From the Department of Hematology, Institut Cochin, INSERM U567, CNRS UMR 8104, Université René Descartes, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France
Received for publication, August 29, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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We have recently shown that a heterotrimeric
Gi protein is coupled to the erythropoietin (Epo)
receptor. The Gi protein constitutively associates in its
heterotrimeric form with the intracellular domain of Epo receptor
(EpoR). After Epo stimulation Gi is released from the
receptor and activated. In the present study we have investigated the
functional role of the heterotrimeric Gi protein bound to EpoR. In Chinese hamster ovary cells expressing EpoR, the
Gi inhibitor pertussis toxin blocked mitogen-activated
protein kinase (MAPK) Erk1/2 activation induced by Epo.
Epo-dependent MAPK activation was also sensitive to the
G The survival, proliferation, and differentiation of erythroid
progenitor cells is regulated by erythropoietin
(Epo)1 (1). The binding of
Epo to its receptor (EpoR), a member of the cytokine receptor
superfamily, initiates a signal cascade that includes activation of the
Jak2 tyrosine kinase, phosphorylation of the EpoR (2-4), activation of
PI3K (5-7), STAT5 (8), and stimulation of the
Ras/Raf/mitogen-activated protein kinases (MAPK) pathway (9-13). The
EpoR recruits several SH2-containing proteins through its intracellular
phosphorylated tyrosines as well as adaptor molecules (for review, see
Ref. 14). We previously developed an expression cloning strategy to
characterize new proteins implicated in EpoR signaling. The strategy
was based on the reactivity of such components with antibodies produced
against proteins reactive with anti-phosphotyrosine (Tyr(P))
antibodies, isolated from the Epo-stimulated hematopoietic cell line
UT7. This led us to isolate a cDNA encoding G Defined signal functions of Gi include inhibition of
adenylyl cyclase, regulation of phospholipase C The MAPKs Erk1/2 play important role in Epo-induced proliferation,
differentiation, and apoptosis (26-28). In hematopoietic cells Epo
activates the Ras/Raf/MEK/MAPK pathway by recruitment of Grb2 either
directly or indirectly via adaptor molecules such as Shc (29, 30) or
SHP-2 (31, 32). This interaction allows the guanine nucleotide release
factor Sos, constitutively bound to Grb2, to convert Ras to an active
GTP-bound form. Furthermore, C3G, an other guanine nucleotide exchange
factor, through its interaction with CrkL, has also been found
participating in Epo-mediated MAPK signaling pathway via Ras (33) and
Rac (34). PI3K and protein kinase C have also been implicated in
Raf/MAPK activation by Epo (35, 36).
Our demonstration that heterotrimeric Gi proteins associate
with EpoR and that receptor activation leads to the activation and
dissociation of Gi from the receptor led us to investigate the downstream targets of Gi. We considered the possibility
that Epo may activate MAPK via the Gi protein bound to
EpoR-like seven-transmembrane receptors. In this study, we
characterized a new Epo-activated MAPK pathway dependent on
Gi. This pathway, similar to the best characterized MAPK
pathway used by GPCR coupled to Gi, is the main pathway for
Epo-dependent MAPK activation in CHO cells. It proceeds
through the Materials--
Purified recombinant human Epo (specific activity
120,000 units/mg) was a kind gift of Dr. M. Brandt (Roche Molecular
Biochemicals). Rabbit polyclonal antibodies against G Plasmid Constructs--
Different constructs of murine EpoR
cytoplasmic regions (Leu-433-Pro-459, Gly-458-Ser-483,
Gly-458-Pro-470, Glu-465-Pro-477) were obtained by PCR and subcloned
in-frame with the MalE protein.
The murine EpoR mutant Transfections--
Stable transfectants of FDCP-1 myeloid cell
line expressing the wild type and mutated forms of EpoR were obtained
by electroporation, as previously described (8). CHO cells were
transfected with expression plasmids coding for full-length EpoR,
Cell Culture and Treatments--
The human erythroleukemic cell
line UT7 was maintained in Immunoprecipitations and MalE Pull-down Assays--
Cells
(1 × 107) were washed in phosphate-buffered saline
containing 50 µM Na3VO4 and lysed
on ice in 150 mM NaCl, 50 mM Tris-HCL (pH 7.5),
1% Brij 98, 1 mM Na3VO4, 2 mM EGTA, 10% glycerol, and protease inhibitors (lysis
buffer). Insoluble material was removed by centrifugation at
15,000 × g for 10 min at 4 °C. For
immunoprecipitation, lysates were incubated with anti-EpoR antiserum
1/250 for 1 h at 4 °C. Protein G-Sepharose (Amersham
Biosciences) was added, and the incubation was pursued for 1 h at
4 °C with rotation. Samples were washed 3 times with lysis buffer
and 2 times with 150 mM NaCl, 50 mM Tris-HCL
(pH 7.5), 0.1% Brij 98, 1 mM
Na3VO4. For MalE fusion protein pull-down
assays, lysates were incubated with 5 µg of MalE fusion protein
prebound to 20 µl of amylose resin beads for 2 h at 4 °C with
rotation. Then, beads were collected and processed as described above
for immunoprecipitation. Proteins were eluted by boiling in SDS sample
buffer, resolved on SDS-PAGE, and subjected to immunoblotting as
described previously (3, 39).
