|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 443-451
Erythropoietin Induces Tyrosine Phosphorylation of Jak2, STAT5A, and
STAT5B in Primary Cultured Human Erythroid Precursors
By
Atsushi Oda,
Kenichi Sawada,
Brian J. Druker,
Katsutoshi Ozaki,
Hina Takano,
Kazuki Koizumi,
Yoshikazu Fukada,
Makoto Handa,
Takao Koike, and
Yasuo Ikeda
From the Division of Hematology, Department of Internal Medicine,
Keio University, Tokyo, Japan; the Blood Center, Keio University,
Tokyo, Japan; The Department of Internal Medicine II, Hokkaido
University School of Medicine Sapporo, Hokkaido 060, Japan; and the
Division of Hematology and Medical Oncology, Oregon Health Sciences
University, Portland, OR.
 |
ABSTRACT |
We examined signaling by erythropoietin in highly purified human
colony forming unit-erythroid cells, generated in vitro from CD34+ cells. We found that erythropoietin induces
tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. Tyrosine
phosphorylation of Jak2 reaches a peak around 10 minutes after
stimulation and is maximum at 5 U/mL of erythropoietin. Tyrosine
phosphorylation of STAT5 is accompanied by the translocation of
activated STAT5 to the nucleus as shown by electrophoretic mobility
shift assay (EMSA) using 32Pi-labeled STAT5 binding site in
the  -casein promoter. Tyrosine phosphorylation STAT1 or STAT3 was
not detected in human erythroid precursors after stimulation with
erythropoietin. Crkl, an SH2/SH3 adapter protein, becomes
coimmunoprecipitated specifically with STAT5 from
erythropoietin-stimulated erythroid cells; although it was shown to
become associated with c-Cbl in the studies using cell lines. Thus,
human erythroid precursors can be expanded in vitro in sufficient
numbers and purity to allow its usage in signal transduction studies.
This report sets a basis for further studies on signaling in primary
cultured human erythroid precursors, which in turn contribute to our
better understanding in the differentiation processes of erythrocytes
and their precursors.
| |
INTRODUCTION |
ERYTHROPOIETIN is a glycoprotein hormone
that is essential for normal erythropoiesis.1-4 In vitro,
erythropoietin is absolutely required for the survival, proliferation,
and differentiation of erythroid precursors, although a recent study
showed that interleukin-6 (IL-6) and its soluble form of the receptor
may also have a similar effect on the erythroid precursors in the
presence of stem cell factor (SCF).5 The effect of
erythropoietin is exerted through binding to the specific receptors on
the surface of responding cells, leading to dimerization of the
receptors and activation of Jak2 tyrosine kinase.6 Whether
Jak2 is preassociated with the erythropoietin receptor before the
ligand binding or not is a controversial issue.7,8 A recent
report suggests that the association is a multistep process, partially
regulated through phosphorylation of the receptor on tyrosine and
serine residues.9 It was reported by many that activation
of Jak2 is accompanied by tyrosine phosphorylation of numerous proteins
including Jak2 itself, STAT proteins, Shc, Vav, and the erythropoietin
receptor, although tyrosine kinases other than Jak2 may also
phosphorylate these proteins.4,6-16 Colony-forming
unit-erythroid (CFU-E) comprises only 0.5% or less of all cells in
normal bone marrow.1,2 Accordingly, most of the studies
cited above have used murine factor-dependent cell lines genetically
engineered to express erythropoietin receptors or murine and human
leukemic cell lines responding to erythropoietin. Although such cells
would undergo differentiation in response to erythropoietin, the
signaling processes in them could be affected by cellular
transformation.
A few studies have used primary erythroid cells from rodents or
chickens.8,15,16 We show here that purified erythroid progenitors can be expanded from CD34+ cells in human
peripheral blood in sufficient numbers to allow the examination of
early signaling by erythropoietin.
| |
MATERIALS AND METHODS |
Reagents.
