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J Biol Chem, Vol. 275, Issue 6, 4398-4406, February 11, 2000
From the Ship1 (SH2 inositol
5-phosphatase 1) has been shown to be a target
of tyrosine phosphorylation downstream of cytokine and immunoregulatory
receptors. In addition to its catalytic activity on
phosphatidylinositol substrates, it can serve as an adaptor protein in
binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of
erythropoiesis, has been shown to activate the tyrosine phosphorylation
of Shc, resulting in recruitment of Grb2. However, the mechanism by
which the erythropoietin receptor (EPO-R) recruits Shc remains unknown.
EPO activates the tyrosine phosphorylation of Ship1, resulting in the
interdependent recruitment of Shc and Grb2. Ship1 is recruited to the
EPO-R in an SH2-dependent manner. Utilizing a panel of
EPO-R deletion and tyrosine mutants, we have discovered remarkable
redundancy in Ship1 recruitment. EPO-R Tyr401 appears
to be a major site of Ship1 binding; however, Tyr429 and
Tyr431 can also serve to recruit Ship1. In addition, we
have shown that EPO stimulates the formation of a ternary complex
consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete
signal transduction pathways. EPO-dependent activation of
ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of
EPO-R deletion mutants. Activation of ERK1/2 was observed in
EPO-R Erythropoietin (EPO)1
exerts its biological activity by binding to its cognate receptor
(EPO-R), a 66-kDa transmembrane protein (1). Binding of EPO unleashes a
conformational change in a preformed EPO-R dimer (2), which leads to
activation of the Janus tyrosine kinase JAK2. The critical role of EPO
(3), EPO-R (3, 4) and JAK2 in erythropoiesis (5, 6) has recently been
elegantly demonstrated, as gene targeting experiments reveal that loss
of any one of these genes leads to a fatal anemia and the embryos fail
to traverse primitive to definitive erythropoiesis.
EPO-dependent JAK2 activation initiates a cascade of
tyrosine phosphorylation events that result in tyrosine phosphorylation of the EPO-R, recruitment of SH2 effector proteins, and subsequent phosphorylation of those proteins. The recruitment of several SH2
domain-containing proteins has been documented, and in many cases, the
site of recruitment on the EPO-R has been established through deletions
or tyrosine mutagenesis of the EPO-R. The cytosolic transcription
factor STAT5 has been shown to bind to EPO-R Tyr343 and
Tyr401 (7, 8); the tyrosine phosphatase Shp2 (9) and the
SOCS family member Cis are recruited to EPO-R Tyr401 (10)
and the tyrosine phosphatase Shp1 to EPO-R Tyr429 and/or
Tyr431 (11); and the 85-kDa subunit of phosphatidylinositol
(PI) 3-kinase has been shown to bind to EPO-R Tyr479
(12).
Several other substrates of EPO-dependent tyrosine
phosphorylation have been described, including Cbl (13), Vav (14), and
Shc (15, 16); however, the mechanism by which the EPO-R recruits these
signaling effectors is unknown. Growth factors and cytokines activate
the Ras signaling pathway through the recruitment of the SH2 adaptor
protein Grb2 and the associated guanine nucleotide release factor SOS.
In earlier studies, we have shown that Grb2-Sos can be recruited to the
EPO-R through direct association with the EPO-R or indirectly via Shp2
or Shc (16). However, direct binding of the EPO-R to Shc was not
observed. In these experiments, we had identified a 145-kDa
phosphoprotein, and with the subsequent identification of Ship1 by
several laboratories (21, 24, 54-56), we became intrigued with the
possibility that EPO-R may recruit Shc through binding to Ship1.
ship1 is expressed exclusively in hematopoietic tissue (17,
18) and developing spermatagonia, whereas the related gene ship2 has a wider tissue distribution (19). Ship proteins
display catalytic activity on phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate substrates. In addition, Ship1,
an SH2 domain-containing protein, can serve as an adaptor protein, as
when tyrosine-phosphorylated, it can bind Shc in a phosphotyrosine-binding domain-dependent manner (20).
Proline-rich sequences found at the carboxyl terminus of Ship1 also
result in recruitment of the SH3 domain of Grb2 (21). In this study, we
show that the EPO-R activates the tyrosine phosphorylation of Ship1 and
that Ship1 binds to the EPO-R in an SH2-dependent fashion
through multiple phosphotyrosine residues, including EPO-R Tyr401, Tyr429, and Tyr431.
Cell Lines and Culture--
Ba/F3 cells expressing wild-type or
mutant EPO-Rs were maintained in RPMI 1640 medium, 10% (v/v) fetal
calf serum, and 50 µM
Various EPO-R constructs were electroporated into Ba/F3 cells.
Individual G418-resistant subclones were isolated by limiting dilution.
The expression of the EPO-R was confirmed by Western blotting, and the
EPO-dependent growth characteristics of each subclone were
examined by performing an XTT assay as described (22).
Generation of EPO-R Mutants--
EPO-R tyrosine mutants were
generated via overlap extension polymerase chain reaction. These
included a series of single tyrosine mutants in which phenylalanine was
substituted for different tyrosine residues in the EPO-R and a series
of add-back mutants to an EPO-R devoid of tyrosine residues.
Oligonucleotide primers were selected that produced a phenylalanine at
amino acids 343, 401, 429, and/or 431. Polymerase chain reaction was
performed using pBSK-EPO-R or selected pBSK-EPO-R tyrosine mutants to
generate an SphI-EcoRI fragment in pCR-Script.
The fidelity of all constructs was confirmed by sequencing both strands
of the 440-base pair fragment. Each SphI-EcoRI
fragment was subcloned into SphI-EcoRI-digested
pBSK-EPO-R. The EPO-R cDNA was then subcloned into pcDNA3.1-neo
using KpnI and EcoRI.
Cytokine Deprivation and Stimulation--
Cells were washed
three times in 10 mM HEPES (pH 7.4) and Hanks' balanced
salts; starved in RPMI 1640 medium supplemented with 10% fetal calf
serum and 50 µM Antibodies--
The anti-phosphotyrosine monoclonal antibody was
generously provided by Dr. Brian Druker, and the anti-Ship1 polyclonal
antibody was generated as described previously (23). Anti-EPO-R,
anti-Grb2, anti-GST, anti-phospho-ERK1/2, and anti-Ship1 antibodies
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Shc polyclonal antibody was obtained from Transduction
Laboratories (Lexington, KY). The anti-ERK1/2 antibody was purchased
from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-phospho-PKB
(Ser473) and anti-PKB antibodies were purchased from
New England Biolabs Inc. (Beverly, MA). A peptide-specific EPO-R
antibody has been described previously (22). Horseradish peroxidase
(HRP)-conjugated protein A or HRP-conjugated sheep
anti-mouse immunoglobulin (Amersham Pharmacia Biotech) was used as the
secondary reagent for immunoblotting.
Immunoprecipitations--
Antibodies were added to 2 mg of
lysates for a 1-h incubation, followed by the addition of a 40-µl
volume of protein A-Sepharose 4B beads (Amersham Pharmacia Biotech),
and incubation was continued for an additional hour. The beads were
washed three times in ice-cold lysis buffer, and immune complexes were
eluted by boiling in Laemmli sample buffer containing 100 mM dithiothreitol. Samples were resolved by
SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a
nitrocellulose membrane for Western blotting.
