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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2578-2585
Erythropoietin Induces the Tyrosine Phosphorylation of GAB1 and Its
Association With SHC, SHP2, SHIP, and Phosphatidylinositol 3-Kinase
By
Carinne Lecoq-Lafon,
Frédérique Verdier,
Serge Fichelson,
Stany Chrétien,
Sylvie Gisselbrecht,
Catherine Lacombe, and
Patrick Mayeux
From the Institut Cochin de Génétique Moléculaire
(ICGM), Institut National de la Santé et de la Recherche
Médicale (INSERM U363), the Service d'Hématologie, the
Laboratoire d'Hématopoïèse, Site Transfusionnel,
Hôpital Cochin, Université René
Descartes, Paris, France; and the Institut National de la Transfusion
Sanguine (INTS), Paris, France.
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ABSTRACT |
Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells.
The 180- and 70-kD proteins have been previously shown to be IRS2 and
the Epo receptor. In this report, we show that the 116-kD protein is
the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the
transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics
and Epo dose-response experiments showed that GAB1 tyrosine
phosphorylation was a direct consequence of Epo receptor activation.
After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase,
the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1
was also associated with the molecular adapter GRB2 in unstimulated
cells, and this association dramatically increased after Epo
stimulation. Thus, GAB1 could be a scaffold protein able to couple the
Epo receptor activation with the stimulation of several intracellular
signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was
also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and
thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1
in UT-7 cells, indicating that this molecule participates in the signal
transduction of several cytokine receptors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE KIDNEY-PRODUCED hormone
erythropoietin (Epo) is absolutely required for the production of
erythrocytes1 by sustaining the survival and proliferation
of the late erythroid progenitors and allowing their terminal
differentiation. Epo interacts with a cell surface receptor that
belongs to the cytokine receptor family.2 Although
stimulation of colony-forming unit-erythroid (CFU-E)
progenitors by Epo allows their differentiation into erythrocytes, the
Epo receptor does not seem to transduce specific differentiation signals but mainly antiapoptotic and proliferative signals (see Socolovsky et al3 for review). Epo binding to its cognate
receptor induces the dimerization of the receptor and the activation of the associated Jak2 tyrosine kinase.4 The Epo receptor is
then tyrosine phosphorylated5-8 and recruits various SH2
domain-containing proteins, thereby leading to the activation of
several intracellular signaling pathways (see Damen and
Krystal9 for a recent review). The activation of the
phosphatidylinositol 3-kinase (PI 3-kinase) by Epo has been previously
reported.10-13 Several mechanisms have been shown to
activate PI 3-kinase in Epo-stimulated cells. PI 3-kinase binds to the
last tyrosine residue of the Epo receptor,14 although the
peptidic sequence following this tyrosine residue does not match the
consensus binding site of the PI 3-kinase SH2-domains.15 However, mutation or deletion of this tyrosine residue does not abrogate Epo-induced PI 3-kinase activation,14,16 and two
alternative mechanisms for PI 3-kinase activation have been reported.
PI 3-kinase could be activated by binding to Vav,17 and we
have previously shown that Epo induced the tyrosine phosphorylation of
the molecular adapter IRS2 and its association with PI
3-kinase.18
GAB1 is another molecular adapter that has been recently cloned and
that exhibits strong homologies with IRS1 and IRS2.19 GAB1
is a 115-kD molecule that seems to play a key role in the intracellular
signaling of the hepatocyte growth factor (HGF) receptor.20-23 Moreover, GAB1 is tyrosine-phosphorylated in
response to epidermal growth factor (EGF),19
insulin,19 nerve growth factor (NGF),23 and
interleukin-6 (IL-6).24 After activation by these
factors, GAB1 has been shown to associate with the molecular adapter
GRB2, the phospholipase C (PLC), the phosphotyrosine phosphatase SHP2, and the PI 3-kinase.19,20,23-25
In this report, we show that GAB1 is strongly tyrosine-phosphorylated
in Epo-stimulated UT-7 cells and in normal human erythroid progenitors.
After tyrosine phosphorylation, GAB1 associates with several signaling
molecules including GRB2, PI 3-kinase, SHC, SHP2, and SHIP.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and
thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1,
suggesting that GAB1 could be a signaling molecule involved in the
mechanism of action of several cytokine receptors.
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MATERIALS AND METHODS |
Cell culture and stimulation.
UT-7 cells26 were maintained by diluting twice weekly in
 minimum essential medium (MEM) containing 10% fetal
calf serum and 2 U/mL Epo. c-mpl-transfected UT-7 cells were obtained
from Drs F. Porteu and I. Dusanter (ICGM, Paris,
France).27 Highly purified recombinant human
Epo (specific activity, 120,000 U/mg) used throughout this study was a
generous gift from Dr M. Brandt (Boehringer Mannheim, Mannheim,
Germany). Before each experiment, the cells were serum-
and growth factor-deprived by overnight incubation in Iscove's
Dulbecco's modified Eagle's medium (DMEM) containing
0.1% deionized bovine serum albumin and 25 µg/mL iron-loaded human
transferrin. Except when otherwise stated, the cells were stimulated in
the same medium for 10 minutes with 10 U/mL recombinant human Epo. The
stimulation was stopped by diluting the cells with ice-cold
phosphate-buffered saline (PBS) containing 50 µmol/L sodium vanadate.
Preparation of cell extracts, immunoprecipitation, and Western blot
analysis.
The cells were solubilized with buffer A containing 1% Nonidet P40
(Boehringer Mannheim) or 1% Brij 98 (Sigma, St Louis, MO; buffer A: 10 mmol/L Tris/HCl, 150 mmol/L NaCl, 5 mmol/L EDTA, 10%
glycerol, 1 mmol/L sodium vanadate, 0.02% NaN3, and
protease inhibitors from Boehringer Mannheim [catalogue no.
1873580]). For the experiments reported here, the same
results were obtained using either Nonidet P40 or Brij 98. After 15 minutes on ice, the extracts were centrifuged for 15 minutes at
27,000g and the supernatants were used for immunoprecipitation.
