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and -
Transcriptional Activity through a Synergy Control MotifMolecular Oncology Group (A.M.T., B.J.W., X.J.Y., V.G.), McGill University Health Centre, and Departments of Biochemistry (A.M.T., V.G.), Medicine (X.J.Y., V.G.), and Oncology (V.G.), McGill University, Montréal, Québec, Canada H3A 1A1
Address all correspondence and requests for reprints to: Vincent Giguère, Molecular Oncology Group, Room H5–42, McGill University Health Centre, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vincent.giguere{at}mcgill.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and 
, regulation by posttranslational modifications is still poorly documented. Here we show that transcriptional repression associated with the ERR amino-terminal domains is mediated through sumoylation at a conserved phospho-sumoyl switch, 
KxEPxSP, that exists within a larger synergy control motif. Arginine substitution of the sumoylatable lysine residue or alanine substitution of a nearby phosphorylatable serine residue (serine 19 in ERR) increased the transcriptional activity of both ERR and -. In addition, phospho-mimetic substitution of the serine residue with aspartate restored the sumoylation and transcriptional repression activity. The increased transcriptional activity of the sumoylation-deficient mutants was more pronounced in the presence of multiple adjacent ERR response elements. We also identified protein inhibitor of activated signal transducer and activator of transcription y as an interacting partner and a small ubiquitin-related modifier E3 ligase for ERR. Importantly, analysis with a phospho-specific antibody revealed that sumoylation of ERR in mouse liver requires phosphorylation of serine 19. Taken together, these results show that the interplay of phosphorylation and sumoylation in the amino-terminal domain provides an additional mechanism to regulate the transcriptional activity of ERR and -. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and β (NR3B1 and NR3B2) were the first orphan NRs identified based on their high level of sequence identity with estrogen receptor (ER) (NR3A1) (2). The ERR subfamily also contains a third isoform (ERR, NR3B3) (3, 4), and all three proteins possess the typical functional domains of NRs. The amino acid sequences of the three ERR isoforms are highly similar, sharing as expected the highest identity in the DNA-binding domain and ligand-binding domain (LBD). However, unlike most related NRs, the three ERR isoforms also share considerable amino acid sequence similarity in their respective amino-terminal domain (NTD), suggesting that the NTD may influence the transcriptional acitivity of the three ERRs by common mechanisms. Although the ERRs have no known natural ligand, the three receptors can be activated in a ligand-independent manner in the presence of coactivator proteins, most notably by members of the steroid receptor coactivator (SRC) and the peroxisome proliferator-activated receptor (PPAR)- coactivator 1 (PGC-1) families (4, 5, 6, 7). Indeed, the elucidation of the crystal structures of ERR LBD bound to a PGC-1 peptide, as well as that of ERR LBD bound to a SRC-1 or receptor-interacting protein-140 peptides, have shown that the two ERRs assume the conformation of ligand-activated NRs in the apparent absence of a ligand, again suggesting that the presence of an agonist ligand may not be an obligatory requirement for the activation of the receptors (8, 9, 10). Thus, posttranslational modifications could play a major role in the control of ERR transcriptional activity. Although ERR has been shown to be a phosphoprotein (11, 12, 13), the current knowledge about ERR posttranslational modifications is still very limited.
Phosphorylation of NRs, as well as their coactivators, is a well-documented mechanism involved in the control of their activities (reviewed in Refs. 14, 15, 16). Similarly, sumoylation, the process of conjugating the small ubiquitin-related modifier (SUMO) protein, has been reported for NRs and coregulators, namely the androgen (17), glucocorticoid (18), progesterone (19, 20), estrogen (21), and mineralocorticoid receptors (22, 23) as well as SF-1 (24, 25), PPAR (26, 27, 28), liver receptor homolog 1 (29), liver X receptors (28), Tr2 (30), the SRC coactivators (19, 31, 32), the histone acetyltransferase p300 (33), and the nuclear receptor corepressor 1 (34). The exact function of sumoylation is still unknown although a growing body of evidence now supports the role of SUMO proteins in the negative regulation of transcription, mainly through corepressor recruitment or clearance-related mechanisms (27, 28, 35, 36, 37, 38).
SUMO proteins are conjugated to a lysine residue within the core consensus site KxE, where represents a hydrophobic residue and x is any residue. Sumoylation is carried out by a set of enzymes that are distinct from those acting on the ubiquitin pathway (39, 40) and consist of a SUMO-activating heterodimeric complex consisting of Aos1 and Uba2 (E1), the single E2-type conjugating enzyme UBC9, and E3-like proteins, which serve to increase the affinity between UBC9 and the substrates by bringing them in a close proximity to UBC9 with a catalytically favorable orientation. In vitro, however, the E3-like activity is not necessary for sumoylation to occur, because E1 and UBC9 are sufficient to induce sumoylation. Three types of SUMO E3 ligases have been described: RanBP2 (a component of nuclear pore complex), the Polycomb protein Pc2, and members of the protein inhibitor of activated signal transducer and activator of transcription (PIAS) family (reviewed in Ref. 41).