Determination of MAPK and Akt Activation--
Erk and Akt
activation were studied using phospho-specific antibodies. Cells
lysates were prepared as described above using lysis buffer containing
1% Nonidet P-40. The protein content was normalized by the micro-BCA
assay (Pierce), and equal amounts of proteins (10 to 50 µg) were
separated by 10% SDS-PAGE. Proteins were transferred to nitrocellulose
membranes and analyzed in immunoblot.
ADP-ribosylation Assay--
To detect G Identification of the Sequence in the EpoR Required for
Gi Binding--
In a previous report we demonstrated that
constitutive association between heterotrimeric Gi protein
and the EpoR occurred through the C-terminal end of the receptor (15).
To characterize more precisely the EpoR domain involved in
Gi binding, we first generated recombinant fusion proteins
between MalE and various EpoR sequences contained in this region (Fig.
1A). Fusion proteins were
incubated with UT7 cell lysates, and bound proteins were analyzed by
immunoblotting with anti-G
To evaluate the role of the identified sequence (465-477) in
vivo we wished to delete this sequence within the EpoR. To avoid perturbation of receptor structure and particularly of Tyr-464 and
Tyr-479 located in the vicinity of Gi binding region, we
constructed an EpoR mutant deleting a shorter region encompassing amino
acids 469-475 ( Epo Stimulates a Gi-dependent
Erk1/2 Activation Pathway in CHO Cells Expressing the
EpoR--
A number of receptors that couple to heterotrimeric G
proteins, including Gi-coupled receptors, have been shown
to stimulate MAPK activation (17, 18, 38). Therefore, we explored the possibility that Gi may participate in MAPK activation
after Epo stimulation. The sensitivity of MAPK activation to the
Gi inhibitor, pertussis toxin, was first examined in
hematopoietic cell lines expressing an endogenous or exogenous EpoR as
well as in normal human erythroid progenitor cells. A weak and
inconstant inhibition of Epo-induced MAPK phosphorylation was observed
with these cells (data not shown). We previously observed that Epo
induced a stronger G protein activation in cell membranes isolated from
epithelial CHO cells expressing EpoR than in UT7 hematopoietic cell
line (15). This led us to hypothesis that a
Gi-dependent Epo-activated MAPK pathway may be
more easily detected in these cells.
The role of Gi in the activation of Erk1/2 by Epo was
investigated in CHO cells stably transfected with the full-length EpoR cDNA (CHO-ER WT). Cells were stimulated with Epo for various times, and the lysates were subjected to immunoblotting with phospho-specific anti-Erk antibodies as shown in Fig. 2.
Epo induced a transient Erk1/2 activation. Preincubation of the cells
with 50 ng/ml pertussis toxin overnight completely inhibited
Epo-induced Erk activation. Total Erk content was unchanged under all
conditions as shown by immunoblotting with anti-Erk antibodies
recognizing both the phosphorylated and the unphosphorylated forms of
Erk 1 and 2. Toxin treatment had no effect on cell viability (data not
shown). These results establish a role for a pertussis toxin-sensitive G protein in Epo-induced Erk activation and show that a
Gi-dependent pathway is the main pathway used
by Epo to activate MAPK in CHO cells.
To ascertain the functional significance of the Gi-EpoR
interaction on MAPK activation we studied the effect of disrupting it.
When stably expressed in CHO cells the EpoR Epo-mediated MAPK Activation in CHO-ER Cells Is Dependent on
G
Epo-mediated MAPK Activation in CHO-ER Cells Is Dependent on JAK2,
Ras, and MEK--
To determine whether Epo-induced JAK2 tyrosine
kinase activation was required for MAPK activation, the EpoR mutant
(W282R) defective in JAK2 binding and kinase activation (43) was stably expressed in CHO cells. In cells expressing the EpoR W282R mutant, Epo-induced MAPK activation was lost (Fig. 4C). Thus, JAK2
activation is required for MAPK activation in these cells. The
requirement for MEK kinase, an upstream activator of MAPK, was also
evaluated using the MEK inhibitor PD98059. As shown in Fig.
4B, Epo-induced MAPK activation was abolished when the cells
were pretreated with the MEK inhibitor before Epo stimulation.
Protein kinase C is activated by Epo and may be implicated in MAPK
activation by Epo (36) and also by Gi coupled to classical GPCR (44). We examined whether protein kinase C might contribute to
Epo-induced MAPK activation in CHO-ER WT cells. Down-regulation of
protein kinase C induced by prolonged exposure of cells to PMA
inhibited MAPK activation in response to PMA but not to Epo (Fig.
5B). Therefore, Epo-stimulated
MAPK activation is independent of a PMA-sensitive protein kinase C. The
best characterized pathway used by Gi-coupled receptors to
activate MAPK requires p21ras activity (45, 46). To investigate
the role of p21ras in Epo-mediated MAPK activation in CHO-ER WT
cells, the effects of the dominant negative mutant RasN17 on
EpoR-stimulated MAPK activation was assessed. As shown in Fig.