Ba/F3 subclones were metabolically labeled with
[35S]methionine/cysteine as described previously (22).
Labeled proteins were immunoprecipitated with an anti-peptide
amino-terminal EPO-R antibody. Labeled proteins were detected by
PhosphorImager analysis.
GST Fusion Protein Binding Experiments--
Lysates (2 mg) were
incubated overnight at 4 °C with GST fusion proteins expressing the
SH2 domain of Ship1 immobilized on glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech). The beads were washed three times in
ice-cold lysis buffer, and proteins were eluted by boiling in Laemmli
sample buffer with dithiothreitol. Samples were resolved by SDS-PAGE
and analyzed by Western blotting.
Western Blotting--
Following the electrophoretic transfer of
proteins to nitrocellulose, the membranes were blocked at room
temperature with 2.5% BSA in Tris-buffered saline (50 mM
Tris (pH 8.0) and 150 mM NaCl) for 1 h. Membranes were
then incubated with an optimal concentration of the primary antibody in
TBST (Tris-buffered saline containing Tween 20) for 1 h at room
temperature, washed four times in TBST, and incubated with the relevant
HRP-conjugated secondary antibody (1:5000 dilution in TBST) for 30 min.
Membranes were washed four times in TBST and visualized by enhanced
chemiluminescence with autoradiographic film (ECL, Amersham Pharmacia
Biotech). For reprobing, membranes were stripped in 62.5 mM
Tris-HCl (pH 6.8), 2% SDS, and 0.1 M Far-Western Blotting--
Anti-EPO-R immunoprecipitates from 2 mg of Ba/F3-EPO-R cells were resolved by SDS-PAGE and transferred to
nitrocellulose membranes. After blocking with 2.5% BSA in
Tris-buffered saline, membranes were serially incubated with eluted GST
fusion proteins (2.5 µg/ml in TBST), anti-GST antibody (0.5 µg/ml
in TBST with 0.05% Triton X-100), and HRP-protein A (1:5000 dilution
in TBST with 0.05% Triton X-100) and subjected to ECL detection.
Transient Transfection--
293T cells were grown in Dulbecco's
modified Eagle's medium with 10% fetal bovine serum. Cells (at 70%
confluence) were transfected with LipofectAMINE (Life Technologies,
Inc.) with various combinations of wild-type EPO-R (2.5 µg),
wild-type Ship1 (2.5 µg), Ship1 R34Q (2.5 µg; generously provided
by Dr. Kodimangalam Ravichandran (University of Virginia,
Charlottesville, VA), empty vector (pEBG, 2.5 µg), and/or JAK2 (0.1 µg). After incubation for 5 h, the transfection solution was
removed, and the cells were cultured in Dulbecco's modified Eagle's
medium for 24 h. The cells were incubated for 18 h in
Dulbecco's modified Eagle's medium + 1 mg/ml BSA prior to stimulation
with no factor or with 10 units/ml EPO for 10 min. Lysates (0.5 mg)
were immunoprecipitated with an anti-Ship1 polyclonal antibody (Santa
Cruz Biotechnology).
Analysis of ERK1/2 Activation--
Lysates (100 µg) were
resolved by SDS-PAGE and transferred to nitrocellulose. After blocking
with 5% skim milk protein in TBST for 3 h at room temperature,
the membrane was incubated with the anti-phospho-ERK1/2 monoclonal
antibody (1:200 dilution in 5% skim milk protein in TBST) overnight at
4 °C, washed three times in TBST, and incubated with HRP-conjugated
sheep anti-mouse immunoglobulin (1:5000 dilution in 5% skim milk
protein in TBST) for 1 h at room temperature. The membrane was
washed three times in TBST and visualized by ECL.
Analysis of PKB Activation--
Lysates (100 µg) were resolved
by SDS-PAGE and transferred to nitrocellulose. After blocking with 5%
skim milk powder in TBST for 1 h at room temperature, the membrane
was incubated with the anti-phospho-PKB polyclonal antibody (1:1000
dilution in 1% BSA in TBST) overnight at 4 °C, washed six times in
TBST, and incubated with HRP-protein A (1:2000 dilution in 2% skim
milk powder) for 1 h at room temperature. The membrane was washed
six times in TBST and visualized by ECL.
The role of Ship1 in cytokine-mediated signaling was examined in
Ba/F3 and DA-3 cells expressing EPO-R as well as in the erythroid cell
line HCD-57. Cell lines were depleted of cytokine and then stimulated
with no factor or with IL-3 or EPO, and immunoprecipitation with a
peptide-specific Ship1 antibody was performed, followed by Western
blotting with the anti-phosphotyrosine monoclonal antibody 4G10 (Fig.
1). IL-3 and EPO stimulated the tyrosine
phosphorylation of Ship1 in Ba/F3-EPO-R (lanes 7 and 8) and DA-3-EPO-R (lanes 10 and
11) cells. Stimulation of HCD-57 cells with EPO also
resulted in the tyrosine phosphorylation of Ship1 (lane
13). IL-3 stimulation of Ba/F3-EPO-R cells (lane
7) and IL-3 (lane 10) or EPO
(lane 11) stimulation of DA-3-EPO-R cells
resulted in the co-immunoprecipitation of a 52-kDa phosphoprotein with
Ship1. Stripping and reprobing of the membrane revealed that this
protein was Shc (data not shown). EPO stimulation of DA-3-EPO-R cells
also resulted in the co-immunoprecipitation of the 72-kDa EPO-R. On
longer exposures, the EPO-R was also shown to co-immunoprecipitate with
Ship1 from Ba/F3-EPO-R and HCD-57 cells (data not shown). The ability
of the EPO-R to associate with Ship1 from the various cell lines was
directly proportional to EPO-R expression. Equal immunoprecipitation of
Ship1 was demonstrated by stripping and reprobing the membrane with an
anti-Ship1 polyclonal antibody (Ship1 immunoblot).
Ship1 was initially described to interact with Grb2 and Shc (21, 24).
To investigate the ability of Ship1 to bind to these adaptor proteins,
immunoprecipitations were performed using peptide-specific Grb2 and
Ship1 antibodies (Fig. 2). DA-3-EPO-R
cells were depleted of cytokine and then stimulated with no factor or
with IL-3 or EPO. As we have previously shown in CTLL-EPO-R cells (16),
Grb2 co-immunoprecipitated Ship1 (145 and 130 kDa), EPO-R (72 kDa), Shp2 (68 kDa), and Shc (52 and 46 kDa) (lane 6)
after EPO stimulation of DA-3-EPO-R cells. IL-3 stimulation resulted in
co-immunoprecipitation of Ship1, Shp2, and Shc with Grb2
(lane 5). Since Grb2 assembled a multimolecular
complex consisting of Ship1, Shp2, Shc, and EPO-R after EPO
stimulation, we were interested in determining whether Shc could be
recruited to the EPO-R through Ship1. As shown in Fig. 1, Ship1
co-immunoprecipitated Shc after IL-3 stimulation (Fig. 2,
lane 8) and Shc and EPO-R after EPO stimulation
(lane 9).
