprising. This raises the nonmutually exclusive possibility that the physical interaction of PIAS1 with hMR sterically prevents or competes for further binding of coregulators required for transcriptional activity. We propose that the dual and additive repressive actions of PIAS could result, in part, from a direct effect on MR, but also through indirect mechanisms on its associated coregulators, presumably via their SUMO modifications (51). Finally, other important dynamic aspects should also be taken into consideration as sumoylation processes may participate in the regulation of target protein stabilization, nucleocytoplasmic shuttling, and retention within subnuclear domains such as in nuclear bodies (24). In summary, we have identified PIAS1 as a novel molecular partner of hMR, which interacts with its NTD. The corepressive activity of PIAS on hMRmediated transactivation seems to rely not only on its

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

capacity to induce sumoylation of hMR and associated coregulators, but also presumably on its direct inhibitory effect. Our findings also emphasize the importance of SUMO modifications as a major posttranslational step for the regulation of hMR function. Further studies are essential to better elucidate the complexity of the molecular mechanisms involved in MR-mediated gene activation.

MATERIALS AND METHODS Construction of Plasmids Human MR N-terminal fragments were obtained by PCR using pRS-hMR as template (55) and Taq platinium highfidelity polymerase (Invitrogen, San Diego, CA) with the following oligonucleotide primers: sense 1, 5⬘-GCAGGCCTCGAGTTATGGAGACCAAAGGCTAC CACAG-3⬘; sense 163, 5⬘-GCAGGCCTCGAGTTTCATTTATGTCTGACTCTGGGAG3⬘; sense 445, 5⬘-GCAG GCCTCGAGTTCCAACAGTAAACCCGTTTCCATT-3⬘; antisense 168, 5⬘-TACCCGGGCC CTCAAGAGTCAGACATAAATGATCTCA-3⬘; antisense 445, 5⬘TACCCGGGCCCTCAT GAACATGAATGCTTGGTTG-3⬘; antisense 602, 5⬘-TACCCGGGCCCTCATATTTTTGAA GGTCTTGAAGATC-3⬘. PCR fragments corresponding to hMR N-terminal amino acids 1–602, 1–167, 445–602, 163– 437, and 163–602 were first inserted by TA-cloning into pGEMTeasy vector (Promega Corp., Madison, WI) for subsequent utilization: XhoI and ApaI digestion fragments were cloned into the yeast expression vector pLexA or the mammalian eukaryotic expression vector pcDNA3.1HISA (CLONTECH, Palo Alto, CA); SalI and XhoI digestion fragments were cloned into the SalI site of the prokaryotic expression vector pGEX-KG. ECoRNR-hPIAS1 cDNA was obtained by PCR using the following oligonucleotide primers: sense, 5⬘AGCGGAACTAAAGCAAATGGTTATG-3⬘; and antisense, 5⬘CTCGAGCTAGATGGATGCCGTGTCCGTGCTGC-3⬘. PCR products were first cloned into pGEMTeasy, before subcloning into the pcDNA3.1HISA vector at the EcoRI and XhoI sites. All plasmids were sequenced to verify frames and nucleotide sequences. hMR was also subcloned from pcDNA3 using HindIII and XhoI sites into pCMV2-Flag at HindIII and SalI sites. Other vectors included N454hMR in pcDNA3 (56), hMR⌬5,6 in pcDNA3 (21), Rous sarcoma virushGR, pSV- ␤ -galactosidase, pFC31-MMTV-Luc, and pGL3-GRE2-TATA-Luc. Yeast Two-Hybrid Screenings and Analysis of Interaction Specificity Yeast two-hybrid screening was performed essentially as previously described (57). Briefly, pLexA-1–602 and pLexA163–437 plasmids were transferred into the L40 yeast strain via the classical lithium acetate procedure and used as bait to screen a human kidney cDNA library (Matchmaker GAL4 two-hybrid) cloned into a pACT2 vector (CLONTECH). Clones were selected on triple selective medium (DO-WLH) containing 60 mM or 5 mM 3-AT for screenings with 1–602 and 163–437 baits, respectively. Positive clones were tested for ␤-galactosidase activities as described below. Plasmids were rescued from His⫹/LacZ⫹ colonies through classical procedures and used to transform AMR70 yeast strain. Specific protein-protein interactions were analyzed by yeast mating tests between rescued plasmids in the AMR70 yeast strain and all NTD hMR fragments cloned into pLexA plasmid in the L40 strain. Selected plasmids were then sequenced.