5A, RasN17 expression inhibited Epo-stimulated
phosphorylation of Erk. In contrast RasN17 expression did not alter the
level of Epo-stimulated Jak2 tyrosine phosphorylation (see Fig.
8A). Taken together these results suggest that in CHO cells
expressing EpoR, Epo acts through Jak2 to activate Ras and Erk1/2
MAPK.
Functional Role of PI3K in Gi-dependent
Epo-induced MAPK Activation--
PI3K has been implicated in the
The requirement for Ras and Jak2 in PI3K activation was then evaluated.
Expression of RasN17 in CHO-ER WT prevented Akt activation after Epo
stimulation (Fig. 8A).
Furthermore the EpoR mutant defective in Jak2 binding and activation
(W282R) failed to activate Akt (Fig. 8B), showing that Jak2
tyrosine kinase and Ras are upstream of PI3K activation. Taken
together, these data demonstrate that the In this study we showed that Epo can activate the Erk1/2 MAPK
pathway via a new Gi-dependent pathway
sensitive to pertussis toxin. The activation of MAPK by EpoR is
mediated by the Our data also demonstrate that PI3K activation by Epo is dependent on
Gi Several candidate tyrosine kinases have been proposed as intermediates
between Four documented pathways have been implicated in Epo-mediated
regulation of the Erk cascade in various hematopoietic cell lines or
erythroid progenitors expressing the EpoR. Three of them, the
Shc/Grb2/Sos pathway (11), the Grb2/Sos pathway (32), and the CrkL/C3G
pathway (33), lead to MAPK activation via Ras. The fourth one involves
PI3K and might require an atypical protein kinase C (36). All these
pathways are initiated through effector binding to phosphorylated
tyrosines located in the intracellular domain of the receptor.
Particularly, tyrosines 460 and 479 have been shown to be involved in
CrkL/C3G and in PI3K-dependent Erk activation by the EpoR,
respectively (36, 40), and tyrosine 464 has a consensus Grb2 binding
motif (32). In CHO cells, MAPK activation by Epo is unlikely to occur
through PI3K binding to tyrosine 479 since PI3K activation is inhibited
by the In hematopoietic cells, Gi binds to the EpoR through the
same EpoR domain required for Gi-dependent Erk
activation in CHO cells, and Gi is activated and
dissociates from the receptor after Epo stimulation (15). However, in
these cells, pertussis toxin has little effect on
Epo-dependent Erk activation. As described above, there are
several different pathways leading to MAPK activation by Epo in
hematopoietic cells. The Gi/PI3K/MAPK pathway may be only
one of them, explaining the relative inefficiency of the toxin in such
cells. The relative contribution of the different pathways leading to
MAPK activation by the EpoR and their interconnections remain poorly
documented and need to be explored further. In contrast with
hematopoietic cells, the Gi-dependent pathway
is the main pathway involved in Epo-dependent MAPK
activation in CHO epithelial cells. Epo-induced EpoR tyrosine
phosphorylation is lower in CHO than in hematopoietic
cells.2 CHO cells may mainly
use the Gi-dependent pathway because of the
inefficiency of MAPK pathways initiated by phosphorylated tyrosines of
the receptor.
In erythroid precursors Gi regulates an Epo-modulated
Ca2+ channel (58, 59). This regulation is mediated by
G competitive inhibitor
ARK1-ct (C-terminal fragment of the
-adrenergic receptor kinase), to the Ras dominant negative mutant
RasN17, and to the phosphoinositide 3-kinase (PI3K) inhibitor LY
294002. A region of 7 amino acids (469-475) in the C-terminal end of
EpoR was shown to be required for Gi binding to EpoR
in vivo. Deletion of this region in EpoR abolished both MAPK and PI3K activation in response to Epo. We conclude that in
Chinese hamster ovary cells, Epo activates MAPK via a novel pathway
dependant on Gi association to EpoR, G
subunit, Ras, and PI3K. The tyrosine kinase Jak2 also contributes to this new pathway, more likely downstream of
and upstream of Ras and PI3K.
This pathway is similar to the best characterized pathway used by seven
transmembrane receptors coupled to Gi to activate MAPK and
may cooperate with other described Epo-dependent MAPK activation pathways in hematopoietic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, the
2 subunit of the heterotrimeric GTP-binding proteins,
and to demonstrate that an heterotrimeric Gi protein is
constitutively associated with EpoR in hematopoietic cell lines as well
as in normal erythroid progenitor cells. The Gi-EpoR
interaction occurs through the C-terminal end of the cytoplasmic domain
of the receptor. After Epo activation the Gi protein is
activated and released from the receptor, most likely with the
concomitant dissociation of Gi
and
subunits
(15).