The SH2 Inositol 5-Phosphatase Ship1 Is Recruited in an
SH2-dependent Manner to the Erythropoietin Receptor*
§,,
, and§**§§
Division of Cellular and Molecular Biology,
Ontario Cancer Institute, the Departments of § Medical
Biophysics and ** Laboratory Medicine and Pathobiology, University of
Toronto, the ¶ Amgen Institute, Toronto, Ontario M5G 2G1, the
Division of Cancer Biology, Sunnybrook and Women's College
Health Sciences Centre, Toronto, Ontario M4N 3MS, and the
Department of Laboratory Medicine and
Pathobiology, Toronto General Hospital, Toronto, Ontario M5G 2M9,
Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
99, which retains only the most proximal tyrosine,
Tyr343. In contrast, EPO-dependent PKB
activation was observed in EPO-R43, but not in EPO-R99. It
appears that EPO-dependent PKB activation is downstream of
a region that indirectly couples to phosphatidylinositol 3-kinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol (RPMI 1640 complete medium) supplemented with 5% (v/v) conditioned medium from
WEHI-3B cells in the presence of 1 mg/ml G418. DA-3-EPO-R cells were
maintained in RPMI 1640 complete medium and 0.5 units/ml human
recombinant EPO. HCD-57 cells were cultured in Iscove's modified
Dulbecco's medium supplemented with 20% fetal calf serum, 50 µM -mercaptoethanol, and 0.5 units/ml human
recombinant EPO.
-mercaptoethanol for 4 h at
37 °C; and then stimulated in the presence or absence of 10 ng/ml
murine recombinant IL-3 or 50 units/ml human recombinant EPO for 10 min
at 37 °C. The cells were washed once in 10 mM HEPES (pH
7.4) and Hanks' balanced salts containing 10 mM sodium
pyrophosphate, 10 mM sodium fluoride, 10 mM
EDTA, and 1 mM sodium orthovanadate and lysed in ice-cold
lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl
(pH 8.0), 150 mM NaCl, 10 mM sodium
pyrophosphate, 10 mM sodium fluoride, 10 mM
EDTA, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 2 mg/ml aprotinin, 2 mg/ml leupeptin,
and 1 mg/ml pepstatin A. After 5 min on ice, the lysates were
centrifuged at 10, 000 × g for 5 min at 4 °C.
-mercaptoethanol
for 30 min at 50 °C; rinsed twice in TBST; and blocked in 2.5% BSA
in Tris-buffered saline prior to primary antibody incubation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (60K):
[in a new window]
Fig. 1.
Ship1 is tyrosine-phosphorylated in
hematopoietic cell lines. Ba/F3-EPO-R (lanes
1-3 and 6-8), HCD-57 (lanes
4, 5, 12, and 13), and
DA-3-EPO-R (lanes 9-11) cells were depleted of
cytokine for 4 h and stimulated with no factor (lanes
1, 4, 6, 9, and
12) or with 10 ng/ml murine IL-3 (lanes
2, 7, and 10) or 50 units/ml human
recombinant EPO (lanes 3, 5,
8, 11, and 13) for 10 min at 37 °C.
Following cell lysis, immunoprecipitation (IP) was performed
with a peptide-specific Ship1 antibody (Ab). Immune
complexes were resolved by SDS-PAGE and transferred to nitrocellulose.
The immunoblot was probed with the anti-phosphotyrosine monoclonal
antibody 4G10 followed by HRP-conjugated sheep anti-mouse IgG and ECL
detection (upper panel). Lysate controls are shown in
lanes 1-5. The membrane was then stripped and
reprobed with an anti-Ship1 polyclonal antibody (lower
panel). Molecular mass standards are indicated in
kilodaltons.
View larger version (53K):
[in a new window]
Fig. 2.
Grb2 and Ship1 recruit similar signaling
complexes after EPO stimulation. DA-3-EPO-R cells were depleted of
cytokine for 4 h and stimulated with no factor (lanes
1, 4, and 7) or with 10 ng/ml murine
IL-3 (lanes 2, 5, and 8) or
50 units/ml human recombinant EPO (lanes 3,
6, and 9) for 10 min at 37 °C. Cell lysates
were immunoprecipitated (IP) with either an anti-Grb2
polyclonal antibody (Ab; lanes 4-6)
or an anti-Ship1 polyclonal antibody (lanes
7-9). Immune complexes were resolved by SDS-PAGE and
transferred to nitrocellulose. The immunoblot was probed with the
anti-phosphotyrosine monoclonal antibody 4G10 and then stripped and
reprobed with antibodies to the EPO-R, Shc, Grb2, and Ship1. Lysate
controls are shown in lanes 1-3. Molecular mass
standards are indicated in kilodaltons.
Since the Ship1 immunoprecipitation experiments demonstrated a ternary
complex between the EPO-R, Ship1, and Shc, the possibility that Ship1
could be recruited to the EPO-R in an SH2-dependent manner
was next tested. Lysates were prepared from DA-3-EPO-R cells, and
in vitro mixing experiments were performed with GST alone
and fused with the Ship1 SH2 domain and a mutated Ship1 SH2 domain
(GST-Ship1 SH2 R34Q) (Fig. 3). The Ship1
SH2 domain was shown to associate with the IL-3 receptor
c chain (140 kDa) (lane 8) after
IL-3 stimulation, and the 72-kDa EPO-R was shown to bind to the Ship1
SH2 domain after engagement of the EPO-R (lane
9). Stripping and reprobing the membrane confirmed that the
72-kDa phosphoprotein in lane 9 was the EPO-R
(lower panel). No phosphoproteins were shown to associate
with GST-Ship1 SH2 R34Q after either IL-3 (lane
11) or EPO (lane 12) stimulation. Only
small amounts of Shc were observed to associate with Ship1, in contrast
to a previous report (25).
|
To examine whether the SH2-dependent association of Ship1
with the EPO-R was direct or indirect, Far-Western blotting experiments were performed (Fig. 4). Lysates from
unstimulated or EPO-stimulated Ba/F3-EPO-R cells were
immunoprecipitated with a peptide-specific EPO-R antibody. Following
SDS-PAGE and transfer to nitrocellulose, the membrane was cut into
strips, and each strip was incubated with GST, GST-Ship1 SH2, or
GST-Ship1 SH2 R34Q (20). Following washing, Western blotting was
performed using an anti-GST antibody followed by HRP-protein A. GST-Ship1 SH2 (lane 6) was shown to bind to the
EPO-R. Neither a GST fusion protein of the Ship1 SH2 domain expressing
an inactivating SH2 domain mutation (R34Q) nor GST alone bound to the
EPO-R. This suggests that Ship1 directly associates with the EPO-R in
an SH2-dependent manner.