Mol Endocrinol, December 2003, 17(12):2529–2542

2539

GST Pull-Down Assay BL21-gold (DE3) Escherichia coli bacteria (Stratagene, La Jolla, CA) were transformed with glutathione S-transferase (GST) N-terminal hMR fusion proteins and cloned into pGEX vector. Bacteria were cultured overnight in Lennox broth ampicillin medium at 37 C, and then diluted 10-fold into fresh medium, cultured at 30 C until OD600 reached 0.6–0.8 for protein induction by 0.3 mM isopropyl-␤-D-thiogalactopyranoside for 2 h. After centrifugation, cells were sonicated into lysis buffer [20 mM Tris-HCl (pH 8), 150 mM NaCl, 5 mM EDTA, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride (PMSF), 2 ␮g/ml leupeptin, 10% glycerol]. After centrifugation, supernatants were incubated with glutathione sepharose 4B gel (Pharmacia Biotech, Piscataway, NJ) for 30 min at 4 C. This gel was washed four times with lysis buffer. In vitro translated proteins by TnT-T7 Quick Coupled Transcription/Translation kit (Promega Corp.) labeled with [35S]methionine (Amersham Pharmacia Biotech, Arlington Heights, IL) were added to immobilized GST-hMR-N-terminal fusion proteins and were incubated for 1 h at 4 C. After four washes with TNEN buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 5 ␮g/ml leupeptin) or HKEN buffer (25 mM HEPES, pH 7.9, 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 5 ␮g/ml leupeptin) interacting proteins were eluted in 2⫻ Laemmli loading buffer, boiled, separated onto 10% acrylamide SDS-PAGE, and then autoradiographed. Liquid ␤-Galactosidase Assay L40 yeast strain transformed with hMR-NTDs or controls (LexA-DBD or unrelated proteins) were grown overnight in liquid medium without tryptophan and uracil at 30 C. Cultures were diluted 100-fold in 4 ml of fresh medium and grown for approximately 15 h until the OD600 reached 0.6–0.8. Cell culture (1 ml) was pelleted for a ␤-galactosidase assay. Yeast pellets were frozen and thawed three times in 500 ␮l Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM ␤-mercapthoethanol). Lysates were incubated for t (time in min) at 30 C with 100 ␮l of 4 mg/ml ortho-nitrophenyl ␤-D-galactopyranoside. Enzymatic reactions were stopped with 250 ␮l of 1 M Na2CO3. OD420 was measured, and ␤galactosidase activities were expressed as arbitrary ␤-galactosidase units calculated according to: arbitrary units (AU) ⫽ OD420/(OD600 ⫻ t). Cell Culture and Transient Transfection RC.SV3 rabbit kidney tubule (58), HK human kidney cortical collecting duct (59), and HeLa cell lines were maintained in DMEM/Ham’s-F12 medium supplemented with 2% dextrancoated charcoal-treated fetal calf serum, 20 mM HEPES, 2 mM glutamine, 100 IU/ml penicillin, 100 ␮g/ml streptomycin, 5 ␮g/ml bovine insulin, 5 ␮g/ml transferrin, 50 nM sodium selenite, and 50 nM dexamethasone in a saturated 5% CO295% air atmosphere at 37 C. All products for cell culture were purchased from Invitrogen. For transient transfections, cells were seeded at 4 ⫻ 105 cells per well into six-well dishes in dexamethasone free medium for 6 h. Plasmids were transfected into cells according to the classical calcium phosphate precipitation method and incubated for 18 h in dexamethasone-free medium. After 24 h hormone incubation period, cells were rinsed twice with cold PBS and recovered in 250 ␮l lysis buffer (25 mM glycylglycine, pH 7.8, 1 mM EDTA, 8 mM magnesium sulfate, 1 mM DTT, 1% Triton X-100, 15% glycerol) at 4 C. Supernatants were used for enzymatic ␤-galactosidase and luciferase assays (100 ␮l each) as previously described (60). Luciferase activities were normalized to ␤-galactosidase activities and expressed as percents of relative luciferase units compared with control.