A subset of consensus SUMO conjugation motifs has recently been extended to include KxExxSP, establishing new sumoylation sites that are phosphorylation dependent and thus referred to as phosphorylation-dependent sumoylation motifs (PDSMs) or phospho-sumoyl switches (42, 43, 44). In addition, this sequence also corresponds to the synergy control (SC) motif Px (0–3)[I/V]K[Q/T/S/L/E/P]Ex (0–3)P, a protein determinant that was identified before the sumoylation consensus and initially proposed to modulate higher-order interactions among transcription factors, including NRs and their coregulators (45, 46). The efficiency of the transcriptional repression exerted by sumoylation of transcription factors within SC motifs has been proposed to depend on the number of consecutive DNA response elements present in the target promoter (47).
Here we present evidence that ERR and - are sumoylated within their respective NTDs and that this modification is induced by phosphorylation of a functional PDSM. Our results show that sumoylation negatively affects ERR and - transcriptional activity without altering subcellular localization, DNA binding properties, or interactions with the coactivator PGC-1. The PDSM within the NTD of the ERRs also controls synergy in the presence of multiple ERR response element (ERRE) and consequently, sumoylation-deficient receptor variants are more potent activators of transcription on the polymorphic ESRRA promoter containing multiple copies of the ERRE. We have also demonstrated that PIASy interacts with and possess an E3-ligase activity toward ERR. Using a phosphorylation-specific antibody, we found that phosphorylation of serine 19 is required for sumoylation of endogenous ERR in mouse liver. Thus, the interplay of phosphorylation and sumoylation at the SC motif provides a novel mechanism to regulate the transcriptional activities of ERR and -.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and - and three (lysines 40, 360, and 439) in ERR (Fig. 1A
). Interestingly, the NTD sumoylation sites (lysine 14 in ERR and 40 in ERR) were found to be embedded within a PDSM motif (42, 43, 48) (Fig. 1B). To determine whether the two ERR isoforms are targets of sumoylation, we first cotransfected human embryonic kidney (HEK)293 cells with either a myc-ERR- or a Flag-ERR-tagged expression vector along with an hemagglutinin (HA)-SUMO2 plasmid. Immunoprecipitation with either anti-myc or anti-Flag antibodies followed by immunoblotting using an anti-HA polyclonal antibody suggested that both ERR (Fig. 1C) and - (Fig. 1D) are modified by SUMO2.
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and -, potential target lysines were mutated to arginines, and the point mutants were subjected to in vitro sumoylation assays with recombinant SUMO1 and SUMO3 proteins. As shown in Fig. 2, A and B, respectively, only the ERR K14R and ERR K40R mutants displayed significantly decreased levels of sumoylation whereas the remaining point mutants (ERR K403R, ERR K360R, and K439R) were sumoylated to a level similar to the wild-type receptors. In the absence of the recombinant E1 activating enzyme, the sumoylated forms were totally absent. The absence of one band in the K403R mutant in comparison to wild-type ERR suggests residual sumoylation of lysine 403 (Fig. 2A).
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and - were transfected in HEK293 cells, along with an HA-SUMO2GG-activated form and UBC9 or with HA-SUMO2
GG and UBC9-C93S dominant-negative forms, and 80 µg of extracts was subjected to Western blot analysis using an anti-ERR or anti-Flag M2 antibody. As shown in Fig. 2
, C and D, slower migrating bands were present when the activated form of SUMO2 and UBC9 were introduced in the HEK293 cells. Consistent with this, the bands were absent when the dominant-negative forms were used, demonstrating that the slower migrating bands were the sumoylated forms of the receptors. Moreover, the slower migrating band was significantly decreased for ERR K14R (Fig. 2C) and absent for ERR K40R mutants (Fig. 2D), confirming that the NTD of both ERR isoforms is the main SUMO attachment site. The residual sumoylation in the ERR K14R lane may suggest a possible modification of lysine 403 (Fig. 2C).
