(Received for publication, August 14, 1995; and in revised form, October 3, 1995)
From the
We report here that the phosphorylation of tyrosine 537 on the
human estrogen receptor (hER) controls the receptor's
dimerization and DNA binding ability. The DNA-binding form of both the
hER from human MCF-7 mammary carcinoma cells and the hER overexpressed
in Sf9 insect cells was isolated using estrogen response element (ERE)
affinity chromatography. Western blot analyses demonstrated that the
DNA-binding form of the hER from MCF-7 or Sf9 cells was (i)
phosphorylated at tyrosine 537, (ii) localized in the nucleus of
estradiol-treated MCF-7 cells with an apparent molecular mass of 67
kDa, and (iii) hyperphosphorylated at serine residue(s). The
non-DNA-binding form of the hER was (i) devoid of phosphorylation at
tyrosine 537, (ii) cytosolic with an apparent molecular mass of 66 kDa,
and (iii) hypophosphorylated at serine residue(s). The
dephosphorylation of the purified hER at phosphotyrosine 537 with a
tyrosine phosphatase eliminated binding to an ERE in a gel mobility
shift assay. The binding of the tyrosine-dephosphorylated hER to an ERE
was restored by the rephosphorylation of tyrosine 537 with Src family
tyrosine kinases, p60 or
p56
. Mutation of tyrosine 537 to phenylalanine
confirmed that the phosphorylation of tyrosine 537 is necessary for the
hER to bind an ERE. An anti-hER antibody restored the binding of the
tyrosine-dephosphorylated hER to an ERE, indicating that the bivalent
anti-hER antibody brought together the two inactive hER monomers. A
far-Western blot confirmed that phosphotyrosine 537 is required for hER
homodimerization.
These experiments establish that dimerization of
the hER and DNA binding are regulated by phosphorylation at tyrosine
537. This is the first demonstration of the regulation of dimerization
of a steroid hormone receptor by phosphorylation. These results are
significant since p60 is overexpressed in
estrogen-dependent breast cancers and may act to enhance the activity
of the hER.
The steroid/thyroid hormone receptors are ligand-dependent
transcription factors that function by binding to hormone response
elements on target genes and regulating transcription(1) . The
processes controlling steroid-specific gene transcription are poorly
understood, although receptor-associated coactivators have been
reported to be involved(2) . Nonetheless, it has been
demonstrated that most steroid/thyroid hormone receptors, including the
human estrogen receptor (hER), ()bind to their hormone
response elements as hetero- or homodimers(3, 4) . It
has been proposed that the dimerization of the steroid/thyroid hormone
receptors is mediated through a leucine zipper motif, a series of
hydrophobic heptad repeats in the carboxyl termini of the
receptors(5, 6) . Recently, Bourguet et al.(7) showed that the ligand-binding domain of human
retinoid X receptor
has a dyad symmetry composed of packed
helices at the receptor's interface, but not a leucine zipper
structure. While the dimerization of most steroid hormone receptors is
required for binding to DNA, accessory proteins and post-translational
phosphorylation have also been suggested to contribute to DNA binding (8, 9, 10) . For example, purification of the
progesterone receptor decreases its DNA binding ability, suggesting
that an accessory protein (e.g. high mobility group protein-1)
is required for high affinity DNA binding(8) . Additionally,
the phosphorylation of steroid/thyroid hormone receptors has been shown
to modulate their DNA binding affinity(11) . The
phosphorylation of the retinoic acid and progesterone receptors
increases, while the phosphorylation of thyroid hormone
receptor-
and nerve growth factor-I-B decreases their
affinity for their respective response elements (12, 13, 14, 15) .