|
To examine this possibility in greater detail, we performed a transient
transfection in 293T cells (Fig. 5). The
EPO-R and JAK2 were expressed with either wild-type Ship1 or the Ship1
SH2 domain R34Q mutant. Tyrosine-phosphorylated wild-type Ship1 was shown to associate with the EPO-R when the cells were stimulated with
EPO (lane 2). No tyrosine phosphorylation of
Ship1 R34Q was observed when this construct was coexpressed with the
EPO-R and JAK2 (lane 4). 293T cells failed to
express Ship1 (Ship1 reprobe; lanes 5 and
6), and the association of the EPO-R and Ship1 in this
cellular environment was dependent on JAK2 (lane
8). The 72-kDa phosphoprotein in the Ship1
immunoprecipitation was confirmed to be the EPO-R (EPO-R reprobe;
lane 2). The experiments in Figs. 3-5 provide
strong evidence that Ship1 directly associates with the EPO-R in an
SH2-dependent fashion.
|
To identify the regions of the EPO-R involved in Ship1 recruitment, we
next examined (i) EPO-dependent tyrosine phosphorylation of
Ship1 and (ii) association of the Ship1 SH2 domain in a panel of EPO-R
deletion mutants expressed in Ba/F3 cells. In addition to the wild-type
EPO-R, four mutants were selected: EPO-R43 (containing 4 Tyr
residues), EPO-R69 (containing 2 Tyr residues), EPO-R99 (containing 1 Tyr residue), and EPO-R99,F1 (containing 0 Tyr residues) (Fig. 6A). All cell
lines (with the exception of EPO-R99,F1) were
capable of EPO-dependent
proliferation as determined by XTT assays (data not shown). Previous
studies have indicated that EPO-R99 is capable of
EPO-dependent mitogenesis when expressed in Ba/F3 cells
(26). The selected subclones were depleted of cytokine and stimulated
in the presence or absence of EPO. Immunoprecipitation with Ship1 was
performed, followed by anti-phosphotyrosine Western blotting (Fig.
6B). EPO stimulation of Ba/F3-EPO-R (lane
8), Ba/F3-EPO-R43 (lane 10), and
Ba/F3-EPO-R69 (lane 12) cells resulted in
robust tyrosine phosphorylation of Ship1. Ba/F3-EPO-R99 cells caused a slight stimulation of Ship1 tyrosine phosphorylation in response to
EPO (lane 14). Ba/F3 or Ba/F3-EPO-R99,F1 cells
showed no increase in Ship1 tyrosine phosphorylation, indicating that
the EPO-dependent increase in tyrosine phosphorylation
requires EPO-R cytoplasmic tyrosines.
|
To examine the ability of the truncated EPO-R mutants to associate with
the Ship1 SH2 domain, in vitro mixing experiments were
performed (Fig. 6C). Lysates identical to those described above were incubated with GST-Ship1 SH2, and tyrosine-phosphorylated proteins were detected. EPO stimulated the association of the wild-type
EPO-R (lane 4), EPO-R43 (lane
6), and EPO-R69 (lane 8) with the
Ship1 SH2 domain. From these experiments, we conclude that EPO-R
Tyr401 is sufficient to couple to EPO-dependent
Ship1 tyrosine phosphorylation and binding of Ship1 to the EPO-R.
All of the EPO-R deletion mutants were capable of activating JAK2, and
all mutants (with the exception of EPO-R99,F1) activate STAT5,
indicating that EPO-R43, EPO-R69, and EPO-R99 activate similar
downstream signaling pathways compared with the wild-type EPO-R (Fig.
6D). Metabolic labeling experiments confirmed expression of
each of the EPO-R deletion mutants in Ba/F3 cells (Fig.
6E).
Previous studies have demonstrated that the binding specificity of the
Ship1 SH2 domain is
pY(Y/D)1X2(L/I/V)3 (27).
Scanning the EPO-R cytoplasmic tyrosine residues, it is evident that
there are four potential Ship1-binding sites, with Tyr401
potentially serving as the optimal binding site (Fig.
7).
|
To investigate the specific tyrosine residues that couple to Ship1
recruitment, a series of EPO-R tyrosine mutants was expressed in Ba/F3
cells. We initially examined a series of single
tyrosine-to-phenylalanine substitutions at Tyr343
(EPO-R-Y7,F1), Tyr401 (EPO-R-Y7,F2), Tyr429
(EPO-R-Y7,F3), and Tyr431 (EPO-R-Y7,F4). All of these
mutants were capable of activating Ship1 tyrosine phosphorylation after
EPO stimulation, and GST-Ship1 SH2 bound to each of these mutants
in vitro (data not shown). As a result, we focused on
another series of EPO-R tyrosine mutants (Fig.
8A). Subclones of the
wild-type EPO-R, EPO-R-Y7,F1, EPO-R-Y6,F1F2, EPO-R-Y5,F1F2F3, and
EPO-R-Y4,F1F2F3F4 were isolated. All EPO-R mutants were shown to
generate EPO-dependent proliferation when analyzed in XTT
assays (data not shown). As described for Fig. 6, we examined the
ability of EPO to stimulate Ship1 tyrosine phosphorylation in each
EPO-R tyrosine mutant (Fig. 8B), tested the ability of
GST-Ship1 SH2 to associate with each EPO-R construct in
vitro (Fig. 8C), and performed far-Western assays to
test the ability of the Ship1 SH2 domain to bind directly to the EPO-R (Fig. 8D). Immunoprecipitations were performed with a
peptide-specific Ship1 antibody, followed by anti-phosphotyrosine
immunoblotting to analyze EPO-dependent Ship1 tyrosine
phosphorylation. EPO stimulated the tyrosine phosphorylation of Ship1
in Ba/F3-EPO-R (Fig. 8B, lane 6),
Ba/F3-EPO-R-Y7,F1 (lane 8), and
Ba/F3-EPO-R-Y6,F1F2 (lane 10) cells. The level of
Ship1 tyrosine phosphorylation decreased in Ba/F3-EPO-R-Y5,F1F2F3 cells
(lane 12) and was low, but detectable in
Ba/F3-EPO-R-Y4,F1F2F3F4 cells (lane 14). No Ship1
tyrosine phosphorylation was observed in Ba/F3 (lane
4) or Ba/F3-EPO-R-F8 (lane 16) cells.
The previous experiments utilizing deletion mutants suggested that
Tyr401 is the major binding site for the Ship1 SH2 domain.
However, EPO stimulation of Ba/F3-EPO-R-Y6,F1F2 cells still resulted in significant tyrosine phosphorylation of Ship1, whereas
EPO-dependent Ship1 phosphorylation was dramatically
reduced in EPO-R-Y5,F1F2F3. Considerable redundancy exists in the
ability of the EPO-R to recruit Ship1. The ability of the EPO-R to
induce detectable Ship1 tyrosine phosphorylation in EPO-R-Y4,F1F2F3F4
cells could be mediated by the binding of Grb2 to EPO-R
Tyr464. Ship1 could be recruited to the EPO-R indirectly
through interaction with the Grb2 SH3 domains.
|
The ability of the Ship1 SH2 domain to associate with the EPO-R tyrosine mutants was examined in an in vitro mixing experiment (Fig. 8C). EPO stimulation of Ba/F3-EPO-R (lane 4), Ba/F3-EPO-R-Y7,F1 (lane 6) and Ba/F3-EPO-R-Y6,F1F2 (lane 8) cells resulted in binding of the EPO-R to the Ship1 SH2 domain as detected by anti-phosphotyrosine immunoblotting. Much weaker binding of EPO-R-Y5,F1F2F3 (lane 10) and EPO-R-Y4,F1F2F3F4 (lane 12) to the Ship1 SH2 domain was detected after EPO stimulation. No 72-kDa phosphoproteins were observed to associate with the Ship1 SH2 domain in the Ba/F3 (lane 2) and Ba/F3-EPO-R-F8 (lane 14) cell lines.
