From the Departments of a Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, d Veterans Affairs Medical Center and Oregon Health Sciences University, Portland, Oregon 97201, e Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706, h Serono Reproductive Biology Institute, Rockland, Massachusetts 02370, i Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia 23298, j Lung Biology Center, University of California, San Francisco, California 94110, and k Biomedial Research Center, Osaka University Medical School, Osaka 565, Japan
Received for publication, April 22, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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In this report we sought to
elucidate the mechanism by which the follicle-stimulating hormone (FSH)
receptor signals to promote activation of the p42/p44 extracellular
signal-regulated protein kinases (ERKs) in granulosa cells. Results
show that the ERK kinase MEK and upstream intermediates Raf-1, Ras,
Src, and L-type Ca2+ channels are already
partially activated in vehicle-treated cells and that FSH does not
further activate them. This tonic stimulatory pathway appears to be
restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase
that associates with ERK in vehicle-treated cells and promotes
dephosphorylation of its regulatory Tyr residue, resulting in ERK
inactivation. FSH promotes the phosphorylation of this phosphotyrosine
phosphatase and its dissociation from ERK, relieving ERK from
inhibition and resulting in its activation by the tonic stimulatory
pathway and consequent translocation to the nucleus. Consistent with
this premise, FSH-stimulated ERK activation is inhibited by the
cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as
well as by inhibitors of MEK, Src, a Ca2+ channel blocker,
and chelation of extracellular Ca2+. These results suggest
that FSH stimulates ERK activity in immature granulosa cells by
relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.
The cytoplasmic p42/p44 mitogen-activated protein kinase
(MAPK)1/extracellular
signal-regulated kinases (ERKs) comprise a critical convergence point
in the signaling pathways initiated by a variety of receptor agonists
that promote cellular differentiation or proliferation. For the classic
receptor tyrosine kinase-initiated pathway, growth factors like
epidermal growth factor (EGF) induce the autophosphorylation of their
receptors and create specific binding sites for Src homology
2-containing proteins such as Grb2 (1). Grb2 complexed to Sos
associates with the receptor tyrosine kinase, and Sos stimulates GDP
release from Ras, leading to Ras activation. Active Ras then binds to
Raf-1, leading to its activation, and Raf-1 in turn catalyzes the
serine phosphorylation and activation of the MAPK/ERK kinase MEK. MEK
then catalyzes the phosphorylation of ERK on regulatory Thr and Tyr
residues, resulting in ERK activation.
Guanine nucleotide-binding protein-coupled receptors (GPCRs) are also
well known activators of ERK; however, there are a variety of pathways
by which GPCRs promote ERK activation. Often, GPCRs such as those
activated by lysophosphatidic acid or angiotensin II promote the
transactivation of a receptor tyrosine kinase as evidenced by its
increased tyrosine phosphorylation (2). Receptor tyrosine kinase
transactivation directs the tyrosine phosphorylation of adaptor
proteins such as Shc, recruitment of the Grb2-Sos complex, and
subsequent Ras activation. It is less clear how GPCRs promote the
tyrosine phosphorylation of the receptor tyrosine kinase, although Src
activation downstream of the G The G protein-regulated second messenger cAMP has also been shown to
both inhibit and activate ERKs, depending on the cell type. In cells
where cAMP inhibits growth factor-stimulated cell proliferation and ERK
activation, cAMP via PKA inhibits Raf-1 activity, although the relevant
PKA substrate has been controversial (7, 8). A recent report shows that
the elusive PKA substrate in this pathway appears to be Src (9). In
fibroblasts, PKA-catalyzed Src phosphorylation directs the activation
of Rap1, which binds and sequesters Raf-1, thereby preventing Ras
activation of Raf-1 (9). Conversely, in PC12 cells, where cAMP
stimulates differentiation, and in HEK293 cells transfected with the
Ovarian granulosa cells comprise a unique cellular model in which the
majority of both the differentiation and proliferation responses to the
agonist follicle-stimulating hormone (FSH) are mediated by cAMP (16).
The FSH receptor is a seven-transmembrane GPCR coupled to adenylyl
cyclase (17) and is expressed exclusively on ovarian granulosa cells in
female mammals (18). FSH stimulates both granulosa cell proliferation
as well as differentiation to a preovulatory phenotype (16). Although
the induction of cyclin D2 can be stimulated in primary granulosa cell
cultures by cAMP (19), the proliferative response to FSH is poorly
understood and likely includes a paracrine component from surrounding
thecal cells since rat granulosa cells do not proliferate in serum-free media in the presence of FSH alone (16, 20). The differentiation response is readily induced in serum-free granulosa cells by FSH and is
characterized by the induction of enzymes required for estrogen and
progesterone biosynthesis, the luteinizing hormone receptor, the type
II regulatory (RII) In this investigation we sought to identify the cellular pathway by
which FSH promotes ERK activation in primary granulosa cells. Results
show that MEK, Raf-1, Ras, and L-type Ca2+
channels are already partially activated in vehicle-treated granulosa cells. This pathway appears to be restrained at the level of ERK by a
100-kDa phosphotyrosine phosphatase (PTP) that associates with ERK in
vehicle-treated cells. FSH promotes the PKA-dependent phosphorylation of this PTP and its dissociation from ERK, leading to
ERK activation and translocation to the nucleus. Consistent with this
premise, FSH-stimulated ERK activation is inhibited by the
cell-permeable PKA-specific inhibitor peptide PKI as well as by
inhibitors of MEK, Src, EGFR tyrosine kinase activity, a Ca2+ channel blocker, and chelation of extracellular
Ca2+. These results suggest that FSH enhances ERK activity
in immature granulosa cells by relieving an inhibition imposed by a
100-kDa PTP.
Materials--
Ovine FSH (oFSH-19) was kindly provided by Dr.
A. F. Parlow of the National Hormone and Pituitary Agency of the
National Institute of Diabetes and Digestive and Kidney Diseases
(Torrence, CA). The following were purchased. H89, AG1478, GF109203X,
8-(4-chlorophenylthio)-cAMP (CPT-cAMP), nifedipine, PP1
(4-amino-5-(4-methylphenyl)-7-(t-butyl)pyradzolol[3,4-d]pyrimidine), A23187, ionomycin, okadaic acid, and phorbol myristic acid (PMA) were
from LC Laboratories (San Diego, CA); PD98059, BayK8644, myristoylated
PKA inhibitor (PKI) 14-22 amide, farnesyltransferase inhibitor 1, and
wortmannin were from Calbiochem; pertussis toxin was from List
Biochemicals Inc. (Campbell, CA); anti-histone H3 phosphorylated on
Ser-10, anti-CREB phosphorylated on Ser-133, anti-phospho-Tyr, Raf-1
Ras binding domain (BD) and Ral GDS-Rap BD glutathione-agarose
conjugates, anti-Rap1, anti-human EGF receptor, and anti-CREB
antibodies were from Upstate Biotechnology (Lake Placid, NY);
anti-MAPK/ERK phosphorylated on Thr-202 and Tyr-204 was from Promega
(Madison, WI); anti-MAPK/ERK antibody was from Zymed
Laboratories Inc. (San Francisco, CA); anti-protein kinase C
(PKC) Granulosa Cell Culture, Immunofluorescence, and Western
Blotting--
Granulosa cells were isolated from ovaries of 26-day-old
Sprague-Dawley rats primed with subcutaneous injections of 1.5 mg of
estradiol-17 Rap Phosphorylation, Ras, Rap, and Raf Activation Assays, and
Immunoprecipitations--
Raf activity was measured by an
immunocomplex kinase assay using purified His6-MEK1 as
substrate (35). After treatment of cells (6 × 106
cells/dish) with vehicle, FSH, or EGF for 2 min, cells were lysed in
buffer A (10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM
To detect Rap1 phosphorylation, cells were incubated for 1 h in
phosphate-free medium then overnight with 0.5 mCi of
32Pi/6 × 106 cells and
treated as indicated. Cells were lysed in buffer B (10 mM
Tris-HCl, pH 7.2, 150 mM NaCl, 50 mM
To detect active Ras or Rap1, cells were treated as indicated, rinsed
with phosphate-buffered saline, lysed in buffer C (25 mM
Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25%
deoxycholate, 10% glycerol, 25 mM NaF, 10 mM
MgCl2, 1 mM EDTA, 1 mM sodium
orthovanadate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) or
buffer B containing protease inhibitors, respectively, and soluble
extract was collected. The soluble extract was incubated with control
GST plus glutathione-agarose, GST-tagged Raf-1 Ras BD, or Ral GDS-Rap
BD glutathione-agarose conjugates for 3 h at 4 °C, and agarose
pellets were collected and washed 4 times with radioimmune
precipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS,
1 mM sodium orthovanadate, 40 mM PMSF), mixed
with SDS sample buffer, and heat-denatured. Active Ras bound to the Ras
BD on Raf and active Rap1 bound to the Rap BD on Ral GDS were detected
by Ras and Rap1 immunoblots, respectively (36, 37).