The hER, like other members of the steroid/thyroid hormone receptor superfamily, undergoes a hyperphosphorylation at serine residues following hormone binding(16) . The dephosphorylation of the hER with potato acid phosphatase reduces but does not eliminate the receptor's affinity for an ERE(16) . The enhanced affinity of the hER for its ERE was found to be mediated by the estradiol-induced phosphorylation of serine 167, the major phosphorylation site on the hER(11) . Casein kinase II specifically phosphorylates serine 167 of the hER(17) . Serine 118 of the hER was identified as a growth factor-regulated phosphorylation site, mediated by mitogen-activated protein (MAP) kinase; however, its role in transcriptional activation of the hER remains to be defined(11) .
An estradiol-independent phosphorylation site
at tyrosine 537 in the carboxyl terminus of the hER has been identified
by amino acid sequencing of P-labeled tryptic peptides of
the hER(18) . Furthermore, the Src family tyrosine kinases,
p60
and p56
, were shown
to specifically phosphorylate tyrosine 537 on the hER, while
protein-tyrosine phosphatase 1B (PTP1B) and Src homology 2
protein-tyrosine phosphatase 1 (SHPTP1) dephosphorylated
phosphotyrosine 537(18) . Interestingly, the tyrosine kinase
activity of p60
in human breast cancers has
been shown to be elevated as compared with other cancers (19) .
The human MCF-7 mammary carcinoma cell line overexpresses
p60
and has been a useful paradigm for
investigating estrogen-dependent processes associated with human breast
cancers(20) . Thus, we sought to gain insight into the
consequences of the phosphorylation of tyrosine 537 on the hER by
p60
.
We show here that the phosphorylation of tyrosine 537 is a regulatory mechanism that controls the capacity of the hER to undergo the monomer to dimer transition. The phosphorylation of tyrosine 537 is also a prerequisite for the estrogen-dependent hyperphosphorylation of the serine residue(s), nuclear retention, and DNA binding of the hER.
Figure 1: Western blot analysis of the wild-type hER and the mutant Y537F hER with the anti-phosphotyrosine and anti-hER antibodies. Proteins in whole cell extracts (WCE) of MCF-7 cells or Sf9 cells expressing the wild-type hER or the mutant Y537F hER were immunoprecipitated (IP) with the anti-hER antibody. The protein extracts were separated by SDS gel electrophoresis and electrotransferred to a PVDF membrane. Whole cell extracts of untreated (lanes 1 and 2) or estradiol-treated (lanes 3 and 4) MCF-7 cells, untreated (lanes 5 and 6) or estradiol-treated (lanes 7 and 8) Sf9 cells expressing the wild-type hER, or untreated Sf9 cells expressing the mutant Y537F hER (lanes 9 and 10) were immunoblotted with the 4G10 anti-phosphotyrosine monoclonal antibody (pY Ab) (lanes 2, 4, 6, 8, 10, and 12). Then, the membrane was stripped and reblotted with the anti-hER antibody (hER Ab) (lanes 1, 3, 5, 7, 9, and 11).
The 66-kDa form of the native hER from untreated MCF-7 cells was recognized by the 4G10 anti-phosphotyrosine monoclonal antibody (Fig. 1, lane 2). However, after estradiol treatment of the MCF-7 cells, the anti-phosphotyrosine antibody recognized only the 67-kDa form and not the 66-kDa form of the hER (Fig. 1, lane 4). These data indicate that only the fraction of the hER in the 66-kDa form, which was phosphorylated at tyrosine, was converted to the 67-kDa form after estradiol treatment of the MCF-7 cells. To further aid in deciphering the function of tyrosine phosphorylation, the wild-type hER and the mutant Y537F hER were expressed in Sf9 cells.
