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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2578-2585
By
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.
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.
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 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.
Cell culture and stimulation.
UT-7 cells26 were maintained by diluting twice weekly in
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- 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- 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.
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.
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
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
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.
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
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.
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TOP
ABSTRACT
INTRODUCTION
(PLC
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).
) 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.
/