Immunoprecipitating antibodies were incubated with the solubilized cell
extracts for 1 hour at 4°C and the mixture was transferred to a
protein G-Sepharose pellet. The suspension was rocked for 1 hour at
4°C. The Sepharose beads were washed twice with buffer A containing
1% detergent and twice with buffer A containing 0.1% detergent. They
were boiled in Laemmli sample buffer and analyzed by Western blot as
previously described,6 except that New England Nuclear
(NEN) Renaissance kit (Boston, MA) was used for revelation.
Antibodies.
We used anti-PI 3-kinase antibodies produced by rabbit immunization
with a mixture of recombinant proteins corresponding to the N- and
C-terminal SH2 domains of p85 fused to GST and commercial anti-PI
3-kinase antibodies from UBI (catalogue no. 06-195, Lake Placid, NY). Anti-GAB1 antibodies (catalogue no. 06-579), anti-Jak2 antibodies (catalogue no. 06-255), anti-SOS antibodies (catalogue no.
06-246), anti-PLC-1 antibodies (catalogue no. 05-163), and anti-IRS2 antibodies (catalogue no. 06-506) were from UBI. Anti-GRB2 antibodies (catalogue no. C-23), anti-PLC-2 antibodies (catalogue no. Q-20), and anti-SHIP antibodies (catalogue no. V-19) were from
Santa Cruz (Santa Cruz, CA). Antiphosphotyrosine
antibodies (4G10) and anti-SHP2 antibodies were generous gifts of Dr B. Drucker (Portland, OR) and Dr A. Ullrich (Martinsried,
Germany), respectively. Anti-Epo receptor antibodies and
anti-SHC antibodies were produced by immunizing rabbits with a
recombinant protein composed of the intracellular domain of the human
Epo receptor or the SH2 domain of the SHC protein fused to GST. We
produced anti-STAT5A and anti-STAT5B antibodies by immunizing rabbits
with peptides corresponding to the 12 last amino acids (AGLFTSARSSLS)
of STAT5A or the 8 last amino acids of STAT5B (QWIPHAQS) coupled to
KLH. These peptidic sequences are specific for STAT5A and STAT5B
respectively. A 1/1 mixture of these antibodies was used for immunoprecipitations.
Amplification and purification of normal human erythroid
progenitors.
Umbilical cord blood units (mean volume, 85 mL) from normal full-term
deliveries were obtained after receiving informed consent of the
mothers from the Obstetrics Unit of the Hôpital
Saint-Vincent-de-Paul (Paris, France). Cord blood units were diluted
with 50 mL PBS and submitted to Ficoll density gradient. Low-density
cells were recovered and CD34+ cells were separated by two
cycles of positive selection using an immunomagnetic procedure (MACS,
CD34 isolation kit; Miltenyi Biotech, Auburn, CA). The
cells were then cultured in serum-free Iscove's DMEM (GIBCO-BRL, Life
Technologies, Grand Island, NY) in the presence of 15%
of a commercial mixture of bovine serum albumin, insulin, and
transferrin (BIT 9500; StemCell Technologies, Vancouver,
CA) and 10 ng/mL IL-3, 10 ng/mL IL-6, and 25 ng/mL stem cell factor
(SCF). Cells were incubated in 5% CO2 in air at 37°C
during 6 days. At day 6, the cells were pelleted by centrifugation and
resuspended in PBS containing 0.8% bovine serum albumin. Monoclonal anti-CD36 IgG1 antibody (Immunotech, Marseille, France) was added at a
final concentration of 1 µg/106 cells and incubated for
30 minutes at 4°C. Cells were washed twice and then incubated with
rat antimouse IgG1 antibody coupled to magnetic microbeads (Miltenyi
Biotech), and CD36+ cells were separated on a MACS column.
A pure erythroid progenitor cell population composed of CFU-E and late
burst forming unit-erythroid (BFU-E) was thus obtained. Indeed, 98% of
the cells were CD36+ and CD71high and less than
3% of the cells were CD14+, CD41+, or
glycophorin A+. More than 96% of the clonogenic colonies
formed by these cells in semisolid culture assays were BFU-E or CFU-E.
These cells were cultured again for 72 hours in the same culture medium
as described above plus 2 U/mL Epo. A dramatic cell proliferation was
observed and led to large numbers of pure erythroid progenitor cells
that were 95% to 100% CD36+ and CD71+. The
glycophorin A marker progressively appeared from days 2 to 3 of
secondary culture. After 72 hours, most cells were immature blasts, and
morphologically recognizable erythroblasts appeared after 4 days of
secondary culture (S.F., manuscript submitted).
PI 3-kinase assays.
PI 3-kinase assays were performed as previously
described.11 Briefly, the cells were stimulated and
solubilized using buffer A containing 1% Nonidet P40, and cell
extracts were immunoprecipitated with anti-GAB1 antibodies and protein
G Sepharose as described above. The Sepharose beads containing
immunoprecipitated proteins were washed twice with buffer A containing
1% Nonidet P40, twice with PBS containing 1 mmol/L sodium vanadate,
twice with a high salt buffer (0.5 mol/L LiCl, 10 mmol/L Tris/HCl, 1 mmol/L sodium vanadate, pH 7.4), and twice with PI 3-kinase buffer (25 mmol/L HEPES, 5 mmol/L MgCl2, 100 mmol/L NaCl, pH 7.4). The
beads were then incubated for 15 minutes at 30°C in 50 µL of PI
3-kinase buffer containing 20 µg of phosphatidylinositol, 20 µg of
phosphatidylserine, 10 µmol/L unlabeled ATP, and 20 µCi of
32P-ATP. The reaction was stopped with 1 mol/L HCl, and
the phospholipids were extracted with methanol/chloroform (1/1
vol/vol). The chloroform extract was washed twice with methanol/1 mol/L
HCl (1/1 vol/vol) and evaporated under vacuum. The dried extracts were
dissolved in 25 µL methanol/chloroform/1 mol/L HCl (100/200/1,
vol/vol/vol), and the phospholipids were separated by thin-layer
chromatography on a silica plate with a mobile phase of
chloroform/methanol/NH4OH/H2O (45/35/3.3/6.7,
vol/vol/vol/vol). Unlabeled phosphatidylinositol monophosphate was run
in an adjacent lane to determine the migration position of
phosphatidylinositol 3 phosphate. After migration, the plate was dried,
exposed to iodine vapor to stain the phosphatidylinositol monophosphate
standard, and exposed 1 to 3 days for autoradiography.