The wild-type hER expressed in Sf9 cells reacted with an anti-hER antibody, confirming the presence of the wild-type hER at 66 and 67 kDa (22) . The 67-kDa form, but not the 66-kDa form, of the wild-type hER from whole cell extracts of Sf9 cells (or following purification of the hER by immunoprecipitation) showed strong reactivity with the anti-phosphotyrosine antibody (Fig. 1, lanes 6 and 8). Antibody 6 immunoprecipitates, before or after estrogen treatment of MCF-7 cells, the 66- or 67-kDa form of the hER with equal efficiency (data not shown). The presence of the 67-kDa form of the wild-type hER was independent of estradiol treatment of the Sf9 cells (Fig. 1, lanes 5 and 7). The lack of estrogen-dependent phosphorylation of the hER in Sf9 insect cells may be due to the 10,000-fold overexpression of the hER in insect as compared with mammalian cells(22) , cell-specific differences in protein kinase regulatory pathways, or loss of regulatory factors during baculoviral infection. An extract from Sf9 cells infected with mock baculovirus showed no reactivity with the anti-hER or the anti-phosphotyrosine antibody at 66 or 67 kDa (data not shown). The mutant Y537F hER expressed in Sf9 cells migrated only at 66 kDa in the absence or presence of estradiol treatment of the Sf9 cells (Fig. 1, lanes 9 and 11) and did not show reactivity with the anti-phosphotyrosine antibody (lanes 10 and 12). These results confirm our earlier findings that the hER is phosphorylated at tyrosine 537 (18) and that the 4G10 anti-phosphotyrosine monoclonal antibody specifically recognizes the phosphorylation at tyrosine 537 on the hER. These data also indicate that the phosphorylation of tyrosine 537 is a necessary prerequisite for the serine hyperphosphorylation of the hER and consequently the formation of the 67-kDa form of the hER.
The
intracellular localization of the 67-kDa form of the hER in MCF-7 cells
was exclusively nuclear after estradiol treatment. Following a 20-min
exposure to [H]estradiol, the nuclear fraction of
the MCF-7 cells contained 90% of the specifically bound
[
H]estradiol, while only 10% was present in the
cytoplasmic fraction (data not shown). Equivalent amounts of cytosolic
and nuclear proteins were separated by SDS gel electrophoresis and
electrotransferred to a PVDF membrane. Western blot analysis with an
anti-hER antibody revealed that the nuclear fraction contained the
67-kDa form of the hER, while the cytosolic fraction contained the
66-kDa form (Fig. 2, lanes 1 and 3).
Consistent with the observations described above, only the 67-kDa form
of the hER reacted with the anti-phosphotyrosine antibody in a Western
blot (Fig. 2, lanes 2 and 4). In the absence
of estradiol treatment of MCF-7 cells, the hER was recovered only in
the cytosolic fraction, demonstrating that estrogen binding promotes
nuclear retention of the hER ( (16) and data not shown). These
results suggest that the biologically active hER is the 67-kDa form
since it is hyperphosphorylated and retained in the nucleus after
estradiol treatment of MCF-7 cells.
Figure 2:
Intracellular localization and tyrosine
phosphorylation of the hER from estradiol-treated MCF-7 cells.
Cytosolic and nuclear fractions were prepared from
[H]estradiol-treated MCF-7 cells. The proteins
(100 µg) were separated by SDS gel electrophoresis and then
transferred to a PVDF membrane. The membrane was immunoblotted with the
anti-phosphotyrosine antibody (pY Ab) (lanes 2 and 4) and then stripped and reblotted with the anti-hER antibody (hER Ab) (lanes 1 and 3).
Figure 3: ERE affinity chromatography of the wild-type hER and the mutant Y537F hER. The ammonium sulfate fractions from estradiol-treated MCF-7 cells (A), untreated Sf9 cells expressing the wild-type hER (B), or Sf9 cells expressing the mutant Y537F hER (C) before chromatography (Pre-chrom.) are shown in lanes 1 and 2. The ammonium sulfate fractions were loaded onto an ERE affinity column and allowed to bind for 30 min at 4 °C. The flow-through fraction or the unbound hER was collected. The ERE affinity column was washed with a Tris buffer containing 150 mM KCl, and the hER eluted with a Tris buffer containing 600 mM KCl. The column fractions were subjected to SDS gel electrophoresis, transferred to a PVDF membrane, and probed with the anti-phosphotyrosine antibody (pY Ab) (lanes 2, 4, and 6). Then, the membrane was stripped and reblotted with the anti-hER antibody (hER Ab) (lanes 1, 3, and 5).