For anti-ERK-agarose pull-downs from total ovarian extracts, soluble
ovarian extracts were prepared in buffer A and incubated (3 h at
4 °C) with anti-ERK-agarose or control ADP-agarose, and agarose
pellets were collected, washed 4 times, mixed with SDS sample buffer,
and heat-denatured. For anti-ERK-agarose pull-downs from soluble
ovarian extracts enriched in PTP-SL, ovaries of 13 estrogen-treated
rats (see above) were homogenized with 15 strokes using a ground glass
homogenizer in buffer D (10 mM Tris-HCl, pH 7.0, 5 mM EDTA, 1 mM EGTA, 0.32 M sucrose,
5 µg/ml pepstatin, 5 µg/ml aprotinin, and 10 µg/ml leupeptin, 50 µg/ml soybean trypsin inhibitor, 10 mM benzamidine, and
10 µg/ml E-64). The homogenate was centrifuged at 105,000 × g for 15 min, supernatant was loaded onto a DEAE-cellulose
column, the column was extensively washed with 10 mM
potassium phosphate, pH 7.0, and PTP-SL was batch-eluted (along with
RII
To detect active, Tyr-phosphorylated EGF receptor, 10 × 106 cells/dish were rinsed in phosphate-buffered saline and
lysed by sonicating for 1 min at 4° C in buffer E (10 mM
potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM
EGTA, 10 mM MgCl2, 50 mM
For PTP-SL immunoprecipitations, cells were treated as indicated,
rinsed, and lysed by sonicating for 1 min at 4° C in buffer F. Insoluble cell debris was removed by centrifugation. The soluble extract was first precleared with protein A+G-agarose and then incubated with anti-PTP-SL antibody plus protein A+G agarose for 4 h at 4° C (31). The agarose pellet was collected, washed with buffer
G, and heat-denatured.
For Ca2+ channel immunoprecipitation, 24 × 106 cells were treated as indicated, rinsed with
phosphate-buffered saline, and frozen at Northern Blot Analysis--
Total RNA was isolated from rat
tissues (brain, liver, and ovary) using TrizolTM. RNA (5 µg each) was
denatured in formamide and formaldehyde and electrophoresed through
formaldehyde-containing 1% agarose gel in NorthernMaxTM MOPS gel
running buffer (Ambion Inc., Austin, TX). RNA was blotted to a
nitrocellulose membrane (Schleicher & Schuell), and the membrane was
cross-linked and hybridized with NorthernMaxTM
prehybridization/ hybridization buffer using PTP-SL cDNA (3 × 106 cpm/ml) as a probe. PTP-SL cDNA (429 base pairs)
corresponding to nucleotides 441-864 was labeled with
[ In-gel PTP Assay--
An in-gel PTP assay in which
[32P]poly(glutamic acid-tyrosine) was incorporated into
the gel was performed (40). [32P]Poly(glutamic
acid-tyrosine) (1 mg) was phosphorylated with 50 units of active Src
(Upstate Biotechnology), partially purified (40), and used at 1 × 106 cpm/ml in gel. After electrophoresis, SDS was removed,
proteins were renatured, and the in-gel phosphatase reaction was
performed by incubating the gel with 50 mM Tris-HCl, pH
8.0, 0.3% Other--
Radioimmunoassays for progesterone and inhibin were
conducted by the Hormone and Neurotransmitter Core Facility at
Northwestern University (P01-HD21921). A commercially available kit
(ICN Biochemicals, Carson, CA) was used for progesterone
radioimmunoassay. Inhibin radioimmunoassay (41) used rat inhibin FSH-stimulated ERK Activation Is PKA-dependent--
We
first investigated the time course of FSH-dependent ERK
activation in serum-free cultures of primary rat granulosa cells. ERK
activation, identified by the phospho-specific ERK antibodies, was
readily detected by 10 min and was reduced by 1 h after FSH addition (Fig. 1). ERK activity remained
low at 48 and 72 h after FSH addition, when granulosa cells had
differentiated to the mature phenotype, as evidenced by the induction
of StAR (Fig. 1), the protein responsible for mobilizing cholesterol
substrate for steroidogenesis (43). Increased phosphorylation of CREB
on Ser-133, an established PKA target (44), was detected by 10 min and
undetectable by 4 h post-FSH. For subsequent studies, cells were
treated for 10 min to detect maximal ERK activation unless otherwise
indicated.
To determine whether or not FSH-stimulated ERK activation was cAMP- and
PKA-dependent, cells were treated with FSH and forskolin (Fig. 2A) or the
cell-permeable cAMP analog CPT-cAMP (Fig. 2B) or with FSH in
the absence and presence of the cell-permeable-selective PKA inhibitor
peptide, Myr-PKI (Fig. 2C). FSH stimulated ERK
phosphorylation 4.1 ± 0.7-fold (n = 6) (Fig.
2A). Forskolin and CPT-cAMP mimicked FSH and activated ERK
3.1 ± 0.7 (n = 5)- and 5.7-fold, respectively. Myr-PKI reduced FSH-stimulated ERK activation by 53 ± 8%
(n = 3). CREB phosphorylation in response to forskolin,
CPT-cAMP, and the PKA inhibitor mirrored that of ERK. These results
support our earlier studies (25) with the PKA inhibitor H89 (45, 46) and confirm that the majority of FSH-stimulated ERK activation in this
granulosa cell model is PKA-dependent.
Treatment of granulosa cells with FSH for 30 min resulted in
localization of the majority of phosphorylated/active ERK in the
nucleus (Fig. 3e) in
conjunction with phosphorylated histone H3 (Fig. 3f). Both
ERK and histone H3 phosphorylations were abrogated by pretreatment of
cells with Myr-PKI (Fig. 3, m and n). These results are consistent with the hypothesis that FSH stimulates ERK
activation to promote changes in gene expression leading to granulosa
cell differentiation.
Cell-permeable cAMP analogs are also known to mimic the long term
responses to FSH, leading to granulosa cell differentiation (16). That
PKA is also required for the differentiation of granulosa cells to a
preovulatory phenotype is evidenced by the ability of the PKA inhibitor
H89 (Fig. 4) and Myr-PKI (not shown) to
block FSH-stimulated induction of AKAP80 (by 98 ± 1.5%,
n = 2). H89 or Myr-PKI also inhibited the induction by
FSH of StAR expression (not shown) and progesterone production (by
97.7 ± 2.3%, n = 2).
FSH-stimulated ERK phosphorylation as well as CREB phosphorylation was
independent of the inhibitory G protein Gi, based on the
inability of pertussis toxin, which inhibits receptor-stimulated Gi activation, to modulate FSH-stimulated ERK activation
(not shown). These results are consistent with a direct action of PKA activated downstream of the FSH receptor to modulate ERK activity rather than the pathway reported for the FSH-stimulated ERK Activation Is Dependent on MEK Activity, but FSH
Does Not Stimulate MEK Activation--
Because ERK activation requires
the upstream kinase MEK, we utilized the MEK inhibitor PD98059 (48) to
confirm that FSH-stimulated ERK activation also required MEK. Results
showed that PD98059 fully blocked (by 100%, n = 2)
acute FSH-stimulated ERK activation (Fig.
5A) but did not affect CREB
phosphorylation (Fig. 5B), consistent with an earlier report
(24). PD98059 also reduced the FSH-stimulated induction of AKAP80 with
granulosa cell differentiation to a mature phenotype (Fig.
5C) by 90 ± 9% (n = 3) but did not
consistently modulate progesterone (22 ± 12% inhibition,
n = 5) or inhibin (3 ± 3% inhibition,
n = 2) secretion or StAR expression (not shown) by
these cells. However, when we evaluated the ability of FSH to stimulate
MEK phosphorylation, results showed that MEK exhibited a detectable
level of phosphorylation that was not enhanced by FSH or forskolin
(10-min treatments) but was strongly increased by EGF (Fig.
5D) and the PKC activator PMA (Fig. 5E).
Consistent with this result, a detailed time course of FSH treatment of
granulosa cells confirmed equivalent MEK phosphorylation at time 0 (i.e. in the absence of FSH) and at 10-20 min post-FSH
addition when FSH stimulated maximal ERK phosphorylation (Fig.
5F). These results show that FSH-stimulated ERK activation
is dependent on MEK activation but that FSH does not stimulate MEK
activation. However, mitogens like EGF or PMA that activate ERK through
mechanisms independent of PKA promote strong MEK activation.
FSH Does Not Enhance B-Raf or Raf-1 Activities--
The presence
in granulosa cells of detectable MEK phosphorylation that was
unaffected by FSH suggested that upstream enzymes in this pathway might
also exhibit detectable activity in the absence of FSH. The activities
of B-Raf and Raf-1 were evaluated in an immunocomplex kinase assay
using recombinant His6-MEK1 as substrate. Results in Fig.
