Linkage of Rapid Estrogen Action to MAPK Activation by ER
-Shc Association and Shc Pathway Activation
Robert X.-D. Song,
Robert A. McPherson,
Liana Adam,
Yongde Bao,
Margaret Shupnik,
Rakesh Kumar and
Richard J. Santen
Departments of Internal Medicine (R.X.-D.S., R.A.M., M.S., R.J.S.)
and Biomolecular Research Facility (Y.B.), University of Virginia
School of Medicine, Charlottesville, Virginia 22908; and Department of
Molecular & Cellular Oncology (L.A., R.K.), University of Texas M.D.
Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Dr. Robert X. Song, Division of Hematology and Oncology, University of Virginia Health Science Center, Charlottesville, Virginia 22901. E-mail:
rs5wf{at}virginia.edu
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ABSTRACT
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E2 rapidly activates MAPK in breast cancer cells, and the
mechanism for this effect has not been fully identified. Since growth
factor-induced MAPK activation involves signaling via the adapter
protein Shc (Src-homology and collagen homology) and its association
with membrane receptors, we hypothesized that breast cancer cells
utilize similar signaling mechanisms in response to E2. In the present
study, we demonstrated that E2 rapidly induced Shc
phosphorylation and Shc-Grb2 (growth factor receptor binding
protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells.
Overexpression of dominant negative Shc blocked the effect of E2 on
MAPK, indicating a critical role of Shc in E2 action. Using selective
inhibitors, we also demonstrated that ER
and Src are upstream
regulators of Shc. A rapid physical association between ER
and Shc
upon E2 stimulation further evidenced the role of ER
on Shc
activation. Mutagenesis studies showed that the phosphotyrosine binding
and SH2 domains of Shc are required to interact with the activation
function 1, but not activation function 2, domain of
ER
. Using a glutathione-S-transferanse-Shc
pull-down assay, we demonstrated that this ER
-Shc association was
direct. Biological consequences of this pathway were further
investigated at the genomic and nongenomic levels. E2 stimulated
MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy
studies showed that E2 rapidly induced formation of membrane ruffles,
pseudopodia, and ER
membrane translocation. The E2-induced
morphological changes were prevented by antiestrogen. Together
our results demonstrate that ER
can mediate the rapid effects of E2
on Shc, MAPK, Elk-1, and morphological changes in breast cancer
cells
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INTRODUCTION
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E2 IS a steroid hormone that plays an
important role in the genesis of human breast cancer and in the growth
of established tumors (1). Classically, E2 elicits genomic
effects on transcription via ER
and ERß, which are mainly located
in the nucleus. As ligand-dependent transcriptional factors, the
hormone-bound ERs interact with estrogen response elements to
stimulate certain genes in E2-responsive tissues and to regulate gene
transactivation (2). The genomic actions of E2 are
characterized by delayed onset of action and sensitivity to inhibitors
of transcription and translation. Many of the E2-induced
transcriptional mechanisms involve ER
, which can be functionally
regulated by posttranslational phosphorylation of serine residues at
the 104, 106, 118, 167, and 236 positions (3, 4, 5, 6). In
addition, tyrosine-537 of ER
has been reported to be phosphorylated
in MCF-7 cells and in ER
-expressing Sf9 insect cells
(7). The biological function of ER
is also regulated by
association with a subset of nuclear proteins, such as coactivators,
corepressors, and integrator proteins (8, 9, 10, 11).
More recently, investigators have recognized rapid nongenomic actions
of E2 on several cellular processes, such as activation of Ras
(12), Raf-1 (13), PKC (14), PKA
(15), Maxi-K channels (16), increments in
intracellular calcium levels (17), and an increase of
nitric oxide (17). Elicited responses depend upon the cell
types studied and the conditions used. In contrast to its
transcriptional effects, the precise nongenomic signaling pathways of
E2, especially the involvement of ER
in this process, are not well
understood and have not been extensively studied
(18, 19, 20).
Several recent reports demonstrated that E2 rapidly activates MAPK in a
number of model systems (13, 21, 22), but the mechanisms
responsible for this are still controversial (23, 24, 25, 26).
Many growth factor receptors on the cell membrane, such as the
receptors for epidermal growth factor (27), nerve growth
factor (28), platelet-derived growth factor
(27), and IGF (29), activate MAPK through a
Shc-mediated pathway. We hypothesized that E2 might co-opt a pathway
using Shc-Grb2-Sos (Src homology-growth factor receptor binding
protein 2-son of sevenless) to activate MAPK. The adapter protein Shc
has no intrinsic kinase domain and transduces signals dependent on the
association with membrane receptors. Three domains mediating
protein-protein interactions have been reported on Shc. Two of these,
the phosphotyrosine binding (PTB) domain in the amino-terminal region
and the Src homology 2 (SH2) domain in the carboxy-terminal region are
separated by a region rich in proline and glycine residues, called the
collagen homology (CH) domain (30). Shc binds rapidly to
the specific phosphotyrosine residues of receptors through its PTB or
SH2 domain and becomes phosphorylated itself on tyrosine residues of
the CH domain (27). The phosphorylated tyrosine residues
on the CH domain provide the docking sites for the binding of the SH2
domain of Grb2 and hence recruit Sos, a guanine nucleotide exchange
protein. This adapter complex formation allows Ras activation via the
Sos protein, leading to the activation of the MAPK pathway (27, 31, 32). Since ER
has been reported to be associated with the
membrane of MCF-7 cells (20), we hypothesized that this
receptor might be involved in mediating E2 action on MAPK activation by
physical interaction with Shc in a manner analogous to that in growth
factor pathways.