The 67-kDa form of the recombinant hER was purified to near homogeneity by ERE affinity chromatography. Coomassie Blue staining indicated that the 67-kDa form of the hER was the only protein present (Fig. 4, lane 1). Western blot analysis with the anti-phosphotyrosine antibody detected only the hER (Fig. 4, lane 2).
Figure 4: Western blot analysis of the 67-kDa form of the hER from Sf9 cells. The wild-type hER from Sf9 cells was subjected to ERE affinity chromatography. The 600 mM KCl eluate fraction (2 µg of protein) from the ERE affinity column was resolved by SDS gel electrophoresis and electrotransferred to a PVDF membrane. The membrane was stained with Coomassie Blue (lane 1) and then immunoblotted with the anti-phosphotyrosine antibody (pY Ab), and the band visualized by chemiluminescence (lane 2).
Figure 5:
Gel mobility shift assay of the 66- and
67-kDa forms of the hER. A whole cell extract (WCE) of Sf9
cells expressing the wild-type hER (lane 1) was incubated with
1 µg of the anti-hER antibody (lane 2) or a 200-fold
excess of unlabeled ERE (lane 3), and then 500 ng of
poly(dIdC) was added for 15 min at 4 °C followed by the
addition of the
P-labeled ERE for 15 min at 4 °C. The
eluate fraction from the ERE affinity column containing the 67-kDa form
of the hER from MCF-7 cells (lane 4), the flow-through
fraction containing the 66-kDa form of the hER (lane 5), the
eluate fraction from the ERE affinity column containing the 67-kDa hER
from Sf9 cells (lane 6), and the flow-through fraction (lane 7) were incubated with 500 ng of poly(dI
dC) for 15
min at 4 °C followed by the addition of the
P-labeled
ERE for 15 min at 4 °C. The final hER concentration in the gel
mobility assays was 10 nM. The protein-DNA complexes were
resolved by nondenaturing gel electrophoresis and visualized by
autoradiography.
The 67-kDa form of the native hER treated with the
tyrosine phosphatase PTP1B resulted in a dose-dependent reduction of
the hERERE complex (Fig. 6A, lanes
2-4) and reactivity with the anti-phosphotyrosine antibody (Fig. 6B, lanes 2-4). With 1 µg of
PTP1B, the hER
ERE complex and the tyrosine phosphorylation of the
hER were completely eliminated (Fig. 6, A and B, lane 4). The tyrosine dephosphorylation by PTP1B
was specific since 1 mM Na
VO
, a
tyrosine phosphatase inhibitor, prevented the loss of the hER
ERE
complex (Fig. 6A, lane 7) and the tyrosine
dephosphorylation of the hER (Fig. 6B, lane
7). The addition of Sf9 mock cytosol (without hER) did not restore
the binding of the tyrosine-dephosphorylated hER to an ERE (data not
shown). The tyrosine-dephosphorylated hER nevertheless migrated as a
67-kDa protein, indicating that PTP1B did not result in serine
dephosphorylation, which is responsible for the 67-kDa form of the hER.
Figure 6:
Tyrosine
dephosphorylation/rephosphorylation of the 67-kDa form of the hER from
MCF-7 cells. A, the 67-kDa form of the hER from
estradiol-treated MCF-7 cells, purified by ERE affinity chromatography,
was dephosphorylated with 0, 0.2, 0.4, or 1.0 µg of PTP1B for 30
min at 37 °C (lanes 1-4, respectively). The PTP1B
(1.0 µg)-dephosphorylated 67-kDa form of the hER was
rephosphorylated with 1 and 3 units of p60(lanes 5 and 6, respectively). The 67-kDa form
of the hER was incubated with 1.0 µg of PTP1B in the presence of 1
mM Na
VO
(lane 7). The hER
samples (10 nM) were then incubated with 500 ng of
poly(dI
dC) for 15 min at 4 °C followed by the addition of the
P-labeled ERE for 15 min at 4 °C. The protein-DNA
complexes were resolved by nondenaturing gel electrophoresis and
visualized by autoradiography. B, shown is the Western blot
analysis of the hER samples. The PVDF membrane was immunoblotted with
the anti-phosphotyrosine antibody (pY Ab), stripped, and
reblotted with the anti-hER antibody (hER
Ab).