6A showed that neither B-Raf
nor Raf-1 activities was increased by FSH. Moreover, although the
commonly high basal level of B-Raf activity (49) was readily detected and not affected by EGF (compare lanes 1 and 3),
Raf-1 activity was detected in vehicle-treated cells and was strongly
enhanced by EGF (compare lanes 4 and 6). These
results indicate that FSH does not stimulate B-Raf or Raf-1 activation.
Rather, a basal level of activity for both kinases was detected.
FSH actually stimulated the phosphorylation of Raf-1 on Ser-259 (Fig.
5F), an established inhibitory phosphorylation site (14,
50). Coincident with the inhibition of Raf-1 activity upon its
phosphorylation on Ser-259, both MEK and ERK phosphorylations decreased
beginning ~30 min post-FSH addition (see Fig. 5F). Based on evidence (a) that Raf-1 is phosphorylated on Ser-259 by
AKT (50), (b) that FSH stimulates AKT
phosphorylation/activation (Ref. 53 and Fig. 5G), and
(c) that FSH-stimulated Raf-1 phosphorylation is inhibited
by the phosphatidylinositol 3-kinase inhibitor wortmannin (not shown),
we can conclude that FSH-stimulated Raf-1 phosphorylation is most
likely downstream of FSH-stimulated AKT.
FSH Does Not Activate Ras or Rap1--
We also evaluated the
activation state of the upstream activators Ras and Rap1 in vehicle-
and FSH-treated cells. Active Ras, indicated by Ras-GTP binding to the
Ras BD on Raf-1 (14), was readily detected in vehicle-treated granulosa
cells (Fig. 6B, lane 1). FSH did not increase the
amount of Ras bound to the Ras BD of Raf-1 (Fig. 6B) (the
reduced binding of Ras to the Ras BD of Raf-1 in response to FSH was
not a consistent observation). Inhibition of the farnesylation of Ras
by the cell-permeable farnesyltransferase inhibitor 1, resulting in
reduced Ras but not Rap-1 function (54), also reduced
FSH-dependent ERK activation by 66 ± 7%
(n = 2) (Fig. 6C), consistent with an
obligatory role for Ras in FSH-stimulated ERK activation. Although
direct activation by cAMP of the guanine nucleotide exchange factors
for Rap1 cannot explain PKA-dependent ERK activation in
granulosa cells especially in the absence of detectable FSH-stimulated
B-Raf activation (12, 13, 55), PKA is reported to phosphorylate Rap1 to
promote its activation (57). To determine whether Rap1 was
phosphorylated in response to FSH treatment granulosa cell ATP pools
were labeled with 32Pi, and cells were
pretreated with or without the PKA inhibitor H89 and then treated with
vehicle or FSH for 10 min followed by a Rap1 immunoprecipitation.
Results showed that although Rap1 was readily immunoprecipitated (Fig.
6D, lower panel), phosphorylated Rap1 was not
detected in vehicle or FSH-treated granulosa cells (Fig. 6D,
upper panel). Moreover, FSH did not stimulate the GTP loading (activation) of Rap1, as evidenced (Fig. 6E)
by the equivalent binding of GTP-Rap1 to the Ral GDS-Rap BD in vehicle
and FSH-treated granulosa cells. Taken together, these results suggest
the existence of a tonic stimulatory pathway leading to modest
activation of Rap1, Ras, Raf-1, and MEK in vehicle-treated granulosa
cells. In the following experiments, we seek to identify upstream
components of this tonic pathway.
Upstream Components of the Tonic Pathway Leading to MEK Activation
Include Src, the EGFR, and Extracellular Ca2+--
We
initially determined whether Src activity contributed to FSH-stimulated
ERK activation. Pretreatment of cells with the Src inhibitor PP1
completely blocked FSH-stimulated ERK activation but did not affect
CREB phosphorylation (Fig.
7A). Blots probed with an
antibody that detects active Src by recognizing unphosphorylated Tyr-532 (designated "dephospho-Src antibody") (32) showed that Src
activity was readily detected in untreated granulosa cells (Fig.
7B, lane 1) and not further activated at 10-min
post-FSH (Fig. 7B, lane 2) or at earlier or later
FSH treatment times (Fig. 5F). Consistent with this result,
the general tyrosine kinase inhibitor genistein completely blocked
FSH-stimulated ERK phosphorylation (Fig.
8A). The EGFR-selective
tyrosine kinase inhibitor AG1478 (58) also completely prevented FSH-,
forskolin-, and EGF-stimulated ERK phosphorylation, whereas only
EGF-stimulated CREB phosphorylation was blocked by AG1478 (Fig.
8B). The effectiveness of AG1478 to inhibit acute
EGF-stimulated tyrosine phosphorylation of the EGFR is shown in Fig.
8C (compare lanes 4 and 8). Inhibition
of the tyrosine kinase activity of nerve growth factor receptor by
AG879 did not affect FSH-stimulated ERK activation (not shown).
However, although a basal level of EGFR tyrosine phosphorylation was
detected in vehicle-treated cells and EGF stimulated the tyrosine
phosphorylation of the EGFR (Fig. 8C), as detected in a
membrane pellet fraction obtained from ~6 × 106
cells, we could not detect any transactivation of the EGFR by FSH or
forskolin (Fig. 8C, compare lanes 1-3).
Equivalent results were obtained when the EGFR was immunoprecipitated
and probed with phospho-Tyr antibody and when tyrosine phosphorylated
proteins were immunoprecipitated with phospho-Tyr antibody and probed
with EGFR antibody (not shown). The EGFR inhibitor AG1478 also reduced the FSH-stimulated induction of AKAP80 by 90 ± 9%
(n = 2) (not shown) but did not affect FSH-stimulated
progesterone secretion (8 ± 8% inhibition, n = 2). These results suggest that EGFR activation is obligatory for
FSH-stimulated ERK activation.
To ascertain whether Src was upstream or downstream of EGFR activity,
we determined whether the EGFR inhibitor AG1478 inhibited Src activity
using the dephospho-Src antibody (which detects active Src) to probe
blots of cell extracts treated with vehicle or FSH (Fig.
8D). Results showed that the dephospho-Src signal was not reduced by AG1478, in contrast to FSH-stimulated ERK phosphorylation, which was reduced by 85 ± 12% (n = 3). Rather,
levels of active Src were elevated in cells pretreated with AG1478. The
basis for this elevation in the active Src signal is not known. This
result suggests that Src activity lies upstream rather than downstream of the EGFR, leading to FSH-stimulated ERK activation.
We also determined whether extracellular Ca2+ contributed
to the ability of FSH to stimulate ERK phosphorylation. Depletion of
extracellular Ca2+ by EGTA strongly reduced FSH-stimulated
ERK phosphorylation (by 86 ± 5%, n = 4), whereas
the effect on CREB phosphorylation was variable (30 ± 20%,
n = 2) (Fig.
9A). The Ca2+
ionophores A23187 (Fig. 8D) and ionomycin (Fig.
9B) also promoted ERK phosphorylation. To determine whether
Ca2+ entry was mediated via Ca2+ channels,
cells were pretreated with the Ca2+ channel inhibitor
nifedipine. Results (Fig. 9C) showed that nifedipine reduced
both basal and FSH-stimulated ERK phosphorylation (by 97 ± 2.5%,
n = 2) but not affect CREB phosphorylation. Consistent with a role for Ca2+ entry into granulosa cells via a
Ca2+ channel, we determined whether FSH stimulated the
phosphorylation on Ser-1928 and the resulting activation of the
The requirement for Ca2+ in the pathway by which FSH
stimulates ERK phosphorylation was shown to be independent of PKC based on the inability of the PKC inhibitor GF109203X to inhibit
FSH-stimulated ERK phosphorylation (Fig. 9F). The
effectiveness of this inhibitor is evidenced by its ability to prevent
PMA-stimulated ERK phosphorylation.
We next determined whether the effect of Ca2+ on
FSH-stimulated ERK activation was upstream or downstream of EGFR
activity. As shown in Fig. 8D, pretreatment of cells with
the EGFR inhibitor AG1478 blocked the ability of the Ca2+
ionophore A23187 to stimulate ERK phosphorylation (by 86 ± 6%,
n = 4), suggesting that Ca2+ is upstream of
the EGFR. We also determined whether the Ca2+ effect was
upstream or downstream of Src. Pretreatment of cells with the Src
inhibitor PP1 reduced the ability of the Ca2+ ionophore
A23187 to stimulate ERK phosphorylation (by 75%, Fig. 9G),
suggesting that the Ca2+ signal is also upstream of Src.
CREB phosphorylation, however, was not affected by PP1.
