Alternative O-Glycosylation/O-Phosphorylation of Serine-16 in Murine Estrogen Receptor beta

POST-TRANSLATIONAL REGULATION OF TURNOVER AND TRANSACTIVATION ACTIVITY*

Xiaogang ChengDagger § and Gerald W. HartDagger

From the Dagger  Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 and the § Graduate Program of the Department of Biochemistry and Molecular Genetics, The University of Alabama at Birmingham, Birmingham, Alabama 35294

Received for publication, November 16, 2000, and in revised form, January 9, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

O-Linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification abundant on nuclear and cytoplasmic proteins. Recently, we demonstrated that the murine estrogen receptor-beta (mER-beta ) is alternatively O-GlcNAcylated or O-phosphorylated at Ser16. Analyses of mER-beta s containing mutations in the three adjacent hydroxyl amino acids at this locus confirmed that Ser16 is the major site of O-GlcNAc modification on mER-beta and that mutants lacking hydoxyl amino acids at this locus are glycosylation-deficient. Pulse-chase studies in transfected Cos-1 cells demonstrate that the turnover rate of the mutant containing a glutamic acid moiety at Ser16, which mimics constitutive phosphorylation at this locus, is faster than that of the wild type receptor. Whereas, the mutant without hydroxyl amino acids at this locus is degraded at a slower rate, indicating that O-GlcNAc/O-phosphate at Ser16 modulates mER-beta protein stability. Luciferase reporter assays also show that the Ser16 locus mutants have abnormal transactivation activities, suggesting that the two alternative modifications at Ser16 on mER-beta may also be involved in transcriptional regulation. DNA mobility shift assays show that the mutants do not have altered DNA binding. Green fluorescence protein constructs of both wild type and mutant forms of mER-beta show that the receptor is nearly exclusively localized within the nucleus. It appears that reciprocal occupancy of Ser16 by either O-phosphate or O-GlcNAc modulates the degradation and activity of mER-beta .


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A large number of proteins, ranging from cytoskeletal components to nuclear proteins, are dynamically modified by O-linked N-acetylglucosamine (O-GlcNAc)1 in virtually all higher eukaryotes, including plants and fungi (for reviews see Refs. 1 and 2). Many O-GlcNAcylated proteins are regulatory proteins, such as transcription factors (3, 4), nucleoporins (5), and cytoskeletal proteins (6, 7). The attachment and removal of O-GlcNAc by specific enzymes (8-13) is rapid and analogous to the dynamics of O-phosphate controlled by kinases and phosphatases (14, 15). O-GlcNAc sites resemble phosphorylation sites, and in some cases the two modifications are mutually exclusive (16). For example, in the case of estrogen receptor beta  (ER-beta ) (17), SV-40 large T antigen (18), and the c-Myc oncogene (19, 20), O-GlcNAc and O-phosphate compete for the same hydroxyl moiety. Like phosphorylation, O-GlcNAcylation is responsive to the cell cycle (21), extracellular signals (22), glucose metabolism (23), and to the growth state of the cell (24). Other studies suggest that O-GlcNAcylation plays important roles in the regulation of transcription and translation (16, 25-27).

The estrogen receptor (ER) is a central component of estrogen regulation (28, 29). Recently ER-beta , a homologue of ER-alpha , was discovered in various species (30-33). ER-beta not only shares many structural and functional features with ER-alpha but also has distinctive characteristics, such as different tissue distribution (31) and differential ligand responsiveness (34). High expression of ER-beta in human brain, cardiovascular system, thymus, bone, kidney, lung, urogenital tract, and gastrointestinal tract suggests that ER-beta has significant roles in cellular functions not previously thought to involve estrogens. Such ER-beta -mediated functions might include effects on memory and reproduction, enhancement of T-cell immunity, vascular lesion protection, and colon cancer protection. In addition, ER-beta appears to be the only estrogen receptor type expressed in the embryonic central nervous system, implying that ER-beta is important to early embryonic development. Transgenic mice with only mER-beta deleted (35) and double mER-alpha /mER-beta knockouts (36) have been generated. Female ER-beta knockout mice develop follicular arrest and anovulation, indicating that both ER-beta and ER-alpha are required for the maintenance of germ and somatic cells in the postnatal ovary. Data from these mice further suggest that several specialized functions are mediated distinctly between ER-alpha and ER-beta . However, because ER-beta was only recently discovered (30-33), much work remains toward elucidating its functions and relationships to ER-alpha .

Previously, we mapped the major O-GlcNAc site to Ser16 near the amino terminus of mER-beta and showed that this same hydroxyl moiety is alternatively modified by O-phosphate (17). Here, we report that these two alternative modifications play distinct roles in modulating mER-beta degradation and function.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

mER-beta O-GlcNAc Site Mutants and Related Plasmid Constructs-- The mER-beta cDNA was engineered using an established method, as described in our previous studies (17). To introduce the appropriate restriction enzyme sites, mER-beta cDNA in pBlueBac 3 (Invitrogen, San Diego, CA) was subcloned into pBlueBacHis2 and excised using XhoI and EcoRI digestion. The resulting fragments were then subcloned into pcDNA3.1(-) for functional studies. All constructs were verified by automated DNA sequencing.