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RESULTS |
Epo induced the association of PI 3-kinase with several
tyrosine-phosphorylated proteins.
To determine which tyrosine-phosphorylated proteins were associated
with PI 3-kinase in Epo-stimulated UT-7 cells, growth factor-deprived
UT-7 cells were stimulated for 10 minutes with Epo and lysed using a
mild detergent. PI 3-kinase was immunoprecipitated and the
immunoprecipitates were analyzed by Western blot using antiphosphotyrosine antibodies. Five phosphotyrosine-containing proteins with molecular masses of 70, 100, 116, 145, and 180 kD were
observed in PI 3-kinase immunoprecipitates
(Fig 1). Two of these proteins have been
previously identified: the 70-kD protein is the activated Epo
receptor14 and the 180-kD protein is the molecular adapter
IRS2.18 The identification of the remaining proteins was
therefore undertaken.
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| Fig 1.
PI 3-kinase associates with several
tyrosine-phosphorylated proteins in Epo-stimulated cells. UT-7 cells
were serum- and growth factor-starved for 18 hours and incubated for 10 minutes in the presence (+) or absence ( ) of 10 U/mL Epo. The
cells were then lysed using 1% Brij 98 and the lysates were cleared by
centrifugation (27000g for 15 minutes). Lysates from
107 cells were immunoprecipitated using anti-PI 3-kinase
antibodies. Immunoprecipitates were analyzed by Western blot using
antiphosphotyrosine antibodies (anti-PY) and anti-PI 3-kinase
(anti-PI3-K) antibodies, successively.
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The 116-kD tyrosine-phosphorylated protein associated with PI
3-kinase was GAB1.
A good candidate for the 116-kD protein is the recently cloned
molecular adapter GAB1. Indeed, GAB1 was shown to associate with PI
3-kinase in cells stimulated with various growth
factors.19,20,23-25 Moreover, the structure of GAB1 is
close to that of IRS2 that we previously showed to associate with PI
3-kinase in Epo-stimulated cells.18 Initial attempts to
probe anti-PI 3-kinase immunoprecipitates with anti-GAB1 antibodies
were unsuccessful due to the low efficiency of the anti-GAB1 antibodies
in Western blot experiments. Consequently, anti-PI 3-kinase
immunoprecipitates were denaturated and reprecipitated with anti-GAB1
antibodies. Figure 2A shows that the 116-kD
tyrosine-phosphorylated protein was directly recognized by anti-GAB1
antibodies. Then, cellular extracts from Epo-stimulated or unstimulated
UT-7 cells were immunoprecipitated with anti-GAB1 antibodies. Western
blot analysis of these immunoprecipitates showed that Epo induced the tyrosine phosphorylation of GAB1 and its association with
tyrosine-phosphorylated proteins of 145, 66, and 52 kD. In most cases,
the subunits of PI 3-kinase are not tyrosine phosphorylated in
stimulated cells and PI 3-kinase activation is realized through its
binding to tyrosine phosphorylated proteins (see Kapeller and
Cantley28 for review concerning PI 3-kinase). To test for
the association of PI 3-kinase with GAB1, the blot was probed with
anti-PI 3-kinase antibodies and this experiment showed that Epo
induced the association of GAB1 with PI 3-kinase (Fig 2B). Reprobing
the blot with anti-GAB1 antibodies showed an electrophoretic shift of
GAB1 that probably reflects the strong level of GAB1 phosphorylation
induced by Epo stimulation (Fig 2B). The lower detection of GAB1 in
extracts from stimulated cells was constantly observed. This probably
corresponded to a decreased affinity of the anti-GAB1 antibodies for
the phosphorylated form of GAB1. Moreover, the association of GAB1 with
signaling proteins (see below) after Epo stimulation could also lower
the accessibility of GAB1 to anti-GAB1 antibodies. Lastly, GAB1
immunoprecipitates were tested for PI 3-kinase activity. Figure 2C
shows that Epo stimulation of UT-7 cells strongly increased the
GAB1-associated PI 3-kinase activity.
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| Fig 2.
GAB1 association with PI 3-kinase in Epo-stimulated
cells. (A) Anti-PI 3-kinase immunoprecipitates were prepared from
Epo-stimulated UT-7 cells as described in Fig 1. Half of the
immunoprecipitates were saved for direct analysis by Western blot and
the remaining was denatured by boiling in nonreducing Laemmli sample
buffer, diluted with solubilization buffer, and reimmunoprecipitated
using anti-GAB1 antibodies. Both samples were analyzed by Western blot
using antiphosphotyrosine (anti-PY) antibodies. (B) UT-7 cells were
serum- and growth factor-starved for 18 hours and incubated for 10 minutes in the presence (+) or absence () of 10 U/mL Epo. The
cells were then lysed using 1% Brij 98 and cleared lysates from
107 cells were immunoprecipitated using anti-GAB1
antibodies. Immunoprecipitates were analyzed by Western blot using
antiphosphotyrosine antibodies (PY). The blot was stripped and reprobed
with anti-PI 3-kinase antibodies and anti-GAB1 antibodies,
successively. (C) Anti-GAB1 immunoprecipitates from UT-7 cells
stimulated for 10 minutes with 10 U/mL Epo or from control cells were
prepared and tested for PI 3-kinase activity as described in Materials
and Methods. The lipid products were separated by thin-layer
chromatography, and the migration position of phosphatidylinositol
3-phosphate (PIP) was determined by comparison with authentic unlabeled
PIP run in adjacent lanes and shown by iodine staining. "Ori"
indicates the origin of migration.