Taken together, these results suggest the existence of a tonic
stimulatory pathway leading to MEK activation in (serum-free) vehicle-treated granulosa cells. That FSH can stimulate ERK
phosphorylation suggests that MEK-stimulated ERK activation must be
suppressed in granulosa cells and that FSH must in some manner overcome
this inhibition. In the following experiments, we investigated the phosphotyrosine phosphatase PTP-SL (60). This phosphatase has been
shown to bind to and inactivate ERK by stimulating the
dephosphorylation of the regulatory Tyr residue (Tyr-204 in ERK1;
Tyr-185 in ERK2) (31, 61). Phosphorylation of PTP-SL by PKA on Ser-231
in the kinase interaction motif inhibits its ability to bind to and
consequently catalyze the dephosphorylation of ERK (61, 62).
A 100-kDa PTPIs Associated with ERK and FSH Stimulates Its
Phosphorylation--
We first determined whether or not PTP-SL was
expressed in rat ovaries. Proteins reactive with anti-PTP-SL antibody
were readily detected at ~220, 180, 100, and 66 kDa in total
granulosa cell extracts (Fig.
10A, lane 1).
Detergent-soluble rat brain (Fig. 10A, lane 2)
and ovarian (Fig. 10B, lane 2) extracts exhibited
prominent anti-PTP-SL-reactive bands at 100 and 66 kDa that were absent when the blot was probed with preimmune serum (Fig. 10B,
lane 1). The 100-kDa anti-PTP-SL-reactive band was retained
in a soluble ovarian extract eluted from DEAE-cellulose with
~0.1-0.15 M salt (Fig. 10C, lane
1) but was undetectable in the 0.18-0.25 M salt DEAE
eluate (lane 2). This result suggests that the 100-kDa
anti-PTP-SL-reactive band is localized to the cytosolic fraction of
granulosa cells.2
We determined whether the soluble 100-kDa anti-PTP-SL-reactive band
exhibited PTP activity. Using the same DEAE extracts as those blotted
in Fig. 10C, lanes 1 and 2, we
performed an in-gel PTP assay in which a 32P-labeled
tyrosine-phosphorylated substrate was incorporated into the gel.
Results confirmed the presence of PTP activity specifically at 100 kDa
only in the samples containing anti-PTP-SL reactivity (Fig.
10C, lanes 3 and 4).
PTP-SL along with striatal-enriched protein-tyrosine phosphatase and
hematopoietic-PTP belong to a subfamily of PTPs grouped on the basis of
sequence conservation and regulation of ERK activity (31, 60, 63-65).
PTP-SL and striatal-enriched protein-tyrosine phosphatase exist as both
membrane and cytosolic forms that are produced by alternative splicing
of the two precursor genes (60, 65). PTP-SL, brain-enriched PTPBR7, and
the PC12-enriched PC12-PTP are all isoforms of the PTP-SL gene produced
by alternative splicing (60, 64, 66). However, the molecular masses for
all the identified family members are <80 kDa. For example, reported
sizes for these proteins are~39 kDa for cytosolic PC12-PTP (66),
~45 kDa for cytosolic PTP-SL, and ~65-80 kDa for membrane-bound
PTP-SL and PTPBR7, respectively (31, 67). Although the ~66-kDa band detected by anti-PTP-SL antibody (Fig. 10, A and
B) might correspond to the membrane-bound isoform of PTP-SL,
the abundant signal at 100 kDa in rat ovarian and brain extracts is
either a new PTP-SL isoform or the product of a closely related but
distinct gene.3 We therefore
determined by Northern blot whether a larger PTP-SL transcript was
detected in rat ovarian and brain extracts. The largest mRNA
reported for PTP-SL is ~4 kilobases (60). Using a cDNA
corresponding to amino acid residues 147-288 of PTP-SL, we detected a
weak signal of ~8.6 kilobases in rat brain and ovarian extracts under
low stringency washing conditions (Fig. 10D). This result is
consistent with the likely presence of a larger PTP-SL-like transcript
in rat ovaries and brain. Taken together, these results show that a
100-kDa PTP-SL-like PTP is present in soluble ovarian extracts.
In the following experiments we determined whether one of the
anti-PTP-SL-reactive proteins in ovarian extracts was complexed with
ERK. We first evaluated whether ovarian PTP-SL-reactive proteins were
pulled down by anti-ERK-agarose. Using a detergent-solubilized ovarian
extract (with anti-PTP-SL reactivity at 66 and 100 kDa), the 100-kDa
PTP was selectively immunoprecipitated by anti-ERK agarose and did not
bind to control ADP-agarose conjugates (Fig. 11A). Most of the 100-kDa
PTP contained in soluble ovarian extracts (which exhibited anti-PTP-SL
reactivity only at 100 kDa) bound to anti-ERK-agarose (Fig.
11B, lane 5); the 100-kDa PTP was only minimally
detected in the anti-ERK-agarose flow-through fraction (lane
3). In contrast, all of the 100-kDa PTP was present in the ADP-agarose flow-through (Fig. 11B, lane 2), and
none was detected in the ADP-agarose eluate (lane 4).
Consistent with these results, total ERK protein was readily detected
in the flow-through fraction from ADP-agarose (lane 2), and
total ERK protein was only detected in anti-ERK-agarose eluate
(lane 5) and not in ADP-agarose eluate (lane 4).
Thus, the majority of the ovarian 100-kDa PTP appears to be complexed
with ERK.
We next determined whether the 100-kDa PTP was phosphorylated in
response to FSH. Granulosa cells in which the cellular ATP pools with
labeled 32Pi were treated with vehicle or FSH
for 15 min then subjected to immunoprecipitation with anti-PTP-SL or
control antibody. Results in Fig. 11C demonstrated increased
phosphorylation of a band at 100 kDa in PTP-SL immunoprecipitates from
FSH-treated cells. Increased phosphorylation of the 100-kDa band was
not detected in control anti-PKC
Finally we determined whether FSH stimulated the dissociation of the
100-kDa PTP from ERK. Granulosa cells were treated for 10 min with
vehicle or FSH. Detergent-enriched cell extracts were then subjected to
anti-ERK-agarose pull-downs followed by a PTP-SL Western blot. Results
showed that the PTP signal at 100 kDa was reduced by 57% in FSH
compared with vehicle-treated cells (Fig. 11E). These
results show that FSH promotes the release of a portion of the ERK from
the 100-kDa PTP.
FSH is obligatory for follicular development beyond the preantral
stage (68). It is well established that cAMP mediates FSH-dependent induction of granulosa cell differentiation
to a preovulatory phenotype (16). Among the genes induced include those
for increased steroidogenesis including P450aromatase and P450side chain cleavage, membrane receptors including those
for luteinizing hormone and prolactin (16), signaling and anchoring proteins such as RII
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
has been implicated in some cells
(3, 4). For those GPCRs whose activated G
subunits promote increased
intracellular Ca2+ and consequent activation of Pyk and Src
leading to EGF receptor (EGFR) transactivation, Src appears to catalyze
the tyrosine phosphorylation of this receptor tyrosine kinase (5).
GPCRs can also stimulate EGFR activation by stimulating the proteolytic
cleavage and resulting release of the soluble EGFR ligand, heparin
binding EGF (6).
2-adrenergic receptor, cAMP via PKA promotes Rap1
phosphorylation and activation of B-Raf, leading to MEK and ERK
activation (10, 11). cAMP can also bind to and directly activate the
Rap1 guanine nucleotide exchange factor EPAC independent of PKA (12,
13), leading to B-Raf and ERK activation (14). In melanocytes, where
cAMP leads to cell differentiation, cAMP independent of PKA promotes
Ras and B-Raf activation, leading to ERK activation independent of Rap1 and EPAC (15). Thus, depending on the cell type, cAMP appears to
utilize a variety of pathways to modulate ERK activity.
subunit of PKA (16, 21), inhibin-
(22), and
an A kinase-anchoring protein AKAP80 (23). We have shown that
FSH-stimulated activation of the immediate early genes c-Fos and serum
glucocorticoid kinase as well as inhibin-
are mediated in part via
the apparently direct phosphorylation of histone H3 on Ser-10 by PKA
(24). Additionally, FSH leads to ERK activation in target ovarian
granulosa cells in an apparently PKA-dependent manner,
based on the ability of the PKA inhibitor H89 to inhibit FSH-stimulated
ERK activation (24, 25). Recent results using a transformed granulosa
cell line support a role for PKA in FSH-stimulated ERK activation (26).