Increased Transcriptional Activity of Sumoylation-Deficient Mutants of ERR and - Requires Multiple DNA Response Elements
To assess whether sumoylation of the two ERR isoforms affects their transcriptional activity, we next transfected HeLa cells with either ERR wild-type forms and NTD KR mutants in the presence or the absence of the coactivator PGC-1 together with the reporter construct 3xESRRApromoter-luciferase (LUC). This reporter is driven by the promoter of the gene encoding ERR (ESRRA) which is a known target of both ERR (50) and - (51). As shown in Fig. 3A, in the presence of PGC-1, the mutant ERR K14R displays a greater transcriptional activity than its wild-type counterpart at both 50 and 100 ng. In Fig. 3B, ERR induced only a modest transcriptional response in the absence of PGC-1. However, a more significant induction of basal LUC activity was observed with the ERR K14R mutant. As previously reported (50, 52), introduction of PGC-1 in HeLa cells stimulated the basal activity of the ESRRA promoter due to the presence of endogenous ERRs in these cells but also stimulated the activity of exogenous ERR. In the presence of PGC-1, the K14R mutant displayed a marked increase in transcriptional activity as compared with the wild-type receptor. Similar results were obtained with ERR and the K40R mutant (Fig. 3C). The increased activity of NTD KR mutants was not caused by differences in protein expression, because wild-type and mutant constructs were expressed at similar levels for both ERR and (Fig. 3, B and C, insets). To avoid interference by endogenous ERR in the transfection assay, we performed the same experiment using ERR-null mouse embryonic fibroblasts (MEFs) immortalized with simian virus 40 large T antigen [ERR-null MEFs-T]. In these cells, introduction of PGC-1 had only a minor effect on the basal activity of the reporter construct (Fig. 3D) and both wild-type ERR and the K14R mutant failed to display basal transcriptional activity on ESRRA-driven reporter. However, both proteins showed a strong transcriptional response to the presence of PGC-1, and the K14R mutant displayed much higher transcriptional activity than the wild-type receptor. To rule out any other interference by endogenous ERR isoforms on the ESRRA-driven reporter, we also assessed the activity of Gal4 DNA-binding domain-ERR and -K14R mutant fusion proteins on a two-copy upstream activating sequence (UAS)-tk-LUC reporter (Fig. 3E). In this context, the sumoylation-deficient ERR K14R mutant displayed a synergistic response to the presence of PGC-1.
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, the consensus motif for sumoylation in both ERR and - also overlaps with an SC motif responsible for regulation of SC in the presence of multiple response elements (45). We therefore wanted to determine whether the SC mechanism was regulating the transcriptional activity of ERR. As shown in Fig. 3F, the greater transcriptional activity of the ERR K14R mutant was observable solely on the 3xERRE-TK-LUC reporter constructs, whereas on the reporter bearing only a single copy of the ERRE, the transcriptional activity was lower and similar for both ERR and ERR K14R. Related to this observation, we have previously demonstrated that a naturally occurring polymorphism within the ESRRA promoter changes the number of consecutive ERREs present in the distal region of the promoter from 1 to 4 (50). We next tested whether changing the sumoylation status of the ERRs affects the regulation of the polymorphic ESRRA promoter. Results presented in Fig. 3, F and G, show that, in the presence of PGC-1, the KR NTD mutants were more potent on the reporter construct driven by the ESRRA promoter containing three copies of the ERR response elements than the reporter containing only two copies of the element. We next tested whether the mutations of lysine 14 and 40 to arginines could, by themselves, change the interaction between the coactivator PGC-1 and the receptor proteins. As shown in Fig. 3I, all four proteins interacted with PGC-1 to a similar level when assayed by glutathione-S-transferase (GST)-pulldowns using equal amounts of immobilized GST and GST-PGC-1/1–250. Taken together, these results demonstrate that sumoylation of ERR and - represses the transcriptional activity of the receptors. Therefore, the regulatory effect of ERR sumoylation would be of greater importance for the individuals expressing promoters bearing multiple copies of the ERRE.
Sumoylation Site Mutants Display Wild-Type Nuclear Localization and DNA Binding Properties
Sumoylation has been shown to affect subcellular localization of transcription factors, an effect that has been associated with repression due to sequestration within nuclear bodies (37, 53). To verify whether sumoylation of ERR affects its cellular localization, we transfected HeLa cells with green fluorescent protein (GFP) constructs for ERR (Fig. 4A, top row) and the K14R mutant (Fig. 4A, middle row). The nuclear localization remained unchanged between the ERR wild-type and K14R mutant (Fig. 4A, first column). Furthermore, no targeting to nuclear bodies was observed for both ERR variants as shown by the absence of colocalization between the HA-SUMO2-induced nuclear bodies (Fig. 4A, second and third columns). Sumoylation is also known to modulate the DNA binding properties of certain transcription factors (36, 37, 53). Thus, we assessed whether sumoylation affects the DNA binding properties of ERR. Using nuclear extracts from HEK293 cells transfected with ERR wild-type, K14R, or K403R mutants, we observed no change in the DNA binding pattern or in supershift generated by an ERR antibody or a purified GST-PGC-1/1–250 fusion protein (Fig. 4B). We also compared the DNA binding pattern of in vitro sumoylated ERR, with both SUMO1 and SUMO3 (Fig. 4C). We observed that sumoylated ERR binding to DNA was as efficient as that of the K14R mutant. The same experiment was then reproduced with the addition of GST-PGC-1 in the DNA binding reaction, and the result showed that the sumoylated form of ERR was similarly supershifted by PGC-1 on DNA (Fig. 4D). Because the SC mechanism is dependent on the presence of more than one copy of the response element, we assessed the DNA binding properties of nonsumoylated and sumoylated ERR on a tandem element probe. We observed no difference in the binding pattern (Fig. 4E) or the intensity of binding (Fig. 4F) with increasing amounts of the nonsumoylated and sumoylated ERR.