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Epo induced the tyrosine phosphorylation of GAB1.
Figure 3 shows the time-course of
Epo-induced GAB1 tyrosine phosphorylation. Tyrosine phosphorylation of
GAB1 was maximal after 10 minutes of Epo stimulation and then started
to decrease, although it remained detectable after 1 hour of
stimulation (Fig 3A). Dose-response experiments showed that the
Epo-induced tyrosine phosphorylation of GAB1 perfectly correlated with
the occupancy of the Epo receptors (Fig 3B). Thus, both the kinetic
experiments and the dose-response curves indicated that the Epo-induced
tyrosine phosphorylation of GAB1 most likely corresponded to a direct
consequence of the Epo receptor activation. In addition to Epo, TPO
also induced the tyrosine phosphorylation of GAB1 in c-mpl-transfected
UT-7 cells (Fig 4). The same
tyrosine-phosphorylated proteins appeared to be associated with
tyrosine-phosphorylated GAB1 in Epo- and TPO-stimulated cells. A low
level of GAB1 tyrosine phosphorylation was also detected in
GM-CSF-stimulated cells (Fig 4). The low efficiency of GM-CSF
stimulation was probably due to the reduced number of high-affinity
GM-CSF receptors at the cell surface of UT-7 cells. Indeed, we detected
only a few hundred high-affinity receptors for GM-CSF, whereas these
cells express approximately 7,000 Epo receptors.29
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| Fig 3.
Characteristics of Epo-induced GAB1 tyrosine
phosphorylation. Serum- and growth factor-deprived UT-7 cells were
stimulated for various times with 10 U/mL Epo (A) or for 10 minutes
with various Epo concentrations (B). Cleared lysates were then prepared
and immunoprecipitated with anti-GAB1 antibodies. Immunoprecipitates
were analyzed by antiphosphotyrosine (anti-PY) antibodies. Assuming an
equilibrium constant of dissociation of 200 pmol/L for the Epo receptor
in UT-7 cells,29 receptor occupancy is 2% for 10 mU/mL
Epo, 18% for 100 mU/mL, 70% for 1 U/mL, 95% for 10 U/mL, and 99.5%
for 100 U/mL. Because apparent binding equilibrium was not achieved
after 10 minutes of incubation for Epo concentrations less than 1 U/mL
(data not shown), receptor occupancy for Epo concentrations of 10 mU/mL
and 100 mU/mL was probably slightly lower than the indicated values.
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| Fig 4.
TPO and GM-CSF induced the tyrosine phosphorylation of
GAB1. UT-7 cells stably transfected with the TPO receptor (c-mpl) and
parental UT-7 cells were stimulated for 10 minutes with 50 ng/mL TPO,
10 U/mL Epo, or 25 ng/mL GM-CSF. Cleared lysates were
immunoprecipitated with anti-GAB1 antibodies and immunoprecipitates
were analyzed by Western blot using antiphosphotyrosine antibodies
(anti-PY).
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GAB1 associates with several signaling proteins in Epo-stimulated
UT-7 cells.
Figure 2B shows that GAB1 was associated with several
tyrosine-phosphorylated proteins in Epo-stimulated cells. Three
tyrosine-phosphorylated proteins were constantly observed in anti-GAB1
immunoprecipitates. The molecular masses of these proteins suggest that
they could be SHC (52 kD), SHP2 and/or the Epo receptor (66 kD), and
SHIP (145 kD). GAB1 was not detected in anti-Epo receptor
immunoprecipitates (Fig 5). In contrast,
anti-SHP2 antibodies recognized a 66-kD protein in GAB1
immunoprecipitates from Epo-stimulated UT-7 cells (Fig 6), and a tyrosine-phosphorylated
protein comigrating with GAB1 was also observed in anti-SHP2
immunoprecipitates from Epo-stimulated cells (data not shown). Thus,
the 66-kD tyrosine-phosphorylated protein associated with GAB1 was
SHP2. The 52-kD protein was recognized by anti-SHC antibodies (Fig 6)
and the association between SHC and GAB1 required the Epo-stimulation
of the cells. Moreover, a tyrosine-phosphorylated protein comigrating
with GAB1 was evidenced in anti-SHC immunoprecipitates from
Epo-stimulated cells (data not shown). Thus, the 52-kD
tyrosine-phosphorylated protein associated with GAB1 in Epo-stimulated
cells was SHC. Probing anti-GAB1 immunoprecipitates with anti-SHIP
antibodies showed that the 145-kD protein was SHIP (Fig 6). In addition
to these tyrosine-phosphorylated proteins, we tested the association of
GAB1 with GRB2. As shown in Fig 6, a low level of association between
GAB1 and GRB2 was seen in resting cells and this association was
strongly increased in Epo-stimulated cells. This result was
reproducibly obtained although the tyrosine phosphorylation of GAB1 was
not detected in resting cells, thus suggesting that the association
between GRB2 and GAB1 could involve two different mechanisms. We did
not detect the presence of Jak2, IRS2, STAT5, Vav, SOS, or PLC in
anti-GAB1 immunoprecipitates (data not shown).
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| Fig 5.
The Epo receptor is not detected in anti-GAB1
immunoprecipitates. Anti-GAB1 and anti-EpoR immunoprecipitates were
prepared from UT-7 cells stimulated for 10 minutes with 10 U/mL Epo or
from control cells. These immunoprecipitates were analyzed by Western
blot using antiphosphotyrosine (anti-PY) antibodies.
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| Fig 6.
Identification of the GAB1-associated proteins. Anti-GAB1
immunoprecipitates were prepared from UT-7 cells stimulated for 10 minutes with 10 U/mL Epo or from control cells and analyzed by Western
blot using antiphosphotyrosine (anti-PY) antibodies. The blot was
stripped and reprobed with anti-SHIP, anti-SHP2, anti-SHC, anti-GRB2,
and anti-GAB1 antibodies, successively.