, and activation of
K+ channels (16). G proteins such as Gq and
Gi also regulate cell growth and differentiation through
the stimulation of a large number of complex cascades, leading to the
activation of MAPKs. Agonist stimulation of seven
transmembrane-spanning G protein-coupled receptors (GPCRs) leads to the
exchange of GDP for GTP on the
subunit of the heterotrimeric G
protein and the subsequent dissociation of the
-GTP and
subunits. Although the main functional properties of G proteins were
initially thought to be essentially determined by the identity of the
subunit,
complexes also regulate a number of effectors. The
mechanisms of the Erk1/2 MAPK activation by GPCR has been explored in
cell lines that are readily transfected, such as COS-7 cells.
The best characterized and probably the main pathway used by GPCR to
activate MAPK via the pertussis toxin (PT)-sensitive Gi
protein depends on the release of free G
subunits acting on a
Ras-dependent pathway (17-19). MAPK activation is
initiated by the G
-mediated tyrosine phosphorylation of Shc and
proceeds through a Shc/Grb2/Sos pathway common to both GPCR and
tyrosine kinase receptors (19). PI3K activity also participates in
G
-mediated MAPK activation upstream of Ras (20-22) or by
stimulating MEK phosphorylation (23). Gi may also regulate
MAPK activation through
subunits. Indeed, activating mutations have
been identified in the G
i2 gene, referred to as
gip2 (24, 25), and MAPK is constitutively activated in
gip2-transformed fibroblasts (24).
subunit of the Gi bound to EpoR and
involves the activation of Jak2, Ras, PI3K, and MEK. Our data also
demonstrate that Gi plays a crucial role for Epo-induced
PI3K activation in CHO cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(M-14), Akt
1/2 (H-136), and Erk (C-16) were purchased from Santa Cruz
Biotechnology. Anti-Jak2 antibodies (06-255) were supplied from Upstate
Biotechnology. Polyclonal antibodies specific to the phospho-forms of
Erk (Thr-202/Tyr-204) and Akt (Ser-473) were supplied from Cell
Signaling Technology. Anti-phosphotyrosine (anti-Tyr(P)) monoclonal
antibodies 4G10 and PY72 were produced from hybridoma cell lines kindly
provided by B. Drucker and B. Sefton. Anti-MalE antiserum and
anti-EpoR antiserum against a fusion protein between glutathione
S-transferase and the cytoplasmic portion of the human EpoR
were described previously (37). Anti-HA peptide monoclonal antibody was
purchased from Roche Molecular Biochemicals. Peroxidase-conjugated
anti-rabbit antibodies were purchased from Amersham Biosciences and
Cell Signaling Technology. MEK1 inhibitor PD98059 was supplied from New
England Biolabs, PI3K inhibitor LY 294002 and PMA were from Sigma,
pertussis toxin was from Alexis, and protease inhibitors were from
Roche Molecular Biochemicals. Amylose resin was supplied from Biolabs. The minigene encoding the C terminus of bovine
ARK1 (
ARK-ct) (38)
was kindly provided by R. Jockers at Institut Cochin, Paris, France.
The expression plasmid pcDNA3/HA-RasN17 was a gift from A. Eychène and the pXM/EpoR-W282R from J. Ihle.
41 was described previously (15). To generate
the
V-L EpoR mutant lacking residues Val-469 to Leu-475 within the
intracellular domain, mutation was introduced into the full-length EpoR
by PCR. The PCR product was digested with HindIII/XbaI and subcloned into the same
restriction sites of a modified pCDNA3 expression vector (15). The
fidelity of all constructs was confirmed by sequencing.
V-L, or W282R EpoR mutants using LipofectAMINE PlusTM
Reagent (Invitrogen) according to the manufacturer's instruction, selected with 20 µg/ml puromycin, and cloned by limiting dilution. EpoR expression was determined using immunoblot analysis of cellular extracts and 125I-Epo binding on whole cells. Cell lines
expressing a similar number of 125I-Epo binding sites of
either wild type or mutated receptors were selected for the study.
Transient transfections of CHO cells were carried out by the same
methodology when 80% confluence was reached using 1 µg of
plasmid/well (10 cm2), and 24 h post-transfection the
cells were harvested and used in experiments as described.
LipofectAMINE transfection of CHO cells consistently resulted in
transfection efficiencies greater than 60% (data not shown).
-minimal essential medium supplemented
with 5% fetal bovine serum, penicillin, streptomycin, 2 mM
L-glutamine, and 2 units/ml Epo. FDCP-1 myeloid cells
expressing EpoR were grown in
-minimal essential medium supplemented
with 5% fetal bovine serum and 2 units/ml Epo. The cells were starved
of Epo by replacing Epo with 3% WEHI-conditioned medium as a
source of interleukin 3 1-2 days before use in the experiments. CHO
cells were grown in Dulbecco's minimal essential medium/nut mix F-12
(Ham's) medium supplemented with 7% fetal bovine serum. Before
stimulation, cells were washed and serum-starved 3-4 h or overnight
when indicated by incubation in Ham's medium in the presence of 0.4%
bovine serum albumin. The cells were stimulated with 10 units/ml Epo or
10 ng/ml PMA. In some experiments PD98059 (30 µM) or LY
294002 (50 µM) were added 30 or 15 min, respectively, before stimulation, and pertussis toxin (50 ng/ml) and PMA (0.6 µg/ml) were added 16 h before stimulation.
i its
ADP-ribosylation was followed by measuring incorporation of
[32P]ADP-ribose in the presence of pertussis toxin
in vitro as previously described (15).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antibodies (Fig. 1B). Recombinant fusion protein containing the EpoR amino acids 458-483 allowed G
binding in vitro, in agreement with previous
data obtained in vivo using EpoR mutants (15). A similar
binding was detected with the MalE-EpoR fusion peptide 465-477,
whereas G
did not bind to EpoR peptides 433-459 and 458-470.
