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Departments of Environmental Medicine and Biophysics (S.F.A.,
D.P.V., A.C.N.) University of Rochester School of Medicine and
Dentistry Rochester, New York 14642
Department of
Pharmacology (M.M., S.S.) Hebrew University of Jerusalem
Faculty of Medicine, School of Pharmacy Jerusalem, 91120,
Israel
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Auricchio and co-workers (7, 8) have proposed that the estradiol binding of the ER is regulated by tyrosine phosphorylation. They reported that the human ER (hER) is phosphorylated on tyrosine 537, and that a purified tyrosine kinase from calf uterus phosphorylates the hER, but only in an estradiol-receptor complex and Ca2+-calmodulin dependent manner (9, 10, 11). They have also isolated a tyrosine phosphatase that dephosphorylated the ER and eliminated estradiol binding (12). However, they have not identified the specific protein kinase(s) or phosphatase(s) involved. Fawell and co-workers (13) have reported that a carboxy-terminal deletion of the mouse ER to amino acid 538 (or 534 of the hER) retained only 25% of its estradiol binding, while deletion to amino acid of 508 (or 504 of the hER) abrogated estradiol binding.
Recently, we identified tyrosine 537 as one of the phosphorylation sites on the native hER from MCF-7 cells and on the recombinant hER from Sf9 insect cells (14). In contrast to the reports of Auricchio and co-workers (7, 8, 9, 10, 11, 12), the phosphorylation of tyrosine 537 on the native hER from MCF-7 cells is not regulated by estradiol (14).
In this report, we propose that the phosphotyrosine and the residues surrounding phosphotyrosine 537 mediate dimerization by a phosphotyrosine of one hER monomer coupling to a phosphotyrosine-binding domain of the complementary hER monomer. We show that phosphorylation of tyrosine residue 537 of the hER induces conformational changes that regulate estradiol binding, hER dimerization, and the positive site-site cooperative [3H]estradiol binding mechanism of the hER.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The PVDF membrane was stripped and reprobed for
tyrosine phosphorylation of the hER with the 4G10 monoclonal
anti-phosphotyrosine antibody. The anti-phosphotyrosine antibody
reacted with the wild type hER, but not with the Y537F hER, indicating
that tyrosine 537 was the only phosphorylated tyrosine residue on the
hER (Fig. 1). Furthermore, the Y537F hER immunopurified with the
anti-hER antibody revealed that it reacted with the anti-hER but not
the anti-phosphotyrosine antibody. A 40% ammonium sulfate fraction of
mock-infected Sf9 cells revealed no reactivity with the anti-hER or
anti-phosphotyrosine antibody at 66 kDa (data not shown).
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). The binding of [3H]estradiol to the
Y537F hER was not enhanced after incubation at 25 C for 1 h and
remained approximately 10-fold less than the wild type hER (data not
shown).
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). The loss of estradiol
binding by the wild type hER was blocked by the addition of 1
mM sodium vanadate, an inhibitor of PTP1B activity,
indicating that the loss of estradiol binding was dependent on the
phosphatase activity. Furthermore, after treatment with PTP1B, the
purified hER did not react with the anti-tyrosine antibody, and
proteolysis products were not detected by Western blot analysis (data
not shown). Treatment of the purified wild type hER with potato acid
phosphatase, a serine/threonine phosphatase, did not alter the
estradiol-binding capacity of the hER (Fig. 3).
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). Estradiol
binding was not restored to the tyrosine-dephosphorylated hER with the
p60c-src in the absence of ATP (data not shown). This is
consistent with our previous finding that the src family tyrosine
kinases incorporate approximately 0.8 mol of
[32P]phosphate/mol of hER (14). The rephosphorylation of
the tyrosine-dephosphorylated hER was specific for phosphotyrosine 537
because casein kinase II, which specifically phosphorylates serine 167
on the hER (17, 18), had no effect on the estradiol-binding capacity.
Phosphorylation of the purified phosphorylated hER with
p60c-src further increased its estradiol-binding capacity
by 20–30%, indicating that purified wild type hER was not completely
phosphorylated on tyrosine 537 (18).