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Epo induces the tyrosine phosphorylation of GAB1 in human erythroid
progenitors.
We detected the expression of GAB1 in all human leukemic cell lines
expressing megakaryocytic or erythroid characteristics such as TF-1,
Ku812, K562, or Mo7E (data not shown). However, the level of expression
of signaling proteins could be abnormally high in these transformed
cell lines, thereby leading to the activation of signaling pathways
that are not activated in normal cells. To verify the physiological
relevance of Epo-induced GAB1 tyrosine phosphorylation, we used normal
erythroid progenitors isolated from human cord blood. More than 95% of
the isolated cells exhibited erythroid progenitor characteristics
(S.F., manuscript submitted). As shown in
Fig 7, Epo also induced the tyrosine
phosphorylation of GAB1 in these human primary cells. In addition, the
tyrosine-phosphorylated proteins associated with GAB1 previously
observed in UT-7 cells were also detectable in anti-GAB1
immunoprecipitates from human erythroid progenitor cells.
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| Fig 7.
Epo induces the tyrosine phosphorylation of GAB1 in
normal erythroid progenitors isolated from human cord blood. A purified
population of human erythroid progenitor cells was prepared as
described in Materials and Methods. The cells were serum- and growth
factor-deprived by overnight incubation in Iscove's DMEM containing
0.4% deionized BSA and 25 µg/mL iron-saturated human transferrin.
The cells were then stimulated (+) or not () for 10 minutes with
10 U/mL Epo. Lysates from 20 × 106 cells were
immunoprecipitated with anti-GAB1 antibodies and analyzed by Western
blot using antiphosphotyrosine (anti-PY) and anti-GAB1 antibodies,
successively.
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DISCUSSION |
Although the stimulation of CFU-E cells by Epo is absolutely required
for the terminal differentiation of these erythroid progenitors in
physiological conditions, recent data strongly suggest that the Epo
receptor does not transduce specific differentiation signals, because
ectopic expression of other cytokine receptors in these CFU-E cells
allows their erythroid differentiation in response to these cytokines
(see Socolovsky et al3 for a recent review). Presumably,
the Epo receptor essentially transduces antiapoptotic and proliferative
signals. PI 3-kinase activation is implicated as a major step in both
mitogenic28 and anti-apoptotic30 signaling pathways. These potencies of PI 3-kinase signaling suggest that PI
3-kinase could be a major intracellular signaling pathway in the
mechanism of action of Epo. PI 3-kinase activation by Epo has been
thoroughly documented previously.10-13 PI 3-kinase
activation most generally involves the association of the SH2 domains
of the regulatory subunit (p85) with tyrosine-phosphorylated proteins. This association both drives the enzyme close to its substrate by
promoting the relocalization of PI 3-kinase to the plasma membrane and
probably causes a conformational change in the regulatory subunit that
increases the enzyme activity.28,31 We observed the
association of PI 3-kinase with five tyrosine-phosphorylated proteins
with molecular masses of 70, 100, 116, 145, and 180 kD. According to
previously published results, the 70-kD protein is the Epo
receptor,10-13 and we recently showed that the 180-kD
protein was IRS2.18
In this report, we show that the 116-kD protein is the molecular
adapter GAB1. GAB1 is strongly tyrosine phosphorylated in Epo-stimulated cells, and this tyrosine phosphorylation perfectly correlated with the tyrosine phosphorylation of Epo receptors in both
kinetics and dose-response studies (Fig 3), strongly suggesting that
the tyrosine phosphorylation of GAB1 is a direct consequence of Epo
receptor activation. Importantly, Epo-induced GAB1 tyrosine phosphorylation was seen not only in the UT-7 erythroleukemia cell
line, but also in normal human erythroid progenitors, demonstrating the
physiological relevance of our observation.
HGF-induced GAB1 tyrosine phosphorylation involves the binding of GAB1
to the tyrosine-phosphorylated form of Met both by a novel
phosphotyrosine binding domain and through GRB2, which links Met by its
SH2 domain and GAB1 by its SH3 domains.20,23 In contrast,
the tyrosine residues of gp130 are not required for IL-6-induced
tyrosine phosphorylation of GAB1 and gp130 is not precipitated by
anti-GAB1 antibodies.24 Our results also indicate that the
tyrosine-phosphorylated form of GAB1 was not associated with the Epo
receptor (see Fig 5), suggesting that the cytokine receptors induce the
tyrosine phosphorylation of GAB1 by a common mechanism, albeit
different from that used by the HGF receptor.
Three potential p85 SH2 domain binding sites with a consensus YXXM
sequence15 (Y447, Y472, and
Y589) are present in GAB1.19 These sites are
involved in insulin- and in NGF-mediated activation of PI
3-kinase.25,32 Because PI 3-kinase only associated with the
tyrosine-phosphorylated form of GAB1 (Fig 2), this association probably
involved these p85 binding sites. In contrast to IRS2, which only
associates with PI 3-kinase and SHIP after Epo
stimulation,18 we observed the association of GAB1 with
several proteins, including GRB2, SHC, SHP2, and SHIP in addition to PI
3-kinase, suggesting that GAB1 could transduce Epo receptor activation
to several intracellular signaling pathways. Similarly to PI 3-kinase,
SHP2 association with GAB1 was only observed in Epo-stimulated cells. A
binding site for the SH2 domains of SHP2 (sequence Y627LDL)
is located in the C-terminal domain of GAB1. Whether this sequence is
responsible for the association of SHP2 with GAB1 is likely, although
this remains to be determined. The association of GRB2 with GAB1
appears to be more puzzling. Indeed, a weak association was detected in
unstimulated cells, but it was strongly enhanced by Epo stimulation.