Western blotting with anti-MalE antibody showed that the amounts of
recombinant proteins bound to amylose resin used in the pull-down
assays were similar. These results indicate that amino acids 465-477
are sufficient to bind G
in vitro. This region does not
include tyrosine residues, which fits well with the constitutive
binding of Gi to EpoR (15).
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Fig. 1.
Characterization of EpoR domain coupling to
Gi. A, schematic representation of EpoR mutants
and peptides used in this study. Top, truncation ( 41) and
deletion (
V469-L475) EpoR
mutants expressed into cells. Bottom, EpoR peptides used as
MalE fusion proteins for in vitro binding studies.
B, association of EpoR intracellular domain with G
in vitro. Lysates from UT7 cells were incubated with
immobilized MalE-EpoR fusion proteins. Bound proteins were resolved by
SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by
anti-G
and anti-MalE immunoblotting. C, FDCP-1 cells
expressing no EpoR (
), wild type EpoR (WT), or the
indicated EpoR mutants were lysed with 1% Brij 98, and
immunoprecipitation was performed with anti-EpoR antiserum.
Immunoprecipitates were subjected to ADP-ribosylation as described
under "Experimental Procedures." The proteins were separated by
SDS-PAGE and transferred to nitrocellulose membrane. G
i
was visualized by autoradiography, and Jak2 was identified by
immunoblotting.
V-L). The EpoR expressed in FDCP-1 cells was
immunoprecipitated from cell lysates, and G
i associated
with EpoR was detected by in vitro ADP-ribosylation assay
(Fig. 1C). In cells transfected with full-length mouse EpoR,
G
i was coprecipitated with anti-EpoR antibodies.
However, G
i was not precipitated with EpoR when mutants
41 (15) or
V-L were expressed. The absence of Gi
binding to the EpoR in cells expressing the
V-L mutant is unlikely
to be due to a general signaling defect of the mutated receptor since the receptor kept the ability to bind the tyrosine kinase Jak2 (Fig.
1C). These results indicate that the sequence
V469PDSEPL475 located in the C-terminal
domain of the EpoR is necessary for Gi/EpoR association
in vivo. This region does not bind any known EpoR effector.
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Fig. 2.
Pertussis toxin treatment inhibits the
phosphorylation of endogenous MAPK following stimulation of EpoR with
Epo. CHO-ER cells expressing WT EpoR were preincubated overnight
at 37 °C in the absence (PT ) or presence
(PT+) of 50 ng/ml pertussis toxin. Subsequently the cells
were stimulated with 10 units/ml Epo for the indicated times and lysed.
The soluble fraction was separated by SDS-PAGE and transferred to
nitrocellulose membranes. Determination of MAPK activity was assessed
by immunoblotting with phospho-specific anti-Erk antibodies
(anti-pErk). The same blot was also probed with antibodies
to Erk1/2 (anti-Erk) to confirm that equal amounts of enzyme
were present in each lane.
V-L mutant, which is
unable to associate with Gi (Fig. 1C), could not
activate Erk after Epo stimulation (Fig.
3A). In contrast, PMA
treatment led to MAPK activation, showing that these cells retained the
ability to activate Erk. To evaluate if the
V-L deletion would
perturb early events of EpoR activation, Jak2 was immunoprecipitated
and subjected to analysis using anti-phosphotyrosine antibodies. Fig. 3B illustrates that the EpoR
V-L mutant had the ability
to associate with the Jak2 tyrosine kinase and that both proteins
became phosphorylated after Epo stimulation. Indeed, when Jak2 was
immunoprecipitated, the phosphorylated forms of Jak2 and EpoR were
detected in immune complexes isolated from cells expressing EpoR WT as
well as EpoR
V-L. Thus, the
V-L deletion has no general effect on
Epo signal transduction. Tyrosines located in the vicinity of the
Val-469-Leu-475 region constitute potential binding sites for
components implicated in Epo-dependent MAPK activation such
as CrkL (40) and Grb2 (32) for Tyr-460 and Tyr-464, respectively.
Therefore, we wanted to determine whether the
V-L deletion did not
alter the phosphorylation of these residues and their ability to bind
their effectors. After Epo activation, phosphorylated EpoR
V-L bound
GST-CrkL and GST-Grb2 as efficiently as phosphorylated EPOR WT did
(data not shown). This strongly suggests that the absence of MAPK
activation in EpoR
V-L is due to a defect in Gi binding
and not to a perturbation of the phosphorylation of proximal tyrosine
residues. Altogether, these data show that the EpoR region that
couples Gi to EpoR is required for Epo-induced Erk
activation in CHO cells and confirm the results obtained with pertussis
toxin treatment.