Next, Far-Western blotting was utilized to discriminate Ship1 binding to the various EPO-R tyrosine mutants (Fig. 8D). Lysates from each cell line were immunoprecipitated with an anti-EPO-R polyclonal antibody. Following electrophoretic transfer, the membrane was incubated with GST-Ship1 SH2, followed by anti-GST Western blotting. Wild-type EPO-R (lane 2) and EPO-R-Y7,F1 (lane 4) strongly bound the Ship1 SH2 domain. Low, but detectable binding of the Ship1 SH2 domain was observed in EPO-R-Y6,F1F2 (lane 6) and EPO-R-Y5,F1F2F3 (lane 8). No binding was observed in EPO-R-Y4,F1F2F3F4 (lane 10) and EPO-R-F8 (lane 12). No binding to GST alone was observed in any of the EPO-R constructs (data not shown). Reprobing the membrane with an anti-EPO-R antibody revealed equal expression of all subclones, except for EPO-R-Y6,F1F2, which was slightly lower.
Ship1 is a complex protein due to its ability to serve as an adaptor
protein in binding to the phosphotyrosine-binding domain of Shc (20)
and to the SH3 domain of Grb2. In addition, its catalytic activity has
been shown to modulate the activation of protein kinase B/AKT in
response to IL-2 (28) and to regulate stem cell
factor-dependent mast cell degranulation (29). To begin to
understand the function of Ship1, we examined EPO-dependent ERK and PKB activation in the panel of EPO-R deletion mutants described
earlier (Fig. 9A). For this
analysis, we included an additional mutant, EPO-R221, which is
unable to bind and activate JAK2 (22). Phosphorylation of ERK1/2 was
observed in all EPO-R deletion mutants, with the exception of
EPO-R221 (lane 15). EPO-R99, which
displayed weak Ship1 tyrosine phosphorylation in response to EPO
stimulation, nevertheless was capable of ERK1/2 activation (lane 12).
|
Phosphorylation of PKB/Akt was detected using an activation-specific
antibody that detected PKB when phosphorylated at Ser473
(Fig. 9B). EPO activated PKB in Ba/F3 cells expressing
wild-type EPO-R (lane 3), EPO-R43
(lane 6), and EPO-R69 (lane
9). No PKB activation was seen in EPO-R99
(lane 12) and EPO-R221 (lane 15).
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Cytokine receptors recruit a multitude of SH2 effector proteins after receptor engagement. Although the precise phosphorylation events have not been mapped, mutagenesis studies have established the binding location of many intracellular signaling proteins. The EPO-R has been shown to activate the tyrosine phosphorylation of Ship1 (21), thus inducing its association with the SH2 adaptor protein Shc (20) and constitutive binding to the Grb2 adaptor protein (21). Using a panel of EPO-R deletion and tyrosine mutants, we have demonstrated that Ship1 is recruited to the EPO-R in an SH2-dependent manner.
Several studies have shown that Ship1 is recruited in an
SH2-dependent manner to immune tyrosine inhibition motifs
expressed on inhibitory receptors, and Ship1 has also been shown to be
recruited to the IL-4 receptor 
chain (30). Earlier studies had
suggested that a 145-kDa phosphoprotein competed with Grb2 for binding
to Shc Tyr(P)317 (31). The initial studies demonstrated
that a pYXN phosphopeptide competed for 145-kDa protein
binding to Shc. Once the 145-kDa protein was identified as Ship1, these
investigators demonstrated that GST-Ship1 SH2 bound to Shc in
vitro and that the Ship1 SH2 domain bound to a Shc
Tyr317 phosphopeptide in BIAcore experiments (25). They
were also unable to demonstrate co-immunoprecipitation of Ship1 and
Grb2 (25). In this study, we have shown that Grb2 can
co-immunoprecipitate a ternary complex that contains Shc and Ship1
(Fig. 2). The Ship1 SH2 domain selectively binds to either the IL-3
receptor c chain or the EPO-R after cytokine stimulation
with the corresponding ligand (Fig. 3). Furthermore, the Ship1 SH2
domain binds directly to the EPO-R in Far-Western experiments (Fig. 4).
We have shown that in cell lines derived to express various EPO-R
deletion and tyrosine mutants, the SH2 domain of Ship1 binds to a
region of the EPO-R that is involved in negative regulation.
There is considerable redundancy in EPO-R residues Tyr343,
Tyr401, Tyr429, and Tyr431, as
shown in Fig. 7. Although Tyr343 is sufficient to activate
the tyrosine phosphorylation and DNA binding of STAT5, other tyrosines
are involved in STAT5 recruitment, most notably Tyr401 (7).
We chose to focus on this region of the EPO-R to identify the
Ship1-binding domain, as all of these sequences represent candidate
Ship1 SH2-binding sites (Fig. 7). Ship1 is tyrosine-phosphorylated in
Ba/F3 cells expressing full-length EPO-R, EPO-R43, EPO-R69, EPO-R-Y7,F1, and EPO-R-Y6,F1F2 after EPO stimulation. However, the
level of tyrosine phosphorylation is dramatically reduced in
EPO-R-Y5,F1F2F3 and EPO-R99. In vitro mixing experiments
indicated that the Ship1 SH2 domain associates with the EPO-R,
EPO-R43, EPO-R69, EPO-R-Y7,F1, and EPO-R-Y6,F1F2. Far-Western
blotting showed that the Ship1 SH2 domain binds directly to the
wild-type EPO-R and EPO-R-Y7,F1. In sum, this indicates that the
preferred binding sites of the Ship1 SH2 domain are tyrosines 401 and/or 429. Shp2 has been shown to bind to Tyr401 (9),
whereas Shp1 associates with Tyr429 (11). It is interesting
that EPO-R Tyr401 is a central site in the recruitment of
several SH2 effector proteins. For example, it is now known that Cis
(10), Ship1 and Shp2 (9), and STAT5 (7, 8) all are capable of binding to this site. Another example of distinct tyrosine sequences recruiting multiple effector proteins has been shown in c-Met Tyr1356,
which recruits phosphatidylinositol 3-kinase, phospholipase C
, Shp2,
and Grb2 (32, 33). In addition, Tie2 recruits Shp2, phosphatidylinositol 3-kinase, Grb2, and Grb7 to Tyr1101
(34, 35).2
In this study, we have provided novel evidence that Ship1 is recruited to a region of the EPO-R that has been shown to be correlated with negative regulation. Although the role of Ship1 in erythropoiesis remains to be resolved, several patients suffering from primary familial and congenital polycythemia have truncation mutations of the EPO-R. Of the mutations that have been identified to date, five patients have deletions from amino acids 414 to 427 of the human EPO-R that delete the 6 carboxyl-terminal tyrosines including EPO-R Tyr429 (37-40), whereas one patient has a deletion that results in loss of the EPO-R Tyr401-binding site (41). The functional basis of primary familial and congenital polycythemia has yet to be resolved. Perhaps primary familial and congenital polycythemia arises through the inability of Ship1 to be recruited to the EPO-R in these patients.