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), indicative
of positive cooperative binding interaction and receptor
homodimerization (20, 21). When the binding assay was performed in the
presence of 0.4 mM phosphotyrosine, the Scatchard plot
appeared linear, and the nH was reduced to 1.03, indicating absence of
positive cooperative binding interaction and conformational changes
associated with receptor homodimerization. This inhibitory effect was
specific to phosphotyrosine, because 0.4 mM phosphoserine
in the binding assay did not affect the positive cooperative binding
interaction of [3H]estradiol with the hER, which remained
highly cooperative (nH = 1.41).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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There are several mechanisms by which tyrosine phosphorylation may regulate the estradiol-binding capacity of the hER. First, the tyrosine phosphorylation of the hER may result in a conformational change in the ligand-binding domain. The tyrosine phosphorylation may promote the formation of an estradiol-binding pocket with a higher affinity for estradiol than the non-tyrosine-phosphorylated hER. Katzenellenbogen and co-workers (22) have suggested amino acids 515 to 535 at the carboxy terminus are important for the formation of the ligand-binding pocket.
A second, and more likely, possibility is that tyrosine 537 phosphorylation, receptor homodimerization, and estrogen binding of the hER are linked. We have demonstrated that the hER requires tyrosine phosphorylation for dimerization through a phosphotyrosyl-SH2 domain-binding mechanism (16, 18). The enhancement of hER dimerization by tyrosine phosphorylation may increase the receptor’s estradiol-binding capacity and affinity by its cooperative estrogen-binding mechanism. The cooperative binding mechanism involves site-site interactions between monomers of the dimeric ER in which estradiol binding by one monomer induces conformational changes in the dimeric receptor that results in an increased affinity of the second monomer for estradiol (20). Therefore, phosphorylation of tyrosine 537 on the hER increases the capacity and affinity for estradiol by a change in the estradiol-binding mechanism, from a noncooperative to a cooperative hormone-binding mechanism, through an acquisition of the receptor to undergo dimerization.
The basal phosphorylation of tyrosine 537, which occurred independently of estrogen binding, is in the hormone-binding region of the receptor that regulates dimerization (16, 18). It has been shown that this phosphorylation is required for binding of hER to an estrogen response element (18). The dimerization of the hER is probably mediated by coupling between phosphotyrosine 537 of the hER and a phosphotyrosine-binding domain (i.e. SH2-like domain) on the hER (16). Further support for this mechanism comes from the ability of phosphotyrosine, but not phosphoserine, to eliminate the estradiol-induced cooperative binding interaction and receptor dimerization. This effect of phosphotyrosine is concentration-dependent (data not shown). It therefore seems that phosphotyrosine competes with the phosphorylated tyrosine 537 to an SH2-like domain in the receptor, thereby hindering the dimerization process.
We hypothesize that the regulation of estradiol binding by tyrosine phosphorylation occurs through the p60c-src family of tyrosine kinases that are coupled to cell-signaling pathways. Very relevant and analogous with these findings are the signal transducers and activators of transcription (STAT) proteins (23). Tyrosine phosphorylation of STATs promotes their homo- and heterodimerization, which allows their translocation to the nucleus and interaction with specific recognition elements to initiate transcription (23). The dimerization of the STAT proteins is mediated by reciprocal coupling between phosphotyrosine on one monomer and a SH2 domain on the opposing monomer (24).
In conclusion, we have demonstrated that phosphorylation of tyrosine 537 is responsible for the regulation of estradiol binding of the hER. We believe that the tyrosine phosphorylation of the hER is regulated by cell-signaling pathways, perhaps in a cell cycle-specific fashion, which controls the hER’s ability to direct estradiol-dependent gene transcription.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Preparation of the hER from Sf9 Cells
The production and expression of the recombinant baculovirus,
AcNPV-hER, carrying the cDNA of the wild type hER, has been described
(15). Whole cell extracts of the Sf9 cells containing the wild type hER
or Y537F hER were prepared as described (14, 15). The whole cell
extracts were made 40% saturated with ammonium sulfate, and the
precipitate was collected by centrifugation. An ammonium sulfate
preparation of the wild type hER in the absence of estradiol was
purified on a ERE-Teflon affinity matrix as previously described with
the inclusion of the phosphatase inhibitors: 50 mM sodium
fluoride, 10 mM sodium pyrophosphate, 1 mM
sodium orthovanadate, and 50 nM okadaic acid (15).