However, the mechanism by which PKA leads to ERK activation in
granulosa cells is not known.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, anti-Raf-1 (C-12), anti-B-Raf (C-19), anti-ERK2-agarose conjugate, anti-AKT, and anti-Rap1 antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA); anti-MEK phosphorylated on Ser-17 and
Ser-221, total MEK, anti-AKT phosphorylated on Thr-308, and
anti-histone H3 phosphorylated on Ser-10 antibodies were from New
England Biolabs/Cell Signaling Technologies (Beverly, MA); anti-StAR
antibody was kindly provided by Dr. Dale Buchanan Hales (27); anti-Ras
and -PKC
antibodies were from Transduction Laboratories (Lexington, KY); recombinant human EGF was from Intergen Co. (Purchase, NY); ADP-agarose was from Sigma; TrizolTM was from Invitrogen. All
other chemicals were from sources previously described (28-30). The
preimmune serum and polyclonal anti-PTP-SL antibody, directed to
GST-mPTP-SL 147-549 (31), were kindly provided by Dr. Rafael Pulido.
An affinity-purified monoclonal anti-PTPBR7 was generated by the Ogata
laboratory by immunizing mice with a maltose binding fusion protein
containing residues 289-656 in the cytoplasmic portion of murine
PTPBR7. An antibody that recognizes active Src unphosphorylated on
Tyr-532 was previously described (32).
on days 23-25 to promote growth of preantral follicles
(23, 29). Cells were either plated on fibronectin-coated 33-mm plastic
dishes (Falcon) at a density of ~5-10 × 106
cells/dish in serum-free medium (29), as indicated, or on coverslips (for immunofluorescence) and treated with indicated additions ~20 h
after plating. Treatments were terminated by aspirating medium and
rinsing cells once with PBS. Total cell extracts were collected by
scraping cells in 0.5 ml of SDS sample buffer (33) followed by heat
denaturation. Protein concentrations were controlled by plating
identical cell numbers per plate in each experiment then loading equal
volumes of total cell extract per gel lane. Equal protein loading was
confirmed by total ERK, PKC
, phosphatidylinositol 3-kinase, or
Ponceau S staining as indicated. Collection of soluble cell extracts is
described below. Granulosa cell proteins were separated by SDS-PAGE (10 or 12% acrylamide in running gel) (34) and transferred to Hybond
C-extra nitrocellulose (Amersham Biosciences). Blots were incubated
with primary antibody overnight at 4° C, and antigen-antibody
complexes were detected by enhanced chemiluminescence (Amersham
Biosciences). For immunofluorescence, cells were treated as indicated,
fixed with 3.7% formaldehyde, permeabilized with 1% Triton X-100 in
PBS, washed, blocked for 1 h in 1% bovine serum albumin in PBS,
incubated overnight at 4° C with anti-histone H3 phosphorylated on
Ser-10 (1:100 dilution, monoclonal antibody from New England
Biolabs/Cell Signaling) and anti-ERK phosphorylated on Thr-202 and
Tyr-204 (1:100 dilution, polyclonal antibody from Promega) in PBS
containing 1% bovine serum albumin. Coverslips were washed and
incubated for 1 h at 37° C with fluorescein
isothiocyanate-conjugated goat anti-rabbit secondary antibody and
TRITC-conjugated goat anti-mouse secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Cells on coverslips were washed and
mounted on slides in VectashieldTM mounting medium (Vector
Laboratories, Burlingame, CA). Slides were analyzed by a Zeiss LFM
510 confocal microscope.
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM sodium
pyrophosphate, 2 mM dithiothreitol, 0.23 mM
phenylmethanesulfonyl fluoride (PMSF), 0.5% Nonidet P-40, and 0.1%
deoxycholate), and insoluble cell debris was removed by centrifugation
at 15,000 × g for 2 min. Raf-1 and B-Raf were
immunoprecipitated overnight from the soluble cell extract as protein A + G-agarose complexes. Washed immunoprecipitates were then incubated
for 20 min at 37° C in a 50-µl reaction mix containing 0.5 µg of
His6-MEK1, 0.1 mM [
-32P]ATP
(10 µCi/tube), 15 mM MgCl2, 1 mM
MnCl2, and 25 mM Tris-HCl, pH 7.4, followed by
the addition of SDS sample buffer (33), heat denaturation, and
SDS-PAGE.
-glycerol phosphate, 1 mM sodium orthovanadate, 0.23 mM PMSF, 5 µg/ml aprotinin, 1% Nonidet P-40, and 0.5%
deoxycholate, 0.1% SDS), insoluble cell debris was removed by
centrifugation, and Rap1 was immunoprecipitated using anti-Rap1
antibody (Santa Cruz Biotechnology) as protein A + G-agarose
complexes. Immunoprecipitates were washed, and immunoprecipitated Rap1
was mixed with SDS sample buffer and heat-denatured. After separation
of proteins in the immunoprecipitated complex by SDS-PAGE, proteins were transferred to nitrocellulose and subjected to
autoradiography and, after decay of radioactivity, to a Rap1 immunoblot.
PKA holoenzyme (38)) with 0.15 M potassium phosphate, all at 4 °C. Eluate was concentrated to 1.15 ml, an aliquot was taken and mixed with SDS sample buffer and heat-denatured (designated "input"), and equal volumes of the eluate were mixed with 0.08 ml of ADP-agarose as a control or anti-ERK-agarose, respectively. After mixing overnight at 4 °C, the flow-through (designated "FT") was collected, mixed with SDS sample buffer, and
heat-denatured, the agarose pellet was washed 4 times with RIPA buffer,
2 times with high salt RIPA buffer (containing 1 M NaCl),
and 2 times with RIPA buffer, and bound proteins were eluted
(designated "eluate") with 0.25 ml of SDS sample buffer and
heat-denatured. For anti-ERK-agarose pull-downs, granulosa cells were
sonicated for 1 min at 4° C in buffer D, extract was centrifuged at
15,000 × g for 5 min, and the supernatant was mixed with 0.06 ml of ADP-agarose or anti-ERK-agarose and rotated for 4 h at 4 °C. The rest of the details are as described above.
-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 2 mM dithiothreitol, 20 µM leupeptin, 100 µg/ml pepstatin). The membrane
pellet was collected by centrifuging lysate at 15,000 × g for 10 min, and the resulting pellet was mixed with SDS
sample buffer, heat-denatured, and probed with anti-phospho-Tyr
antibody. Alternatively, granulosa cell extracts prepared by sonicating
cells in buffer F (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, 1 µg/ml aprotinin, 100 mM NaF, 2 mM sodium
orthovanadate, and 20 mM sodium pyrophosphate) were
subjected to immunoprecipitations with either anti-phospho-Tyr or
anti-EGFR antibody; immunoprecipitates were washed with buffer G (20 mM Hepes, pH 7.0, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) and, after SDS-PAGE and transfer to nitrocellulose, probed with anti-EGFR or anti-phospho-Tyr antibodies, respectively.
70 °C. Using an antibody
(anti-FP1) that recognizes the 210-kDa
subunit of class C
L-type Ca2+ channel independent of its
phosphorylation state, the
1C subunit was
immunoprecipitated from cell extracts solubilized in buffer H (1%
Triton X-100, 10 mM EDTA, 10 mM EGTA, 25 mM sodium pyrophosphate, 25 mM sodium fluoride,
10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM
p-nitrophenyl phosphate, 1 µM
microcystin, 1 µg/ml pepstatin, 10 µg/ml leupeptin, 20 µg/ml
aprotinin, 200 nM PMSF, and 8 µg/ml each calpain
inhibitors I and II). The precipitated proteins were washed and mixed
with SDS sample buffer, subjected to SDS-PAGE, and transferred to
nitrocellulose (39). Blots were first probed with affinity-purified
anti-CH3P antibody to detect the relative level of phosphorylation at
Ser-1928 on the
1C subunit. Blots were then stripped and
reprobed with anti-FP1 antibody to detect total
1C
subunit precipitated (39).
-32P]dCTP by RediprimeTM II DNA-labeling kit
(Amersham Biosciences). The blot was washed 2 times with 2× SSC
(1× SSC = 0.15 M NaCl and 0.015 M sodium
citrate) and 0.1% SDS at room temperature and subjected to autoradiography.
-mercaptoethanol, 0.04% Tween 40, 1 mM EDTA,
and 4 mM dithiothreitol for 6 h at room temperature.
The gel was then stained, dried, and subjected to autoradiography. PTP
activity was detected as regions on the autoradiogram from which
32P was removed.
(1-27) as the standard and for iodination and sheep anti-Tyr-27 rat
inhibin
(antibody (Ab) 795). DEAE-cellulose chromatography,
cAMP-agarose affinity chromatography, and RII overlay assays were
performed as previously described (38). Results were analyzed using
Student's t test (p
0.05) (42) and are
presented as the means ± S.E. (n > 3) or as the
means ± range (n = 2).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of FSH-stimulated phosphorylation
of ERK and CREB and expression of StAR. Granulosa cells were
treated for the indicated times with 50 ng/ml FSH followed by
preparation of total cell extracts, as described under "Experimental
Procedures." After SDS-PAGE and transfer of proteins to
nitrocellulose, blots were probed with the indicated antibodies to
phosphorylated (PH) and/or total p42/p44 MAPK/ERK
(ERK), PH-CREB, and StAR. Results are representative of two
independent experiments. Results for PH-ERK and -CREB at times 0, 10 min and 1 and 4 h were previously reported (24).