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on Serine 19 Is Essential for Sumoylation of Lysine 14). We then assessed the effect of ERR S19A and S19D mutations (Fig. 5, A and B) as well as the ERR S45A and S45D mutations (Fig. 5C) on the transactivation properties of the receptors. Using the 3xESRRApromoter-LUC reporter construct in HeLa cells (Fig. 5, A and C) or the two-copy UAS-tk-LUC reporter in COS-1 cells (Fig. 5B), we observed that the ERR S19A and ERR S45A mutants displayed increased transcriptional activity compared with their wild-type counterparts. In contrast, the phospho-mimetic mutants ERR S19D and ERR S45D had similar transcriptional activity as the wild-type receptors. This result is in agreement with the sumoylation level of wild-type and mutant ERRs. HEK293 cells were transfected with the ERR point mutants along with an HA-SUMO2GG-activated form and UBC9 or with HA-SUMO2GG and UBC9-C93S dominant-negative forms, and 80 µg of extracts were subjected to Western blot analysis using a anti-ERR or to an anti-FlagM2 agarose beads immunoprecipitation and Western analysis with anti-flag antibody. We observed that the sumoylation capacity was impaired in a similar manner as the KR mutants and after alanine mutations of serine 19 for ERR (Fig. 5D) and of serine 45 for ERR (Fig. 5E). Conversely, the phospho-mimetic mutations of these serines to aspartic acid residues restored the capability for sumoylation for both ERR (Fig. 5D) and - (Fig. 5E). Taken together, these results support a phosphorylation-dependent sumoylation mechanism of the ERR and - NTDs.
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sumoylation. Of potential SUMO E3 ligase candidates tested, we observed that PIASy was the most effective in promoting the sumoylation of ERR in cultured cells (Fig. 6A). HEK293 cells were cotransfected with pCMV5-Flag-PIASy and myc-ERR wild-type or K14R mutant expression plasmid. The coexpression of Flag-PIASy with ERR resulted in covalent modification of myc-ERR with endogenous SUMO. Considering that in vitro, both SUMO1 and SUMO3 were able to modify the ERRs, the endogenous SUMO modifier in this setting could be either one of the SUMO isoforms. After immunoprecipitation on FlagM2 agarose, PIASy was shown to interact with both the nonsumoylated and the sumoylated form of ERR (Fig. 6B). In a similar manner, PIASy also interacted with an apparent similar affinity with the ERR sumoylation-deficient mutant K14R (Fig. 6B). The cotransfection of flag-PIASy along with the ERR and phospho-sumoyl switch mutants markedly stimulated the sumoylation of ERR. In agreement with the role associated with serine 19, the S19A mutant showed no modified form in the presence of the ligase, whereas the S19D mutation restored the modification (Fig. 6C). Considering the high potential for ERR multisite phosphorylation events suggested by large-scale studies of HeLa cells and mouse liver nuclear phosphoproteins (57, 58), mutants of the adjacent serine 22 (S22A and S22D) were also tested for sumoylation levels and showed no difference in comparison with the wild-type receptor (Fig. 6C). Interestingly, the transcriptional activity of the mutants (Figs. 5A and 6D) shows good correlation with the levels of sumoylation observed in Fig. 6C.
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in Vivo phospho-sumoyl switch was functional in vivo. Therefore, we generated a custom-made rabbit antiserum directed against the phosphorylated S19 of ERR. The relative affinity of the anti-ERR and the anti-ERR pS19 antisera was assessed by dot blot against the immobilized phosphorylated synthetic peptide antigen and its nonphosphorylated homolog. We observed that the anti-ERR recognized this particular epitope very weakly in comparison with the anti-ERR pS19 antisera. Also, the anti-ERR pS19 antisera, although detecting slightly the nonphosphorylated antigen, displayed a strong preference for the immobilized phospho-peptide (data not shown).
Mouse liver extracts prepared in a phosphorylation- and sumoylation-preserving buffer were subjected to parallel immunoprecipitations using the anti-ERR as well as the anti-ERR pS19 antisera along with corresponding preimmune serum. Subsequent Western blot analysis with the anti-ERR antiserum confirmed the high levels of ERR protein in mouse liver and the effective immunoprecipitation of the nonsumoylated form of ERR by the anti-ERR antiserum and in a weaker manner, by the anti-ERR pS19 antibody (Fig. 7A). In both immunoprecipitation conditions (Fig. 7A, left side with anti-ERR and right side with anti-ERR pS19), the higher band corresponding to sumoylated ERR could not be detected by the anti-ERR. This is because the anti-ERR displays a weaker recognition of the serine 19 epitope involved in the present mechanism (Fig. 7A). To overcome this, a commercially available anti-ERR antibody recognizing the C terminus of hERR (amino acids 339–364) was used to validate the identity of the different bands. Western blot analysis with this antibody revealed a weak higher band after immunoprecipitation with the anti-ERR antibody (Fig. 7B, left side). Interestingly, the higher band corresponding to sumoylated ERR was more abundant when the immunoprecipitation was performed using the anti-ERR pS19 antibody (Fig. 7B, right side).