Altered Sites I (Promega, Madison, WI) was used to mutate mER-beta Ser16. mER-beta cDNA in pcDNA3.1(-) was subcloned into pAlter I via XbaI and EcoRI sites. The mutagenesis primers (shown in antisense; S16A, CCTTCCAGGTTACCGACGGCGGCGGGAACACTGTAGTTC; S16E, CCTTCCAGGTTACCGACCTCGGCGGGAACACTGTAGTTC) containing changes (with underline) of the Ser16 to Ala or Glu along with the changes of two adjacent Ser15 and Thr17 to Ala and Val were synthesized and used, respectively, in the mutagenesis process. The mutants were named as S16A and S16E accordingly, as illustrated below in Fig. 1. The mutated cDNAs were verified by automated sequencing with serial internal primers covering the entire coding region and subcloned back into pcDNA3.1(-) and other plasmids for functional studies.

To study the subcellular localization of mER-beta , mER-beta cDNA in pAlter I was digested with BglII and EcoRI and subcloned into GFP vector pEGFP-C1 (CLONTECH, Palo Alto, CA) in-frame. The final engineered mER-beta cDNAs in pEGFP-C1 were verified by automated DNA sequencing. To immunoprecipitate mER-beta expressed in mammalian cell lines, a FLAG tag encoding the peptide epitope, DYKDDDDK, was incorporated into the carboxyl-terminal end of mER-beta cDNA using polymerase chain reaction (37). All constructs were verified by automated DNA sequencing.

Characterization of the Glycosylation of the mER-beta O-GlcNAc Mutant-- Expression and purification of mER-beta proteins from Sf9 cells were described previously (17). The O-GlcNAc moieties on purified mER proteins were labeled with galactosyltransferase by transferring [3H]galactose from UDP-[3H]galactose, as described previously (38). The tritium-labeled protein was separated from unincorporated UDP-[3H]galactose using a 1.5- × 30-cm Sephadex G-50 column in 50 mM ammonium formate, 0.1%(w/v) SDS. For tritium images, the protein was resolved on 10% SDS-PAGE gel, and the gel was stained with Coomassie Blue R-250, impregnated with 1 M salicylic acid for 30 min, dried under vacuum, and exposed to x-ray film at -70 °C for 2 days. For tryptic map comparison, the labeled proteins were digested with 2% (w/w) trypsin (sequencing grade, Roche Molecular Biochemicals, Indianapolis, IN) in 0.1 M ammonium bicarbonate (pH 7.4) at 37 °C overnight. Separation of [3H]galactose-labeled tryptic mER glycopeptides was achieved on a C2/C18 reversed phase column (3.2 × 100 mm, Amersham Pharmacia Biotech, Piscataway, NJ) using a 0-60%(v/v) gradient of acetonitrile in 0.1%(w/v) trifluoroacetic acid over 90 min at a flow rate 0.1 ml/min on a SMART system (Amersham Pharmacia Biotech). Fractions were collected (0.2 ml/fraction) and counted.

Tissue Culture and Transfection-- Estrogen receptor-deficient Cos-1 cells are maintained in phenol red-free Dulbecco's modified Eagle's medium/F-12 medium (Life Technologies Inc., Gaithersburg, MD) supplemented with 5% (w/v) charcoal/dextran-stripped fetal bovine serum (estrogen-depleted). Cos-1 cells were transfected using the liposome method (Life Technologies Inc.). At 24 h post-transfection, transfected cells were changed with fresh medium. 17beta -Estradiol (E2)was added to the medium at a final concentration of 20 nM, unless otherwise indicated.

Pulse-chase 35S Labeling in Vivo-- For pulse-chase analyses, Cos-1 cells (in 100-mm dishes) were transfected with wild type or mutant mER-beta . At 40-48 h post-transfection, the cells were rinsed with methionine-cysteine-free medium once, incubated at 37 °C for 30 min, then supplemented with 120 µCi of labeling mix (35S Express label mix, PerkinElmer Life Sciences, Boston, MA) in the same medium. At desired time points, cells were placed into complete medium containing no label for the chase. Cells were harvested at various time points and lysed with 1 ml of lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1%(v/v) Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 10 µg/ml leupeptin) on ice for 1 h. The supernatants obtained after centrifugation at 10,000 × g for 10 min at 4 °C were stored at -80 °C or used immediately for immunoprecipitation.