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Fig. 2.
FSH-stimulated p42/44 MAPK/ERK activation is
mimicked by forskolin and CPT-cAMP and inhibited by Myr-PKI. In
panel A, granulosa cells were treated for 10 min with 50 ng/ml FSH or 10 µM forskolin (For). After
SDS-PAGE and transfer of proteins to nitrocellulose, total cell
extracts were probed with the indicated antibodies. Results are
representative of more than five experiments. PH,
phosphorylated. In panel B, granulosa cells were treated for
10 min with 50 ng/ml FSH or 1 mM CPT-cAMP, and total cell
extracts were analyzed as in panel A. In panel C,
granulosa cells were pretreated for 60 min with vehicle
(Veh) or 50 µM Myr-PKI amide followed by
treatment with vehicle or 50 ng/ml FSH for 10 min. After SDS-PAGE and
transfer of proteins to nitrocellulose, blots were first stained with
Ponceau S to confirm equal protein loading per lane and then probed
with the indicated antibodies. For the rest of the details see the
legend to Fig. 1. Results are representative of three separate
experiments.
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Fig. 3.
FSH-stimulated phospho-ERK is predominately
localized to the nucleus of granulosa cells. Granulosa cells on
coverslips were pretreated 30 min with vehicle (Veh) or 50 µM Myr-PKI and then treated 30 min with vehicle or 50 ng/ml FSH. Cells were then subjected to immunofluorescence using
anti-PH-ERK (polyclonal antibody, Promega) and anti-phosphorylated
(PH)-histone H3 (monoclonal antibody, New England
Biolabs/Cell Signaling), as detailed under "Experimental
Procedures." Phase contrast image of cells is also shown.
Combined shows the image of PH-ERK plus
phosphorylated-histone H3. Results are representative of two separate
experiments.
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Fig. 4.
FSH-dependent induction of AKAP80
is prevented by H89. Granulosa cells were pretreated 60 min with
vehicle (Veh) or 10 µM H89 (in 50%
Me2SO) and then treated for 72 h in the absence and
presence of 50 ng/ml FSH. Total cell extracts were subjected to
SDS-PAGE, and proteins were transferred to Immobilon then subjected to
an RII overlay assay to detect AKAPs, as detailed under "Experimental
Procedures." Results are representative of two experiments.
2-adrenergic
receptor in which the PKA-phosphorylated
2-adrenergic
receptor preferentially promotes activation of Gi and
consequent ERK activation via G
(47).
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Fig. 5.
FSH-stimulated ERK activation is
MEK-dependent, but FSH does not stimulate MEK
activation. In panels A and B, granulosa
cells were pretreated for 90 min with 50 µM PD98059 or
vehicle (Veh) followed by treatment for 10 min with vehicle
or 50 ng/ml FSH and preparation of total cell extracts as in Fig. 1.
Results in panel A are representative of two experiments.
PH, phosphorylated. In Panel C, after
pretreatments for 90 min with vehicle or 50 µM PD98059,
cells were treated for 72 h with vehicle or 50 ng/ml FSH. For the
rest of the details see the legend to Fig. 4. Results are
representative of three separate experiments. In panels D
and E, granulosa cells were treated for 10 min with vehicle,
50 ng/ml FSH, 10 µM forskolin, 25 ng/ml EGF, or 10 nM PMA as indicated. Total cell extracts were probed, as
described in the legend to Fig. 1. Results for panel C are
representative of four experiments. In panels F and
G, granulosa cells were treated for the indicated times with
50 ng/ml FSH, and total cell extracts were probed with the indicated
antibodies. Phosphorylated-Raf1 antibody is directed to Ser-259, an
inhibitory phosphorylation site. Results are representative of two
experiments. The data in panel F showing FSH-stimulated ERK
and CREB phosphorylation have been previously reported (24).
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Fig. 6.
FSH does not stimulate Raf-1, B-Raf, Ras, or
Rap1 activation. In panel A, granulosa cells were
treated for 10 min with 50 ng/ml FSH or 25 ng/ml EGF, B-Raf and Raf-1
were immunoprecipitated from soluble extracts of cells lysed in buffer
A, and Raf activities were measured in an immunocomplex kinase assay
using His6-MEK1 as substrate, as detailed under
"Experimental Procedures." Veh, vehicle; IP,
immunoprecipitate. Results are representative of two separate
experiments. In panel B, granulosa cells were treated for 2 min with vehicle, 50 ng/ml FSH, or 25 ng/ml EGF, cells were lysed in
buffer C, and soluble cell extracts were mixed with GST-tagged Ras BD
of Raf-1 conjugated to agarose as detailed under "Experimental
Procedures." Washed agarose pellets were subjected to SDS-PAGE, and
proteins were blotted to nitrocellulose; blots were probed with
anti-Ras antibody. Total protein in cell extracts was assessed by
anti-PKC antibody reactivity (lower panel). Results are
representative of three separate experiments. In panel C,
granulosa cells were preincubated for 60 min with vehicle or 10 µM farnesyltransferase inhibitor 1 (FTase Inh)
then treated for 10 min with vehicle or 50 ng/ml FSH. Total cell
extracts were probed with the indicated antibodies. Results are
representative of two separate experiments. In panel D,
granulosa cells were incubated overnight with 0.5 mCi of
32Pi to label ATP pools, pretreated for 1 h with vehicle or 10 µM H89 (in 50% Me2SO),
then treated for 10 min with vehicle or 50 ng/ml FSH. Cells were then
lysed in buffer B, Rap1 was immunoprecipitated, and proteins in the
Rap1 immunoprecipitate were subjected to SDS-PAGE and transferred to
nitrocellulose for subsequent autoradiography and, after
32P decay, to Western blotting to detect immunoprecipitated
Rap1. In panel E, granulosa cells were treated for 10 min
with vehicle or 50 ng/ml FSH, cells were lysed in RIPA buffer, and
soluble extracts were precleared with glutathione-agarose and then
subjected to control GST or Ral-GDS-Rap1 BD GST pull downs, as
described in panel B. Blots were probed with anti-Rap1
antibody. Results are representative of two separate experiments.
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Fig. 7.
FSH does not activate Src but Src activity is
required for FSH to activate ERK. In panel A, granulosa
cells were pretreated 30 min with vehicle (Veh) or 15 µM PP1 then treated for 10 min with vehicle or 50 ng/ml
FSH. Total cell extracts were probed with the indicated antibodies.
Results are representative of three independent experiments. In
panel B, granulosa cells were treated with vehicle or FSH
for 10 min, and total cell extracts were probed with the indicated
antibodies. Results are representative of three experiments.
PH, phosphorylated.
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Fig. 8.
FSH-stimulated ERK activation is dependent on
EGFR activity. In panel A, granulosa cells were
preincubated for 30 min with vehicle (Veh) or 30 µg/ml
genistein then treated for 10 min with vehicle or 50 ng/ml FSH. Total
cell extracts were probed with the indicated antibodies. Results are
representative of two experiments. PH, phosphorylated. In
panel B, granulosa cells were preincubated for 15 min with
vehicle or 250 nM AG1478 then treated for 10 min with
vehicle, 50 ng/ml FSH, 10 µM forskolin, or 25 ng/ml EGF.
Total cell extracts were probed with the indicated antibodies. Results
are representative of more than five experiments. In panel
C, granulosa cells were preincubated 15 min without or with 250 nM AG1478 and then treated for 5 min with 50 ng/ml FSH, 10 µM forskolin, or 25 ng/ml EGF. Cells were sonicated in
(detergent-free) buffer E, and a membrane pellet was collected, as
detailed under "Experimental Procedures." Proteins in the total
pellet fraction were separated by SDS-PAGE, blotted to nitrocellulose,
and probed with phospho-Tyr (PH-Y) antibody. Results are
representative of three experiments. In panel D, granulosa
cells were pretreated 15 min with vehicle or 250 nM AG1478
then treated for 10 min with vehicle, 50 ng/ml FSH, or 125 µM A23187 (A23). Blots were probed with the indicated
antibodies. Results are representative of three experiments.
subunit of class C L-type Ca2+ channel (39). To
this end, cells were treated for 10 min with vehicle or FSH, and the
1C subunit of the L-type Ca2+
channel was immunoprecipitated from cell extracts using an antibody (anti-FP1) that recognizes total
1C subunit
L-type Ca2+ channel independent of its
phosphorylation state. The resulting blots were probed first with an
affinity-purified antibody that detects
1C subunit
phosphorylated at Ser-1928 (anti-CH3P), stripped, and reprobed with an
antibody that detects total
1C subunit protein at 210 kDa (anti-FP1) (39). Results showed that the
1C subunit L-type Ca2+ channel was already phosphorylated
in vehicle-treated granulosa cells and that FSH did not enhance its
phosphorylation (Fig. 9D). Consistent with results that
suggest that L-type Ca2+ channels are
phosphorylated and open in vehicle-treated cells, the
L-type Ca2+ channel agonist BayK8644 did not
enhance ERK phosphorylation over levels in vehicle-treated cells (Fig.