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pS19 antiserum (Fig. 7C) and anti-SUMO2 (Fig. 7D) antibodies revealed only a SUMO2-modified form of ERR when immunoprecipitation was performed with the phospho-specific antisera and not with the anti-ERR. Also, in agreement with the sumoylation-promoting role of serine 19, Fig. 7C (right side) demonstrates that the pool of serine 19-phosphorylated ERR is completely sumoylated, as depicted by the shift in molecular weight observed. The relative abundance of the sumoylated vs. the nonsumoylated species is usually low. The detection of endogenously sumoylated proteins is therefore difficult. The anti-ERR pS19 antiserum also slightly detected the lower ERR band in the input lanes (Fig. 7C). The reason for this is the slight recognition of the nonphosphorylated ERR, in agreement with the much lower affinity for the nonphosphorylated peptide (data not shown). However, after enrichment for the serine 19-phosphorylated ERR by immunoprecipitation with the anti-ERR pS19, the only band detected is the sumoylated ERR.
Furthermore, the higher molecular weight band observed in Fig. 7, B and C (right side), comigrates with the one observed when Western blot analysis was performed with the anti-SUMO2 antibody (Fig. 7D, right side). Taken together these results not only indicate that ERR is sumoylated in vivo but demonstrate the requirement of serine 19 phosphorylation for efficient sumoylation of the NTD.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and - as bona fide targets of sumoylation. We showed that the NTDs of ERR and - contain a phosphorylation-dependent sumoylation consensus motif KxExxSP, also known as PDSM (42, 43, 44), that is conserved from Drosophila to humans and is also present in the ERRβ isoform. Moreover, the PDSM is embedded within a SC motif (Fig. 1B), an extended motif determinant proposed to regulate higher-order interactions among transcription factors (45). Our study demonstrated that the three overlapping motifs are functional, placing the ERR isoforms in a unique category within the superfamily of NRs.
Sumoylation is generally associated with transcriptional repression. In agreement with this observation, we have shown that mutation of the main SUMO acceptor sites in the ERR and - NTDs increases their transcriptional activities. Importantly, the greater transcriptional activity of the NTD KR mutants can be observed only in the presence of PGC-1. This reinforces the notion that ERR is a major conduit of PGC-1 activity (5, 6, 50, 59, 60, 61). In fact, the presence of a PGC-1 coactivator family member appears to be crucial for ERR activation and has been proposed to act as a protein ligand for the ERRs (62). The apparent dependency on PGC-1 for the effect observed in this study mirrors the importance of this cofactor in ERR function. Furthermore, because PGC-1 activates ERR function via the LBD (63), our results suggest that intramolecular interactions between the NTD and the LBD may play an important role in controlling ERR transcriptional activity.
SUMO conjugation has been shown to negatively regulate the activity of transcription factors through the regulation of different molecular properties of the target protein. These effects seem to be specific to each target protein and vary from reduction of DNA binding to alteration of protein stability or sequestration to subnuclear bodies (35, 36, 37). For the ERRs, it seems that properties such as localization, coactivator recruitment, DNA binding, and protein stability are not affected by sumoylation. Therefore, these may not account for the repressive effect of sumoylation suggested by the increased transcriptional activity of the mutated ERR proteins (Figs. 3 and 5). Instead, we showed that sumoylation regulates ERR transcriptional activity via a SC mechanism. Indeed, the increased transcriptional activity of the ERR NTD sumoylation-deficient mutants on the ESRRA promoter containing three copies of the ERR response element suggests that sumoylation of the SC motif could have a direct impact on the expression of ERR itself. Thus, sumoylation of ERR may be an important component for the fine tuning of the autoregulatory loop regulating ERR expression in the presence of PGC-1. This regulatory mechanism is also likely to influence the expression of other ERR target genes that harbor multiple ERREs in their promoter/regulatory regions.
In the absence of a known natural ligand, the regulation of ERR and - transcriptional activities by posttranslational modifications becomes of crucial importance. To our knowledge, the only identified phosphorylation site within the ERRs so far is threonine 124 of ERR, which lies within a consensus PKC
phosphorylation site (12). In addition, ERR has also been shown to be phosphorylated after epidermal growth factor treatment in MCF-7 cells (64) and hyperphosphorylated in BT-474 cells, a human breast cancer cell line overexpressing the oncogene ErbB2 (11). Moreover, a large-scale characterization of HeLa cell nuclear phosphoproteins confirmed the phosphorylation status of the endogenous ERR, identifying serine 19 as one of multiple phosphorylated residues within the NTD by tandem mass spectroscopy (57). Using phosphorylation-mimicking mutants of ERR and -, we have provided evidence for the importance of the phosphorylation status of serines 19 and 45 for sumoylation of the ERR lysine 14 and ERR lysine 40, respectively (Fig. 6). The negative charges created by the phosphorylation events close to the SUMO acceptor site can be compared with the recently identified negatively charged amino acid-dependent sumoylation motif (NDSM). The NDSM mechanism relies on the presence of negatively charged amino acids in close proximity to the sumoylation site to enhance the sumoylation. The ERR isoforms also possess such an acidic patch, although it is located outside of the limit of the identified NDSM (48). The main difference with the ERRs occurs in the possibilities offered by a regulated phosphorylation event as opposed to the fixed enhancement provided by the presence of a negatively charged glutamate residue.