2540

Mol Endocrinol, December 2003, 17(12):2529–2542

Targeted Mutagenesis Lysine to arginine mutations were obtained by PCR-based targeted single-point mutation using the QuikChange SiteDirected Mutagenesis Kit (Stratagene) with pcDNA3-hMR as a matrix. Mutated plasmids were sequenced to verify the correct mutation and were digested with HindIII and XhoI for subcloning at the same sites into a new pcDNA3 vector. Sumoylation Assays and Nickel Pull Down Sumoylation assays on [35S]methionine-labeled in vitro translated proteins, prepared using the TnT-T7 Quick Coupled Transcription/Translation kit (Promega Corp.), were performed using the protocol of Desterro et al. (28). Recombinant in vitro sumoylation assays (20 ␮l total volume, 60 min incubation at 37 C) were carried out as previously described (47) in TMA buffer (20 mM Tris, pH 7.5, 2 mM MgCl2, 5 mM ATP) containing 1 ␮g recombinant SUMO-1, 100 ng E1 (Aos1/Uba2), 50 ng E2 (Ubc9) and in the presence or absence of 180 ng PIAS proteins. Reactions were stopped by the addition of Laemmli buffer, and products were visualized by autoradiography. In vivo sumoylation assays were carried out using the nickel pull-down technique, as described previously (49). Purified and total extracts were separated by SDS-PAGE and analyzed by Western blot using an anti-FLAG M2 antibody (Sigma Chemical Co., St. Louis, MO). Immunofluorescence Detection HeLa cells were cotransfected with pSG5-SUMO and pCMV2-Flag-hMR or Flag-K12345 plasmids according to Lipofectamine (Invitrogen) standard procedures. After 24 h of aldosterone treatment, cells were fixed for 10 min with 10% formalin solution (Sigma) and permeabilized with 0.5% Triton X-100 in PBS for 15 min. MRs were detected by incubating coverslips with a primary anti-Flag antibody (rabbit polyclonal M2, Sigma) and then with a secondary antirabbit antibody coupled to fluorescein isothiocyanate. SUMO was detected with anti-GMP1 (mouse monoclonal, Zymed Laboratories, Inc., South San Francisco, CA) and an antimouse antibody coupled with Texas Red. Fluorescence microscopy was performed using a DMRB microscope (Leica Corp., Deerfield, IL) equipped with a CoolSnapFX charge-coupled device camera (Princeton Instruments, Princeton, NJ) controlled by MetaVue software (Universal Imaging Corp., Dowingtown, PA). Statistical Analysis Statistical analysis was performed using t test for unpaired comparisons (InStat, version 2.01; GraphPad Software, San Diego, CA). P ⬍ 0.05 was considered to be significant.

Acknowledgments We thank Gilles Hetet and Colette Galand for their excellent technical assistance in yeast two-hybrid meanders and Franck Letourneur and Nicolas Lebrun for their help in sequencing. We also thank Marie-Liesse Asselin, Marc Pallardy, and Ke Shuai for providing us with plasmids, and Shoshana Sztal-Mazer for her careful reading of the manuscript.

Received July 30, 2003. Accepted September 8, 2003. Address all correspondence and requests for reprints to: Marc Lombes, M.D., Ph.D., Institut National de la Sante´ et de la Recherche Me´ dicale, U478, Faculte´ de Me´ decine Xavier Bichat, 16 rue Henri Huchard, 75870 Paris cedex 18, France. E-mail: [email protected]

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

This work was supported by the INSERM. L.P.-L.T. is the recipient of a fellowship from the Ministe`re de l’Education Nationale de la Recherche et de la Technologie and from La Ligue Nationale contre le Cancer (LNCC). This work was also supported in part by grants from the Association de la Recherche contre le Cancer, LNCC and La Fondation de France (to A.D.). Part of this work has been presented as oral communication during the 85th annual meeting of The Endocrine Society, June 19–22, 2003, Philadelphia, PA.