9E).
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Fig. 9.
FSH-stimulated ERK activation is dependent on
extracellular Ca2+. In panel A, granulosa
cells were pretreated 60 min with vehicle (Veh) or 3 mM EGTA then treated 10 min with vehicle or 50 ng/ml FSH.
Total cell extracts were probed with the indicated antibodies. Results
are representative of four experiments. PH, phosphorylated.
In panel B, cells were treated for 10 min with vehicle, 50 ng/ml FSH, or 4 µM ionomycin (IONO). Total
cell extracts were probed with the indicated antibodies. Results are
representative of three experiments. In panel C, cells were
pretreated for 30 min with vehicle or 10 µM nifedipine
then treated for 10 min with vehicle or 50 ng/ml FSH. Total cell
extracts were probed with indicated antibodies. Results are
representative of two experiments. In panel D, cells were
treated with vehicle or 50 ng/ml FSH for 10 min, membrane proteins were
extracted in buffer H, and the 1C subunit of the
L-type Ca2+ channel at 210 kDa was
immunoprecipitated with an antibody (anti-FP1) that recognizes
1C subunit independent of its phosphorylation state, as
detailed under "Experimental Procedures." The resulting blot was
probed with anti-CH3P antibody, which detects phosphorylation at
Ser-1928 on the
1C subunit, stripped, and reprobed for
total
1C subunit with anti-FP1 antibody. In panel
E, granulosa cells were treated for 10 min with vehicle, 50 ng/ml
FSH, or 1 µM BayK8644. Total cell extracts were probed
with the indicated antibodies. In panel F, granulosa cells
were pretreated for 30 min with vehicle or 5 µM GF109203X
(GFX) then treated 10 min with vehicle, 50 ng/ml FSH, and 10 nM PMA. Blots of total cell extracts were probed with the
indicated antibodies. Results are representative of three experiments.
In panel G, cells were pretreated for 30 min with 15 µM PP1 then 10 min with vehicle or 125 µM
A23187. Total cell extracts were probed with the indicated antibodies.
Results are representative of two experiments.
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Fig. 10.
A 100-kDa PTP reactive with anti-PTP-SL
antibody is present in a cytosol fraction of ovarian extracts. In
panel A, total granulosa cell extract (see legend to Fig. 1)
and detergent-soluble rat brain extracts (prepared in buffer A) from
~24-day-old rats were probed with anti-PTP-SL antibody. Results are
representative of three experiments. IB, immunoblot. In
panel B, detergent-soluble ovarian extracts (prepared in
buffer A) from ~24-day-old rats were probed with preimmune serum or
anti-PTP-SL antibody. In panel C, partially purified soluble
ovarian extracts were probed for PTP-SL immunoreactivity (lanes
1 and 2) and for PTP activity (lanes 3 and
4). To obtain the partially purified extracts, ovaries of 28 estrogen-treated rats were homogenized in (detergent-free) buffer D. A
high speed supernatant was obtained by centrifugation of the homogenate
at 105,000 × g and loaded onto a DEAE-cellulose
column. Fractions eluting with ~0.1-0.15 M potassium
phosphate (peak 2, representing the major peak of PKA activity (38)) or
~0.18-0.25 M salt (peak 3) were separately pooled,
concentrated to 1 ml, and then incubated overnight with cAMP-agarose.
For lanes 1 and 2, an aliquot (50 µl) of the
flow-through that did not bind to cAMP-agarose was mixed with SDS
sample buffer, heat-denatured, subjected to SDS-PAGE, blotted to
nitrocellulose, and probed with anti-PTP-SL antibody. Lane 1 is from DEAE peak 2, and lane 2 from DEAE peak 3. For the
rest of details, see "Experimental Procedures." For lanes
3 and 4, the same samples were subjected to an in-gel
PTP assay. Results are representative of three separate experiments.
In panel D, RNA in the indicated rat tissues was
subjected to a Northern blot probed with PTP-SL cDNA corresponding
to amino acid residues 147-288.
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Fig. 11.
FSH stimulates the phosphorylation of the
100-kDa PTP in granulosa cells. In panel A, ovarian
extracts (1.36 mg protein) in (detergent-enriched) buffer A were
incubated with control ADP-agarose or anti-ERK agarose. Agarose pellets
were washed, heat-denatured, and subjected to SDS-PAGE, blotted to
nitrocellulose, and probed with anti-PTP-SL antibody. Results are
representative of two experiments. IB, immunoblot. In
panel B, soluble ovarian extracts were prepared as described
in the legend to Fig. 10C in detergent-free buffer D, loaded
onto a DEAE-cellulose column, and batch-eluted with 0.15 M
potassium phosphate; the eluate was concentrated to 1.15 ml. After
removing an aliquot (Input), equal volumes of concentrated
eluate were mixed overnight with control ADP-agarose and anti-ERK
agarose. The flow-through (FT) that did not bind to agarose
was collected, agarose was washed, and bound proteins eluted with
SDS-sample buffer, as detailed under "Experimental Procedures."
After SDS-PAGE and blotting to nitrocellulose, blots were probed with
the indicated antibodies. The percentage of original input volume mixed
with agarose conjugates that was loaded onto SDS-PAGE is indicated.
Results are representative of two experiments. In panels C
and D, cellular ATP pools were prelabeled with
32Pi, and granulosa cells were pretreated
(panel D) for 60 min with vehicle (Veh) or 10 µM H89 and then treated for 15 min with vehicle or 50 ng/ml FSH. Cell extracts were prepared in detergent-enriched buffer F
and then subjected to immunoprecipitation (IP) with the
indicated antibodies (Ab), as detailed under "Experimental
Procedures". After SDS-PAGE, gels were dried and exposed to film.
Results in each panel are representative of at least two separate
experiments. In panel E, granulosa cells were pretreated for
15 min with 1 µM okadaic acid, a Ser/Thr protein
phosphatase 2A preferential inhibitor (56), then treated for 10 min
with vehicle or FSH, sonicated for 1 min in (detergent-enriched) buffer
F, and sonicate was centrifuged at 15,000 × g for 5 min, and the supernatant was mixed with 0.06 ml of anti-ERK-agarose for
4 h at 4° C. IB, immunoblot. For the rest of details
see the panel A legend. Results are representative of two
separate experiments.
immunoprecipitations (Fig.
11C). Phosphorylation of the 100-kDa band in PTP-SL
immunoprecipitates was stimulated by forskolin and blocked by
pretreatment of cells with the PKA inhibitor H89 (Fig. 11D).
These results suggest that FSH via PKA promotes the phosphorylation of
the 100-kDa PTP in granulosa cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and AKAP80 (23), and hormones such as inhibin-
(22). As depicted in Fig.
12, FSH via cAMP promotes activation of
PKA (29), leading to the translocation of the PKA catalytic subunit to
the nucleus (29, 69), and phosphorylation of CREB (24, 70). FSH via PKA
also promotes phosphorylation of histone H3 (24, 29) and induction of
the immediate early genes serum glucocorticoid kinase (71) and c-Fos
(72). Because the majority of the actions of cAMP are mediated by PKA,
it has been assumed that the actions of FSH to induce genes leading to the preovulatory phenotype are dependent on PKA. In support of this
hypothesis, we previously reported that the association of phosphorylated histone H3 with promoters of serum glucocorticoid kinase, inhibin-
, and c-Fos was blocked by the PKA inhibitor H89
(24). Induction of AKAP80 as well as StAR expression and inhibin-
and progesterone secretion are also inhibited by the PKA inhibitor H89
(see "Results").
View larger version (43K):
[in a new window]
Fig. 12.
Schematic model of FSH-stimulated,
PKA-dependent activation of ERK in granulosa cells.
Results support the existence of a tonic pathway leading from
Ca2+ entry through activated L-type
Ca2+ channels to Src, the EGFR, Ras, Raf-1, and MEK in
granulosa cells. ERK activity is limited by its association with a PTP,
and this inhibition is relieved upon PKA-dependent
phosphorylation (P) of a PTP, resulting in FSH-stimulated
ERK phosphorylation. FSH via cAMP/PKA does not appear to modulate Rap
or B-Raf activation, as indicated by the × and dotted
lines. Asterisks mark active enzymes; C is
the catalytic subunit of PKA.