Our data, together with other recent reports showing phosphorylation-dependent sumoylation of MEF2 family members (43, 65, 66, 67), HSF-1 (54), and PPAR (55) strengthen this model as a common signaling-dependent regulating mechanism for sumoylation of transcription factors and demonstrate the functionality of the phospho-sumoyl switch in vivo. Furthermore, the observation that PPAR is the only member of the NR family sharing this PDSM with the three ERR isoforms also suggests a potential link between phosphorylation-dependent sumoylation and metabolic control by NRs. As both ERR and - have emerged as essential regulators of energy metabolism (13, 52, 59, 60, 68, 69, 70, 71, 72, 73, 74, 75), the identification of the signaling pathways regulating ERR sumoylation and the study of how phosphorylation-dependent sumoylation specifically affects the expression of metabolic genes will be of importance for our understanding of pathologies such as cardiovascular diseases, diabetes, and obesity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ESRRApromoter-LUC, 2xESRRApromoter-LUC, and the 3xESRRApromoter-LUC reporter constructs as well as the pCMX-ERR expression plasmid have been previously described (50). The expression vectors for wild-type SUMO2 and wild-type UBC9 were previously described (49, 76). The shorter forms of SUMO2, one terminated with the diglycine motif (SUMO2GG) acting as a constitutively activated protein as well as one nonconjugatable form terminated before the diglycine motif (SUMO2GG) acting as a dominant negative, were generated by PCR and the purified products were cloned into pcDNA3.1 derivatives (Invitrogen, Carlsbad, CA). Point mutants of ERR (K14R, K403R, S19A, S19D, S22A, S22D), ERR (K40R, K360R, K439R, S45A, S45D), and UBC9 (C93S) were made by site-directed mutagenesis. The DNA fragments were sequenced and subcloned into pCMX-myc for ERR constructs, pCMX-Flag for ERR constructs, and pcDNA-HA for UBC9 (C93S). The wild-type and mutant versions of ERR and - were also subcloned into pCMX-Gal4 and pEGFP-C1 (CLONTECH Laboratories, Inc., Palo Alto, CA). The expression vector pcDNA3/HA-hPGC-1 was provided by A. Kralli (77). The plasmids pGEX2T-PGC-1/1–250 and pCMV5-Flag-PIASy, -PIASx, -PIASxβ, and -PIAS3 were described previously (64, 78, 79).
Cell Culture and Transient Transfections
COS-1, HeLa, and HEK293 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum. Cells were plated in 12-well or 10-cm plates 16–18 h before transfection with Fugene 6 (Roche Diagnostics, Mannheim, Germany). MEFs were freshly isolated as previously described (80) using ERR-null mice embryos (81) and were immortalized by inducing a stable expression of the simian virus 40 large T antigen (ERR-null MEF-T).
Reporter Gene Assays
Cells were harvested on ice 48 h after transfection for determination of LUC and β-galactosidase activities. Each transfection was performed in duplicate at least three times.
Fluorescence Microscopy
HeLa cells were plated on glass coverslips 16 h before transfection with GFP and HA-SUMO2 expression constructs for 48 h. Cells were washed three times with PBS, fixed, and permeabilized on ice with 2% paraformaldehyde-0.2% Triton X-100 in PBS for 15 min. The cells were then washed three times in PBS, quenched with 50 mM NH4Cl in 1x PBS for 10 min, rinsed twice with PBS, and blocked with 5% BSA in PBS for 2 h at room temperature. After a 30-min incubation at room temperature with -HA monoclonal antibody (Roche), the cells were washed three times with 1x PBS and then incubated with -mouse Alexa555 (Molecular Probes, Inc., Eugene, OR) for 30 min at room temperature and rinsed three times with PBS. The cells were then subjected to staining with 4'6-diamidino-2-phenylindole (Sigma Chemical Co., St. Louis, MO) rinsed again, and mounted on glass slides with Immu-Mount (Thermo Fisher Scientific, Inc., Waltham, MA). Cells were analyzed under a Zeiss epifluorescence confocal microscope (Carl Zeiss, Thornwood, NY).
GST Pull-Down Assay
Equal amounts of bacterially expressed GST or GST-PGC-1/1–250 protein containing the NR interaction motifs immobilized on glutathione sepharose beads were combined with 10 µl of 35S-labeled ERR as well as ERR and NTD KR mutant proteins produced with the TNT T7 coupled reticulocyte lysate system (Promega Corp., Madison, WI) in 500 µl of GST binding buffer (20 mM Tris, pH 7.5; 100 mM KCl; 0.1 mM EDTA; 0.05% Nonidet P-40; 10% glycerol; 1 mg/ml BSA; 1 mM phenylmethylsulfonyl fluoride; protease inhibitor tablet complete mini (Roche) for 1 h at 4 C. The beads were washed five times with cold binding buffer, and the immobilized proteins were eluted by boiling in 2x sample buffer. The eluted proteins were resolved on SDS-PAGE, and the fixed and dried gels were visualized by autoradiography.