To test our hypothesis, we used MCF-7 and its variant LTED cells. LTED
cells were developed from parental MCF-7 cells growing long-term in
estrogen-depleted medium (33). This maneuver causes a 5-
to 10-fold up-regulation of ER
expression (33). In the
present study, we demonstrated that E2 rapidly stimulated both MAPK and
Shc phosphorylation and induced the association of Shc with ER
.
Using selective inhibitors and a dominant mutant Shc, we further
demonstrated that Shc is upstream of MAPK and that the rapid actions of
E2 are ER
-dependent and involve ER
/Shc association. Furthermore,
a direct physical association of ER
with Shc was evidenced by
protein pull-down assay. Our results show that the PTB and SH2 domains
of Shc interacted with the AF-1 region of ER
. Tyrosine-537 of ER
was not involved in this interaction. Measurement of Elk-1 activation
and morphological changes by confocal microscopy provided further
evidence of the rapid, nongenomic role of E2 in these cells. Together,
our data suggest that the nongenomic effects of E2 on Shc and MAPK
activation are mediated by the classical ER
receptor, that E2
can induce an association of ER
with Shc, and that Shc appears to be
a crucial step for the activation of MAPK.
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RESULTS
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Effects of Selective Inhibitors and Dominant-Negative Shc
on E2-Induced MAPK Phosphorylation
Prior studies reported that E2 rapidly activated MAPK in
MCF-7 cells (34). To investigate the upstream signals
mediating E2 action on MAPK, we first confirmed that E2 increased MAPK
activation in our MCF-7 cells by measuring MAPK phosphorylation. Figure 1A
shows that E2 increased both p42 and
p44 MAPK phosphorylation in a time-dependent manner with the peak
at 15-min treatment. We then questioned whether the effects of E2 on
MAPK involved ER
, MEK (MAPK kinase), and Src tyrosine kinase. As
shown in Fig. 1B
, the antiestrogen ICI, the MEK inhibitor
PD98059, and a pan Src inhibitor PP2 all blocked E2-induced MAPK
phosphorylation, implying that ER
, MEK, and Src family members are
required for this step. PP2 alone abolished basal MAPK phosphorylation,
suggesting the role of Src in maintaining basal MAPK function (Fig. 1B
).

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Figure 1. MAPK Phosphorylation in MCF-7 Cells
A, E2-induced MAPK phosphorylation. MCF-7 cells were treated with
vehicle or E2 at 10-10 M for the times
indicated. The phosphorylation status of MAPK (p42 and p44) from
whole-cell lysates was assessed using an antibody recognizing dual
phosphorylated MAPK (top panel). Levels of total MAPK
protein were determined on the same blot to control for loading
variations (bottom panel). The positions of
phosphorylated and total MAPK proteins are indicated at the
right. B, Effects of pathway-selective inhibitors on
E2-induced MAPK phosphorylation. MCF-7 cells were pretreated with 23
µM PD98059, 10-9 M ICI 182 780
(ICI), and 5 nM PP2 for 30 min. Cells were then treated
with vehicle or 10-10 M E2 for 15 min. The
phosphorylated (top panel) and total MAPK (bottom
panel) were determined. C, Overexpression of mutant Shc blocked
E2-induced MAPK phosphorylation. MCF-7 cells were either not
transfected or transfected with the mutant Shc (ShcFFF) or control
vectors (pEBG) for 2 d. Then cells were treated with vehicle or
10-10 M E2 for 15 min. The phosphorylated
(top panel) and total MAPK (middle panel)
were detected. Both GST-ShcFFF and endogenous Shc expression are shown
(bottom panel). The above experiments have been repeated
three times, and one of the representative experiments is shown.
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Activation of MAPK through classical growth factor signaling pathways
involves the phosphorylation and activation of the adapter protein Shc.
To test whether Shc is necessary for E2-induced MAPK phosphorylation,
we transiently transfected MCF-7 cells with a dominant-negative Shc
mutant. The mutant is a glutathione-S-transferase (GST)
fusion protein with phenylalanines substituted for tyrosines at the
239/240 and 317 positions of the Shc CH domain (ShcFFF), rendering it
defective in signaling to MAPK (35, 36). Expression of
ShcFFF decreased E2-induced MAPK phosphorylation compared with
untransfected and empty vector (pEBG)-transfected cells (Fig. 1C
). Together, these results indicated that ER
, Src, and Shc are all
upstream components regulating MAPK activation.
E2 Rapidly Induces Shc Pathway Activation
To further confirm the involvement of Shc in the E2 rapid
signaling pathway, we evaluated this step in both MCF-7 and LTED cells
that express a 10-fold elevation of ER
levels. As shown in
the top panel of Fig. 2A
, E2
at 10-10 M increased p52
kDa Shc phosphorylation in a time-dependent manner with the
maximum response at 3 min. When LTED cells were compared with MCF-7
cells, Shc was phosphorylated to a greater extent in LTED cells
(left in Fig. 2A
) than parental MCF-7 cells
(right in Fig. 2A
) under basal conditions and increased
proportionately in response to E2. To validate the results, the
polyvinylidene difluoride (PVDF) membranes were reprobed
with anti-Shc antibodies. The results show a similar amount of Shc
proteins of both p46 and p52 kDa in MCF-7 and LTED cells (Fig. 2A
, bottom panels). E2-induced Shc phosphorylation was also
dose-dependent with the optimal dose at 10-10
M (data not shown).