The rephosphorylation of the tyrosine-dephosphorylated hER by
p60 resulted in a dose-dependent increase in the
hER
ERE complex (Fig. 6A, lanes 5 and 6). The action of the p60
was not observed in
the absence of ATP (data not shown). A Western blot confirmed that the
tyrosine phosphorylation of the hER by the p60
restored
reactivity with the anti-phosphotyrosine antibody (Fig. 6B, lanes 5 and 6). Western
blot analysis with the anti-hER antibody confirmed the presence of the
hER in all the samples (Fig. 6B). These results
indicate that the binding of the native hER to an ERE is dependent on
the phosphorylation of tyrosine 537.
The purified recombinant
wild-type hER showed identical characteristics, with respect to
tyrosine phosphorylation and binding to an ERE, as the native hER. The
tyrosine dephosphorylation of the recombinant wild-type hER (Fig. 7B, lanes 1 and 2) eliminated
ERE binding (Fig. 7A, lanes 1 and 2).
The tyrosine dephosphorylation of the recombinant wild-type hER was
inhibited with 1 mM NaVO
(Fig. 7B, lanes 11 and 12) and
prevented the loss of the hER
ERE complex by inhibiting the
activity of the tyrosine phosphatases (Fig. 7A, lanes 11 and 12).
Figure 7:
Phosphorylation and ERE binding of the
67-kDa form of the hER from Sf9 cells. A, the purified 67-kDa
form of the hER from Sf9 cells was dephosphorylated with 1 µg of
PTP1B (lane 1) or 5 µg of SHPTP1 (lane 2) and
incubated with 1 µg of PTP1B and 1 mM
NaVO
(lane 11) or 5 µg of SHPTP1
and 1 mM Na
VO
(lane 12). The
tyrosine-dephosphorylated hER was rephosphorylated with 5 units of
p60
(lanes 3 and 7), 50
units of p56
(lanes 4 and 8),
50 ng of casein kinase II (CKII) (lanes 5 and 9), or 50 ng of MAP kinase (MAPK) (lanes 6 and 10). The hER samples (10 nM) were then
incubated with 500 ng of poly(dI
dC) for 15 min at 4 °C
followed by the addition of the
P-labeled ERE for 15 min
at 4 °C. The protein-DNA complexes were resolved by nondenaturing
gel electrophoresis and visualized by autoradiography. B, the
treated hER samples were separated by SDS gel electrophoresis and
electrotransferred to a PVDF membrane for Western blot analysis. The
membrane was immunoblotted with the anti-phosphotyrosine antibody (pY Ab) and then stripped and reblotted with the anti-hER
antibody (hER Ab). The bands were visualized by
chemiluminescence.
Casein kinase II or MAP kinase
activity did not restore the ERE binding of the
tyrosine-dephosphorylated hER (Fig. 7A, lanes
5, 6, 9, and 10). However, ERE binding
was restored after the 67-kDa form of the purified recombinant hER
(that was dephosphorylated with PTP1B or SHPTP1) was rephosphorylated
with p60 or p56
(Fig. 7A, lanes 3, 4, 7, and 8). The
restoration of ERE binding was specific since p60
or
p56
in the absence of ATP did not restore the
hER
ERE complex (data not shown). Western blot analysis with the
anti-phosphotyrosine antibody confirmed the phosphorylation of tyrosine
537 by p60
or p56
, but not by casein
kinase II or MAP kinase (Fig. 7B, lanes
3-10).
