Estradiol-Binding Mechanism and Binding Capacity of the Human Estrogen Receptor Is Regulated by Tyrosine Phosphorylation
Steven F. Arnold1,
Michal Melamed,
Daria P. Vorojeikina,
Angelo C. Notides and
Shlomo Sasson2
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
 |
ABSTRACT
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We have investigated the effects of
tyrosine phosphorylation on the estradiol-binding mechanism and binding
capacity of the human estrogen receptor (hER). The wild type hER and a
point mutant form of the hER, in which tyrosine 537 was mutated to
phenylalanine (Y537F hER), were expressed in Sf9 insect cells. The wild
type hER, but not the Y537F hER, reacted with a anti-phosphotyrosine
monoclonal antibody, indicating that tyrosine 537 was the only tyrosine
phosphorylated on the hER. Scatchard and Hill analyses of the the
binding interaction of [3H]estradiol with the
wild type hER indicated that the addition of millimolar
phosphotyrosine, but not tyrosine, phosphate, or phosphoserine,
abolished the cooperative binding mechanism of the hER. These
observations are consistent with the idea that phosphotyrosine blocks
dimerization and site-site interactions between the hER monomers. The
wild type hER bound 10-fold more
[3H]estradiol than the Y537F hER. Treatment
of the purified wild type hER with a tyrosine phosphatase decreased the
binding capacity of the hER by approximately 90%, whereas, a
serine/threonine phosphatase had no effect. The estrogen-binding
capacity of the tyrosine-dephosphorylated hER was completely restored
by rephosphorylation of tyrosine 537 with
p60c-src, a tyrosine kinase. These results
indicate that p60c-src can restore estrogen
binding to the tyrosine-dephosphorylated hER and that dimerization and
cooperative site-site interaction of the hER occur via a
phosphotyrosine-binding interaction.
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INTRODUCTION
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The estrogen receptor (ER) is a member of the steroid/thyroid
hormone receptor superfamily of ligand-activated transcription factors
(1). The steroid hormone receptors bind hormone response elements and
direct steroid hormone-specific gene transcription (2). Therefore,
factors that affect the hormone binding of the steroid hormone
receptors will also modulate hormone-directed transcription. For
example, the hormone-binding capacity of the glucocorticoid receptor
appears to be regulated by posttranslational phosphorylation (3). Munck
and Brinck-Johnsen (4) made the initial observation that the
hormone-binding capacity of the glucocorticoid receptor is modulated by
intracellular ATP levels. These investigators have proposed a cyclic
model in which the phosphorylation state of the glucocorticoid
receptor, perhaps in a cell cycle-dependent manner, regulates hormone
binding (5). In addition, the glucocorticoid and progesterone
receptors, complexed with the heat shock proteins, facilitate hormone
binding (6).
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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Expression of the Y537F hER Mutant in Sf9 Cells
Tyrosine 537 of the hER was mutated to phenylalanine, and the
resulting mutant receptor, Y537F hER, was expressed in Sf9 insect cells
using the baculovirus expression system. An ammonium sulfate
precipitate of the wild type hER and the Y537F hER were separated by
SDS-gel electrophoresis and electrotransferred to a poly(vinylidene)
difluoride (PVDF) membrane for a Western blot. The anti-hER antibody
recognized the wild-type hER and the Y537F hER at 66 kDa (Fig. 1
). 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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Figure 1. A Western Blot of the Wild Type hER and the Y537F
hER from Sf9 Cells
Y537F hER (lanes 1 and 3) and wild type hER (lanes 2 and 4) were
separated by SDS-gel electrophoresis and electrotransferred to a PVDF
membrane. The membrane was probed with an anti-hER antibody
(left panel) then stripped and reprobed with an
anti-phosphotyrosine antibody (right panel).
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Estradiol Binding of the Wild Type hER as Compared with the Y537F
hER
The effect of tyrosine phosphorylation on the estradiol-binding
capacity of the hER was investigated by the comparing the ability of
the wild type hER and the Y537F hER to bind
[3H]estradiol. Increasing amounts (25100 µg protein
of ammonium sulfate precipitate) of the wild type hER or the Y537F hER
were incubated with [3H]estradiol for 12 h at 4 C
and the [3H]estradiol-hER complex was measured by the
hydroxyapatite (HAP) assay. The wild type hER bound approximately
10-fold more [3H]estradiol than the Y537F hER (Fig. 2
). 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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Figure 2. Binding of [3H]Estradiol by the Wild
Type hER or the Y537F hER
Twenty five to 100 µg protein of the 40% ammonium sulfate fraction
of the Sf9 cell expressing the wild type hER or the Y537F hER (each
containing 30 fmol/µg protein) were incubated with 100 nM
[3H]estradiol or [3H]estradiol plus a
200-fold excess of unlabeled estradiol for 12 h at 4 C. The
[3H]estradiol-hER complex was adsorbed to HAP and washed,
and the radioactivity was measured. Each value represents the
specifically bound [3H]estradiol and is representative of
four determinations in two separate experiments.