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Fig. 3.
MAPK phosphorylation requires the integrity
of Gi binding domain of the EpoR. CHO-ER cells
expressing WT EpoR or mutant V-L EpoR were serum-depleted and
stimulated with 10 units/ml Epo or 10 ng/ml PMA for the indicated time
(A) or for 5 min with Epo (B) and lysed.
A, phosphorylated Erk and total Erk were detected by
immunoblotting as in Fig. 2. B, Jak2 was immunoprecipitated,
and phosphorylated Jak2 and EpoR were identified by immunoblotting with
anti-phosphotyrosine (pTyr) antibodies.
--
Given the sensitivity of Epo action to pertussis toxin,
we first examined whether Epo-mediated Erk1/2 activation may be
secondary to inhibition of adenylyl cyclase by Gi. Indeed,
in some cells a rise in cAMP has been shown to inhibit the Ras/MAPK
pathway at the level of Ras/Raf interaction (41). Therefore, a
Gi-mediated fall in cAMP level may relieve Raf inhibition
by protein kinase A and positively regulate MAPK. Pretreatment of
CHO-ER WT cells with the cell-permeable cAMP derivative dibutyryl-cAMP,
which mimics the effect of cAMP, failed to prevent Epo-induced MAPK activation (data not shown). This suggests that MAPK activation does
not proceed through
i-mediated inhibition of adenylyl cyclase.
subunits derived from PT-sensitive heterotrimeric G proteins
were shown to mediate Ras-dependent MAPK activation (17, 18, 42). MAPK activation through Gi-coupled receptors such as the
2A-adrenergic receptor and the lysophosphatidic
acid receptor is sensitive to inhibition by the C-terminal fragment of
the
-adrenergic receptor kinase (
ARK1-ct).
ARK1-ct interacts
with free
and acts as a competitive inhibitor of
G
-mediated signals but does not affect G
i-mediated
signaling, allowing distinguishing between
i and
pathways (38). To determine whether the PT-sensitive phosphorylation of
Erk1/2 stimulated by Epo was mediated by G
, we studied the effect
of Epo on CHO-ER WT cells transiently transfected with the
ARK1-ct
peptide. As shown in Fig. 4A
phosphorylation of endogenous Erk1/2 following Epo stimulation was
significantly reduced compared with cells transfected with control
plasmid, although the total Erk amount was not modified. We conclude
that Epo-induced MAPK activation in CHO-ER cells occurs through the release of
subunits of heterotrimeric Gi
protein.
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Fig. 4.
The phosphorylation of endogenous Erk-1/2
after stimulation of EpoR with Epo is dependent on
, MEK, and Jak2. CHO-ER WT cells
were transfected with the minigene encoding the C terminus of bovine
ARK1 (
ARK-ct) or empty vector. 24 h later the
cells were serum-starved for 4 h (A). CHO-ER WT cells
were serum-starved and preincubated (+) or not (
) with 30 mM PD98059 for 30 min (B). CHO-ER cells
expressing WT EpoR or mutant W282R EpoR were serum-depleted
(C). Subsequently the cells were stimulated with 10 units/ml
Epo for 5 min and lysed. The soluble fraction was analyzed by
anti-phospho-Erk and anti-Erk immunoblotting.
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Fig. 5.
Contribution of Ras and not protein kinase C
to Epo-dependent MAPK activation. CHO-ER WT cells were
transfected with various amounts of HA-RasN17 as indicated. The total
plasmid DNA amount was kept constant by adding pCDNA empty vector.
24 h later the cells were serum-starved for 4 h and
stimulated with Epo(+) or not ( ) for 5 min (A). CHO-ER WT
cells were serum-depleted and preincubated or not for 16 h with
0.6 µg/ml PMA followed by stimulation with 10 units/ml Epo or 10 ng/ml PMA for 5 min (B). Erk activation was assessed by
immunoblotting with phosphospecific anti-Erk antibodies. The same blot
was also probed with anti-Erk antibodies.
-dependent activation of MAPK by
Gi-coupled receptors (21, 22). Therefore, we next examined whether PI3K also contributed to Epo-induced MAPK activation in CHO-ER
WT cells. Pretreatment of the cells with LY 294002, a PI3K inhibitor,
blocked Epo-stimulated activation of the PI3K, as detected by
antibodies specific to the phospho-form of the PI3K effector Akt (Fig.
6A). LY 294002 also strongly
decreased the activation of MAPK by Epo without modification of the
total Erk content (Fig. 6A). These data show that PI3K
contributes to Epo-dependent MAPK activation in CHO-ER WT
cells. Because MAPK activation is essentially Gi-dependent in these cells, they also suggest
that PI3K is a component of the Gi-dependent
Erk1/2 pathway. To determine whether Gi is required for
PI3K activation, Epo-induced Akt phosphorylation was compared in CHO-ER
WT expressing the full-length receptor or the
V-L mutant defective
in Gi interaction and MAPK activation. As shown in Fig.
7A the deletion mutant had
lost the ability to activate Akt in response to Epo. As expected,
expression of the
competitor
ARK1-ct also inhibited Akt
phosphorylation by EpoR WT (Fig. 7B).
View larger version (33K):
[in a new window]
Fig. 6.
PI3K is necessary for MAPK
phosphorylation. Inhibition of MAPK activation with PI3K
inhibitor. CHO-ER WT cells were serum-starved and preincubated (+) or
not ( ) with 50 µM LY 294002 for 15 min. Subsequently
the cells were stimulated with 10 units/ml Epo for the indicated time
and lysed. Erk activation was assessed by immunoblotting with
phosphospecific anti-Erk antibodies (anti-pErk) and PI3K
activation by immunoblotting with phosphospecific anti-Akt antibodies
(anti-pAkt). The same blot was probed with antibodies to
Erk1/2 (anti-Erk) and Akt (anti-Akt) to confirm
that equal amounts of enzymes were present in each lane.
View larger version (17K):
[in a new window]
Fig. 7.
PI3K activation requires the integrity of the
Gi binding domain in EpoR and .
A, PI3K activation requires the integrity of Gi
binding domain of the EpoR. CHO-ER cells expressing WT EpoR or mutant
V-L EpoR were serum-depleted and stimulated with Epo for the
indicated time. B, PI3K activation is dependent on
.
CHO-ER WT cells were transfected with the minigene encoding the C
terminus of bovine
ARK1 (
ARK-ct) or empty vector.
24 h later the cells were serum-starved for 4 h, stimulated
with 10 units/ml Epo for 5 min, and lysed. Phosphorylated Akt and total
Akt present in cell lysates were detected by immunoblotting.
subunit of
Gi plays a crucial role in Epo-mediated PI3K activation and
that PI3K is a component of the Epo-activated
Gi-dependent MAPK activation pathway downstream
of Jak2 and Ras.
View larger version (20K):
[in a new window]
Fig. 8.
PI3K activation is dependent on Jak2 and Ras
and Jak2 activation is independent on Ras. A, Ras is
required for PI3K activation but not for Jak2 activation. CHO-ER cells
WT were transfected with 0.75 µg of HA-RasN17 or empty vector.
24 h later the cells were serum-starved for 4 h, stimulated
with Epo (+) or not ( ) for 5 min, and lysed. Jak2 was
immunoprecipitated (IP), and phosphorylated Jak2 was
identified by immunoblotting with anti-phosphotyrosine (PY).
Total Jak2 in immunoprecipitates was also determined by anti-Jak2
antibodies (top). Phosphorylated Akt (pAkt),
total Akt, phosphorylated Erk (pErk), total Erk, and
HA-RasN17 present in cell lysates were visualized by immunoblotting as
indicated (bottom). B, Jak2 is required for PI3K
activation. CHO-ER cells expressing WT EpoR or EpoR mutant W282R were
serum-depleted, stimulated with Epo (+) or not (
) for 5 min, and
lysed. Phosphorylated and total Akt, phosphorylated EpoR
(REpo), and phosphorylated and total Erk present in cell
lysates were analyzed by immunoblotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits and not the
subunit of
Gi. Indeed, MAPK phosphorylation in response to Epo was
inhibited by the coexpressed
competitor
ARK1 polypeptide. The
marked inhibition observed with these agents suggests that the
pathway is the main pathway for Epo-mediated MAPK activation in CHO
cells expressing the EpoR. Classical G protein-coupled receptors, (17,
18, 42, 47) and some tyrosine kinase receptors, such as the receptors
for insulin-like growth factor-1 and fibroblast growth factor (48, 49),
activate Erk1/2 through mechanisms involving
subunits derived
from pertussis toxin-sensitive G proteins in neuronal cells, epithelial
cells, and fibroblasts. To our knowledge our study represents the first
demonstration of a functional role of G
subunits in cytokine
receptor signaling. Signaling from Gi to MAPK more often
involves
subunits of heterotrimeric G proteins acting on a
Ras-dependent pathway (42, 45). Likewise, Epo-induced MAPK
activation is consistent with this general mechanism, since it is
dependent on Ras as shown by its inhibition by a dominant negative
mutant of Ras (RasN17).
subunits and is required for Erk activation
downstream of Ras in CHO cells. Indeed (i) Epo-induced Akt
phosphorylation was inhibited by expression of the
ARK1-ct or
RasN17, (ii) the EpoR mutant defective in Gi binding lost
the ability to phosphorylate both Erk1/2 and the PI3K effector Akt, and
(iii) the PI3K inhibitor LY 294002 inhibited MAPK activation by Epo.