Immunoprecipitation-- Immunoprecipitation was performed as described previously (37). Briefly, cell lysates with 5 × 107 dpm 35S total labeling were precleared with 20 µl of a 50% (v/v) slurry of protein G-agarose at 4 °C for 3 h. The precleared supernatants were then mixed with 20 µl of 50% (v/v) slurry of anti-FLAG M2 beads (Sigma Chemical Co., St. Louis, MO) and rotated at 4 °C for 2 h. The beads were washed, resuspended in 50 µl of 2× SDS-PAGE loading buffer, boiled, and loaded onto a 10%(w/v) SDS-PAGE gel. The gel was fixed and stained with Coomassie Blue R-250, destained, dried, and exposed to x-ray film. Relative density values were determined using a digital imaging system IS1000 (Alpha Innotech Corp., San Leandro, CA).

Luciferase Activity Assays-- All transfections were done with Cos-1 cells at 50-60% confluence in 12-well plates. For each well, 0.2 µg of mER-beta plasmid DNA in pcDNA3.1(-), containing either wild type or one of the mutants, along with 0.2 µg of pcDNA3.1(-)-lacZ, 5 ng of pERE-TK-Luc (a gift from Dr. Gilles B. Tremblay, McGill University, Montreal, Canada) were transfected into Cos-1 cells. At 24 h post-transfection, 17beta -estradiol was added with fresh medium at a final concentration of 20 nM and cells were further incubated overnight. Lysates were made by washing cells with phosphate-buffered saline buffer (pH 7.4) twice and lysed in reporter lysis buffer (Promega, Wisconsin, MI). Aliquots of lysates were assayed using commercial luciferase and galactosidase kits (Promega). Each mER-beta sample was transfected in triplicate.

Electrophoretic Mobility Shift Assay-- To determine the DNA binding activity of the ER-beta mutants, extracts of mER-transfected Cos-1 cells were made according to published methods (39). The assay was carried out in binding buffer containing 20 mM HEPES, pH 7.4, 50 mM KCl, 1 mM dithiothreitol, 10% glycerol, 50 nM estradiol, 0.5 mg/ml bovine serum albumin, 50 ng/µl poly[d(I·C)/d(I·C)], and protease inhibitors. 32P-Labeled double-stranded oligonucleotide probe (1 ng) containing a consensus estrogen response element (ERE) sequence from chicken vitellogenin (5'-CTAGAAAGTCAGGTCACAGTGACCTGATCATT-3') was used in 20-µl reaction volumes. Preincubation with all the reaction components except the labeled probe was conducted on ice for 15 min. After addition of the labeled probe, the reaction was allowed to incubate on ice for an additional 15 min. The ER·ERE complex was then resolved on native 6% polyacrylamide gels. The gel was fixed, dried, and exposed to x-ray film at -80 °C. The antibody against ER-beta (CalBiochem, La Jolla, CA) and excess unlabeled ERE probe were added as indicated.

Characterization of Subcellular Localization-- To examine subcellular localization of mER-beta , the GFP fusion constructs of mER-beta cDNA were transfected into mammalian cells lines, as described above. After 1 day, fresh media and 17beta -estradiol at the final concentration of 20 nM were added. Fluorescence images were recorded using a digital camera (Hamamatsu, Tokyo, Japan).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mutation of the O-GlcNAc/O-Phosphate Locus on mER-beta -- Our previous studies demonstrated that mER-beta is modified alternatively by O-GlcNAc or O-phosphate at Ser16 (17). To reveal biological roles of the O-GlcNAcylation/O-phosphorylation on mER-beta , we mutated Ser16 into either Ala or Glu along with Ser15 and Thr17 into Ala and Val to generate two mutants, designated mER-beta S16A and S16E, respectively, as illustrated in Fig. 1. The S16E mutant is designed to mimic the constitutive phosphorylation of Ser16 (40, 41). Ser15 and Thr17 were concomitantly mutated to eliminate the possibility for promiscuous O-glycosylation/O-phosphorylation at this locus, which might occur even when the observed major acceptor site (Ser16) is eliminated.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   mER-beta mutants used in this study. Wild-type mER-beta cDNA was mutated at the Ser16 by changing either Ser right-arrow Ala or Ser right-arrow Glu along with changing Ser15 (Ser right-arrow Ala) and Thr17 (Thr right-arrowVal). The two mutants were named S16A and S16E, respectively. All constructs were engineered to incorporate a FLAG tag at their carboxyl terminus.