To confirm that the phosphorylation of tyrosine
537 is required for the hER to bind an ERE, the ERE binding properties
of the mutant Y537F hER were assayed. As expected, a whole cell extract
of Sf9 cells expressing the wild-type hER or the purified 67-kDa form
of the recombinant wild-type hER produced a hERERE complex (Fig. 8, lanes 1 and 2). However, at 10 or 40
nM mutant Y537F hER, a hER
ERE complex was not formed (Fig. 8, lanes 3 and 4).
Figure 8:
Gel mobility shift assay of the mutant
Y537F hER. Whole cell extracts (WCE) from Sf9 cells expressing
the wild-type hER (10 nM) (lane 1), the purified
67-kDa wild-type hER (10 nM) (lane 2), 10 nM mutant Y537F hER (lane 3), or 40 nM mutant Y537F
hER (lane 4) were incubated with 500 ng of poly(dIdC)
for 15 min at 4 °C followed by the addition of the
P-labeled ERE for 15 min at 4 °C. The protein-DNA
complexes were resolved by nondenaturing gel
electrophoresis.
An ``antibody
rescue experiment'' (27) suggested that the
phosphorylation of the hER at tyrosine 537 regulates the monomer to
dimer transition of the receptor (Fig. 9). The premise of the
antibody rescue experiment is that the anti-hER antibody is bivalent
and recognizes two molecules of hER. Therefore, the anti-hER antibody
can bring together two inactive hER monomers and facilitate dimer
formation and DNA binding. The purified 67-kDa form of the recombinant
hER was incubated in the absence or presence of the anti-hER antibody
or a nonspecific antibody. The anti-hER antibody, but not the
nonspecific antibody, supershifted the hERERE complex (Fig. 9, lanes 1, 2, and 4). The 67-kDa form
of the recombinant hER after dephosphorylation with PTP1B did not form
a hER
ERE complex (Fig. 9, lane 5). Interestingly,
we observed the appearance of a supershifted complex (hER
Ab
hER
ERE) when the dephosphorylated hER was incubated with
the anti-hER antibody (Fig. 9, lane 6). The hER
Ab
hER
ERE complex was eliminated with an excess of unlabeled
ERE (Fig. 9, lanes 3 and 9), but not unlabeled
glucocorticoid response element (lane 8), indicating that the
complex was ERE-specific. A supershifted hER Ab
hER
ERE
complex was not formed with the anti-hER antibody when hER peptide 6,
which was used to generate the anti-hER antibody, was added to the gel
shift reaction (22) . In addition, a nonspecific antibody did
not produce a complex with the dephosphorylated hER (data not shown).
Thus, the anti-hER antibody facilitated the formation of the hER dimer
and allowed ERE binding. Predictably, the mutant Y537F hER did not form
a hER
ERE complex, whereas the addition of the anti-hER antibody
to the mutant Y537F hER gel shift reaction produced a hER
Ab
hER
ERE complex (Fig. 9, lanes 10 and 11). These results suggest that the phosphorylation of
tyrosine 537 on the hER is required for dimerization of the hER.
Figure 9:
Anti-hER antibody-induced dimerization and
ERE binding of the hER. Ten nM purified 67-kDa wild-type hER
from Sf9 cells was incubated alone (lane 1) or with the
anti-hER antibody (hER Ab) (lane 2), the anti-hER
antibody and a 200-fold excess of unlabeled ERE (lane 3), or a
nonspecific antibody (lane 4). Ten nM purified 67-kDa
wild-type hER from Sf9 cells dephosphorylated with PTP1B was incubated
alone (lane 5) or with the anti-hER antibody (lane
6), the anti-hER antibody and 5 µg of hER peptide 6 (22) used to generate the anti-hER antibody (lane 7),
the anti-hER antibody and a 200-fold excess of a glucocorticoid
response element (GRE) (lane 8), or the anti-hER
antibody and a 200-fold excess of unlabeled ERE (lane 9). A
whole cell extract of Sf9 cells expressing the mutant Y537F hER (10
nM) was incubated alone (lane 10) or with the
anti-hER antibody (lane 11), the anti-hER antibody and 5
µg of hER peptide 6 (lane 12), the anti-hER antibody and a
200-fold excess of a glucocorticoid response element (lane
13), or the anti-hER antibody and a 200-fold excess of unlabeled
ERE (lane 14). The treated hER samples were incubated with 500
ng of poly(dIdC) for 15 min at 4 °C followed by the addition
of the
P-labeled ERE for 15 min at 4 °C. The
protein-DNA complexes were resolved by nondenaturing gel
electrophoresis and visualized by autoradiography. hER Ab-hER-ERE denotes the supershifted anti-hER antibody complexed to two
molecules of hER, which in turn is bound to the
P-labeled
ERE.