SDS-Gel Electrophoresis and Western Blot Analysis
Ammonium sulfate preparations, containing approximately 25 µg
of protein, of the wild-type hER and the Y537F hER were added to
Laemmli sample buffer and separated on a 10% acrylamide SDS-gel at 30
mA for 5 h (19). The SDS-gel was electrotransferred to a PVDF
membrane (Millipore, Bedford, MA). The membrane was blocked in 2%
(wt/vol) BSA for 3 h at room temperature, then probed with
anti-hER antibody 6 (15). The bands were visualized by chemiluminesence
using the enhanced chemiluminescence (ECL) system (Amersham, Arlington
Heights, IL). The PVDF membranes were stripped for reprobing in 62.5
mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol,
and 2% SDS for 30 min at 50 C, then rinsed with PBS and reprobed with
the horseradish peroxidase-conjugated monoclonal 4G10
anti-phosphotyrosine antibody (1:1000 dilution) for 3 h.
Estradiol Binding of the hER
Ammonium sulfate preparations of the wild type hER or the Y537F
hER (30 fmol/µg protein, for the experiment shown in Fig. 2
) or the
purified wild type hER (15 fmol, for the experiment shown in Fig. 3)
were added to the binding buffer consisting of: 20 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 100
mM KCl, 50 mM sodium fluoride, 10
mM sodium pyrophosphate, 1 mM sodium
orthovanadate, 50 nM okadaic acid, 10% (vol/vol) glycerol,
0.5 mM leupeptin, and 0.2 mM PMSF. The
concentration of the Y537F hER was estimated by comparing the Y537F hER
to known quantities of the purified wild type hER on a Western blot.
The purified wild type hER was determined by its specific
[3H]estradiol binding, SDS-gel electrophoresis, and
quantitative protein determination (data not shown). Mock-infected Sf9
whole cell extract or bovine 
-globulin was added to the binding
buffer to give a final protein concentration of 1.5 mg/ml.
[3H]Estradiol at a final concentration of 100
nM was added, while the nonspecific binding was measured by
a parallel incubation with [3H]estradiol plus a 200-fold
excess of estradiol for 12 h at 4 C. After the incubation, 100
µl of a 50% slurry of HAP in the binding buffer was added and
allowed to adsorb the hER for 40 min at 4 C. The HAP was washed three
times with 0.5 ml of the binding buffer. The HAP pellets were suspended
in 0.5 ml ethanol, scintillation fluid was added, and the radioactivity
was measured.
The effect of phosphotyrosine and phosphoserine on the
equilibrium-binding mechanism of [3H]estradiol to the
wild type hER was determined according to Melamed et al.
(21). Ammonium sulfate precipitates of the hER were dissolved in TDEE
buffer (40 mM Tris-HCl, pH 7.4, I mM
dithiothreitol, 1 mM EDTA, 1 mM EGTA)
containing 0.1 mM PMSF. 0.2 mM leupeptin, 1
µg/ml pepstatin, 10% (vol/vol) glycerol, 100 mM KCl, 1
mM orthovanadate, 1 mM sodium pyrophosphate,
and 10 mg/ml bovine -globulin. Tubes containing 200 µl of hER
preparation were preincubated in duplicates without or with 0.4
mM phosphotyrosine or phosphoserine and varying
[3H]estradiol concentrations (0.5 to 60 nM)
and incubated for 1 h at 25 C. Nonspecific binding was measured by
a parallel incubation of the receptor with radioactive estradiol in the
presence of a 200-fold excess of unlabeled estradiol. Nonspecific
binding was less then 5% of total [3H]estradiol binding.
During the incubation period, a 50-µl sample from each tube was
removed to determine the total [3H]estradiol
concentration. At the end of the incubation, the tubes were cooled on
ice for 5 min; 100 µl of 1% (wt/vol) charcoal and 0.01% (wt/vol)
dextran 500 suspension in TDEE buffer were then added and incubated for
10 min at 4 C. The suspension was then centrifuged, and a 100-µl
aliquot of the supernatant was removed for scintillation counting.