The constitutive association could result from the binding of the SH3
domain(s) of GRB2 to proline-rich sequences located in the C-terminal
part of the GAB1 molecule. These sequences have been shown to be
responsible for most of the association of GAB1 with the HGF
receptor.20,23 According to this hypothesis, we have
observed the binding of GAB1 to a GST-GRB2 fusion protein containing a
deletion in the SH2 domain (data not shown). The Epo-induced
association between GAB1 and GRB2 could be realized through several
mechanisms that involve the SH2 domain of GRB2. This association could
be direct through binding of the GRB2 SH2 domain to a consensus
sequence (YKND) present in the N-terminal part of GAB1 or indirect
through SHP2 and/or SHC. Indeed, these two proteins possess tyrosine
residues able to bind, once phosphorylated, the SH2 domain of GRB2.
According to this hypothesis, these GAB1-GRB2 complexes could be able
to bind SOS through the SH3 domains of GRB2. This association to SOS
could explain the GAB1-mediated activation of ERK that has been
previously reported in IL-6-stimulated cells.24 However, we did not detect SOS1 in anti-GAB1 immunoprecipitates, possibly because of the low efficiency of our anti-SOS antibodies. We also observed the association of SHC with GAB1. This is the first
demonstration of an association between these two proteins. Whether SHC
associated with GAB1 directly or through another GAB1-associated
protein such as GRB2 or SHIP remains to be determined.
We have identified the 145-kD protein present in anti-GAB1
immunoprecipitates as SHIP. SHIP was observed in all immunoprecipitates containing PI 3-kinase, namely the anti-IRS2,18 anti-GAB1
(this report), anti-PI 3-kinase, and anti-Epo receptor
immunoprecipitates (data not shown). The presence of SHIP in the PI
3-kinase-containing complexes should greatly increase the production
of phosphatidylinositol 3,4 bisphosphate and the disappearance of
phosphatidylinositol 3,4,5 trisphosphate. SHIP appears to be a negative
modulator of intracellular signaling in most cases. However,
SHIP / mice have a reduced number of bone
marrow CFU-E, and it is not established whether SHIP has a positive or
negative role in Epo signaling.33 On a molecular basis, the
phosphatidylinositol 3,4 bisphosphate production could increase Akt
activation,34 which could exert an antiapoptotic function
through the phosphorylation of the proapoptotic protein
Bad.35,36 However, the disappearance of
phosphatidylinositol 3,4,5 could also lead to the inhibition of
signaling pathways activated through this second messenger. Overall,
our results show that GAB1 could constitute a scaffold protein,
allowing the formation of a multimolecular complex containing proteins
able to activate several intracellular pathways, including PI 3-kinase
in Epo-stimulated cells.
It is now clearly established that PI 3-kinase could be activated in
Epo-stimulated cells by several mechanisms that involve or do not
involve the tyrosine residues of the Epo receptor. Moreover, the PI
3-kinase inhibitor wortmannin inhibits the Epo-induced proliferation of
DA-3 cells expressing wild-type Epo receptors as well as Epo receptors
devoid of Tyr479,14 demonstrating the
physiological relevance of all these mechanisms. Although in
experimental conditions these pathways appear to be redundant, it
should be kept in mind that normal erythroid progenitors express lower
levels of Epo receptors that the cell lines used as experimental models
and that they have to respond in vivo to low Epo concentrations. In
these conditions, PI 3-kinase activation by these different pathways
could be additive and required to allow a cellular response to a low
level of Epo stimulation. Moreover, the importance of the last tyrosine
residue of the Epo receptor that directly binds PI 3-kinase is not
clearly established. Indeed, it has been reported that this tyrosine
residue of the Epo receptor induces a signal transduction pathway
sufficient for proliferation and differentiation of fetal liver CFU-E
in mice.37 However, truncation of the Epo receptor that
also removes this tyrosine residue causes hypersensitivity to Epo and
benign erythrocytosis in humans.38 In addition, mice
expressing Epo receptors truncated after the first tyrosine residue of
intracellular domain exhibit normal hematological parameters despite
the removal of the PI 3-kinase binding site (J.N. Ihle,
personnal communication of unpublished results). Because
it is now demonstrated that all of these receptors induce PI 3-kinase
activation, the role of PI 3-kinase in Epo-induced proliferation and/or
survival of erythroid progenitors has to be carefully addressed.
| |
FOOTNOTES |
Submitted October 22, 1998; accepted December 11, 1998.
C.L.-L. and F.V. contributed equally to this work.
Supported by grants from the Association pour la Recherche sur le
Cancer (ARC Contract No. 1373) and from the Ligue Nationale Contre le
Cancer. F.V. is supported by the GLAXO WELLCOME Laboratories.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Patrick Mayeux, PhD, ICGM,
INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint Jacques,
F75014 Paris, France; e-mail: mayeux{at}cochin.inserm.fr.
| |
REFERENCES |
1.
Wu H, Liu X, Jaenish R, Lodish HF:
Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
Cell
83:59, 1995[Medline]
[Order article via Infotrieve]
2.
D'Andrea AD, Lodish HF, Wong GG:
Expression cloning of the murine erythropoietin receptor.
Cell
57:277, 1989[Medline]
[Order article via Infotrieve]
3.
Socolovsky M, Lodish HF, Daley GQ:
Control of hematopoietic differentiation: Lack of specificity in signaling by cytokine receptors.
Proc Natl Acad Sci USA
95:6573, 1998[Free Full Text]
4.
Witthuhn B, Quelle FW, Silvennoinen O, Yi T, Tang B, Muira O, Ihle JN:
JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following Epo stimulation.
Cell
74:227, 1993[Medline]
[Order article via Infotrieve]
5.
Damen J, Mui ALF, Hughes P, Humphries KR, Krystal G:
Erythropoietin-induced tyrosine phosphorylation in a high erythropoietin-receptor expressing lymphoid cell line.
Blood
80:1923, 1992[Abstract/Free Full Text]
6.
Dusanter-Fourt I, Casadevall N, Lacombe C, Muller O, Billat C, Fischer S, Mayeux P:
Erythropoietin induces the tyrosine phosphorylation of its own receptor in human erythropoietin-responsive cells.