REFERENCES 1. Nuclear Receptor Nomenclature Committee 1999 A unified nomenclature system for the nuclear receptor superfamily. Cell 97:161–163 2. Bonvalet JP 1998 Regulation of sodium transport by steroid hormones. Kidney Int Suppl 65:S49–S56 3. Arriza JL, Simerly RB, Swanson LW, Evans RM 1988 The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response. Neuron 1:887–900 4. Lombes M, Alfaidy N, Eugene E, Lessana A, Farman N, Bonvalet JP 1995 Prerequisite for cardiac aldosterone action. Mineralocorticoid receptor and 11␤-hydroxysteroid dehydrogenase in the human heart. Circulation 92:175–182 5. Funder JW, Pearce PT, Smith R, Smith AI 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583–585 6. Farman N, Rafestin-Oblin ME 2001 Multiple aspects of mineralocorticoid selectivity. Am J Physiol Renal Physiol 280:F181–F192 7. Lombes M, Kenouch S, Souque A, Farman N, RafestinOblin ME 1994 The mineralocorticoid receptor discriminates aldosterone from glucocorticoids independently of the 11␤-hydroxysteroid dehydrogenase. Endocrinology 135:834–840 8. Couette B, Fagart J, Jalaguier S, Lombes M, Souque A, Rafestin-Oblin ME 1996 Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure. Biochem J 315: 421–427 9. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486 10. Zennaro MC, Keightley MC, Kotelevtsev Y, Conway GS, Soubrier F, Fuller PJ 1995 Human mineralocorticoid receptor genomic structure and identification of expressed isoforms. J Biol Chem 270:21016–21020 11. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344 12. McKenna NJ, Xu J, Nawaz Z, Tsai SY, Tsai MJ, O’Malley BW 1999 Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol 69:3–12 13. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736 14. Voegel JJ, Heine MJ, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J 15:3667–3675 15. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:2735–2744 16. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ, Stallcup MR 1998 Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32.

33.

34.

(GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 12:302–313 Hittelman AB, Burakov D, Iniguez-Lluhi JA, Freedman LP, Garabedian MJ 1999 Differential regulation of glucocorticoid receptor transcriptional activation via AF-1associated proteins. EMBO J 18:5380–5388 Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ, O’Malley BW 1999 A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97:17–27 Yang L, Guerrero J, Hong H, DeFranco DB, Stallcup MR 2000 Interaction of the ␶2 transcriptional activation domain of glucocorticoid receptor with a novel steroid receptor coactivator, Hic-5, which localizes to both focal adhesions and the nuclear matrix. Mol Biol Cell 11: 2007–2018 Hellal-Levy C, Fagart J, Souque A, Wurtz JM, Moras D, Rafestin-Oblin ME 2000 Crucial role of the H11–H12 loop in stabilizing the active conformation of the human mineralocorticoid receptor. Mol Endocrinol 14:1210–1221 Zennaro MC, Souque A, Viengchareun S, Poisson E, Lombes M 2001 A new human MR splice variant is a ligand-independent transactivator modulating corticosteroid action. Mol Endocrinol 15:1586–1598 Fuse H, Kitagawa H, Kato S 2000 Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF1). Mol Endocrinol 14:889–899 Kitagawa H, Yanagisawa J, Fuse H, Ogawa S, Yogiashi Y, Okuno A, Nagasawa H, Nakajima T, Matsumoto T, Kato S 2002 Ligand-selective potentiation of rat mineralocorticoid receptor activation function 1 by a CBPcontaining histone acetyltransferase complex. Mol Cell Biol 22:3698–3706 Jackson PK 2001 A new RING for SUMO: wrestling transcriptional responses into nuclear bodies with PIAS family E3 SUMO ligases. Genes Dev 15:3053–3058 Govindan MV, Warriar N 1998 Reconstitution of the Nterminal transcription activation function of human mineralocorticoid receptor in a defective human glucocorticoid receptor. J Biol Chem 273:24439–24447 Hu X, Lazar MA 1999 The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402:93–96 Iniguez-Lluhi JA, Pearce D 2000 A common motif within the negative regulatory regions of multiple factors inhibits their transcriptional synergy. Mol Cell Biol 20:6040–6050 Desterro JM, Thomson J, Hay RT 1997 Ubch9 conjugates SUMO but not ubiquitin. FEBS Lett 417:297–300 Hollenberg SM, Evans RM 1988 Multiple and cooperative trans-activation domains of the human glucocorticoid receptor. Cell 55:899–906 Hovland AR, Powell RL, Takimoto GS, Tung L, Horwitz KB 1998 An N-terminal inhibitory function, IF, suppresses transcription by the A-isoform but not the Bisoform of human progesterone receptors. J Biol Chem 273:5455–5460 Jenster G, van der Korput HA, Trapman J, Brinkmann AO 1995 Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J Biol Chem 270:7341–7346 Chamberlain NL, Whitacre DC, Miesfeld RL 1996 Delineation of two distinct type 1 activation functions in the androgen receptor amino-terminal domain. J Biol Chem 271:26772–26778 Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, Shuai K 1998 Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci USA 95:10626–10631 Tan J, Hall SH, Hamil KG, Grossman G, Petrusz P, Liao J, Shuai K, French FS 2000 Protein inhibitor of activated STAT-1 (signal transducer and activator of transcrip-