Far-Western blot analysis established that the tyrosine
phosphorylation of the hER was necessary for dimerization. Dimer
formation was maximal when both monomers of the hER were phosphorylated
at tyrosine 537. Dimer formation was also observed when one of the
monomers of the hER dimer was tyrosine-phosphorylated; however, the
extent of dimerization was reduced to more than one-half. The
far-Western blot analysis utilized the purified recombinant hER that
was either P-labeled at tyrosine 537 with p60
(
P-Y537 hER) (Fig. 10A) or
P-labeled at serine 167 with casein kinase II as a probe (
P-S167 hER) (Fig. 10B). The
P-Y537 hER showed strong protein-protein interaction with
the purified recombinant wild-type hER immobilized on a PVDF membrane (Fig. 10A, lane 1). However, far-Western
analysis of the hER that was tyrosine-dephosphorylated prior to SDS gel
electrophoresis showed a reduced interaction with
P-Y537
hER (Fig. 10A, lanes 2 and 3). An
immunoblot with the anti-tyrosine antibody confirmed that the
PVDF-immobilized hER was tyrosine-dephosphorylated (Fig. 10C, lanes 2, 3, 6,
and 7). One mM Na
VO
inhibited
the activity of PTP1B and SHPTP1 (Fig. 10C, lanes 4 and 5) and prevented the reduction of interaction with
P-Y537 hER (Fig. 10A, lanes 4 and 5). The hER was tyrosine-dephosphorylated with PTP1B or SHPTP1
and then rephosphorylated with casein kinase II, MAP kinase,
p60
, or p56
. The tyrosine
phosphorylation, but not the serine phosphorylation, of the hER
restored the full dimerization potential to the dephosphorylated hER (Fig. 10A, lanes 8 and 9 versus lanes 6 and 7). Far-Western blot analysis with the
P-S167 hER confirmed that hER dimerization was dependent
on tyrosine and not serine phosphorylation (Fig. 10B).
An immunoblot of the PVDF membrane with the anti-hER antibody showed
that the amount of hER in each lane was equivalent (Fig. 10D).
Figure 10:
Dimerization of the hER is maximal when
both monomers are tyrosine-phosphorylated. The recombinant wild-type
hER, purified in the presence of the phosphatase inhibitors and
therefore fully phosphorylated (lane 1), was then
dephosphorylated with the tyrosine phosphatase PTP1B or SHPTP1 in the
absence (lanes 2 and 3) or presence (lanes 4 and 5) of NaVO
, an inhibitor of
tyrosine phosphatases. The dephosphorylated hER was then
rephosphorylated with casein kinase II (CKII) (lane
6), MAP kinase (MAPK) (lane 7),
p60
(lane 8), or p56
(lane 9). The hER samples (1.5 pmol each) were
subjected to SDS gel electrophoresis and then electrotransferred to a
PVDF membrane. The hER, purified in the absence of phosphatase
inhibitors and therefore partially dephosphorylated, was
P-phosphorylated with p60
(A) or casein kinase II (B) and used as a probe
for assaying monomer-monomer interaction of the hER. The PVDF membranes
were also immunoblotted with the anti-tyrosine antibody (pY
Ab) (C) or the anti-hER antibody (hER Ab) (D).