mER-beta O-GlcNAc Site Mutant Is Glycosylation-deficient-- To assess the glycosylation state of mER-beta O-GlcNAc site mutants, the protein of the S16A mutant expressed and purified from insect Sf9 cells was probed for O-GlcNAc using galactosyltransferase and UDP-[3H]galactose (38). As shown in Fig. 2A, the protein of the S16A mutant was labeled approximately 5-fold less than the wild type, suggesting that the mutant S16A protein is deficient in O-GlcNAc. To further compare tryptic maps between the wild type and the S16A mutant, labeled proteins were digested with trypsin and the tryptic glycopeptides were resolved on a reverse phase column. As shown in Fig. 2B, tryptic glycopeptide maps from the S16A mutant (top panel) showed only baseline levels of radioactivity, whereas the wild type (bottom panel) showed the same major tryptic glycopeptide (retention time = 42 min) and smaller labeled peaks at retention times of 20, 36, and 54 min, respectively, previously seen for mER-beta (17). Previous analyses (17) of the smaller labeled peaks in (Fig. 2B, bottom panel) indicate that they likely result from incomplete proteolysis. This conclusion is supported by their disappearance in the mutant (Fig. 2B, top panel). Thus, we conclude that the mER-beta mutants at the Ser16 locus are glycosylation-deficient.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   The Ser16 to Ala mutant is poorly glycosylated in vivo. A, the wt and the S16A mutant mER-beta were expressed and purified from insect Sf9 cells. Purified proteins were enzymatically labeled with UDP-[3H]galactose using galactosyltransferase. [3H]Galactose-labeled proteins were resolved by 10% SDS-PAGE gel electrophoresis. Gels were impregnated with 1 M salicylic acid, dried, and exposed to x-ray film at -80 °C for 2 days. Scintillation counting of the bands indicated that the band from wild-type contained ~1.77 × 106 dpm and that from the mutant contained ~3.61 × 105 dpm. B, [3H]galactose-labeled proteins were digested with trypsin. Tryptic peptides were resolved on a C2/C18 reversed phase column. The column was developed with a 90-min linear gradient of 0-60% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid from 6 to 96 min at a flow rate of 0.1 ml/min. Eluates were collected and counted. Upper panel, the S16A mutant tryptic profile; lower panel, the wt tryptic profile. Note: Smaller labeled peaks likely result from incomplete proteolysis of the major glycosylation site.

The O-Glycosylation/O-Phosphorylation Site Mutants of mER-beta Have Altered Turnover Rates-- PEST regions in proteins, enriched with Pro, Glu, Ser, and Thr, have been proposed to be responsible for the rapid degradation of certain proteins (42, 43). Our earlier studies documented that O-GlcNAcylation sites on mER-alpha (44) and mER-beta (17) are in regions of the proteins that have high PEST scores. This observation suggests that one likely role of O-GlcNAc on ER is to modulate ER protein stability. Therefore, we directly examined the relative turnover rates in vivo of the wt and mutant mER-beta proteins using pulse-chase analyses. Transfected Cos-1 cells were pulse-labeled with 35S-labeled amino acids in vivo for 3 h and chased for up to 6 h. Quantitative results averaged from three independent experiments are shown in Fig. 3. Compared with wt, the degradation of the S16A mutant appears to be slower, whereas the degradation of the S16E mutant appears to be much faster. Assuming rough linearity, the wt mER-beta turned over rapidly in the presence of estrogen, with an average half-life of 7-8 h, similar to the range of ER-alpha reported previously (45). In contrast, the S16A mutant has a prolonged average half-life of about 15-16 h, and the S16E mutant has a shortened average half-life of about 4-5 h. These findings suggest that O-phosphorylation on the Ser16 of mER-beta results in accelerated degradation of mER-beta as mimicked by the S16E mutant, whereas O-glycosylation, which blocks phosphorylation, would be predicted to result in stabilization of the protein.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   The modification state of Ser16 of mER-beta affects the protein's turnover rate. Cos-1 cells transfected with either the wild type or mutated mER-beta were metabolically labeled with 35S-protein Express label for 3 h and then chased with unlabeled rich medium for various time points as indicated in the figure. Cell lysates were prepared as described under "Materials and Methods." Samples, containing ~5 × 107 dpm per lysate, were immunoprecipitated with anti-FLAG antibody M2, and isolated proteins were resolved by electrophoresis on 10% SDS-PAGE gels. Gels were fixed, dried, and exposed to x-ray film for 30 min. 35S images from three separate experiments were processed with an IS-1000 Digital imaging system. The density values of mER-beta bands from every time point are converted into percentages relative to the zero time point of each sample. The average percentages along with standard deviations are plotted.