Mol Endocrinol, December 2003, 17(12):2529–2542

35.

36.

37.

38. 39.

40. 41.

42.

43.

44.

45.

46. 47. 48.

49.

50.

51.

52.

2541

tion-1) is a nuclear receptor coregulator expressed in human testis. Mol Endocrinol 14:14–26 Moilanen AM, Karvonen U, Poukka H, Yan W, Toppari J, Janne OA, Palvimo JJ 1999 A testis-specific androgen receptor coregulator that belongs to a novel family of nuclear proteins. J Biol Chem 274:3700–3704 Kotaja N, Aittomaki S, Silvennoinen O, Palvimo JJ, Janne OA 2000 ARIP3 (androgen receptor-interacting protein 3) and other PIAS (protein inhibitor of activated STAT) proteins differ in their ability to modulate steroid receptordependent transcriptional activation. Mol Endocrinol 14: 1986–2000 Tan JA, Hall SH, Hamil KG, Grossman G, Petrusz P, French FS 2002 Protein inhibitors of activated STAT resemble scaffold attachment factors and function as interacting nuclear receptor coregulators. J Biol Chem 277:16993–17001 Fagart J, Wurtz JM, Souque A, Hellal-Levy C, Moras D, Rafestin-Oblin ME 1998 Antagonism in the human mineralocorticoid receptor. EMBO J 17:3317–3325 Kumar R, Thompson EB 2003 Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions. Mol Endocrinol 17:1–10 Rogerson FM, Fuller PJ 2003 Interdomain interactions in the mineralocorticoid receptor. Mol Cell Endocrinol 200: 45–55 He B, Kemppainen JA, Wilson EM 2000 FXXLF and WXXLF sequences mediate the NH2-terminal interaction with the ligand binding domain of the androgen receptor. J Biol Chem 275:22986–22994 Thompson J, Saatcioglu F, Janne OA, Palvimo JJ 2001 Disrupted amino- and carboxyl-terminal interactions of the androgen receptor are linked to androgen insensitivity. Mol Endocrinol 15:923–935 Webb P, Anderson CM, Valentine C, Nguyen P, Marimuthu A, West BL, Baxter JD, Kushner PJ 2000 The nuclear receptor corepressor (N-CoR) contains three isoleucine motifs (I/LXXII) that serve as receptor interaction domains (IDs). Mol Endocrinol 14:1976–1985 Cohen RN, Brzostek S, Kim B, Chorev M, Wondisford FE, Hollenberg AN 2001 The specificity of interactions between nuclear hormone receptors and corepressors is mediated by distinct amino acid sequences within the interacting domains. Mol Endocrinol 15:1049–1061 Makowski A, Brzostek S, Cohen RN, Hollenberg AN 2003 Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor. Mol Endocrinol 17:273–286 Kahyo T, Nishida T, Yasuda H 2001 Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol Cell 8:713–718 Schmidt D, Muller S 2002 Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc Natl Acad Sci USA 99:2872–2877 Sachdev S, Bruhn L, Sieber H, Pichler A, Melchior F, Grosschedl R 2001 PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestration into nuclear bodies. Genes Dev 15:3088–3103 Seeler JS, Marchio A, Losson R, Desterro JM, Hay RT, Chambon P, Dejean A 2001 Common properties of nuclear body protein SP100 and TIF1␣ chromatin factor: role of SUMO modification. Mol Cell Biol 21:3314–3324 Jimenez-Lara AM, Heine MJ, Gronemeyer H 2002 PIAS3 (protein inhibitor of activated STAT-3) modulates the transcriptional activation mediated by the nuclear receptor coactivator TIF2. FEBS Lett 526:142–146 Kotaja N, Karvonen U, Janne OA, Palvimo JJ 2002 The nuclear receptor interaction domain of GRIP1 is modulated by covalent attachment of SUMO-1. J Biol Chem 277:30283–30288 Poukka H, Karvonen U, Janne OA, Palvimo JJ 2000 Covalent modification of the androgen receptor by small