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Figure 2. E2-Induced Shc Pathway Activation in MCF-7 and Its
Variant Cells
A, Effects of E2 on Shc phosphorylation. Both LTED and
MCF-7 cells were treated with vehicle or 10-10
M E2 for the times indicated and then extracted as
described in Materials and Methods. Shc phosphorylation
was measured by immunoprecipitation (IP) of Shc protein and detection
of tyrosine phosphorylation (top panel) on immunoblot
(IB). To normalize Shc protein loading, the membranes were stripped and
reprobed with polyclonal anti-Shc antibodies (bottom
panel). Nonspecific monoclonal or polyclonal IgG were employed
in all immunoprecipitation (IP) steps of the following experiments
as negative controls. B, Effects of E2 on Shc-mediated adapter protein
complex formation. LTED cells were treated with vehicle or E2 at
10-10 M for 3 min. The association of Grb2/Shc
and Grb2/Sos was measured by immunoprecipitation of Grb2 and detection
of Shc (left top panel) or Sos (middle top
panel) on Western blots. The same membrane was reprobed with
antibodies against Grb2 to detect Grb2 protein loading
(left and middle bottom panels). The
Grb2/Sos association was further confirmed by reciprocal antibody
method (right panel). C, Effects of the selective
inhibitors on E2-induced Shc phosphorylation in MCF-7 cells. MCF-7
cells were pretreated with 5 nM PP2, 23 µM
PD98059, and 10-9 M ICI for 30 min. Cells then
were challenged with vehicle or 10-10 M E2 for
3 min. The phosphorylation of Shc was determined as described in
Materials and Methods (top panel). The
membrane was reprobed with anti-Shc antibodies to show Shc protein
loading (bottom panel). Above experiments were done
three times and a representative experiment is shown.
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Classical pathways of Shc activation by growth factors involve Shc
association with the adapter proteins, Grb2 and Sos. Accordingly, we
examined the effects of E2 on Shc-Grb2-Sos complex formation. LTED
cells were used in this study because of their enhanced Shc
phosphorylation in response to E2. As shown in Fig. 2B
, Grb2/Shc
(left panel) and Grb2/Sos (middle and
right panels) complexes were faintly apparent in the
vehicle-treated group, but greatly increased by
10-10 M E2 treatment at 3
min. Together, these results suggest that E2 is capable of activating
the Shc-mediated signaling pathway in MCF-7 cells, which is regulated
by the level of ER
expression.
Effects of Selective Signaling Pathway Inhibitors on E2-Induced Shc
Phosphorylation
Because Shc is responsible for the activation of MAPK by E2, we
wished to determine components responsible for Shc phosphorylation.
Additionally, we wished to demonstrate a role for ER
in this process
and to exclude MAPK as the cause of Shc phosphorylation. Shc has been
reported to be a substrate of c-Src in HEK-293 cells (37).
Accordingly, we examined the effects of PP2, ICI, and PD98059 on
E2-induced Shc phosphorylation. In the presence of the inhibitors,
MCF-7 cells were stimulated with vehicle or
10-10 M E2 for 3 min, and the status
of Shc phosphorylation was examined. Figure 2C
shows that both PP2 and
ICI effectively inhibited E2-induced Shc phosphorylation, indicating
that Src family kinases and ER
are required for Shc activation. As
expected, PD98059 did not influence Shc phosphorylation status,
suggesting that MAPK functions downstream of Shc. No effects of
these inhibitors were apparent in the absence of E2 stimulation. These
results indicate that both ER
and Src are upstream components of Shc
functionality, and their involvement is required for Shc
phosphorylation.
E2-Stimulated ER
Association with Shc
We postulated that ER
might mediate effects of E2 on Shc by
directly or indirectly associating with the Shc adapter protein. This
concept was based on the fact that activated Shc transduces signals by
associating with membrane receptors and our evidence that ICI
blocked E2-induced Shc phosphorylation in MCF-7 cells. To test this
hypothesis, we immunoprecipitated Shc protein and detected ER
on
Western blots. As shown in Fig. 3
, E2
rapidly induced the association of ER
with Shc in both LTED and
MCF-7 cells (top panel). The induction was seen as early as
1 min of E2 treatment and reached a plateau from 3 to 10 min.
Compared with MCF-7 cells, ER
/Shc complexes were present basally in
LTED cells, which express 5- to 10-fold higher levels of ER
, and
greatly increased in response to E2 treatment. Consistency of protein
loading was confirmed by reprobing the membrane with anti-Shc antibody
(bottom panel). Thus, the association between ER
and Shc
was observed not only in MCF-7 cells, but it was greatly enhanced in
ER
up-regulated LTED cells. The results were also confirmed by the
immunoprecipitation of ER
and the detection of Shc on immunoblot
(data not shown). Together, our data suggest that as an upstream signal
of Shc, the level of ER
expression affects the level of Shc
phosphorylation and ER
/Shc association.
Study of Molecular Basis of ER
/Shc Interactions
To study the molecular basis of the interacting domains on ER
and Shc, various GST-tagged Shc and ER
mutants were employed (Fig. 4A
). Furthermore, to
minimize factors specific to breast cancer cells, COS-1 cells that do
not have detectable endogenous ER
were used and electroporated with
different ER
and Shc expressing vectors (Fig. 4A
). First, we
examined the Shc-interacting domain(s) on ER
by electroporating
wild-type Shc (ShcWT) with either wild-type ER
expressing vector
(HEGO) or a mutant ER
expressing vector in which phenylalanine was
substituted for tyrosine in the 537-position (Y537F). As shown in Fig. 4B
, the Y537F mutation did not alter the Shc/ER
complex formation
compared with wild-type ER
, suggesting that Y537 of ER
is not the
site interacting with Shc. Coexpression of either the truncated
activation function 1 (AF-1) or activation function 2 (AF-2)
domains of ER
with ShcWT in COS-1 cells confirmed that it is the
AF-1, but not AF-2 (contains Y537), domain of ER
that interacts with
Shc (Fig. 4, C and D). E2 increased the association of AF-1
with Shc (Fig. 4C
). The mechanism for this is not known and was
unexpected, since AF-1 does not contain a binding site for E2. We
postulate that E2 might exert effects on AF-1 or Shc binding proteins
independently of ER
, leading to an increase of AF-1/Shc
association.