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Estradiol Binding of the Purified Wild Type hER
To eliminate the possibility that the tyrosine to phenylalanine
mutation produced a deleterious conformational change in the hER that
was responsible for the loss of estradiol binding, the estradiol
binding and tyrosine phosphorylation of the wild type hER, purified to
apparent homogeneity by ERE-affinity chromatography (15, 16), was
investigated. The purified wild type hER reacted with
anti-phosphotyrosine antibody, indicating it was phosphorylated on
tyrosine 537 (14, 16). Dephosphorylation of the wild type hER with
PTP1B, a tyrosine phosphatase, reduced the estrogen-binding capacity of
the receptor by 90% (Fig. 3
). 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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Figure 3. Elimination of [3H]Estradiol Binding
by the Purified Wild Type hER
The purified wild type hER was dephosphorylated with potato acid
phosphatase (PAP) or PTP1B, a phosphotyrosine phosphatase, then assayed
for [3H]estradiol binding. Each value is an average of
three determinations in two separate experiments. The 100% value was
1250 ± 125 dpm.
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Conditions for the stoichiometric in vitro phosphorylation
of the hER by the src family tyrosine kinase, p60c-src, on
tyrosine 537 have been reported (14). The estradiol binding of the
tyrosine-dephosphorylated hER was increased 80% by rephosphorylation
with p60c-src and ATP (Fig. 4
). 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 2030%, indicating that purified wild type hER was not completely
phosphorylated on tyrosine 537 (18).

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Figure 4. Restoration of [3H]Estradiol Binding
to the Purified Wild Type hER
The tyrosine-dephosphorylated hER was rephosphorylated with casein
kinase II or p60c-src and then assayed for
[3H]estradiol binding. Each value is an average of three
determinations in two separate experiments. The 100% value was
1250 ± 120 dpm.
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Effect of Phosphotyrosine on the Equilibrium-Binding Mechanism of
[3H]Estradiol with of the Wild-Type hER
ERs from various species including hER bind estradiol in a
positive cooperative manner (20). [3H]Estradiol binding
with 10 nM wild type hER shows a convex Scatchard plot and
a Hill coefficient (nH) of 1.39 (Fig. 5
), 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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Figure 5. Equilibrium Binding Analysis of
[3H]Estradiol Binding with hER.
The receptor (10 nM) was incubated without or with 0.4
mM phosphotyrosine or phosphoserine and
[3H]estradiol (0.560 nM) as described in
Materials and Methods. The binding data were analyzed and
presented according to Scatchard and Hill.
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DISCUSSION
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This study demonstrates that the tyrosine 537 to phenylalanine
mutation of the hER severely reduced the receptors ability to bind
estradiol. Purified wild type hER dephosphorylated at tyrosine 537 also
displayed reduced estradiol-binding capacity. The estradiol binding of
the wild type hER was restored by site-specific phosphorylation on
tyrosine 537 with the tyrosine protein kinase,
p60c-src.
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 receptors
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 hERs ability to direct estradiol-dependent gene
transcription.
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MATERIALS AND METHODS
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Materials
17ß-[6,7-3H(N)]estradiol (45.6 Ci/mmol) was
purchased from DuPont/New England Nuclear (Boston, MA). Leupeptin was
obtained from Peninsula Laboratories (Belmont, CA). The potato acid
phosphatase was from Boehringer Mannheim (Indianapolis, IN). The 4G10
monoclonal anti-phosphotyrosine antibody, p60c-src (1 pmol
PO4 transferred/min/mg), and PTP1B (4 nmol PO4
removed/min/mg) were from Upstate Biotechnology (Lake Placid, NY). The
casein kinase II (3 µmol PO4 transferred/min/mg) was a
generous gift of Drs. D. W. Litchfield and E. G. Krebs. Pepstatin,
phenylmethylsulfonyl fluoride (PMSF), phosphotyrosine, and
phosphoserine were from Sigma (St. Louis, MO). All other chemicals were
reagent grade.
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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We thank Drs. D. W. Litchfield and E. G. Krebs for the casein
kinase II. We also thank Drs. J. D. Obourn and N. J. Koszewski for
helpful discussions.
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FOOTNOTES
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Address requests for reprints to: Angelo C. Notides, Department of Environmental Medicine, Box EHSC, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
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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