2542 Mol Endocrinol, December 2003, 17(12):2529–2542

53.

54.

55.

56.

ubiquitin-like modifier 1 (SUMO-1). Proc Natl Acad Sci USA 97:14145–14150 Liu W, Wang J, Yu G, Pearce D 1996 Steroid receptor transcriptional synergy is potentiated by disruption of the DNA-binding domain dimer interface. Mol Endocrinol 10: 1399–1406 Lombes M, Binart N, Oblin ME, Joulin V, Baulieu EE 1993 Characterization of the interaction of the human mineralocorticosteroid receptor with hormone response elements. Biochem J 292:577–583 Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Housman DE, Evans RM 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268–275 Deppe CE, Heering PJ, Viengchareun S, Grabensee B, Farman N, Lombes M 2002 Cyclosporine a and FK506 inhibit transcriptional activity of the human mineralocorticoid receptor: a cell-based model to investigate partial aldosterone resistance in kidney transplantation. Endocrinology 143:1932–1941

Pascual-Le Tallec et al. • PIAS1 Is a Corepressor of hMR

57. Nicolas G, Fournier CM, Galand C, Malbert-Colas L, Bournier O, Kroviarski Y, Bourgeois M, Camonis JH, Dhermy D, Grandchamp B, Lecomte MC 2002 Tyrosine phosphorylation regulates ␣ II spectrin cleavage by calpain. Mol Cell Biol 22:3527–3536 58. Vandewalle A, Lelongt B, Geniteau-Legendre M, Baudouin B, Antoine M, Estrade S, Chatelet F, Verroust P, Cassingena R, Ronco P 1989 Maintenance of proximal and distal cell functions in SV40-transformed tubular cell lines derived from rabbit kidney cortex. J Cell Physiol 141:203–221 59. Prie D, Friedlander G, Coureau C, Vandewalle A, Cassingena R, Ronco PM 1995 Role of adenosine on glucagon-induced cAMP in a human cortical collecting duct cell line. Kidney Int 47:1310–1318 60. Zennaro MC, Le Menuet D, Lombes M 1996 Characterization of the human mineralocorticoid receptor gene 5⬘-regulatory region: evidence for differential hormonal regulation of two alternative promoters via nonclassical mechanisms. Mol Endocrinol 10:1549–1560

Growth Hormone Society Research Fellowships 2004 The Growth Hormone Research Society intends to award two Research Fellowships in 2004 to promote international exchange of scientific and clinical research expertise in any area of GH-related research. Awards of US $40,000 will provide personal salary support and travel costs for a post-doctoral scientist or clinical training fellow wishing to pursue a research project for one year in a host institution of their choice. For further details and an application form, see the GRS website, http://www.GHresearchsociety.org or contact the GRS office at [email protected] The closing date for receipt of applications is March 1, 2004.

Suggest Documents