The role of Ship1 in hematopoietic function is complex. Gene targeting
experiments revealed that mice lacking functional Ship1 suffer from
splenomegaly and myeloid infiltration into the lung (42). Increased
myeloid progenitors were found in the bone marrow and spleen of
knockout animals (42). Bone marrow-derived mast cells from
Ship1
/ mice show increased degranulation in response to
Steel factor (29). The numbers of colony forming unit-erythroid were
observed to be lower in Ship1/ mice (42).
Ship1 may modulate several signal transduction pathways in response to
EPO. First, it serves as an adaptor protein in the SH3-dependent binding of Grb2 (21) and the
activation-dependent binding of Shc (20), which may couple
Ship1 to Ras activation. Second, through its catalytic activity, Ship1
may regulate PKB/Akt activity (28), as it has been shown that mast
cells derived from Ship1/ mice have elevated PKB
activity (23). Third, as has been shown for Steel
factor-dependent mast cell degranulation (29), loss of
Ship1 may result in increases in calcium influx due to elevated levels
of phosphatidylinositol 3,4,5-trisphosphate, which has been shown to be
an agonist of calcium channel action.
We have shown that Ship1 recruitment of the EPO-R does not directly
correlate with ERK1/2 activation. Deletion mutants of the EPO-R
containing only Tyr343 have been shown to be competent for
mitigating EPO-dependent mitogenesis, differentiation, and
cell survival pathways (26, 43, 44). In this cellular context, EPO
stimulates STAT5 tyrosine phosphorylation (7, 8, 45, 46) and ERK
activation (47), but abrogates Ship1 recruitment. Several studies have
shown that PI 3-kinase is upstream of PKB activation (48). The p85
subunit of PI 3-kinase is recruited to the carboxyl-terminal tyrosine, EPO-R Tyr479 (12). However, activation of PKB does not
correlate with binding of PI 3-kinase to the EPO-R (Fig.
9B). PI 3-kinase can be recruited indirectly through other
signaling effectors such as Cbl (49). Therefore, it appears that
EPO-dependent PKB activation requires more proximal
elements located within the carboxyl-terminal 100 amino acids of the
EPO-R cytoplasmic tail. Several studies have shown that EPO stimulates
a calcium influx through a voltage-independent calcium channel
(50-53). We have recently shown that EPO-R Tyr460 couples
to EPO-dependent calcium influx (36). Since Ship1 appears to be recruited to a region of the EPO-R distinct from that involved in
calcium influx, it would appear that Ship1 may not directly affect
calcium signaling in erythroid progenitor cells. The effects of Ship1
on these and other yet to be identified signaling pathways await to be
dissected in erythroblasts isolated from gene-targeted animals.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Monique Yoakim and Ben Neel for the gift of EPO-R-F8 and Kodimangalam Ravichandran for the GST-Ship1 SH2 R34Q construct. We appreciate helpful comments on the manuscript from Jane McGlade, Sonya Penfold, and members of the Barber laboratory.
| |
FOOTNOTES |
|---|
* This work was supported in part by Medical Research Council of Canada Grant MT 13612 and by the National Cancer Institute of Canada and the University of Toronto Faculty of Medicine Dean's Research Funds (all to D. L. B.). This work was performed in partial fulfillment of the requirements for a Ph.D. degree from the University of Toronto (J. M. M.).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.
§§ Supported by a special fellowship from the Leukemia Society of America. Performed this work in partial fulfillment of the requirements for a Ph.D. degree from the University of Toronto. To whom correspondence should be addressed: Ontario Cancer Inst., Div. of Cellular and Molecular Biology, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-4455; Fax: 416-946-2065; E-mail: dbarber@oci.utoronto.ca.
2 Jones, N., Master, Z., Jones, J., Bouchard, D., Sasaki, H., Daly, R., and Dumont, D. J. (1999) J. Biol. Chem. 274, 30896-30905.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: EPO, erythropoietin; EPO-R, erythropoietin receptor; JAK, Janus tyrosine kinase; STAT, signal transducer and activator of transcription; PI, phosphatidylinositol; IL, interleukin; GST, glutathione S-transferase; PKB, protein kinase B; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; XXT, sodium 3,3'-[1[(phenylamino) carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid; BSA, bovine serum albumin.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | D'Andrea, A. D., Lodish, H. F., and Wong, G. G. (1989) Cell 57, 277-285[CrossRef][Medline] [Order article via Infotrieve] |
| 2. |
Livnah, O.,
Stura, E. A.,
Middleton, S. A.,
Johnson, D. L.,
Jolliffe, L. K.,
and Wilson, I. A.
(1999)
Science
283,
987-990 |
| 3. | Wu, H., Liu, X., Jaenisch, R., and Lodish, H. F. (1995) Cell 83, 59-68[CrossRef][Medline] [Order article via Infotrieve] |
| 4. |
Lin, C.-S.,
Lim, S.-K.,
D'Agati, V.,
and Costantini, F.
(1996)
Genes Dev.
10,
154-164 |
| 5. | Neubauer, H., Cumano, A., Muller, M., Wu, H., Huffstadt, U., and Pfeffer, K. (1998) Cell 93, 397-409[CrossRef][Medline] [Order article via Infotrieve] |
| 6. | Parganas, E., Wang, D., Stravopodis, D., Topham, D. J., Marine, J. C., Teglund, S., Vanin, E. F., Bodner, S., Colamonici, O. R., van Deursen, J. M., Grosveld, G., and Ihle, J. N. (1998) Cell 93, 385-395[CrossRef][Medline] [Order article via Infotrieve] |
| 7. |
Klingmuller, U.,
Bergelson, S.,
Hsiao, J. G.,
and Lodish, H. F.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8324-8328 |
| 8. | Gobert, S., Chretien, S., Gouilleux, F., Muller, O., Pallard, C., Dusanter-Fourt, I., Groner, B., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1996) EMBO J. 15, 2434-2441[Medline] [Order article via Infotrieve] |
| 9. |
Tauchi, T.,
Damen, J. E.,
Toyama, K.,
Feng, G.-S.,
Broxmeyer, H. E.,
and Krystal, G.
(1996)
Blood
87,
4495-4501 |
| 10. |
Verdier, F.,
Chretien, S.,
Muller, O.,
Varlet, P.,
Yoshimura, A.,
Gisselbrecht, S.,
Lacombe, C.,
and Mayeux, P.
(1998)
J. Biol. Chem.
273,
28185-28190 |
| 11. | Klingmuller, U., Lorenz, U., Cantley, L. C., Neel, B. G., and Lodish, H. F. (1995) Cell 80, 729-738[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Damen, J. E.,
Cutler, R. L.,
Jiao, H.,
Yi, T.,
and Krystal, G.
(1995)
J. Biol. Chem.
270,
23402-23408 |
| 13. |
Barber, D. L.,
Mason, J. M.,
Fukazawa, T.,
Reedquist, K. A.,
Druker, B. J.,
Band, H.,
and D'Andrea, A. D.