FSH also promotes the activation of ERK (24-26) and downstream RSK2 (24, 25). The PKA dependence of ERK activation in response to FSH in primary granulosa cells was conclusively established in this report. The goal of these studies was to identify the cellular mechanism by which PKA, activated downstream of the FSH receptor, promoted ERK activation. For these studies we used a serum-free primary granulosa cell culture model to identify the signaling pathway by which FSH activates ERK. Utilizing this model, our results show that ERK activation appears to be obligatory for the induction of AKAP80, based on the ability of the MEK inhibitor PD98059 to block this response to FSH. ERK activation, however, does not appear to be necessary for other PKA-mediated responses of granulosa cells to FSH, such as progesterone and inhibin secretion. PKA-dependent histone H3 phosphorylation is also independent of ERK activation (24).
Our results support the existence of a tonic stimulatory pathway leading to partial MEK but not ERK activation in (serum-free) vehicle-treated granulosa cells. This conclusion is based on evidence that FSH does not promote the activation of MEK, yet MEK inhibition with PD98059 blocks the ability of FSH to activate ERK (shown herein) as well as downstream RSK2 (24). We ruled out the well described pathways by which cAMP/PKA stimulates Rap1 activation leading to activation of B-Raf, MEK, and ERK (10, 11, 55) based on the inability of FSH to activate Rap1, B-Raf, or MEK (Fig. 12). Rather, MEK as well as the upstream activator Raf-1 exhibit detectable activity in the absence of FSH, and inhibition of Ras actions inhibits FSH-stimulated ERK activation. This tonic pathway leading to Ras activation appears to include Ca2+ entry, at least in part, through phosphorylated and activated L-type Ca2+ channels, leading to the activation of Src, the EGFR, and Ras followed by activation of Raf-1 and MEK (Fig. 12). However, we do not know what stimulates phosphorylation of the Ca2+ channel and consequent Ca2+ entry into granulosa cells. Notably, mitogens like EGF and PMA that activate ERK independent of PKA promote strong activation of MEK.
We have shown that granulosa cell extracts express a soluble 100-kDa PTP that is recognized by an antibody developed against PTP-SL. Moreover, a portion of the ovarian ERK is complexed with the 100-kDa PTP, based on the ability of anti-ERK-agarose to selectively pull-down ERK complexed to this PTP. PTP-SL has been shown to complex with ERK in other cell types, resulting in ERK dephosphorylation on the regulatory Tyr residue (Tyr-204 in ERK1) and consequent ERK inactivation (31, 61). It has also been established that PTP-SL is a PKA substrate, phosphorylated on Ser-231 (62). Similarly, striatal-enriched protein-tyrosine phosphatase and hematopoietic-PTP, two other members of this subfamily of PTPs, are both phosphorylated on the equivalent Ser by PKA (62, 73, 74). PKA-catalyzed phosphorylation of these PTPs results in release of ERK from the PTP and consequent ERK activation (62, 73, 74). Our results show that FSH as well as the adenylyl cyclase activator forskolin stimulates the phosphorylation of the 100-kDa PTP in granulosa cell extracts and that this phosphorylation is inhibited by pretreatment of cells with the PKA inhibitor H89. We also demonstrate in anti-ERK pull-down assays that FSH treatment of granulosa cells stimulates the release of the 100-kDa PTP from ERK. Thus, these functional characteristics of the 100-kDa PTP suggest that this protein belongs to the PTP-SL subfamily of PTPs that regulate ERK activities. However, there are no reports of a 100-kDa isoform of PTP-SL. The 100-kDa PTP most likely represents either a new PTP-SL isoform or the product of a closely related gene.
Taken together, our results support the hypothesis that PKA acts to stimulate ERK activity in granulosa cells by relieving the inhibition of ERK imposed by the 100-kDa PTP. With PKA-dependent phosphorylation of this PTP, ERK is released from its complex with the PTP in granulosa cell cytosol, becomes phosphorylated/activated as a consequence of a tonic pathway, and translocates to the nucleus (see Fig. 3). That granulosa cell ERK in its active conformation phosphorylates substrate proteins ultimately to alter gene expression is suggested by our data showing that the MEK inhibitor PD98059 reduces the ability of FSH to enhance expression of AKAP80.
We hypothesized the existence of a tonic stimulatory pathway leading to
MEK activation based not only on inhibitor data but also on activity
assays and/or epitope-specific antibodies that detect the active
conformation of proteins for Src, Raf-1, MEK, and the 1C
subunit of the L-type Ca2+ channel. The tonic
pathway leading to MEK activation appears to be initiated by the entry
of extracellular Ca2+. Because FSH has been shown to
increase the entry of Ca2+ into granulosa cells (52), one
would predict that ERK would remain active as a result of the positive
feedback actions of Ca2+ coupled with actions of PKA to
maintain the 100-kDa PTP in a phosphorylated conformation. However,
FSH-stimulated ERK activation is transient (see Fig. 5F).
This is in part attributable to the phosphorylation of Raf-1 on Ser-259
most likely by AKT (50) activated in response to FSH, resulting in the
inactivation of Raf-1 (14, 50) and consequent reduction in the
phosphorylation of MEK.
The ability of EGTA, the L-type Ca2+ channel blocker nifedipine, the Src inhibitor PP1, the EGFR tyrosine kinase inhibitor AG1478, and the Ras inhibitor to completely prevent FSH-stimulated ERK activation indicates that the tonic pathway involving extracellular Ca2+, Src, the EGFR, and Ras is the predominant route to the ERK regulated by FSH in granulosa cells rather than an alternative pathway such as one involving Rap1. This conclusion is substantiated by our inability to detect enhanced MEK phosphorylation in response to FSH.
This is the first report demonstrating agonist-stimulated modulation of
ERK activity in conjunction with agonist-stimulated PKA-dependent phosphorylation of a PTP in a physiological
cell model. Thus, these studies support a physiological role for this PTP in regulating ERK activity in an intact cellular model in response
to an extracellular ligand.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the gift of anti-PTP-SL polyclonal antibody and critical advice from Dr. Rafael Pulido, Instituto de Investigaciones Citologicas, 46010 Valencia, Spain.
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FOOTNOTES |
---|
* This work was funded by National Institutes of Health Grants P01 HD21921 (to M. H.-D.), R01 HD36408 (to D. W. C.), and R01 DK52825 (to P. D.), United States Army U. S. Army Medical Research and Materiel Command Grant DAMD 17-00-1-0386 and Northwestern University Program in Endocrinology, Diabetes, and Hormone Action T32 DK07169 (to L. M. S.). Preliminary results were presented at the 83rd Annual Endocrine Society Meeting, June 20-23, 2001, Denver, CO.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b These authors contributed equally to this work.
c Present address: Wyeth Ayerst Women's Health Research Institute, 145 King of Prussia Rd., Radnor, PA 19087.
f Present address: Vollum Institute, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97291.
g Present address: Dept. of Pharmacology, University of Iowa College of Medicine, 51 Newton Rd., Iowa City, IA 55242.
l To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-7459; Fax: 312-503-0566; E-mail: mhd@northwestern.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M203901200
2
The 100-kDa PTP was selectively detected in
DEAE-cellulose eluates that corresponded to the elution position
(~0.1-0.15 M salt) of the RII PKA holoenzyme (38). We
therefore determined whether this PTP or ERK was complexed with PKA R
subunits or associated AKAPs. Upon cAMP-agarose affinity purification
of R subunits from this PKA peak, the 100-kDa PTP eluted exclusively in
the flow-through fraction and did not bind to the cAMP-agarose, as
evidenced by the absence of detectable PTP-SL signal in the fractions
eluted from cAMP-agarose by 75 mM cAMP or by SDS (not
shown). Similarly, ERK protein was also detected only in the
flow-through fraction and did not bind to cAMP-agarose (not shown). We
also determined whether any AKAPs as identified by an RII overlay assay
were detected in anti-ERK-agarose eluates. Results showed that
characteristic ovarian AKAPs (38) were readily detected in ADP-agarose
and anti-ERK-agarose flow-through fractions and none were detected in
anti-ERK-agarose eluates (not shown). These results suggest that the
100-kDa PTP and ERK are not associated directly with the PKA R subunit
or with an associated AKAP.
3 Neither the 66- nor the 100-kDa proteins in total granulosa cell extracts is recognized by anti-PTPBR7, whereas this antibody detected a predominant ~70-kDa band in rat brain likely corresponding to PTPBR7.
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; PKA, protein kinase A; PKI, heat-stable PKA inhibitor; FSH, follicle-stimulating hormone; MEK, MAPK/ERK kinase; StAR, steroidogenic acute regulatory protein; EGFR, epidermal growth factor (EGF) receptor; CREB, cAMP-response element-binding protein; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; PMA, phorbol myristic acid; AKAP, A-kinase anchoring protein; RIPA, radioimmune precipitation assay buffer; GPCR, G protein-coupled receptor; PTP, protein-tyrosine phosphatase; R, regulatory subunit of PKA; PMSF, phenylmethylsulfonyl fluoride; BD, binding domain; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; GDS, guanine nucleotide dissociation stimulator; Akt, also known as protein kinase B.
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