In Vitro Sumoylation Assay
[35S]-myc-ERR, K14R, and K403R and [35S]-flag-ERR, K40R, K360R, and K439R proteins were produced using the TNT T7-coupled reticulocyte lysate system (Promega) and subjected to in vitro sumoylation reactions with E1 and E2 purified enzymes along with SUMO1 or SUMO3 purified proteins (LAE BIO, Rockville, MD). Briefly, 2 µl of [35S]-myc-ERR, [35S]-flag-ERR, and KR variant in in vitro translation reactions were combined with 150 ng of purified human SAE1/SAE2 (E1), 1 µg of purified human UBC9, and 1 µg of purified human SUMO1 or SUMO3 proteins, and then incubated in a sumoylation buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, and 2.5 mM ATP at 37 C for 1 h. The control reaction was performed under the same conditions but the purified E1 enzyme was omitted to prevent sumoylation from occurring. Reactions were stopped by boiling in reducing sodium dodecyl sulfate sample buffer for separation by SDS-PAGE and detection by autoradiography.
In Cells and in Vivo Sumoylation Assay
HEK293 cells were transfected with the specified ERR or - constructs along with the activated HA-SUMO2GG and UBC9 to favor the sumoylation of targets or with the dominant-negative forms HA-SUMO2GG and UBC9 (C93S) to inhibit sumoylation. About 48 h after transfection, cells were lysed in buffer S (15 mM Tris-HCl, pH 6.7; 0.5% sodium dodecyl sulfate; 3% glycerol, 0.8x PBS; 4% Nonidet P-40; 0.1% β-mercaptoethanol) containing 25 mM N-ethylmaleimide, 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 50 mM sodium fluoride, and 1x complete miniprotease inhibitor tablet (Roche). The lysates were sonicated at power 5 for 15 sec using a VirSonic 100 (VirTis, Gardiner, NY) sonicator. Protein concentration was determined by Bradford assay and 80 µg of lysates were used for Western blot analysis or 250 µg of lysates were used for immunoprecipitation with a monoclonal anti-myc antibody (Roche) for ERR constructs and an anti-flag M2 resin (Sigma) for ERR constructs. For endogenous ERR sumoylation, 2-month-old male C57/BL6 mouse liver extract was prepared in buffer K (20 mM phosphate buffer, pH 7; 150 mM NaCl; 0.1% Nonidet P-40; 5 mM EDTA) containing 25 mM N-ethylmaleimide, 20 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 50 mM sodium fluoride, 25 mM β-glycerol phosphate, and 1x complete miniprotease inhibitor tablet (Roche). Briefly, mouse livers were homogenized in buffer K for 5 sec with a polytron-type homogenizer. The crude lysate was incubated for 1 h at 4 C with rotation followed by centrifugation at 13,000 rpm for 5 min at 4 C. The supernatant was then quantified by Bradford assay and 2 mg of whole-cell liver extract was used for immunoprecipitation with a previously described anti-hERR polyclonal antiserum raised in our laboratory against the whole N terminus (first 74 amino acids) (73) or with an anti-ERR pS19 (described below). The anti-SUMO2/3 antibody (no. AB3876) was obtained from Chemicon International (Temecula, CA).
EMSA
EMSAs were performed as previously described (82) using the consensus ERR response element (ERRE) probe (5'-TCGACGCTTTCAAGGTCATATCCG-3') and a tandem probe containing two ERRE elements from the ERR promoter endogenous sequence (5'-CCGTGACCTTCATTCGGTCACCGCAGTGACCTTCAT-3'). The ERRE sequences are underlined. HEK293 cells were transfected with 10 µg of pCMX-myc-ERR, K14R, or K403R expression vectors for 48 h after which nuclear extracts were prepared as previously described (83). About 2 µg of extract was used per EMSA reaction. For PGC-1 supershift experiments, 2 µg of nuclear extracts was mixed with 2 µg of purified GST-PGC-1/1–250. EMSA with in vitro sumoylated proteins was performed as described above for in vitro sumoylation after which the complete reaction (20 µl) or increasing amounts of the reaction (5, 10, 20 µl) were used for the EMSA reaction.
Generation of the pS19 Phospho-Specific Antibody
The anti-ERR pS19 rabbit antiserum was custom generated by Chemicon International against the phosphorylated Ser19 peptide (CPLYIKAEPApSPD) conjugated to keyhole limpet hemocyanin. To assess specificity of the antisera for the phosphorylated epitope, a dot blot analysis was performed by spotting 0.2 to 1 µg of synthetic phosphorylated and the correponding nonphosphorylated peptides on nitrocellulose membranes followed by Western blot analysis using the previously described general anti-ERR antiserum and the new anti-ERR pS19 phospho-antiserum.