(1997)
Blood
89,
3166-3174 |
| 14. |
Miura, O.,
Miura, Y.,
Nakamura, N.,
Quelle, F. W.,
Witthuhn, B. A.,
Ihle, J. N.,
and Aoki, N.
(1994)
Blood
84,
4135-4141 |
| 15. |
Damen, J. E.,
Liu, L.,
Cutler, R. L.,
and Krystal, G.
(1993)
Blood
82,
2296-2303 |
| 16. |
Barber, D. L.,
Corless, C. N.,
Xia, K.,
Roberts, T. M.,
and D'Andrea, A. D.
(1997)
Blood
89,
55-64 |
| 17. |
Geier, S. J.,
Algate, P. A.,
Carlberg, K.,
Flowers, D.,
Friedman, C.,
Trask, B.,
and Rohrschneider, L. R.
(1997)
Blood
89,
1876-1885 |
| 18. |
Liu, Q.,
Shalaby, F.,
Jones, J.,
Bouchard, D.,
and Dumont, D. J.
(1998)
Blood
91,
2753-2759 |
| 19. | Pesesse, X., Deleu, S., De Smedt, F., Drayer, L., and Erneux, C. (1997) Biochem. Biophys. Res. Commun. 239, 697-700[CrossRef][Medline] [Order article via Infotrieve] |
| 20. |
Lamkin, T. D.,
Walk, S. F.,
Liu, L.,
Damen, J. E.,
Krystal, G.,
and Ravichandran, K. S.
(1997)
J. Biol. Chem.
272,
10396-10401 |
| 21. |
Damen, J. E.,
Liu, L.,
Rosten, P.,
Humphries, R. K.,
Jefferson, A. B.,
Majerus, P. W.,
and Krystal, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1689-1693 |
| 22. |
Barber, D. L.,
DeMartino, J. C.,
Showers, M. O.,
and D'Andrea, A. D.
(1994)
Mol. Cell. Biol.
14,
2257-2265 |
| 23. |
Liu, Q.,
Sasaki, T.,
Dumont, D. J.,
and Penninger, J. M.
(1999)
Genes Dev.
13,
786-791 |
| 24. |
Lioubin, M. N.,
Algate, P. A.,
Tsai, S.,
Carlberg, K.,
Aebersold, A.,
and Rohrschneider, L. R.
(1996)
Genes Dev.
10,
1084-1095 |
| 25. |
Liu, L.,
Damen, J. E.,
Hughes, M. R.,
Babic, I.,
Jirik, F. R.,
and Krystal, G.
(1997)
J. Biol. Chem.
272,
8983-8988 |
| 26. |
D'Andrea, A. D.,
Yoshimura, A.,
Youssoufian, H.,
Zon, L. I.,
Koo, J.-W.,
and Lodish, H. F.
(1991)
Mol. Cell. Biol.
11,
1980-1987 |
| 27. |
Osborne, M. A.,
Zenner, G.,
Lubinus, M.,
Zhang, X.,
Songyang, Z.,
Cantley, L. C.,
Majerus, P.,
Burn, P.,
and Kochan, J. P.
(1996)
J. Biol. Chem.
271,
29271-29278 |
| 28. |
Aman, M. J.,
Lamkin, T. D.,
Okada, H.,
Kurosaki, T.,
and Ravichandran, K. S.
(1998)
J. Biol. Chem.
273,
33922-33928 |
| 29. | Huber, M., Helgason, C. D., Scheid, M. P., Duronio, V., Humphries, R. K., and Krystal, G. (1998) EMBO J. 17, 7311-7319[CrossRef][Medline] [Order article via Infotrieve] |
| 30. |
Zamorano, J.,
and Keegan, A. D.
(1998)
J. Immunol.
161,
859-867 |
| 31. |
Liu, L.,
Damen, J. E.,
Cutler, R. L.,
and Krystal, G.
(1994)
Mol. Cell. Biol.
14,
6926-6935 |
| 32. | Ponzetto, C., Bardelli, A., Zhen, Z., Maina, F., dallaZonca, P., Giordano, S., Graziani, A., Panayotou, G., and Comoglio, P. M. (1994) Cell 77, 261-271[CrossRef][Medline] [Order article via Infotrieve] |
| 33. |
Zhu, H.,
Naujokas, M. A.,
Fixman, E. D.,
Torossian, K.,
and Park, M.
(1994)
J. Biol. Chem.
269,
29943-29948 |
| 34. | Huang, L., Turck, C. W., Rao, P., and Peters, K. G. (1995) Oncogene 11, 2097-2103[Medline] [Order article via Infotrieve] |
| 35. |
Kontos, C. D.,
Stauffer, T. P.,
Yang, W. P.,
York, J. D.,
Huang, L.,
Blanar, M. A.,
Meyer, T.,
and Peters, K. G.
(1998)
Mol. Cell. Biol.
18,
4131-4140 |
| 36. |
Miller, B. A.,
Barber, D. L.,
Bell, L. L.,
Beattie, B. K.,
Zhang, M.-Y.,
Rothblum, L. I.,
and Cheung, J. Y.
(1999)
J. Biol. Chem.
274,
20465-20472 |
| 37. |
DelaChapelle, A.,
Traskelin, A.-L.,
and Juvonen, E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
4495-4499 |
| 38. |
Sokol, L.,
Luhovy, M.,
Guan, Y.,
Prchal, J. F.,
Semenza, G. L.,
and Prchal, J. T.
(1995)
Blood
86,
15-22 |
| 39. | Furukawa, T., Sakaue, M., Otsuka, T., Narita, M., Koike, T., Kishi, K., Utsumi, H., and Aizawa, Y. (1996) Blood 88, 145a (abstr.) |
| 40. |
Kralovics, R.,
Indrak, K.,
Stopka, T.,
Berman, B. W.,
Prchal, J. F.,
and Prchal, J. T.
(1997)
Blood
90,
2057-2061 |
| 41. | Kralovics, R., Sokol, L., Tze, L., Guan, Y., Prchal, J. F., and Prchal, J. T. (1995) Blood 86, 18a (abstr.) |
| 42. |
Helgason, C. D.,
Damen, J. E.,
Rosten, P.,
Grewal, R.,
Sorensen, P.,
Chappel, S. M.,
Borowski, A.,
Jirik, F.,
Krystal, G.,
and Humphries, R. K.
(1998)
Genes Dev.
12,
1610-1620 |
| 43. |
Iwatsuki, K.,
Endo, T.,
Misawa, H.,
Yokouchi, M.,
Matsumoto, A.,
Ohtsubo, M.,
Mori, K. J.,
and Yoshimura, A.
(1997)
J. Biol. Chem.
272,
8149-8152 |
| 44. |
Socolovsky, M.,
Dusanter-Fourt, I.,
and Lodish, H. F.
(1997)
J. Biol. Chem.
272,
14009-14012 |
| 45. | Damen, J. E., Wakao, H., Miyajima, A., Krosl, J., Humphries, R. K., Cutler, R. L., and Krystal, G. (1995) EMBO J. 14, 5557-5568[Medline] [Order article via Infotrieve] |
| 46. | Quelle, F. W., Wang, D., Nosaka, T., Thierfelder, W. E., Stravopodis, D., Weinstein, Y., and Ihle, J. N. (1996) Mol. Cell. Biol. 16, 1622-1631[Abstract] |
| 47. |
Bergelson, S.,
Klingmuller, U.,
Socolovsky, M.,
Hsiao, J. G.,
and Lodish, H. F.