Because MAPK activation by Epo is mainly
Gi-dependent in CHO cells, these data also
suggest that the
-dependent PI3K activation by Epo is
upstream of MAPK and does not form a separate pathway. Similarly, PI3K
was shown to be involved in Gi-dependent MAPK
activation by platelet-derived growth factor in airway smooth muscle
cells (50, 51). PI3K has a critical role linking G protein-coupled
receptors and G
to the MAPK-signaling pathway. Different
mechanisms could be implicated in this process. Previous reports
implicate PI3K
or -
(21, 52) and PI3K
(22) in the activation
of MAPK by classical Gi-coupled receptors upstream of Sos
and Ras activation (21, 22). PI3K
could represent the link between
and a downstream tyrosine kinase required for
Ras-dependent MAPK activation (22). However, this mechanism
does not apply here since we show that PI3K is downstream of Ras. PI3K
could also contribute to the Gi-initiated MAPK pathway
without Ras requirement (23).
and Ras/MAPK activation including c-Src family kinases
(53), Syk (54), and Pyk2 (55). Alternatively, Gi can
activate MAPK through transactivation of the epidermal growth factor
tyrosine kinase receptor (56). Activated c-Src or epidermal growth
factor receptor mediate Ras/MAPK activation through tyrosine
phosphorylation of Shc and its subsequent association with Grb2 (53,
56). An essential component for Gi-dependent Erk activation by EpoR is the tyrosine kinase Jak2. Indeed CHO cells
expressing the W282R EpoR mutant defective in Jak2 binding and
activation have lost the ability to activate MAPK. Epo-induced PI3K
activation was also inhibited in these cells, suggesting that PI3K is
located downstream of Jak2 in this pathway. Ras is probably positioned
downstream of Jak2, since RasN17 expression had no effect on Jak2
phosphorylation. Jak2 contribution to MAPK activation is independent of
Gi binding to EpoR and subsequent Gi
dissociation from the receptor. Indeed the
V-L mutation in EpoR,
which disrupts Gi binding to EpoR, did not alter Jak2
phosphorylation by Epo and its binding to the receptor. Furthermore in
32D cells, we previously showed that Epo-induced Gi
dissociation from the EpoR is not affected when the Jak2-deficient EpoR
mutant is expressed (15). Therefore, Jak2 is more likely acting
downstream of
release and upstream of Ras and PI3K. In addition
to Jak2, the tyrosine kinases Syk and Lyn have been implicated in EpoR
activation (39, 57), but their contribution to
Gi-dependent MAPK activation remains to be determined.
competitor
ARK-ct. The region of the EpoR required for
Gi binding in vivo and
Gi-dependent MAPK phosphorylation by Epo (amino
acids 469-475) does not include tyrosines. Thus,
Gi-dependent Erk activation may involve a
different mechanism. The possibility that the absence of Erk activation
in the
V-L EpoR deleted of the Gi binding domain resulted from a conformational change affecting the nearby tyrosines is
highly unlikely. Indeed Epo-induced CrkL and Grb2 interaction is
preserved in the
V-L mutant. Furthermore, pertussis toxin and
ARK-ct also inhibit Epo-induced MAPK activation.
i2, and tyrosine 460 in EpoR is critical for
Epo-stimulated Ca2+ influx in CHO and in Ba/F3
hematopoietic cells (60). Tyrosine 460 is not required for
Gi interaction with the EpoR in vitro, and
Gi-dependent Erk activation involves the
subunits of Gi. These data suggest that different
Gi-dependent pathways lead to calcium (through
i2) and MAPK (through
) activation by EpoR. Epo
function in non-hematopoietic cells is not unique to epithelial cells.
Indeed, myoblasts, neural cells, and endothelial cells have been shown
to express EpoR (61-63). Epo has anti-apoptotic function in neural
cells and in brain (64, 65) and is implicated in brain development and
neurogenesis (66, 67). Epo has also been implicated in angiogenesis
(68) and may regulate muscle cell proliferation and differentiation
(62). Although the intracellular pathways activated by Epo in
non-hematopoietic cells are still poorly documented, Epo was shown to
activate MAPK and Jak2 in neural and in muscle cells (62, 65, 69).
Further investigations would allow the determination of the function
and contribution of the Gi-dependent pathway to
MAPK activation pathway in hematopoietic and non-hematopoietic cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. A. Eychène for
providing HA-RasN17 vector, Dr. J. Ihle for pXM/EpoR-W282R vector, and
Dr. R. Jockers for the ARK-ct vector and for helpful discussions. We
thank J. Garcia for advice with the use of HA-RasN17 vector and Dr.
Sylvie Gisselbrecht for continuous support.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Comité de Paris of the Ligue Nationale Contre le Cancer, associate laboratory No. 8.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.
To whom correspondence should be addressed. Fax: 33-1-40-51-65-10;
E-mail: duprez@cochin.inserm.fr.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M208834200
2 P. Mayeux, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
Epo, erythropoietin;
EpoR, Epo receptor;
MAPK, mitogen-activated protein kinase;
Erk, extracellular signal-regulated kinase;
PI3K, phosphoinositide 3-kinase;
GPCR, seven transmembrane-spanning G protein-coupled receptor;
G
protein, heterotrimeric GTP-binding protein;
PT, pertussis toxin;
MalE, maltose-binding protein;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
CHO, Chinese
hamster ovary;
PMA, phorbol 12-myristate 13-acetate;
HA, hemagglutinin;
ARK1-ct, C-terminal fragment of the
-adrenergic receptor
kinase.
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