The O-Glycosylation/O-Phosphorylation Site Mutants of mER-beta Have Altered Transactivation Activities-- The fact that the major O-GlcNAc site on mER-beta is located within the transactivation domain of the protein led us to examine the role of O-GlcNAc/O-phosphate at this site in ER-mediated transcriptional activation. The transactivation activities of the mutants were measured by cotransfection of mER-beta cDNAs with an ERE-linked luciferase reporter gene in Cos-1 cells. As summarized in Fig. 4, the S16E mutant has elevated transactivation activities compared with wt-mER-beta . However, the S16E mutant is not further stimulated by estrogen, suggesting that the S16E is constitutively active under these conditions. In contrast, the S16A has only basal activity with minor stimulation by estrogen. The relatively modest level of stimulation seen in this system may reflect the lack of appropriate coactivators in the Cos-1 cells used. Nonetheless, it appears that the alternative post-translational modification of Ser16 of mER-beta is not only crucial to achieve normal levels of ER-mediated transactivation but also is important to estrogen responsiveness of the receptor.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 4.   Modification of Ser16 of mER-beta modulates the protein's transactivation activity. Cos-1 cells were cotransfected with the reporter plasmid ERE-TATA-TK-Luc, internal control beta -galactosidase expression plasmid, and the expression vector (V) containing wt or mutant mER-beta cDNA. At 24 h post-transfection, 20 nM 17beta -estradiol (E2) in fresh medium was added, and the cells were incubated overnight. Cells were then harvested, and luciferase assays were performed. All activities were normalized with activities obtained from equal numbers of cells cotransfected with beta -galactosidase. The relative luciferase activity was set as 1 for the activity in cells transfected with the vector alone.

Mutation of the O-Glycosylation/O-Phosphorylation Site on mER-beta Does Not Affect DNA Binding-- To compare the binding of the wild type and mutant forms of mER-beta to the DNA response elements, we tested cell extracts from mER-beta -transfected Cos-1 cells using electrophoretic mobility shift assays. As shown in Fig. 5, the two mutants are able to form the same ER·ERE complexes as the wild type. Differences in intensity of the complexes result from the relative amount of mER-beta in each nuclear extract as determined by Western blotting using an antibody to the FLAG epitope on each recombinant protein (data not shown). Note that the extract from the S16E mutant contains less mER-beta protein, probably due to its rapid rate of degradation, even though the experiments were normalized for transfection efficiency (Fig. 3).


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 5.   Modification of Ser16 of mER- beta  does not affect the protein's DNA binding. The mER-beta cDNAs were transfected into Cos-1 cells by the liposome method. Cell extracts were prepared for the electrophoretic mobility shift assay. The vitellogenin estrogen response element (ERE) DNA probe was labeled with 32P using the Klenow fragment of DNA polymerase. The specificity of the assay was demonstrated by cold probe competition (100-fold excess) and mER-beta antibody blocking of the interaction. Note: mER-beta levels are less in the S16E mutant due to its rapid degradation (see text and Fig. 3).

Mutant and wt mER-beta Proteins Are Localized Exclusively in the Nucleus-- Although ER-alpha is known to be mainly localized in the nucleus, the subcellular localization of the homologue ER-beta has not been studied. To investigate the subcellular distribution of ER-beta , we fused mER-beta to GFP. The localization of GFP-fused wt mER-beta was then studied in several different mammalian cell lines. As shown in Fig. 6, virtually all of the receptor is restricted to the nucleus in monkey kidney Cos-1 cells, independent of the presence or the absence of its cognate ligand estrogen. The same distribution of the wt receptor was also observed for other cell lines, including HeLa and MCF-7 cells (data not shown).


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 6.   mER- beta  localizes exclusively to nucleus. mER-beta cDNAs were subcloned into pGFP-C1 vector, and fusion constructs were transfected into Cos-1 cells by the liposome method. 20 nM 17beta -estradiol (E2) was added at 24 h post-transfection. Images were observed at 48 h post-transfection and recorded using a Hamamatsu digital camera with settings of 5 s for fluorescent images (right column) and 0.2 s for phase images (left column). wt, cells transfected with wild-type mER-beta fused to GFP; GFP, cells transfected with GFP construct alone; wt + E2, cells transfected with wild-type mER-beta grown in the presence of E2; GFP + E2, cells transfected with GFP construct alone and grown in the presence of E2.

Earlier studies on nuclear pore proteins suggested that O-GlcNAcylation is likely involved in nuclear transport (5, 46-48). Because the functionality of the estrogen receptor relies on its proper subcellular localization, we also examined the subcellular localization of the two glycosylation/phosphorylation site mutants as GFP fusion proteins. Both mutants are also exclusively localized to the nucleus (data not shown), suggesting that modifications at Ser16 are not involved in mediating mER-beta 's transport into the nucleus.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Since the discovery of the O-GlcNAc modification, several functional roles have been postulated, such as modulation or mediation of protein·protein interactions, regulation of nuclear transport, transient regulation of phosphorylation site availability, and modulation of protein turnover (1, 2). In this study, we provide evidence that O-GlcNAc on mER-beta has a reciprocal relationship with phosphorylation by capping a phosphorylation site that modulates mER-beta degradation and is also important for the receptor's transactivational activity.