Statistics
One-way ANOVA followed by Bonferonni post-test analysis were performed using GraphPad InStat software (GraphPad Software, Inc., San Diego, CA). Where indicated, *** is P < 0.0001 and ** is P < 0.001.
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ACKNOWLEDGMENTS |
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This work was supported by the Canadian Institutes of Health Research (CIHR) and the Canadian Cancer Society through the National Cancer Institute of Canada. A.M.T. is a recipient of a Canadian Institutes of Health Research graduate scholarship.
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FOOTNOTES |
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First Published Online December 6, 2007
Abbreviations: ER, Estrogen receptor; ERR, estrogen-related receptor; ERRE, ERR response element; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; LBD, ligand binding domain; LUC, luciferase; MEFs, mouse embryonic fibroblasts; NDSM, negatively charged amino acid-dependent sumoylation motif; NR, nuclear receptor; NTD, amino-terminal domain; PDSM, phosphorylation-dependent sumoylation motif; PGC-1, PPAR- coactivator 1; PPAR, peroxisome proliferator-activated receptor; PIAS, protein inhibitor of activated signal transducer and activator of transcription; SC, synergy control; SRC, steroid receptor coactivator; SUMO, small ubiquitin-related modifier; UAS, upstream activating sequence.
Received for publication July 20, 2007. Accepted for publication November 27, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(PGC-1) coactivates the cardiac-enriched nuclear receptors estrogen-related receptor- and -. Identification of novel leucine-rich interaction motif within PGC-1. J Biol Chem 277:40265–40274. J Biol Chem 278:9013–9018 (ERR): crystal structure of ERR ligand binding domain in complex with peroxisome proliferator-activated receptor coactivator-1. J Biol Chem 279:49330–49337 ligand binding domain in three functional states reveal the molecular basis of small molecule regulation. J Biol Chem 281:37773–377811 transcriptional activities are regulated in part via the ErbB2/HER2 signaling pathway. Mol Cancer Res 5:71–85. Cancer Res 65:6120–6129 is a transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene. Mol Cell Biol 17:5400–5409[Abstract] hinge region regulates its transcriptional activity. Mol Endocrinol 19:2671–2684 is modulated by SUMO-1 modification. J Biol Chem 279:29551–29557. Nature 437:759–763[CrossRef][Medline]. Mol Cell 25:57–70[CrossRef][Medline] inhibits transcriptional synergy through its PIASy-enhanced modification by SUMO-1 or SUMO-3. J Biol Chem 278:9134–9141 (ERR) promoter dictates PGC-1 control of ERR expression. J Biol Chem 279:18504–18510 and peroxisome proliferator-activated receptor- coactivator-1 regulate estrogen-related receptor- gene expression via a conserved multi-hormone response element. J Mol Endocrinol 34:473–487 directs peroxisome proliferator-activated receptor signaling in the transcriptional control of energy metabolism in cardiac and skeletal muscle. Mol Cell Biol 24:9079–9091 is negatively regulated by SUMO conjugation in the amino-terminal domain. Genes Cells 9:1017–1029 and GABPA/β specify PGC-1-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc Natl Acad Sci USA 101:6570–6575 (ERR) functions in PPAR coactivator 1 (PGC-1)-induced mitochondrial biogenesis. Proc Natl Acad Sci USA 101:6472–6477 coactivator 1 (PGC-1) signaling by an estrogen-related receptor (ERR) ligand. Proc Natl Acad Sci USA 101:8912–8917 coactivator 1β/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity. Proc Natl Acad Sci USA 100:12378–12383-cofactor interactions. Mol Endocrinol 21:62–76 site can dictate mode of binding and PGC-1 activation of target promoters. Mol Endocrinol 20:302–310 in the control of mitochondrial fatty acid β-oxidation during brown adipocyte differentiation. J Biol Chem 272:31693–31699 is a repressor of phosphoenolpyruvate carboxykinase gene transcription. J Biol Chem 281:99–106 (ERR) is a transcriptional regulator of apolipoprotein A-IV and controls lipid handling in the intestine. J Biol Chem 279:52052–52058 coactivates PDK4 gene expression via the orphan nuclear receptor ERR: a mechanism for transcriptional control of muscle glucose metabolism. Mol Cell Biol 25:10684–10694 is essential for adaptive thermogenesis. Proc Natl Acad Sci USA 104:1418–1423 and . Cell Metabolism 5:345–356[CrossRef][Medline] directs and maintains the transition to oxidative metabolism in the post-natal heart. Cell Metabolism 6:16–24 is required for the bioenergetic and functional adaption to cardiac pressure overload. Cell Metabolism 6:25–37[CrossRef][Medline]. J Biol Chem 277:13918–139251 gene in breast cancer cells. Mol Endocrinol 19:1584–1592. Mol Cell Biol 23:7947–7956NURSA Molecule Pages Link:
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