This study provides the first evidence that the tyrosine
phosphorylation of the hER regulates the capacity of the receptor to
homodimerize and bind an ERE. We have presented evidence, from several
different experimental approaches, that the phosphorylation at tyrosine
537 of the hER is a prerequisite for ERE binding. We observed that the
nuclear form, but not the cytosolic form, of the hER is
tyrosine-phosphorylated. Only the nuclear hER bound an ERE in a gel
mobility shift assay. The dephosphorylation of the purified hER with a
tyrosine phosphatase results in the loss of ERE binding, while
rephosphorylation with p60 or p56
, but
not with serine-protein kinases, restores ERE binding. These
observations were confirmed by the inability of the mutant Y537F hER to
bind an ERE. The antibody rescue experiments show that ERE binding is
restored to the tyrosine-dephosphorylated hER or the mutant Y537F hER
by virtue of the ability of the anti-hER antibody to promote dimer
formation. Far-Western blot analysis demonstrated that the tyrosine
phosphorylation of at least one of the hER monomers, but preferably
both, is required for dimer formation. Overall, our results indicate
that the phosphorylation of tyrosine 537 facilitates the transition of
the hER from the monomer to dimer form.
The reactivity of the
anti-phosphotyrosine antibody with the native hER was equivalent in the
absence (i.e. the 66-kDa form of the hER) or presence (i.e. the 67-kDa form of the hER) of estradiol treatment of
MCF-7 cells, confirming earlier observations that the phosphorylation
of tyrosine 537 is not increased by estradiol treatment(18) .
In a previous report, the ERE-Teflon matrix containing 500 nmol of
double-stranded ERE/ml of Teflon fiber bound both the 66- and 67-kDa
forms of the hER(22) . In the experiments described here, the
ERE content was only 1-5% of that previously used and
consequently revealed selectivity for the 67-kDa form of the hER.
Recently, tyrosine phosphorylation has been reported to regulate the
dimerization of the signal transducer and activators of transcription
(STAT) family of DNA-binding proteins(9, 10) . The
STAT proteins dimerize through the association of a phosphorylated
tyrosine residue on one monomer and with a SH2 domain on the opposing
monomer(9, 10) . This mechanism of dimerization allows
for an additional level of control of the activity of a transcription
factor. A computer search of the amino acid sequence of the hER has
failed to reveal a region of significant homology to the SH2 domain of
p60.
We hypothesize that the 67-kDa form of the hER
is the transcriptionally active form of the receptor. In the absence of
estradiol treatment of MCF-7 cells, the 66-kDa form of the native hER
exists as two populations of receptors: one that is phosphorylated at
tyrosine 537 and one that is not. After estradiol treatment of MCF-7
cells, only the population of hER that is phosphorylated at tyrosine
537 will undergo serine hyperphosphorylation, as monitored by the
production of the up-shifted 67-kDa form of the hER. We ()and others (28) have shown that the
phosphorylation of tyrosine 537 is required for the hER to bind
estradiol in vitro and in vivo. Numerous mechanisms
can account for the nuclear retention of the 67-kDa form of the native
hER; estradiol has been shown to increase the binding of the calf
estrogen receptor to an ERE by isocratic elution chromatography (29) , although the influence of hormone binding is not
observed with the ERE affinity column or gel mobility shift analysis.
We have shown that the phosphorylation of serine 167 on the hER
increases the affinity of the hER for an ERE(11) . In addition,
the hER associates in an estradiol-dependent fashion with
transcriptional proteins, such as transcriptional intermediary factor 1
(TIF1)(2) . Nonetheless, the DNA binding ability of the hER is
regulated at two levels of post-translational phosphorylation: first,
the phosphorylation of tyrosine 537 is required for hER binding to an
ERE, and second, the phosphorylation of serine 167 increases the hER
(already phosphorylated at tyrosine 537) affinity for an ERE.
In
conclusion, the post-translational phosphorylation of tyrosine 537 on
the hER is required to produce the biologically active hER.
Furthermore, the oncogenic potential of protein-tyrosine kinases, such
as p60, may alter the activity of the hER in
estrogen-dependent breast cancer.