Previous studies on eukaryotic initiation factor 2-associated protein p67 and on the transcription factor Sp1 showed that O-GlcNAc removal from both p67 and Sp1 targets them for rapid degradation by the proteasome (25, 49). Studies on the mutated forms of mER-beta not only provide additional evidence for O-GlcNAc regulating protein degradation but also suggest that the saccharide may act by blocking the addition of phosphate, which itself targets the protein for rapid degradation. To further understand the relative roles of O-GlcNAc versus O-phosphate at Ser16, we compared the wt mER-beta to both the S16A and the S16E mutants, the latter of which mimics constitutive phosphorylation (40, 41, 50, 51). The simplest interpretation of the data is that the S16E mutant behaved as the phosphorylated form of the protein resulting in accelerated degradation, whereas the S16A mutant behaved analogous to the "capped" glycosylated form of the protein that slowed degradation. Because many of the known O-GlcNAc sites are located near proline residues, glycosylation sites adjacent to acidic amino acids will have high intrinsic PEST scores, whereas others may be dependent upon phosphorylation to target PEST-mediated degradation (42, 43). It has been suggested that phosphorylation can change Ser or Thr residues into negatively charged residues so as to convert some imperfect PST sequences into PEST degradation signals. In contrast, O-GlcNAcylation could prevent these phosphorylation effects by either competing at the same hydroxyl directly or by changing the protein conformation indirectly to mask the charged regions, as has been suggested for p53 (52).

The N terminus of mER-beta , which harbors the major O-GlcNAc site, mediates the receptor's a transactivation functions, which in turn activates target genes (53, 54). Our luciferase reporter data show that the extent of transactivation is dependent upon the modification of the hydroxyl group of Ser16. These data suggest that O-GlcNAc/O-phosphate at this site directly plays a role in modulating mER-beta -mediated transactivation. Earlier studies suggested that O-GlcNAcylation modulates transactivation by mediating the appropriate protein·protein interactions of many transcription factors such as Sp1 (3, 26, 27). Recent in vivo studies showed that the concentration of these transcriptional activator proteins is regulated by the proteasome-mediated degradation pathway, and the rate of degradation of activators by the proteasome correlates with activation domain potency in vivo (55). Consistent with these earlier reports on other transcription factors, the alternate O-GlcNAc/O-phosphorylation of mER-beta appears to be involved in both degradation and transactivation functions of the molecule.

Based on our in vivo [32P]orthophosphate labeling studies (data not shown), Ser16 is one of several phosphorylation sites on mER-beta . It is likely that Ser16 is a regulatory site with typically low occupancy and rapid cycling. Earlier studies on transcription factor Sp1 have suggested that, upon glucose starvation, Sp1 undergoes rapid deglycosylation and becomes more susceptible to proteasome degradation (49). Recently, it has been reported that ER-alpha is rapidly degraded by the 26 S proteasome upon estrogen stimulation (56). O-GlcNAc transferase activity is exquisitely sensitive to concentrations of UDP-GlcNAc/UDP, which are in turn highly sensitive to energy metabolism (11, 23). Thus, in energy-rich conditions, O-GlcNAc levels would be expected to increase, in turn preventing degradation of certain proteins, such as ER-beta .

Our green fluorescence protein fusion results revealed that there is no significant redistribution of mER-beta in mammalian cells induced by the mutations, excluding the possibility that nuclear localization requires either phosphorylation or O-GlcNAcylation at the Ser16 of mER-beta . However, we did observe some minor nuclear pattern changes, such as a clustering of ER-beta within the nucleus upon estrogen treatment (Fig. 6). This clustering phenomenon is similar to that seen for ER-alpha using a similar approach (57).

The occurrence of O-GlcNAcylation sites in the key regulatory regions of some oncogenes and tumor suppressors, such as c-Myc, p53, and SV-40 large T-antigen (18-20, 52), reinforces the potential regulatory significance of this modification. If a reciprocal relationship between O-GlcNAcylation and O-phosphorylation is found to be common, then it will be important to carefully evaluate the respective roles of these distinct modifications. Generally, this will not be possible by direct site-directed mutagenesis approaches but rather will require novel methods, such as the chemi-enzymatic synthesis of site-specifically modified glyco- and phospho-forms of these regulatory proteins (58-60). The interplay between O-GlcNAc and O-phosphate on mER-beta Ser16 demonstrated in this study provides another excellent example where both of these modifications work in a coordinated manner to regulate the activity of a key regulatory protein.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Gilles B. Tremblay for providing mER-beta cDNA and for the related ERE-reporter plasmids and for technical advice with respect to luciferase activity studies. We thank Sai Iyer for his work and evaluation of the transactivation experiments. Finally, we thank the members of the Hart laboratory for helpful discussion and critical reading for the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R01 CA42486 (to G. W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205-2185. Tel.: 410-614-5993; Fax: 410-614-8804; E-mail: gwhart@jhmi.edu.

Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010411200

    ABBREVIATIONS

The abbreviations used are: O-GlcNAc, O-linked N-acetylglucosamine; mER-beta , murine estrogen receptor-beta ; PAGE, polyacrylamide gel electrophoresis; galactosyltransferase, Galbeta (1-4)galactosyltransferase; ERE, estrogen response element; GFP, green fluorescence protein; E2, 17beta -estradiol; wt, wild type.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Comer, F. I., and Hart, G. W. (2000) J. Biol. Chem. 275, 29179-29182[Free Full Text]
2. Hart, G. W. (1997) Ann. Rev. Biochem. 66, 315-335[CrossRef][Medline] [Order article via Infotrieve]
3. Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133[Medline] [Order article via Infotrieve]
4. Reason, A. J., Morris, H. R., Panico, M., Marais, R., Treisman, R. H., Haltiwanger, R. S., Hart, G. W., Kelly, W. G., and Dell, A. (1992) J. Biol. Chem. 267, 16911-16921[Abstract/Free Full Text]
5. Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, R. S., Gerace, L., and Hart, G. W. (1987) J. Cell Biol. 104, 1157-1164[Abstract]
6. Hagmann, J., Grob, M., and Burger, M. M. (1992) J. Biol. Chem. 267, 14424-14428[Abstract/Free Full Text]
7. Arnold, C. S., Johnson, G. V. W., Cole, R. N., Dong, D. L. Y., Lee, M., and Hart, G. W. (1996) J. Biol. Chem. 271, 28741-28744[Abstract/Free Full Text]
8. Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990) J. Biol. Chem. 265, 2563-2568[Abstract/Free Full Text]
9. Haltiwanger, R. S., Blomberg, M. A., and Hart, G. W. (1992) J. Biol. Chem. 267, 9005-9013[Abstract/Free Full Text]
10. Kreppel, L. K., Blomberg, M. A., and Hart, G. W. (1997) J. Biol. Chem. 272, 9308-9315[Abstract/Free Full Text]
11. Kreppel, L. K., and Hart, G. W. (1999) J. Biol. Chem. 274, 32015-32022[Abstract/Free Full Text]
12. Lubas, W. A., Frank, D. W., Krause, M., and Hanover, J. A. (1997) J. Biol. Chem. 272, 9316-9324[Abstract/Free Full Text]
13. Dong, D. L.-Y., and Hart, G. W. (1994) J. Biol. Chem. 269, 19321-19330[Abstract/Free Full Text]
14. Krebs, E. G. (1993) Angew. Chem. Int. Ed. Engl. 32, 1122-1129
15. Fischer, E. H. (1993) Angew. Chem. Int. Ed. Engl. 32, 1130-1137
16. Kelly, W. G., Dahmus, M. E., and Hart, G. W. (1993) J. Biol. Chem. 268, 10416-10424[Abstract/Free Full Text]
17. Cheng, X., Cole, R. N., Zaia, J., and Hart, G. W. (2000) Biochemistry 39, 11609-11620[CrossRef][Medline] [Order article via Infotrieve]
18. Medina, L., Grove, K., and Haltiwanger, R. S. (1998) Glycobiology 8, 383-391[Abstract/Free Full Text]
19. Chou, T.-Y., Dang, C. V., and Hart, G. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4417-4421[Abstract]
20. Chou, T.-Y., Hart, G. W., and Dang, C. V. (1995) J. Biol. Chem. 270, 18961-18965[Abstract/Free Full Text]
21. Chou, C.-F., and Omary, M. B. (1994) J. Cell Sci. 107, 1833-1843[Abstract/Free Full Text]
22. Kearse, K. P., and Hart, G. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1701-1705[Abstract]
23. Han, I., Oh, E.-S., and Kudlow, J. E. (2000) Biochem. J. 350, 109-114[CrossRef][Medline] [Order article via Infotrieve]
24. Roquemore, E. P., Chevrier, M. R., Cotter, R. J., and Hart, G. W. (1996) Biochemistry 35, 3578-3586[CrossRef][Medline] [Order article via Infotrieve]
25. Datta, B., Ray, M. K., Chakrabarti, D., Wylie, D. E., and Gupta, N. K. (1989) J. Biol. Chem. 264, 20620-20624[Abstract/Free Full Text]
26. Roos, M. D., Su, K. H., Baker, J. R., and Kudlow, J. E. (1997) Mol. Cell. Biol. 17, 6472-6480[Abstract]
27. Du, X.-L., Edelstein, D., Rossetti, L., Fantus, I. G., Goldberg, H., Ziyadeh, F., Wu, J., and Brownlee, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12222-12226[Abstract/Free Full Text]
28. Parker, M. G. (1995) Vitam. Horm. 51, 267-286[Medline] [Order article via Infotrieve]
29. Muramatsu, M., and Inoue, S. (2000) Biochem. Biophys. Res. Commun. 270, 1-10[CrossRef][Medline] [Order article via Infotrieve]
30. Kuiper, G. G., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5925-5930[Abstract/Free Full Text]
31. Kuiper, G. J. M., and Gustafsson, J.-A. (1997) FEBS Lett. 410, 87-90[CrossRef][Medline] [Order article via Infotrieve]
32. Mosselman, S., Polman, J., and Dijkema, R. (1996) FEBS Lett. 392, 49-53[CrossRef][Medline] [Order article via Infotrieve]
33. Tremblay, G. B., Tremblay, A., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Labrie, F., and Giguere, V. (1997) Mol. Endocrinol. 11, 353-365[Abstract/Free Full Text]
34. Barkhem, T., Carlsson, B., Nilsson, Y., Enmark, E., Gustafsson, J., and Nilsson, S. (1998) Mol. Pharmacol. 54, 105-112[Abstract/Free Full Text]
35. Krege, J. H., Hodgin, J. B., Couse, J. F., Enmark, E., Warner, M., Mahler, J. F., Sar, M., Korach, K. S., Gustafsson, J. A., and Smithies, O. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15677-15682[Abstract/Free Full Text]
36. Couse, J. F., Hweitt, S. C., Bunch, D. O., Sar, M., Walker, V. R., Davis, B. J., and Korach, K. S. (1999) Science 286, 2328-2331[Abstract/Free Full Text]
37. Kramer, M. F., and Coen, D. M. (1999) Curr. Prot. Mol. Biol. 3, 15.0.3-15.8.8
38. Roquemore, E. P., Chou, T.-Y., and Hart, G. W. (1994) Methods Enzymol. 230, 443-460[Medline] [Order article via Infotrieve]
39. Kumar, V., and Chambon, P. (1988) Cell 55, 145-156[Medline] [Order article via Infotrieve]
40. Liu, Z. P., Galindo, R. L., and Wasserman, S. A. (1997) Genes Dev. 11, 3413-3422[Abstract/Free Full Text]
41. Chen, D. S., Pace, P. E., Coombes, R. C., and Ali, S. (1999) Mol. Cell. Biol. 19, 1002-1015[Abstract/Free Full Text]
42. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368[Medline] [Order article via Infotrieve]
43. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem. Sci. 21, 267-271[CrossRef][Medline] [Order article via Infotrieve]
44. Jiang, M.-S., and Hart, G. W. (1997) J. Biol. Chem. 272, 2421-2498[Abstract/Free Full Text]
45. Pakdel, F., Le Goff, P., and Katzenellenbogen, B. S. (1995) J. Steroid Biochem. Mol. Biol. 46, 663-672
46. Schindler, M., Hogan, M., Miller, R., and DeGaetano, D. (1987) J. Biol. Chem. 262, 1254-1260[Abstract/Free Full Text]
47. Hanover, J. A., Cohen, C. K., Willingham, M. C., and Park, M. K. (1987) J. Biol. Chem. 262, 9887-9894[Abstract/Free Full Text]
48. Hanover, J. A. (1992) FASEB J. 6, 2288-2295[Abstract/Free Full Text]
49. Han, I., and Kudlow, J. E. (1997) Mol. Cell. Biol. 17, 2550-2558[Abstract]
50. Jaffe, L., Ryoo, H. D., and Mann, R. S. (1997) Genes Dev. 11, 1327-1340[Abstract]
51. Ashcroft, M., Kubbutat, M. H. G., and Vousden, K. H. (1999) Mol. Cell. Biol. 19, 1751-1758[Abstract/Free Full Text]
52. Shaw, P., Freeman, J., Bovey, R., and Iggo, R. (1996) Oncogene 12, 921-930[Medline] [Order article via Infotrieve]
53. Shibata, H., Spencer, T. E., Onate, S. A., Jenster, G., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1997) Recent Prog. Horm. Res. 52, 141-164[Medline] [Order article via Infotrieve]
54. Edwards, D. P. (1999) Vitam. Horm. 55, 165-218[Medline] [Order article via Infotrieve]
55. Molinari, E., Gilman, M., and Natesan, S. (1999) EMBO J. 18, 6439-6447[Abstract/Free Full Text]
56. Nawaz, Z., Lonard, D. M., Dennis, A. P., Smith, C. L., and O'Malley, B. W. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1858-1862[Abstract/Free Full Text]
57. Htun, H., Holth, L. T., Walker, D., Davie, J. R., and Hager, G. L. (1999) Mol. Biol. Cell 10, 471-486[Abstract/Free Full Text]
58. Hecht, S. M., Alford, B. L., Kuroda, Y., and Kitano, S. (1978) J. Biol. Chem. 253, 4517-4520[Abstract]
59. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., and Schultz, P. G. (1989) Science 244, 182-188[Medline] [Order article via Infotrieve]
60. Liu, D. R., Magliery, T. J., Pastrnak, M., and Schultz, P. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10092-10097[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.