HEPES, sodium dodecyl sulfate (SDS), 2-mercaptoethanol (2-ME), sodium
orthovanadate, bovine serum albumin (BSA), DNase, chicken egg albumin,
Iscove's Modified Dulbecco's Medium (IMDM), propidium iodide (PI),
protein A-Sepharose, protein G-Sephalose, isopropyl -D-thiogalactopyranoside (IPTG), Triton X-100, and Tris
(hydroxymethyl) aminomethane (Tris) were purchased from Sigma (St
Louis, MO). Polyvinylidene difluoride (PVDF) membranes (pore size, 0.45 µm) were from Millipore Corporation (Bedford, MA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) molecular standards and 32Pi were from Amersham (Arlington Heights,
IL) or BioRad (Richmond, CA). Double stranded Poly (dI-dC) was from
Pharmacia Biotech (Milwaukee, WI). Enhanced chemiluminescence (ECL)
reagents including secondary antibodies were purchased from Amersham.
Insulin (porcine sodium, activity United States Pharmacopoeia [USP]
U/mg) was purchased from Calbiochem and Behring
Diagnostics, La Jolla, CA). Antiphosphotyrosine murine monoclonal
antibody (4G10) was used as described.17-19 STAT3, Crkl and
Jak2 antisera, and anti-STAT1 and glutathione S-transferase (GST)
monoclonal antibodies were from Santa Cruz (Santa Cruz, CA). An
anti-STAT5 monoclonal antibody was from Transduction Laboratories (Lexington, KY). Nitroblue tetrazolium chloride (NBT) and 5-bromo-4 chloro-3-indolyl phosphate p-toluidine salt (BCIP) were from GIBCO BRL
(Gaithersburg, MD). For EMSA, an anti-STAT5 antiserum (a
kind gift from Dr Hiroshi Wakao, University of Tokyo, Tokyo, Japan), described previously, was used.20 STAT5A and STAT5B
antisera were from R&D systems (Minneapolis, MN). Recombinant
erythropoietin, IL-6, granulocyte-macrophage colony-stimulating factor
(GM-CSF), and SCF were kindly donated from Kirin Brewery
Co Ltd (Tokyo, Japan). Erythropoietin was also kindly donated from
Chugai Pharmaceutical Co Ltd (Tokyo, Japan). Vitamin B 12 and folic acid were from Sankyo Pharmaceutical Co (Tokyo, Japan) and
Takeda Pharmaceutical Co (Osaka, Japan), respectively. Human IL-3
(108 chronic myelogenous leukemia U/mg) was from Amgen
Biologicals (Thousand Oaks, CA). Dynabeads M 450 coated with goat
antimouse IgG were from Dynal Inc (Great Neck, NY). Fetal calf serum
(FCS), penicillin, and streptomycin were from Flow Laboratories Inc
(McLean, VA).
Ex vivo generation of erythroid progenitor cells.
Recombinant human granulocyte colony-stimulating factor (G-CSF; Chugai
Pharmaceutical Co and Kyohwa Hakkoh Pharmaceutical Co, Tokyo, Japan)
was administered to healthy adult volunteers (who had previously signed
consent forms approved by the Hokkaido University School of Medicine
and the Hokkaido Red Cross Blood Center Committee for the Protection of
Human Subjects), as described elsewhere.21 The mobilized
peripheral blood (PB) CD34+ cells were isolated with
immunomagnetic beads, as described in detail
elsewhere.22,23 The cells were then cryopreserved and stored until use in a tank with liquid nitrogen. The frozen PB CD34+ cells were thawed, suspended in IMDM
containing 30% heat inactivated and 100 U/mL DNase, and were
centrifuged at 400g for 5 minutes at 4°C. The cells were
washed two times with IMDM containing 20% FCS, and resuspended in IMDM
containing 0.3% deionized bovine serum albumin (BSA). The cells were
then cultured in liquid phase, as described elsewhere with minor
modification.24 In brief, the cells ranging from 0.5 × 104 to 2.0 × 104 cells/mL were
suspended in a mixture containing 20% FCS, 10% heat-inactivated
pooled human AB serum, 1% BSA, 10 µg/mL insulin, vitamin B 12 at 10 µg/mL, and folic acid at 15 µg/mL, with recombinant human IL-3 at
100 U/mL, recombinant human SCF at 100 ng/mL, and erythropoietin
(180,000 U/mg) at 4 U/mL, in the presence of 5 × 10 5 mol/mL 2-ME, penicillin at 50 U/mL and
streptomycin at 50 U/mL, and IMDM in a 50 mL polystyrene flask (Corning
Costar Corp, Cambridge, MA). After incubation for 8 days at 37°C in
a 5% CO2/95% atmosphere, the cells were collected, washed
twice with IMDM containing 0.3% BSA, and were factor starved in IMDM
containing 20% FCS for 12 hours (day-8
cells).25-27
Semisolid culture of progenitors.
Day-8 cells were incubated in triplicate at a concentration of 500 cells/mL in flat-bottomed, 48-well, tissue culture plates (Linbro, Flow
Laboratories) with 0.25 mL serum-free fibrin clots, as described with
erythropoietin at 2 U/mL with or without SCF at 100 ng/mL and IL-3 at
100 U/mL.25 After 7 days of incubation at 37°C in a 5%
CO2/95% atmosphere, the clots were fixed and stained with
benzidine-hematoxylin.26 Erythroid colony forming cells (ECFC) were defined as cells that gave rise to colonies of 2 to 49 hemoglobinized cells after 7 days of culture of day-8
cells.27
Flow cytometry.
Phenotyping of the purified cells and day-8 cells was analyzed by
fluorescence-activated cell sorting vantage (Becton Dickinson, Mountain
View, CA) run by Lysis II, as reported previously.28 In
brief, the cells were washed twice with staining medium (SM; phosphate
buffered saline [PBS] supplemented with 0.05% NaN3 and 3% FCS). After counting the number of cells, 1 × 106
cells were incubated with HPCA-2 (8G12, fluorescein
isothiocyanate-conjugated [FITC] or phycoerythrin-conjugated [PE],
Becton Dickinson), glycophorin A (Gly A FITC; DAKO Japan Co, Kyoto,
Japan), My7 (CD13 PE; Coulter Immunology, Hialeah, FL), and CD117
(c-kit PE; Immunotech, Marseilles, France) for 20 minutes on ice. The
cells were washed with SM twice and resuspended in 1 mL of SM
containing 5 µg/mL of PI. Cell aggregates and dead cells were
excluded using forward and side scatter and PI staining,
respectively, and 10,000 events were analyzed.
Immunoprecipitation, gel electrophoresis, and Western blotting.
After starvation, day-8 cells were stimulated with erythropoietin.
Cells were lysed by the addition of an equal amount of lysis buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride
(PMSF), 10 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 0.8 µg/mL leupeptin, 2% Triton X-100 (vol/wt), pH 7.4). After 20 minutes
on ice, the lysates were centrifuged at 10,000g (at 4°C)
for 20 minutes. The supernatant was removed and precleared with
preimmune serum and protein A-Sepharose or protein
G-Sephalose (40 µL of 50% slurry) for 1 hour. Antisera were then
added and incubated for 2 to 3 hours on ice. Protein A-Sepharose (40 µL of 50% slurry) was added and incubated for 1 hour. The immune complexes were washed with 1 mL of cold washing buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L PMSF, 10 mmol/L EGTA, 1 mmol/L sodium
orthovanadate, 0.8 µg/mL leupeptin, 1% Triton X-100 (vol/wt), pH
7.4) three times and then resuspended in Laemmli's sample buffer (10%
glycerol, 1% SDS, 5% 2-ME, 50 mmol/L Tris-HCl (pH 6.8),29 and 0.002% bromophenol blue),24 with 10 mmol/L EGTA and 1 mmol/L sodium orthovanadate. After boiling at 95°C for 5 minutes,
one-dimensional electrophoresis was performed on SDS 10% or 7.5 to
15% polyacrylamide gels as described.30 Separated proteins
were electrophoretically transferred from the gel onto PVDF membranes
or nitrocellulose in a buffer containing Tris (25 mmol/L), glycine (192 mmol/L) and 20% methanol at 0.2 amps for 12 hours at room temperature. To block residual protein binding sites, membranes were
incubated in TBST (Tris-buffered saline [TBS]; 10 mmol/L Tris, 150 mmol/L NaCl, pH 7.6 with 0.1% Tween 20) with 10% chicken egg albumin. The blots were washed with TBST and incubated overnight with primary antibodies at a final concentration of 1.0 µg/mL in TBST. The primary
antibody was removed and the blots were washed four times in TBST and
incubated with horseradish peroxidase-conjugated or alkaline
phosphatase-conjugated second antibodies diluted 1:3000 in TBST. Blots
were then washed four times in TBST. Antibody reactions were detected
with NBT/BCIP or chemiluminescence according to the manufacturer's
instructions.
EMSA.
Nuclear extracts (40 µL, containing 10 to 20 µg protein) were
prepared from day-8 cells (2 × 107 cells) by lysis
followed by high salt extraction as described previously.20
Two microliters of nuclear extracts were mixed with 20 µL of binding
buffer [10 mmol/L Tris-HCl, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
1,4 dithiothreitol, 0.1% NP-40, 5% glycerol, 1 mg/mL
bovine serum albumin, and 2 mg/mL poly(dI-dC), pH 7.5] containing
50,000 cpm end-labeled bovine -casein promoter probe (5 -AGATTCTAGGAATTCAAATC-3). The mixture was incubated for
30 minutes at room temperature. Complexes were separated on 5%
nondenaturing polyacrylamide gels in 0.25 × Tris borate EDTA
buffer and detected by autoradiography.
TF-1 cell.
Human IL-3 and GM-CSF-dependent TF-1 cell line was kindly provided by
Dr Hisamaru Hirai (University of Tokyo, Tokyo, Japan).30 Cells were maintained in IMDM containing 10% fetal calf serum (FCS)
and 1 ng/mL human GM-CSF. Before stimulation with GM-CSF or IL-3, cells
were incubated in IMDM containing 10% FCS without exogenous IL-3 or
GM-CSF for 18 hours. After two washes, they were incubated in PBS (pH
7.4) at 37°C.
GST binding assays.
The GST-SH2 domain of Crkl fusion protein (GST-Crkl-SH2) was generated
as previously described.30,31 The GST-fusion construct was
transformed into Escherichia coli DH-5 and protein
expression was induced by 0.5 mmol/L IPTG to exponentially growing
cells. The GST-Crkl-SH2 was isolated from sonicated bacterial lysates using glutathione sepharose beads. A total of 2.5 µg of GST-fusion proteins were incubated with 50 µL of glutathione sepharose beads in
bacterial lysis buffer (150 mmol/L NaCl, 16 mmol/L
Na2HPO4, 4 mmol/L
NaH2PO4, [pH 7.3] containing 10 µg of
aprotinin, 1 mmol/L orthovanadate, 1 mmol/L PMSF, and 0.1%
-mercaptoethanol). The beads were washed three times with PBS and
were incubated with cell lysates for 3 hours on ice. Proteins were
separated by SDS-PAGE and transferred onto PVDF membranes for
immunoblot analysis.
| |
RESULTS |
The purified fraction from mobilized PB contained 96.7% ± 1.6% (n = 3, mean ± SD) CD34+ cells. Cultivation of PB
CD34+ cells for 8 days in serum-containing medium with
erythropoietin, IL-3, and SCF resulted in a 120 ± 49-fold expansion
of the total cell number (day-8 cells, n = 8) with a viability of 96% ± 3%. A total of 71% ± 13% cells expressed the specific
erythroid marker Gly A, whereas CD34 and myelomonocytic marker CD13
were almost absent, as shown in Table 1. In
morphologic analysis by May-Grunwald-Giemsa staining, day-8 cells
predominantly consisted of immature erythroid cells with a purity of
92% ± 6%,27 whereas most of the remaining cells
showed a basophilic/eosinophilic-like feature. The maturation level of
erythroid progenitor cells closely associates with colony stimulating
factor (CSF) requirements for erythroid development.24 When
CSF requirements of day-8 cells were analyzed in serum-free medium, the
ECFC required erythropoietin alone but not IL-3 and SCF for erythroid
development, which indicates that ECFC in day-8 cells predominantly
consists of mature erythroid progenitor cells. The erythroid progenitor
cells that gave rise to aggregates with 8 to 49 erythroblasts and that
were equivalent to CFU-E, consisted 78.8% ± 2.5%, whereas the
rest of ECFC gave rise to aggregates with 2 to 7 erythroblasts.
We then set out to examine some early signaling events in these human
erythroid precursor cells. When Jak2 was immunoprecipitated from
lysates of erythroid cells before and after stimulation with erythropoietin (10 U/mL), we saw the inducible tyrosine phosphorylation of Jak2 (Fig 1A, upper panel). Jak2 was
equally immunoprecipitated before and after stimulation (Fig 1A, lower
panel). Time course experiments of Jak2 tyrosine phosphorylation showed
tyrosine phosphorylation within 1 minute that reached a peak within
approximately 10 minutes after addition of erythropoietin and then
decreased (10 U/mL; Fig 1B, upper panel). Erythropoietin induced
protein tyrosine phosphorylation in erythroid cells in a dose-dependent
manner (0.5 to 5 U/mL; Fig 1C, upper panel). Next, we examined whether STAT5A and B become tyrosine phosphorylated in erythroid cells stimulated with erythropoietin (10 U/mL, for 10 minutes). A STAT5 monoclonal antibody recognizes both STAT5A and STAT5B
(Fig 2, lower panel). The latter moved
faster than STAT5A in the gel. Both STAT5A and STAT5B became tyrosine
phosphorylated (Fig 2, upper panel). The thin tyrosine phosphorylated
band, which moved slower than STAT5B, may be STAT5A, because STAT5B
antisera are weakly cross reactive with STAT5A according to the
manufacturers' instructions. After tyrosine phosphorylation, STAT5
dimerizes and translocates to the nucleus where it activates or
represses transcription.12,13 We next examined whether
treatment of the erythroid cells with erythropoietin (10 U/mL, for 20 minutes) resulted in the binding of STAT5 to a phosphorylated probe
from the -casein promoter by EMSA (Fig
3). Nuclear extracts were prepared from the erythroid cells before and
after stimulation with erythropoietin (10 U/mL, for 20 minutes).
Erythropoietin induced the electrophoretic shift of the end-labeled
probe in the erythroid cells (Fig 3, shift). STAT5 antisera
supershifted the DNA-protein complex and bound to the labeled probe
(Fig 3, super shift), indicating that STAT5 is activated by
erythropoietin. In contrast, neither STAT3 nor STAT1 was tyrosine
phosphorylated in the erythroid cells stimulated by erythropoietin
(Fig 4, upper panels; 10 U/mL, for 10 minutes), whereas IL-6-induced tyrosine phosphorylation of STAT3 in
TF-1 was readily detected under the same experimental
conditions.11,12,32 Needless to say, we cannot rule out
slight erythropoietin-induced tyrosine phosphorylation of STAT1 and
STAT3 under the threshold of our detection system.
View larger version (23K):
[in this window]
[in a new window]
View larger version (33K):
[in this window]
[in a new window]
View larger version (34K):
[in this window]
[in a new window]
| Fig 1.
(A, upper panel) Tyrosine phosphorylation of Jak2 in
human erythroid cells stimulated by erythropoietin (10 U/mL). Day-8
cells were lysed by the addition of an equal amount of a buffer
containing 2% Triton X-100 before and after exposure to erythropoietin
(10 U/mL). Jak2 was immunoprecipitated with specific Jak2 antisera. Immune complexes were resuspended in SDS sample buffer. Tyrosine phosphorylation of Jak2 was detected by 4G10 as described in the Materials and Methods. Bands were visualized by chemiluminescence. (A,
lower panel) The same nylon membrane used in A was stripped of the
antibody and reprobed for Jak2. Bands were visualized by NBT/BCIP.
Lanes are the same as in (A). (B, upper and lower panels) The time
course of tyrosine phosphorylation of Jak2. Tyrosine phosphorylation of
Jak2 was detected as described in (A). Lane 1, resting erythroid cells.
Lanes 2 to 5, 1 minute, 5 minutes, 10 minutes, and 30 minutes after
exposure to erythropoietin (10 U/mL). (B, upper and lower panels) The
time course of tyrosine phosphorylation of Jak2. Day-8 cells were
stimulated with erythropoietin (10 U/mL) for the indicated time.
Tyrosine phosphorylation of Jak2 was detected as described in (A). (C,
upper and lower panels) The dose response of tyrosine phosphorylation
of Jak2. Day-8 cells were stimulated with different concentrations of
erythropoietin for 10 minutes. Tyrosine phosphorylation of Jak2 was
detected as described in (A).
{/ANNT;4224n;;0n;0n}A
|
|
View larger version (54K):
[in this window]
[in a new window]
| Fig 2.
(upper panel) Tyrosine phosphorylation of STAT5 in the
erythroid day-8 cells stimulated by erythropoietin (10 U/mL) for 10 minutes. The erythroid cells were lysed by the addition of an equal
amount of a buffer containing 2% Triton X-100 before and after
exposure to erythropoietin (10 U/mL). STAT5 was immunoprecipitated with STAT5A or STAT5B antisera as indicated. Immune complexes were
resuspended in SDS sample buffer. Tyrosine phosphorylation of STAT5 was
detected by 4G10 as described in Fig 1A. Bands were visualized by
chemiluminescence. (lower panel) The same nylon membrane was stripped
of the antibody and reprobed for STAT5 with an anti-STAT5 monoclonal
antibody. Bands were visualized by chemiluminescence.
|
|
View larger version (51K):
[in this window]
[in a new window]
| Fig 3.
Erythropoietin activates STAT5 in erythroid cells. Growth
factor-deprived day-8 cells were stimulated with 10 U/mL erythropoietin and nuclear extracts were prepared. Nuclear extracts were subject to
EMSA by using a -casein probe. Lane 1, nuclear extracts from unstimulated cells; lane 2, nuclear extracts from
erythropoietin-stimulated cells; lane 3, the same as in lane 2, except
the nuclear extracts were incubated with STAT5 antisera before EMSA.
Shift indicates probe complex in the absence of the antibodies. Super
Shift indicates the supershifted band in the presence of STAT5
antisera.
|
|
View larger version (48K):
[in this window]
[in a new window]
View larger version (28K):
[in this window]
[in a new window]
| Fig 4.
(A, upper panel) Tyrosine phosphorylation of STAT3 was
not tyrosine phosphorylated in the erythroid cells stimulated with erythropoietin for 10 minutes. The erythroid cells were lysed by the
addition of an equal amount of a buffer containing 2% Triton X-100
before and after exposure to erythropoietin (10 U/mL). STAT3 was
immunoprecipitated with specific anti-STAT3 antisera. As a positive
control, TF-1 cells were stimulated with IL-6 (100 ng/mL) for 10 minutes. Immune complexes were resuspended in SDS sample buffer.
Tyrosine phosphorylation of STAT3 was detected by 4G10 as described in
Fig 1A. Bands were visualized by chemiluminescence. Lane 1, resting
erythroid cells; lane 2, 10 minutes after exposure to erythropoietin
(10 U/mL); lane 3, resting TF-1 cells, lane 4, 10 minutes after
exposure to IL-6 (100 ng/mL). (lower panel) The same nylon membrane was
stripped of the antibody and reprobed for STAT3. Bands were visualized
by chemiluminescence. Lanes are the same as in upper panel. (B)
Anti-STAT1 was used instead of STAT3 antisera.