J Biol Chem
267:10670, 1992[Abstract/Free Full Text]
7.
Miura O, D'Andrea A, Kabat D, Ihle JN:
Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.
Mol Cell Biol
11:4895, 1991[Abstract/Free Full Text]
8.
Yoshimura A, Lodish HF:
In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.
Mol Cell Biol
12:706, 1992[Abstract/Free Full Text]
9.
Damen JE, Krystal G:
Early events in erythropoietin-induced signaling.
Exp Hematol
24:1455, 1996[Medline]
[Order article via Infotrieve]
10.
Damen JE, Mui ALF, Puil L, Pawson T, Krystal G:
Phosphatidylinositol 3-kinase associates, via its Src homology 2 domains, with the activated erythropoietin receptor.
Blood
81:3204, 1993[Abstract/Free Full Text]
11.
Mayeux P, Dusanter-Fourt I, Muller O, Mauduit P, Sabbah M, Drucker B, Vainchencker W, Fischer S, Lacombe C, Gisselbrecht S:
Erythropoietin induces the association of phosphatidylinositol 3' kinase with a tyrosine phosphorylated complex containing the erythropoietin receptor.
Eur J Biochem
216:821, 1993[Medline]
[Order article via Infotrieve]
12.
He TC, Zhuang H, Jiang N, Waterfield MD, Wojchowski DM:
Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.
Blood
82:3530, 1993[Abstract/Free Full Text]
13.
Miura O, Nakamura N, Ihle JN, Aoki N:
Erythropoietin-dependent Association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.
J Biol Chem
269:614, 1994[Abstract/Free Full Text]
14.
Damen J, Cutler RL, Jiao H, Yi T, Krystal G:
Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the p85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.
J Biol Chem
270:23402, 1995[Abstract/Free Full Text]
15.
Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanafusa H, Schaffhausen B, Cantley LC:
SH2 domains recognize specific phosphopeptide sequences.
Cell
72:767, 1993[Medline]
[Order article via Infotrieve]
16.
Gobert S, Porteu F, Pallu S, Muller O, Sabbah M, Dusanter-Fourt I, Courtois G, Lacombe C, Gisselbrecht S, Mayeux P:
Tyrosine phosphorylation of the erythropoietin receptor: Role for differentiation and mitogenic signal transduction.
Blood
86:598, 1995[Abstract/Free Full Text]
17.
Shigematsu H, Iwasaki H, Otsuka T, Ohno Y, Arima F, Niho Y:
Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity.
J Biol Chem
272:14334, 1997[Abstract/Free Full Text]
18.
Verdier F, Chrétien S, Billat C, Gisselbrecht S, Lacombe C, Mayeux P:
Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2: An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.
J Biol Chem
272:26173, 1997[Abstract/Free Full Text]
19.
Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ:
A Grb2-associated docking protein in EGF- and insulin-receptor signalling.
Nature
379:560, 1996[Medline]
[Order article via Infotrieve]
20.
Bardelli A, Longati P, Gramaglia D, Stella MC, Comoglio PM:
Gab1 coupling to the HGF/Met receptor multifunctional docking site requires binding of Grb2 and correlates with the transforming potential.
Oncogene
15:3103, 1997[Medline]
[Order article via Infotrieve]
21.
Fixman ED, Holgado-Madruga M, Nguyen L, Kamikura DM, Fournier TM, Wong AJ, Park M:
Efficient cellular transformation by the Met oncoprotein requires a functional Grb2 binding site and correlates with phosphorylation of the Grb2-associated proteins, Cbl and Gab1.
J Biol Chem
272:20167, 1997[Abstract/Free Full Text]
22.
Weidner KM, Di Cesare S, Sachs M, Brinkmann V, Behrens J, Birchmeier W:
Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis.
Nature
384:173, 1996[Medline]
[Order article via Infotrieve]
23.
Nguyen L, Holgado-Madruga M, Maroun C, Fixman ED, Kamikura D, Fournier T, Charest A, Tremblay ML, Wong AJ, Park M:
Association of the multisubstrate docking protein Gab1 with the hepatocyte growth factor receptor requires a functional Grb2 binding site involving tyrosine 1356.
J Biol Chem
272:20811, 1997[Abstract/Free Full Text]
24.
Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K, Nakajima K, Hibi M, Hirano T:
Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase.
Mol Cell Biol
18:4109, 1998[Abstract/Free Full Text]
25.
Holgado-Madruga M, Moscatello DK, Emlet DR, Dieterich R, Wong AJ:
Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor.
Proc Natl Acad Sci USA
94:12419, 1997[Abstract/Free Full Text]
26.
Komatsu N, Nakauchi H, Miwa A, Ishihara T, Eguchi M, Moroi M, Okada M, Sato Y, Wada H, Yamata Y, Suda T, Miura Y:
Establishment and characterization of a human leukemic cell line with megakaryocytic features: Dependency on granulocyte-macrophage colony stimulating factor, interleukin 3, or erythropoietin for growth and survival.
Cancer Res
51:341, 1991[Abstract/Free Full Text]
27.
Porteu F, Rouyez MC, Cocault L, Benit L, Charon M, Picard F, Gisselbrecht S, Souyri M, Dusanter-Fourt I:
Functional regions of the mouse thrombopoietin receptor cytoplasmic domain: Evidence for a critical region which is involved in differentiation and can be complemented by erythropoietin.
Mol Cell Biol
16:2473, 1996[Abstract]
28.
Kapeller R, Cantley LC:
Phosphatidylinositol 3-kinase.
Bioessays
16:565, 1994[Medline]
[Order article via Infotrieve]
29.
Hermine O, Mayeux P, Titeux M, Mitjavila MT, Casadevall N, Guichard J, Komatsu N, Suda T, Miura Y, Vainchenker W, Breton-Gorius J:
Granulocyte-macrophage colony-stimulating factor and erythropoietin act competitively to induce two different programs of differentiation in the human pluripotent cell line UT-7.
Blood
80:3060, 1992[Abstract/Free Full Text]
30.