(1998)
J. Biol. Chem.
273,
2396-2401 |
| 48. | Coffer, P. J., Jin, J., and Woodgett, J. R. (1998) Biochem. J. 335, 1-13 |
| 49. |
Odai, H.,
Sasaki, K.,
Iwamatsu, A.,
Hanazono, Y.,
Tanaka, T.,
Mitani, K.,
Yazaki, Y.,
and Hirai, H.
(1995)
J. Biol. Chem.
270,
10800-10805 |
| 50. |
Miller, B. A.,
Cheung, J. Y.,
Tillotson, D. L.,
Hope, S. M.,
and Scaduto, R. C., Jr.
(1989)
Blood
73,
1188-1194 |
| 51. | Cheung, J. Y., Elensky, M. B., Brauneis, U., R. C., Scaduto, J., Bell, L. L., Tillotson, D. L., and Miller, B. A. (1992) J. Clin. Invest. 90, 1850-1856 |
| 52. | Miller, B. A., Bell, L. L., Lynch, C. J., and Cheung, J. Y. (1994) Cell Calcium 16, 481-490[CrossRef][Medline] [Order article via Infotrieve] |
| 53. |
Cheung, J. Y.,
Zhang, X.-Q.,
Bokvist, K.,
Tillotson, D. L.,
and Miller, B. A.
(1997)
Blood
89,
92-100 |
| 54. | Kavanaugh, W., Pot, D., Chin, S., Deuterreinhard, M., Jefferson, A., Norris, F., Masiarz, F., Couseens, L., Majerus, P., and Williams, L. (1996) Curr. Biol. 6, 438-445[CrossRef][Medline] [Order article via Infotrieve] |
| 55. | Liu, Q., and Dumont, D. J. (1997) Genomics 39, 109-112[CrossRef][Medline] [Order article via Infotrieve] |
| 56. |
Odai, H.,
Sasaki, K.,
Iwamatsu, A.,
Nakamoto, T.,
Ueno, H.,
Yamagata, T.,
Mitani, K.,
Yazaki, Y.,
and Hirai, H.
(1997)
Blood
89,
2745-2756 |
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 P. M. Sharma, H.-S. Son, S. Ugi, W. Ricketts, and J. M. Olefsky Mechanism of SHIP-Mediated Inhibition of Insulin- and Platelet-Derived Growth Factor-Stimulated Mitogen-Activated Protein Kinase Activity in 3T3-L1 Adipocytes Mol. Endocrinol., February 1, 2005; 19(2): 421 - 430. [Abstract] [Full Text] [PDF]
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J. M.-Y. Ho, M. H.-H. Nguyen, J. K. Dierov, K. M. Badger, B. K. Beattie, P. Tartaro, R. Haq, B. W. Zanke, M. P. Carroll, and D. L. Barber TEL-JAK2 constitutively activates the extracellular signal-regulated kinase (ERK), stress-activated protein/Jun kinase (SAPK/JNK), and p38 signaling pathways Blood, July 30, 2002; 100(4): 1438 - 1448. [Abstract] [Full Text] [PDF]
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R. Haq, A. Halupa, B. K. Beattie, J. M. Mason, B. W. Zanke, and D. L. Barber Regulation of Erythropoietin-induced STAT Serine Phosphorylation by Distinct Mitogen-activated Protein Kinases J. Biol. Chem., May 3, 2002; 277(19): 17359 - 17366. [Abstract] [Full Text] [PDF]
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C. P. Miller, D. W. Heilman, and D. M. Wojchowski Erythropoietin receptor-dependent erythroid colony-forming unit development: capacities of Y343 and phosphotyrosine-null receptor forms Blood, February 1, 2002; 99(3): 898 - 904. [Abstract] [Full Text] [PDF]
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K. Kirito, K. Nakajima, T. Watanabe, M. Uchida, M. Tanaka, K. Ozawa, and N. Komatsu Identification of the human erythropoietin receptor region required for Stat1 and Stat3 activation Blood, January 1, 2002; 99(1): 102 - 110. [Abstract] [Full Text] [PDF]
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M. K. Henry, J. T. Lynch, A. K. Eapen, and F. W. Quelle DNA damage-induced cell-cycle arrest of hematopoietic cells is overridden by activation of the PI-3 kinase/Akt signaling pathway Blood, August 1, 2001; 98(3): 834 - 841. [Abstract] [Full Text] [PDF]
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D. L. Barber, B. K. Beattie, J. M. Mason, M. H.-H. Nguyen, M. Yoakim, B. G. Neel, A. D. D'Andrea, and D. A. Frank A common epitope is shared by activated signal transducer and activator of transcription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation Blood, April 15, 2001; 97(8): 2230 - 2237. [Abstract] [Full Text] [PDF]
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P. Bruhns, F. Vely, O. Malbec, W. H. Fridman, E. Vivier, and M. Daeron Molecular Basis of the Recruitment of the SH2 Domain-containing Inositol 5-Phosphatases SHIP1 and SHIP2 by Fcgamma RIIB J. Biol. Chem., November 22, 2000; 275(48): 37357 - 37364. [Abstract] [Full Text] [PDF]
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M. E. March, D. M. Lucas, M. J. Aman, and K. S. Ravichandran p135 Src Homology 2 Domain-containing Inositol 5'-Phosphatase (SHIPbeta ) Isoform Can Substitute for p145 SHIP in Fcgamma RIIB1-mediated Inhibitory Signaling in B Cells J. Biol. Chem., September 22, 2000; 275(39): 29960 - 29967. [Abstract] [Full Text] [PDF]
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A. Arai, E. Kanda, Y. Nosaka, N. Miyasaka, and O. Miura CrkL Is Recruited through Its SH2 Domain to the Erythropoietin Receptor and Plays a Role in Lyn-mediated Receptor Signaling J. Biol. Chem., August 24, 2001; 276(35): 33282 - 33290. [Abstract] [Full Text] [PDF]
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X. Pesesse, V. Dewaste, F. De Smedt, M. Laffargue, S. Giuriato, C. Moreau, B. Payrastre, and C. Erneux The Src Homology 2 Domain Containing Inositol 5-Phosphatase SHIP2 Is Recruited to the Epidermal Growth Factor (EGF) Receptor and Dephosphorylates Phosphatidylinositol 3,4,5-Trisphosphate in EGF-stimulated COS-7 Cells J. Biol. Chem., July 20, 2001; 276(30): 28348 - 28355. [Abstract] [Full Text] [PDF]
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T. C. Wagner, S. Velichko, D. Vogel, M. R. S. Rani, S. Leung, R. M. Ransohoff, G. R. Stark, H. D. Perez, and E. Croze Interferon Signaling Is Dependent on Specific Tyrosines Located within the Intracellular Domain of IFNAR2c. EXPRESSION OF IFNAR2c TYROSINE MUTANTS IN U5A CELLS J. Biol. Chem., January 4, 2002; 277(2): 1493 - 1499. [Abstract] [Full Text] [PDF]
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