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Figure 4. Study of Shc/ER Interaction
A, Schematic ER and GST-tagged Shc constructs for
eukaryotic expression. In left panel, HEGO is wild-type
ER -expressing vector. Y537F is a vector expressing mutant ER in
which the tyrosine-537 was mutated into phenylalanine. The AF-1 vector
encodes amino acids 1367 of ER and AF-2 encodes 179595. In the right panel, the GST-Shc fusion protein-expressing
vectors are shown. The oval region in the N terminus of
Shc corresponds to the GST fusion protein. The white,
gray, and diagonally lined rectangles represent
the PTB, CH, and SH2 domains of Shc, respectively. B, Examination of
tyrosine-537 of ER interaction with Shc. Wild-type Shc vector
(ShcWT) was cotransfected with either HEGO or Y537F
ER -expressing vectors into COS-1 cells. Cells were then treated with
vehicle or 10-10 M E2 for 5 min, and the
ER /Shc association was assessed. C and D, The AF-1 domain of ER
is required to interact with Shc. COS-1 cells were cotransfected with
GST-tagged wild-type Shc (ShcWT) and different ER constructs as
indicated for 3 d. Cells were then treated with vehicle or
10-10 M E2 for 5 min. The ER -Shc association (top panels) was measured by immunoprecipitation of GST and detection of ER on immunoblot using either anti-C (panel C) or anti-N-terminal (panel D) ER antibodies. The membrane was reprobed with anti-GST antibodies to
normalize the protein loading (bottom panels). To
confirm the positions of AF-1 and AF-2, the ER-transfected COS-1 cell
extracts were loaded on the same gels, and the expression of
immunoprotein bands of HEGO, AF-1, and AF-2 is shown on the
right sides of the top panels. E, Mapping
the ER -interacting domains on Shc. Wild-type ER (HEGO) was
transiently transfected into COS-1 cells with different GST-tagged Shc
expression vectors as indicated. On d 3, cells were extracted, and the
interactions between ER and wild-type Shc (top left
panel) or mutated Shc proteins (top right panel)
were measured as described in Materials and Methods. The
same membranes were stripped and reprobed with anti-ER antibody to
normalize the protein loading (bottom panels).
Quantitation of the GST-Shc immunoprotein bands normalized by ER
protein loading is shown as fold induction. All figures are
representative blots of the study that was performed three times. F,
GST pull-down assay of ER -Shc interaction. GST-tagged fusion
proteins expressing Shc-selective domains were purified by the beads of
Glutathione Sepharose 4B. Beads (15 µl) conjugated with or without
GST, GST-SH2, or GST-PTB+CH domains of Shc were incubated with 5 ng
recombinant full-length human ER protein overnight. To control
the specificity, the full-length human ER was also incubated with
anti-vitamin D receptor, anti-ER , or nonspecific immunoglobulin
(IgG) as indicated. After washing the beads three times, the eluted
proteins were resolved on 10% SDS-PAGE, transferred onto a PVDF
membrane, and detected with anti-ER antibody (top
panel). The gel was stained with Coomassie blue to show the
positions of the Shc fusion proteins (bottom panel). One
of four experiments is shown.
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Next we further mapped the ER
-interacting site(s) on Shc. When ShcWT
was coexpressed with ER
(HEGO) in COS-1 cells, we demonstrated an
association of GST-Shc protein with ER
under basal conditions and
further enhancement of this association with 5 min of E2 treatment
(Fig. 4E
, top left panel). We then used selective
domain-expressing Shc mutants to examine which domains of Shc were
necessary for this association. Both the PTB (ShcPTB)- and the SH2
(ShcSH2) domain-expressing proteins formed complexes with ER
, which
further increased in intensity upon E2 stimulation (Fig. 4E
, top
right panel). Proteins that only expressed the GST or CH domain of
Shc failed to interact with ER
. After normalizing the GST-Shc
immunoprotein bands (Fig. 4E
, top panels) for ER
protein
loading (Fig. 4E
, bottom panels), we show that the Shc/ER
association induced by E2 is 2- to 3-fold higher than the control in
cells expressing PTB or SH2 and is similar to that in cells containing
ShcWT.
Shc can be a substrate of c-Src that associates with ER
in MCF-7
cells after E2 treatment (38). It would be of interest to
know whether c-Src is involved in this ER
-Shc complex in our breast
cancer system. Using several different anti-c-Src (B-12, N-16, and
SRC2, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
anti-ER
antibodies (D-12 and MC-20, Santa Cruz Biotechnology, Inc.), we did not detect c-Src in ER
-Shc complexes of either
MCF-7 or LTED cells (data not shown). To further address the molecular
basis of Shc-ER
interaction, we tested whether this interaction is
direct or mediated by third-party proteins using GST-pull-down assays.
Both GST-tagged SH2 and PTH+CH domain fusion proteins of Shc were
produced at the size of 37 kDa and 68 kDa (Fig. 4F
, bottom
panel). The 62-kDa recombinant full-length ER
was pulled down
by both GST-tagged Shc proteins (Fig. 4F
, top panel). As
negative control, the beads and GST protein alone did not pull down the
ER
protein. The specificity was confirmed by immunoprecipitation and
detection of ER
with anti-ER
antibodies, but not by the
nonspecific or anti-vitamin D receptor antibodies. Together, our
results demonstrate that the interaction between ER
and Shc is
direct and does not require the presence of any other proteins.
Biological Effects of E2-Induced Nongenomic Pathway Activation
To further provide evidence that the ER
-Shc-MAPK pathway is
biologically relevant, we used two approaches. First, we evaluated the
role of MAPK on the activation of Elk-1, which is a transcriptional
factor phosphorylated and activated by MAPK (39). Second,
we examined the rapid effects of E2 on morphological changes of MCF-7
cells.
To study Elk-1 activation by E2, a well characterized reporter system
utilizing both GAL4-Elk-1 and GAL4-luciferase vectors (40)
was cotransfected into MCF-7 cells. Figure 5
shows that E2 increased Elk-1
activation in a dose-dependent manner at 6 h as shown by
luciferase assay. The E2 doses higher than 10-12
M significantly stimulated Elk-1 activation, indicating the
involvement of MAPK in E2 action.

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Figure 5. E2-Stimulated MAPK Substrate Elk-1 Activation
A transient transfection method was used to study MAPK-mediated Elk-1
activation. MCF-7 cells were cotransfected with GAL4-Elk-1 and
GAL4-E1B-Luciferase vectors. Twenty-four hours after transfection,
cells were stimulated with vehicle or different doses of E2 for 6
h. Luciferase activity for each lysate was determined. The data are
presented as mean ± SD of three experiments. *,
P < 0.05 compared with untreated control.
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To further provide direct biological evidence of E2 stimulation of
Shc-MAPK pathways, effects of E2 on cell morphology were examined. We
reasoned that E2 might elicit effects similar to those induced by
exogenous growth factors since it used a pathway normally activated by
growth factors, namely the Shc-MAPK pathway. Our coauthors recently
used the confocal microscopic method to demonstrate that heregulin
induces several rapid cellular effects, such as formation of membrane
ruffles, filopodia, pseudopodia, actin polymerization, and loss of
focal adhesion kinase plaques (41). Using identical
methods, we studied rapid cell morphological changes and their
substructures using LTED cells due to their strong responses with
respect to ER
/Shc association and Shc phosphorylation upon E2
treatment.
Under basal conditions (Fig. 6A
), LTED
cells exhibited a low level of membrane ruffles (red),
indicating minimal actin polymerization sites. ER
(green)
appeared to be predominantly in the nucleus with some also present in
the cytoplasm. Beginning at 10 min (data not shown) and increasing by
20 min, E2 induced dramatic morphological effects, including the
formation of additional membrane ruffles and cell shape alterations
(Fig. 6B
). Even more dramatic was the formation of pseudopodia shown in
Fig. 6C
as an arm-like extension of the cell membrane with a fist-like
structure at its terminus. To demonstrate that ER
mediated these
morphological responses, the effects of E2 were abrogated by
coadministration of the pure antiestrogen ICI along with E2 (Fig. 6D
).
ICI alone resulted in no morphological changes (Fig. 6E
).
Use of confocal microscopy and immunofluorescence provided a dynamic
means of assessing ER
location and alterations in response to E2.
Accordingly, we focused on the region contiguous to the cell membrane.
The insets in Fig. 6F
show enlarged areas indicated by
arrows in panels AE. Under basal conditions, minimal
green staining could be observed along the cell membrane of
the cells (Fig. 6A
, inset a, right panel). In
marked contrast, E2 appeared to translocate ER
into the region along
the membrane ruffles as indicated by the strong appearance of
green staining (Fig. 6B
, inset b,right panel). As
shown by merging the red (actin) and green
(ER
) views, the ER
appeared as yellow (Fig. 6B
, inset b, left panel), indicating colocalization with actin
in the membrane ruffles. Striking also was the translocation of
the ER
into the fist-like region of the pseudopodia as shown by both
the green staining (Fig. 6C
, inset c, right
panel) and yellow merged views (Fig. 6C
, inset c,
left panel). ICI blocked E2-induced ER
membrane
translocation with little effect by it alone (Fig. 6
, D and E;
inset d, right and left panels; and inset
e, right and left panels). The immunofluorescence
staining for vinculin (blue) represents the focal adhesion
contacts of the cells that are not highly visible.
The morphological changes demonstrated in Fig. 6
represent examples of
consistent changes observed in many cells. To establish that these
changes were statistically significant, we used an integrated measure
of membrane changes called dynamic membrane formation (DM). This
integrated parameter includes both membrane ruffles and pseudopodia. As
shown in Fig. 7
, the E2-induced increase
in DM was statistically significant and could be blocked by ICI.
Collectively, these results demonstrated that E2 rapidly induced
specific changes on cell morphology in addition to its effects on
membrane-mediated Shc-MAPK activation.

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Figure 7. Summary of Dynamic Membrane Formation in LTED Cells
The changes in ruffles and pseudopodias are combined under the term,
dynamic membrane (DM), formation. Quantitative results from 12
individual cells are summarized. *, P <
0.05.
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DISCUSSION
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Our studies demonstrated that MCF-7 breast cancer cells utilize a
classical growth factor signaling pathway to mediate the nongenomic
effects of E2. This pathway involves the direct association of ER
with Shc, the phosphorylation of Shc, and the formation of Shc-Grb2-Sos
complexes. Shc is critical for E2-induced MAPK activation. Using a
mutagenesis approach, we demonstrated that the SH2 and PTB domains of
Shc are the sites interacting with ER
and that the AF-1 region of
ER
is required to interact with Shc.
Evidence of the biological relevance of this pathway is inferred from
the demonstration that Elk-1, one of the mediators of MAPK, was
increased by E2. More importantly, we obtained direct biological
evidence of the nongenomic effects of E2 by demonstrating morphological
changes with confocal microscopy. These studies showed that E2 rapidly
induced formation of membrane ruffles and pseudopodia, but also
translocation of ER
to the membrane and into perimembrane regions of
pseudopodia. Finally our results demonstrated that
E2, mediated by ER
, activated a classical
growth factor-signaling pathway involving Shc adapter proteins.
The adapter protein Shc mediates the effects of many growth factors to
activate MAPK. However, several recent studies demonstrated different
mechanisms whereby E2 can induce MAPK activation. McDonnell
and associates (34) showed that calcium is required for
E2-induced MAPK activation in MCF-7 cells, and this event is ER
dependent. E2-induced MAPK activation was also reported to be
independent of ER
or ERß but required the existence of epidermal
growth factor receptor (EGFR) and GRP30, a G protein-coupled receptor
homolog in an ER
-negative breast cancer cell line MDA-MB-231
(25). EGFR expression in MCF-7 cells is very low, and
Parsons and colleagues (42) have reported that EGFR
protein is not detectable in MCF-7 cells either by immunoprecipitation
or by immunoblotting methods. In our system, we do not detect EGFR in
either MCF-7 or its variant cells (data not shown).
In the present study, we demonstrated that ER
, as a key mediator, is
required for E2 action on Shc/ER
association and Shc and MAPK
phosphorylation. Approximately 0.5% of total ER
was estimated to be
associated with Shc after E2 stimulation as shown by an ER
-Shc
coimmunoprecipitation study (data not shown). The activation of MAPK
with E2 is less than that by growth factors, as reported by other
investigators (34). Our unpublished data also show that
MAPK phosphorylation induced by 10 ng/ml TGF
was 35-fold higher than
untreated controls, compared with 2.5-fold induction induced by
10-10 M E2. ER
-dependent MAPK
activation has been reported by many laboratories using MCF-7 and other
cell lines treated with E2 (39, 43). The
differences regarding ER
involvement in MAPK activation under
varying conditions are unknown. Nevertheless, it is not surprising
that E2 can activate multiple rapid signaling pathways in different
cell lines due to the fact that it can exert nongenomic effects on
nitrous oxide, PKC, PKA, and MAPK. Detailed examination of E2 induction
through each of these mechanisms will be required before a full
understanding of the biological relevance of these various pathways
emerges.
A single time point of Shc phosphorylation induced by E2 was first
reported in MCF-7 cells (13). We extended these
observations by conducting detailed time studies and showed the effects
as early as 1 min after E2 treatment. ER
is required for Shc
phosphorylation, and the association of ER
with Shc appears to be
important for Shc activation. It has been suggested that PTB and SH2
are two distinct binding domains, which may link to phosphorylated
tyrosine residues in a different manner (44). Both PTB and
SH2 domains of Shc are required to associate with EGFR
(45), but Neuregulin-activated ErbB2 and ErbB3 bind only
to PTB domain of Shc (46). The functional roles of the PTB
or SH2 sites in their interaction with ER
are now under
investigation.
The PTB and SH2 domains of Shc interact with phosphorylated tyrosine
residue(s) of other proteins. The exact tyrosine residue of the ER
responsible for binding to Shc is unknown. Since tyrosine-537 of human
ER
may serve as a constitutively phosphorylated site
(7), we considered it a possible docking site for Shc.
However, our data show that the AF-1 domain of ER
, but not the AF-2
or tyrosine-537 region, interacts with Shc, indicating that there might
be other tyrosine residues phosphorylated on the N-terminal ER
. We
postulated that 1 of 10 potential tyrosine residues in the AF-1 region
(amino acids 1179) might be involved in the association with Shc,
such as tyrosine 43, 52, 54, 60, 73, 80, 130/131, 139, and 150. Further
investigation of these sites is now being conducted in our
laboratory.
ER
has been reported to associate functionally with many cell
membrane proteins, such as IRS-1, the p85 subunit of PI3K, and caveolin
(47, 48, 49). c-Src has been reported to physically associate
with ER
of bovine uterus, but there is no direct evidence that
ER
-associated c-Src is responsible for Shc phosphorylation
(13). The present study demonstrated that the Src
inhibitor PP2 blocked Shc phosphorylation induced by E2. This evidence
suggests that a Src protein family member is functionally involved in
E2-induced Shc phosphorylation, even though Src is not detectable in
the ER
-Shc complex. It is possible that other Src family members are
involved in phosphorylating Shc or that the Src-ER
complex is
unstable during immunoprecipitation.
The breast cancer models used in our studies involve parental MCF-7 and
its variant LTED cells. When compared with its parental cells, LTED
cells show an increased growth rate in E2-deprived media and respond to
very low concentrations of E2 when grown in nude mice
(50). ER
, but not ERß, is detectable in our MCF-7 and
its variant cells (33, 50). The presence of increased
amounts of ER
appears to amplify the components examined in the
ER
-Shc signaling pathway. Whenever effects, such as ER
/Shc
association and Shc phosphorylation, were compared in LTED and MCF-7
cells, the responses were qualitatively similar but quantitatively
greater in the variants. For this reason, we used this variant for
demonstrating certain effects such as Grb2/Sos interactions and
morphological changes by confocal microscopy.
Our studies demonstrated rapid, nongenomic effects of E2 by two
separate methods, biochemical assessment of the Shc-MAPK pathway and
morphological effects on cell shape, membrane ruffles, pseudopodia, and
ER
translocation. Both effects could be blocked by the antiestrogen
ICI, indicating that Shc-MAPK activation and morphological changes were
all initiated by ER
. Even though we do not yet have direct evidence
that the observed E2 effects on cell structure are mediated by
activation of the Shc and MAPK pathway, the role of Shc and MAPK on
cell adhesion and motility has been demonstrated in several cell lines
(51, 52). The interaction of Shc with membrane integrin in
MCF-7 cells was reported to regulate the locomotion and cell adhesion
of MCF-7 cells (53). We postulate that the E2-induced
membrane signaling pathway activation, especially ER
-involved
activation of Shc, might be linked to cell morphology changes in our
cell models.
The suggestion that ER
can reside in or near the cell membrane has
been controversial in the past (54). Recent transfection
studies provided additional support that ER
can be present in the
cell membrane (55). Razandi et al.
(55) transfected ER
into Chinese hamster ovary (CHO)
cells and demonstrated that 3% of expressed ER
was associated with
cell membranes. Immunochemical studies with several anti-ER
antibodies on fixed cells including MCF-7 cells also suggested
the presence of membrane ER
(20). Our observation using
confocal microscopy showed that E2 increased the amounts of ER
in
the region of the cell membrane. Our findings provide the first
evidence that E2 can functionally influence the amount of ER
translocated into the perimembranous area. At the present time, we do
not have direct morphological evidence that Shc is associated with this
membrane-associated ER
, but we plan to examine this issue in the
future.
In conclusion, we demonstrated that the signaling pathway mediating the
rapid action of E2 involved the interaction of Shc with ER
, Shc
activation, and the phosphorylation of MAPK. The PTB and SH2 domains of
Shc as well as AF-1 domain of ER
are the sites mediating the
interaction. Using selective pathway inhibitors and a dominant negative
Shc mutant, we confirmed that ER
-mediated Shc activation resulted in
MAPK phosphorylation in MCF-7 cells. As evidence of functionality, E2
rapidly stimulated cell morphological changes and induced activation of
the MAPK substrate, Elk-1. Additional studies are now required to
provide further proof of the role of each of the signaling molecules in
the biological events observed.
 |
MATERIALS AND METHODS
|
---|
Reagents and Plasmids
Tissue culture supplies were obtained from Fisher Scientific (Pittsburgh, PA). Improved MEM (IMEM) with or
without phenol red (zinc option, Richters modification) and FBS were
products of Life Technologies, Inc. (Gaithersburg, MD). E2
was obtained from Steraloids (Wilton, NH). ICI was from AstraZeneca,
Inc. (Wilmington, DE). E2 and ICI were dissolved in ethanol with
a final concentration of ethanol in medium of less than 0.01%. PP2 and
PD98059 were from Calbiochem (La Jolla, CA) and dissolved
in dimethylsulfoxide. Full-length ER
recombinant protein was
purchased from Affinity BioReagents, Inc. (Golden, CO).
Protease inhibitors, leupeptin, pepstatin, and aprotinin, were
purchased from Sigma (St. Louis, MO).
Mammalian expression vectors encoding human 52-kDa forms or
partial regions of Shc with GST fusion proteins were gifts from Dr.
Ravichandran Kodi (University of Virginia, Charlottesville, VA) and
have been described previously (46). The amino acid
sequences encoded by the different Shc constructs are: full-length
(1471), PTB (17203), CH (233377), and SH2 (377471). The
full-length human ER
expression vector (HEGO) was a gift from Dr.
Rosalie Uht (University of Virginia) and has been previously reported
(56). The ER
expression vectors of AF-1, AF-2, and the
Y537F mutant in which tyrosine 537 was mutated into phenylalanine were
constructed in our laboratory by PCR and site-directed mutagenesis
(QuickChange, Stratagene, La Jolla, CA) as previously
described (57). The fusion gene GAL4-Elk-1 and its
reporter gene GAL4-E1B-Luc were generously provided by Dr. Michael J.
Weber (University of Virginia) and have been described previously
(40).
Cell Culture
The MCF-7 cells and COS-1 cells were maintained in 5%
FBS-IMEM and 10% FBS-DMEM, respectively. The LTED cells were developed
from MCF-7 cells by growing long term (i.e. 6 months to 2
yr) in phenol red-free IMEM supplemented with 5% Dextran-coated
charcoal-stripped FBS and have been extensively characterized with
respect to growth characteristics, responsiveness to E2, and ER content
(33, 50). As determined by ligand binding assays as well
as mRNA and Western blots, these cells contain 5- to 10-fold higher
levels of endogenous ER
than the parental MCF-7 cells. The content
of ERß is negligible to absent as demonstrated by Western blot and
PCR (data not shown). All cell lines were routinely cultured in a
humidified 95% air-5% CO2 incubator at 37
C.
Cell Stimulation and Lysate Preparation
For rapid E2-induced signaling pathway studies, cells
cultured in IMEM containing 1% dextran-coated charcoal-stripped FBS
were treated at 37 C with vehicle or E2. Time and doses are indicated
in the figure legends. If inhibitors were used, cells were pretreated
with the inhibitors for 30 min and then challenged with E2. Cells were
washed once with ice-cold PBS containing 1 mM
Na2VO4 and extracted with
binding buffer (50 mM Tris, pH 8.0, 150 mM
NaCl, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 25
mM NaF, 2 mM
Na2VO4, and 10 µg/ml of
each aprotinin, leupeptin, and pepstatin). Cell lysates were
centrifuged at 14,000 x g for 10 min at 4 C to pellet
insoluble material. The supernatant of cell extract was analyzed for
protein concentration by a DC protein assay kit based on the Lowry
method (Bio-Rad Laboratories, Inc.).
Immunoprecipitation and Immunoblotting
Immunoprecipitation and immunoblotting were carried out as
described previously (58). Briefly, 1 mg of cell lysate
from each treatment was immunoprecipitated using one of the following
antibodies: 1 µg monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology, Inc.), 1 µg monoclonal anti-ER
antibody
(D-12, Santa Cruz Biotechnology, Inc.), 1.2 µg
polyclonal anti-Grb2 antibody (C-23, Santa Cruz Biotechnology, Inc.), 1 µg polyclonal anti-GST antibody, and 1 µg
monoclonal anti-Sos antibody (Transduction Laboratories, Inc., Lexington, KY). Incubations proceeded for 4 h at room
temperature or overnight at 4 C in the presence of 35 µl of 50%
slurry protein-G or protein-A Sepharose beads (Life Technologies, Inc.). The beads were washed three times in cold binding
buffer. The proteins eluted from the beads were analyzed on 10%
SDS-polyacrylamide gels and transferred to PVDF membranes. The PVDF
membranes were probed with one of the following primary antibodies:
horseradish peroxidase-conjugated monoclonal antiphosphotyrosine
antibody (PY20, Transduction Laboratories, Inc.),
polyclonal anti-ER
antibody (HC-20, Santa Cruz Biotechnology, Inc.), polyclonal anti-Shc (Transduction Laboratories, Inc.), polyclonal anti-GST antibodies (Z-5, Santa Cruz Biotechnology, Inc.), monoclonal anti-Grb2 (Transduction Laboratories, Inc.), monoclonal anti-GST, monoclonal anti-ER
N-terminal antibody (COIM2133, Fisher Scientific),
polyclonal anti-C-terminal ER
antibodies (MC-20, Santa Cruz Biotechnology, Inc.), and polyclonal anti-Sos antibodies
(Transduction Laboratories). After washing the PVDF membrane, the
immunoblots were incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 h [donkey against rabbit IgG from
Pierce Chemical Co. (Rockford, IL) or sheep against mouse
IgG from Amersham Pharmacia Biotech (Piscataway, NJ)] and
further developed using the chemiluminescence detection system
(Pierce Chemical Co.). The reciprocal studies for all
protein-protein interactions were conducted and the same results were
confirmed. All experiments were done at least three times.
GST Pull-Down Assay
Prokaryotic vectors expressing both PTB+CH and SH2
domains of Shc were gifts from Dr. Ravichandran Kodi (University of
Virginia), expressed in Escherichia coli BL21 and purified
with a GST-glutathione affinity system (Amersham Pharmacia Biotech). GST, GST-ShcPTB+CH, and GST-ShcSH2 proteins were
induced, solubilized, and bound to Glutathione beads. To test the
protein interaction, 15 µl of the suspension were incubated in
binding buffer with 5 ng of human full-length ER
expressed and
purified from Baculovirus. After incubation, the beads were washed
three times with the buffer. Bound proteins were eluted with 40 µl of
2x SDS-PAGE buffer, electrophoretically separated in 10% SDS gel, and
transferred on the PVDF membrane. The membrane was probed with
monoclonal anti-ER
antibody, and the immunoprotein bands were
visualized as described above.
Electroporation and Transfection
COS-1 cells were electroporated exactly as described by Roy
et al. (59) with 5 µg each of Shc constructs
and 5 µg of each ER
-expressing vectors. Cells were then incubated
in DMEM supplemented with 10% FBS for 48 h. The medium was
changed again with serum-free and phenol-free DMEM for 1 d. Four
hours before E2 treatment, the medium was changed again with fresh
serum-free and phenol-free medium. Cells were then treated with E2 for
5 min and extracted with binding buffer. At this step, the samples were
either stored at -80 C or prepared for protein-protein association
assay. To study MAPK-mediated Elk-1 activation, MCF-7 cells were
transiently transfected with both 2 ng of GAL4-Elk-1 and 0.1 µg of
GAL4-E1B-Luc reporter vectors encoding GAL-Elk fusion protein and
GAL-luciferase, respectively. Transfection was performed with Effectene
transfection reagent (QIAGEN, Valencia, CA)
according to company specifications; 24 h after transfection,
cells were treated with different doses of E2 for 6 h. Luciferase
activity was measured for each lysates, and samples were normalized by
ß-galactosidase cotransfected with above vectors. All transfections
were conducted at least three times and the data are presented as
mean ± SD.
Confocal Analysis of E2-Induced Cell Morphology Changes
Cells were fixed in paraformaldehyde followed by chilled acetone
and blocked in normal goat serum followed by incubation with monoclonal
antibody against vinculin (Sigma) and with polyclonal
antibodies against ER
(Santa Cruz Biotechnology, Inc.).
For triple costaining of filamentous actin, ER
and vinculin and 0.1
µM Alexa 546-conjugated phalloidin were included during
incubation with the Alexa 633-goat antimouse and Alexa 488 goat
antirabbit secondary antibodies. Slides were mounted, and analyzed by a
LSM inverted microscope (Carl Zeiss, Thornwood, NY)
(60). Each image represents Z sections at the same
cellular level and magnification. Confocal analysis was performed using
a Carl Zeiss laser scanning confocal microscope and
established methods, involving processing of the same section for each
detector and comparing pixel by pixel. Localization of ER
with
ruffles is demonstrated by the development of yellow color
due to red and green overlapped pixels. The
effect of E2 on the changes of filamentous actin in terms of dynamic
membrane was measured based on the formation of ruffles and pseudopodia
from 12 cells in each treatment. Extensive application of identical
methods for analysis of effects of heregulin has been recently
published by Adam et al. (41).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Ravichandran Kodi for supplying the Shc-expressing
constructs and Dr. Michael J. Weber for providing us with the Elk-1
vector. We also thank Dr. Rosalie Uht for her gift of ER
-expressing
vector.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants CA-65622 (to R.J.S.), CA-80066
(to R.K.), and DK-57082 (to M.A.S.).
Abbreviations: AF-1 and AF-2 domains, Activation function 1 and
2 domains; CH domain, collagen homology domain; EGFR, epidermal
growth factor receptor; Grb2, growth factor receptor binding protein 2;
GST, glutathione-S-transferase; HEGO, human ER
expression vector; ICI, ICI 182 780; IMEM, improved MEM; LTED cells,
MCF-7 cells growing long-term in estrogen-depleted medium; MEK, MAPK
kinase; PTB domain, phosphotyrosine binding domain; PVDF,
polyvinylidene difluoride; Shc, Src-homology and collagen homology;
ShcWT, wild-type Shc; Sos, Son of Sevenless; Src, Src family of
tyrosine kinases; c-Src, p60 of Src.
Received for publication February 20, 2001.
Accepted for publication September 6, 2001.
 |
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