Franke TF, Kaplan DR, Cantley LC:
PI3K: Downstream AKTion blocks apoptosis.
Cell
88:435, 1997[Medline]
[Order article via Infotrieve]
31.
Rordorf-Nikolic T, van Horn DJ, Chen D, White MF, Backer JM:
Regulation of phosphatidylinositol 3'-kinaseby tyrosyl phosphoproteins: Full activation requires occupancy of both SH2 domains in the 85 kDa regulatory subunit.
J Biol Chem
270:3662, 1995[Abstract/Free Full Text]
32.
Rocchi S, Tartare-Deckert S, Murdaca J, Holgado-Madruga M, Wong AJ, Van Obberghen E:
Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules.
Mol Endocrinol
12:914, 1998[Abstract/Free Full Text]
33.
Helgason CD, Damen JE, Rosten P, Grewal R, Sorensen P, Chappel SM, Borowski A, Jirik F, Krystal G, Humphries RK:
Targeted disruption of SHIP leads to hemopoietic perturbations, lung pathology, and a shortened life span.
Genes Dev
12:1610, 1998[Abstract/Free Full Text]
34.
Franke TF, Kaplan DR, Cantley LC, Toker A:
Direct regulation of the AKT proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.
Science
275:685, 1997
35.
Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME:
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
Cell
91:231, 1997[Medline]
[Order article via Infotrieve]
36.
del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G:
Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.
Science
278:687, 1997[Abstract/Free Full Text]
37.
Klingmuller U, Wu H, Hsiao JG, Toker A, Duckworth BC, Cantley LC, Lodish HF:
Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors.
Proc Natl Acad Sci USA
94:3016, 1997[Abstract/Free Full Text]
38.
De La Chapelle A, Traskelin A, Juvonen E:
Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis.
Proc Natl Acad Sci USA
90:4495, 1993[Abstract/Free Full Text]
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|
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|
|
C. R. Maroun, D. K. Moscatello, M. A. Naujokas, M. Holgado-Madruga, A. J. Wong, and M. Park
A Conserved Inositol Phospholipid Binding Site within the Pleckstrin Homology Domain of the Gab1 Docking Protein Is Required for Epithelial Morphogenesis
J. Biol. Chem.,
October 29, 1999;
274(44):
31719 - 31726.
[Abstract]
[Full Text]
[PDF]
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C. Guillard, S. Chretien, R. Jockers, S. Fichelson, P. Mayeux, and V. Duprez
Coupling of Heterotrimeric Gi Proteins to the Erythropoietin Receptor
J. Biol. Chem.,
January 12, 2001;
276(3):
2007 - 2014.
[Abstract]
[Full Text]
[PDF]
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L. S. Lock, I. Royal, M. A. Naujokas, and M. Park
Identification of an Atypical Grb2 Carboxyl-terminal SH3 Domain Binding Site in Gab Docking Proteins Reveals Grb2-dependent and -independent Recruitment of Gab1 to Receptor Tyrosine Kinases
J. Biol. Chem.,
September 29, 2000;
275(40):
31536 - 31545.
[Abstract]
[Full Text]
[PDF]
|
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K. J. Cowan, D. A. Law, and D. R. Phillips
Identification of Shc as the Primary Protein Binding to the Tyrosine-phosphorylated beta 3 Subunit of alpha IIbbeta 3 during Outside-in Integrin Platelet Signaling
J. Biol. Chem.,
November 10, 2000;
275(46):
36423 - 36429.
[Abstract]
[Full Text]
[PDF]
|
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M. Kong, C. Mounier, J. Wu, and B. I. Posner
Epidermal Growth Factor-induced Phosphatidylinositol 3-Kinase Activation and DNA Synthesis. IDENTIFICATION OF Grb2-ASSOCIATED BINDER 2 AS THE MAJOR MEDIATOR IN RAT HEPATOCYTES
J. Biol. Chem.,
November 10, 2000;
275(46):
36035 - 36042.
[Abstract]
[Full Text]
[PDF]
|
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H. Sakamoto, T. Kitamura, and A. Yoshimura
Mitogen-activated Protein Kinase Plays an Essential Role in the Erythropoietin-dependent Proliferation of CTLL-2 Cells
J. Biol. Chem.,
November 10, 2000;
275(46):
35857 - 35862.
[Abstract]
[Full Text]
[PDF]
|
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M. von Lindern, M. P.-v. Amelsvoort, T. van Dijk, E. Deiner, E. van den Akker, S. van Emst-de Vries, P. Willems, H. Beug, and B. Lowenberg
Protein Kinase C alpha Controls Erythropoietin Receptor Signaling
J. Biol. Chem.,
October 27, 2000;
275(44):
34719 - 34727.
[Abstract]
[Full Text]
[PDF]
|
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S. Ali and S. Ali
Recruitment of the Protein-tyrosine Phosphatase SHP-2 to the C-terminal Tyrosine of the Prolactin Receptor and to the Adaptor Protein Gab2
J. Biol. Chem.,
December 8, 2000;
275(50):
39073 - 39080.
[Abstract]
[Full Text]
[PDF]
|
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R. J. Ingham, L. Santos, M. Dang-Lawson, M. Holgado-Madruga, P. Dudek, C. R. Maroun, A. J. Wong, L. Matsuuchi, and M. R. Gold
The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase
J. Biol. Chem.,
April 6, 2001;
276(15):
12257 - 12265.
[Abstract]
[Full Text]
[PDF]
|
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F. Verdier, P. Walrafen, N. Hubert, S. Chretien, S. Gisselbrecht, C. Lacombe, and P. Mayeux
Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors
J. Biol. Chem.,
June 9, 2000;
275(24):
18375 - 18381.
[Abstract]
[Full Text]
[PDF]
|
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N. Kashige, N. Carpino, and R. Kobayashi
Tyrosine phosphorylation of p62dok by p210bcr-abl inhibits RasGAP activity
PNAS,
February 29, 2000;
97(5):
2093 - 2098.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION