 |
INTRODUCTION |
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
(ER-
) (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-
, a homologue of ER-
, was
discovered in various species (30-33). ER-
not only shares many
structural and functional features with ER-
but also has distinctive
characteristics, such as different tissue distribution (31) and
differential ligand responsiveness (34). High expression of ER-
in
human brain, cardiovascular system, thymus, bone, kidney, lung,
urogenital tract, and gastrointestinal tract suggests that ER-
has
significant roles in cellular functions not previously thought to
involve estrogens. Such ER-
-mediated functions might include effects
on memory and reproduction, enhancement of T-cell immunity, vascular
lesion protection, and colon cancer protection. In addition, ER-
appears to be the only estrogen receptor type expressed in the
embryonic central nervous system, implying that ER-
is important to
early embryonic development. Transgenic mice with only mER-
deleted
(35) and double mER-
/mER-
knockouts (36) have been generated.
Female ER-
knockout mice develop follicular arrest and anovulation,
indicating that both ER-
and ER-
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-
and ER-
. However, because ER-
was only
recently discovered (30-33), much work remains toward elucidating its
functions and relationships to ER-
.
Previously, we mapped the major O-GlcNAc site to
Ser16 near the amino terminus of mER-
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-
degradation and function.
 |
MATERIALS AND METHODS |
mER-
O-GlcNAc Site Mutants and Related Plasmid
Constructs--
The mER-
cDNA was engineered using an established
method, as described in our previous studies (17). To introduce the
appropriate restriction enzyme sites, mER-
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-
Ser16. mER-
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-
, mER-
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-
cDNAs in pEGFP-C1
were verified by automated DNA sequencing. To immunoprecipitate mER-
expressed in mammalian cell lines, a FLAG tag encoding the peptide
epitope, DYKDDDDK, was incorporated into the carboxyl-terminal end of
mER-
cDNA using polymerase chain reaction (37). All constructs
were verified by automated DNA sequencing.
Characterization of the Glycosylation of the mER-
O-GlcNAc
Mutant--
Expression and purification of mER-
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.
17
-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-
. 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-
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, 17
-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-
sample was transfected in triplicate.
Electrophoretic Mobility Shift Assay--
To determine the DNA
binding activity of the ER-
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-
(CalBiochem, La Jolla, CA) and excess unlabeled ERE probe were added as indicated.
Characterization of Subcellular Localization--
To examine
subcellular localization of mER-
, the GFP fusion constructs of
mER-
cDNA were transfected into mammalian cells lines, as
described above. After 1 day, fresh media and 17
-estradiol at the
final concentration of 20 nM were added. Fluorescence
images were recorded using a digital camera (Hamamatsu, Tokyo, Japan).
 |
RESULTS |
Mutation of the O-GlcNAc/O-Phosphate Locus on mER-
--
Our
previous studies demonstrated that mER-
is modified alternatively by
O-GlcNAc or O-phosphate at Ser16
(17). To reveal biological roles of the
O-GlcNAcylation/O-phosphorylation on mER-
, we
mutated Ser16 into either Ala or Glu along with
Ser15 and Thr17 into Ala and Val to generate
two mutants, designated mER-
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- mutants used in
this study. Wild-type mER- cDNA was mutated at the
Ser16 by changing either Ser Ala or Ser Glu along
with changing Ser15 (Ser Ala) and Thr17
(Thr Val). The two mutants were named S16A and S16E, respectively.
All constructs were engineered to incorporate a FLAG tag at their
carboxyl terminus.
|
|
mER-
O-GlcNAc Site Mutant Is Glycosylation-deficient--
To
assess the glycosylation state of mER-
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-
(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-
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- 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-
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-
(44) and mER-
(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-
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-
turned over rapidly in the presence of estrogen, with an
average half-life of 7-8 h, similar to the range of ER-
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-
results in accelerated degradation of mER-
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- affects the protein's turnover
rate. Cos-1 cells transfected with either the wild type or mutated
mER- 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- 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-
Have
Altered Transactivation Activities--
The fact that the major
O-GlcNAc site on mER-
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-
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-
. 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-
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- modulates the protein's transactivation
activity. Cos-1 cells were cotransfected with the reporter plasmid
ERE-TATA-TK-Luc, internal control -galactosidase expression plasmid,
and the expression vector (V) containing wt or mutant
mER- cDNA. At 24 h post-transfection, 20 nM
17 -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
-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-
Does Not Affect DNA Binding--
To compare the binding of the wild
type and mutant forms of mER-
to the DNA response elements, we
tested cell extracts from mER-
-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-
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-
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- does not affect the protein's DNA
binding. The mER- 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- antibody blocking of the interaction.
Note: mER- levels are less in the S16E mutant due to its rapid
degradation (see text and Fig. 3).
|
|
Mutant and wt mER-
Proteins Are Localized Exclusively in the
Nucleus--
Although ER-
is known to be mainly localized in the
nucleus, the subcellular localization of the homologue ER-
has not
been studied. To investigate the subcellular distribution of ER-
, we
fused mER-
to GFP. The localization of GFP-fused wt mER-
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- localizes
exclusively to nucleus. mER- cDNAs were subcloned into
pGFP-C1 vector, and fusion constructs were transfected into Cos-1 cells
by the liposome method. 20 nM 17 -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- fused to GFP; GFP, cells transfected with
GFP construct alone; wt + E2, cells transfected with
wild-type mER- 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-
's transport
into the nucleus.
 |
DISCUSSION |
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-
has a reciprocal
relationship with phosphorylation by capping a phosphorylation site
that modulates mER-
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-
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-
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-
, 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-
-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-
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-
. 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-
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-
.
Our green fluorescence protein fusion results revealed that there is no
significant redistribution of mER-
in mammalian cells induced by the
mutations, excluding the possibility that nuclear localization requires
either phosphorylation or O-GlcNAcylation at the
Ser16 of mER-
. However, we did observe some minor
nuclear pattern changes, such as a clustering of ER-
within the
nucleus upon estrogen treatment (Fig. 6). This clustering phenomenon is
similar to that seen for ER-
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-
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.