Determination of specific [3H]estradiol binding and
Scatchard (25) and Hill (26) analyses were performed as previously
described (21). Stability of the receptor was assayed as described
previously (21).
In Vitro Tyrosine Dephosphorylation/Phosphorylation
of the Purified Recombinant hER
The in vitro dephosphorylation of the purified wild
type hER (15 fmol in 20 µl) was carried out in phosphatase buffer
[50 mM HEPES, pH 7.4, 150 mM NaCl, and 5%
(vol/vol) glycerol]. Five microliters of PTP1B, conjugated to agarose
beads, were added to the receptor and incubated for 30 min at 37 C. The
products in the supernatant were recovered by centrifugation at
15,000 x g at 4 C and subsequent washing of the beads
with phosphatase buffer. In other experiments, 10 µl of potato acid
phosphatase were added for 1 h at 4 C, and the estradiol binding
was performed as described above. The in vitro
rephosphorylation reactions were done as follows: the purified
dephosphorylated wild type hER (15 fmol) was suspended in 50 µl of
p60c-src reaction buffer (20 mM Tris HCl, pH
7.4, and 50 mM MgCl2) or 50 µl casein kinase
II (CKII) reaction buffer (50 mM Tris HCl, pH 7.6, and 10
mM MgCl2) and 1 mM ATP. Next, 3 U
of p60c-src or 1 U of CKII were added to initiate the
reaction. The reaction was carried out for 15 min at 30 C and was
terminated by placing the tubes at 4 C, after which the estradiol
binding was performed as described above.
Site-Directed Mutagenesis of the hER
Oligonucleotide site-directed mutagenesis of the hER was
performed essentially according to the method of Kunkel (27). A
single-stranded template was prepared from M13mp19 containing
the hER cDNA grown in Escherichia coli strain CL236. A 28-bp
primer that contained a mutation to change tyrosine 537 to
phenylalanine and a novel restriction site, XhoI, was used.
The oligonucleotide primer was phosphorylated for 45 min at 37 C by T4
polynucleotide kinase (Bio-Rad, Hercules, CA) in 50 mM
Tris, pH 7.5, 10 mM MgCl2, 5 mM
dithiothreitol, and 1 mM ATP. The phosphorylated
oligonucleotide and the UTP-containing DNA templates were mixed at a
5:1 molar ratio in 10 mM Tris, pH 7.5, and 5 mM
MgCl2 for the annealing reaction, heated to 70 C, and then
slowly cooled to room temperature. A polymerase reaction was carried
out in 20 mM Tris, pH 7.5, 10 mM
MgCl2, 5 mM dithiothreitol, 500
µM deoxynucleoside triphosphates, 1 mM ATP,
2.5 U of the Klenow fragment of DNA polymerase, 1 U of T4 DNA ligase,
and 2 µg of gene 32 T4 protein and was carried out for 16 h at
16 C. The double-stranded DNA was used to transform E. coli
strain NM 1193. All mutants were verified by the restriction enzyme
digestion with XhoI and DNA sequencing. The mutated cDNA of
the hER was cloned into the EcoRI site of the pVL1393
baculovirus transfer vector. The orientation of the cloned fragment was
confirmed by a digestion with BglII. The resulting
baculovirus vector, AcNPV-Y537F, was purified by CsCl centrifugation
and transfected into Sf9 cells using the BaculoGold Linearized
Baculovirus DNA (PharMingen, San Diego, CA.) and Lipofectin Reagent
(GIBCO BRL, Grand Island, NY).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by NIH Grants HD-06707 and ES-01247 (to A.C.N.), NIH Training Grant T32ES 07026 (to S.F.A.), and a grant from The Israel Cancer Association (to S.S.)
1 Current address: Center for Bioenvironmental Research, Tulane
University, New Orleans, Louisiana 70112. 
2 The authors dedicate this paper to their beloved friend,
Angelo C. Notides, who passed away November 10, 1996.
Received for publication September 26, 1996. Revision received October 24, 1996. Accepted for publication October 28, 1996.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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