|
|
We have recently observed that Crkl, a 39-kD SH2/SH3 adapter protein,
becomes associated with STAT5 in human platelets after stimulation by
thrombopoietin (submitted). Given the similarity between
thrombopoietin and erythropoietin signal transductions, we examined
phosphotyrosine immunoblots of the Crkl immunoprecipitates from
erythropoietin-treated erythroids, which show the presence of a protein
with a molecular weight of approximately 95 to 100 kD
(Fig 5A). This protein was reactive with an
anti-STAT5 monoclonal antibody (Fig 5B, upper panel). Before and after
stimulation the same amount of Crkl was immunoprecipitated from
erythroid cells (Fig 5B, lower panel). To further confirm the
specificity of Crkl-STAT5 interaction, the effects of phenylphosphate
and Crkl immunizing peptide were examined. More STAT5 was
coimmunoprecipitated from erythropoietin-stimulated cells than
starved (resting) cells (Fig 5C, upper panel). Phenylphosphate (20 mmol/L), which is known to disrupt phosphotyrosine-dependent protein
interactions and was added after the lysis of erythropoietin-stimulated
cells, inhibited the coimmunoprecipitation of STAT5 with Crkl (Fig 5C, upper panel) but not immunoprecipitation of Crkl (lower panel). Crkl
immunizing peptide inhibited immunoprecipitation of Crkl and STAT5,
suggesting that the Crkl antisera is specific.
View larger version (24K):
[in this window]
[in a new window]
View larger version (26K):
[in this window]
[in a new window]
View larger version (63K):
[in this window]
[in a new window]
View larger version (31K):
[in this window]
[in a new window]
| Fig 5.
(A and B) Crkl immunoprecipitates from
erythropoietin-stimulated erythroid cells contain a tyrosine
phosphorylated 95- to 100-kD protein, which is also recognized by a
STAT5 monoclonal antibody. Erythroid cells were lysed by the addition
of an equal amount of a buffer containing 2% Triton X-100 before and
after exposure to eythropoietin (10 U/mL for 10 minutes). Crkl was
immunoprecipitated with specific Crkl antisera. The Crkl
immunoprecipitates were divided into two. Tyrosine phosphorylated
proteins (A), STAT5 (B, upper panel), and Crkl (B, lower panel) in the
Crkl immunoprecipitates were detected as described in Fig 1. (C)
Crkl-STAT5 coimmunoprecipitation was inhibited by phenylphosphate
(PP, 20 mmol/L) or Crkl immunizing peptide (20 µg/mL).
Erythroid cells were divided equally to four samples. Three samples
were incubated wtih eythropoietin (10 U/mL for 10 minutes). Crkl was
immunoprecipitated with specific Crkl antisera. STAT5 (upper panel) and
Crkl (lower panel) in the Crkl immunoprecipitates were detected as
described in (A) and (B). (D) Bacterially expressed the SH2 domain of
Crkl (GST-Crkl-SH2) binds to STAT5 from erythropoietin-stimulated
erythroid cells. Lysates from starved ( ) erythroid cells or cells
stimulated with 10 U/mL of erythropoietin for 10 minutes (+) were
analyzed for binding to GST-Crkl-SH2. Bound proteins were separated by
SDS-PAGE, transferred to PVDF membranes and immunoblotted with STAT5
(upper panel) or GST (lower panel) monoclonal antibodies.
|
|
Because phenyl phosphate inhibited the coimmunoprecipitation of STAT5
with Crkl and we only see tyrosine phosphorylation of STAT5 in
erythroid cells, we suspected that the SH2 domain of Crkl may be
responsible for the binding of Crkl to the phosphorylated STAT5.
Accordingly, we examined the binding of STAT5 to bacterially expressed
GST-Crkl-SH2 protein (Fig 5D). Lysates from starved ()
erythroid cells or cells stimulated with 10 U/mL of erythropoietin for
10 minutes (+) were incubated with GST-Crkl-ST2. The SH2 domain showed
only a trace amount of binding to STAT5 from starved cells with a
significant increase using lysates from erythroid cells.
| |
DISCUSSION |
In this study, we examined erythropoietin-signaling in human erythroid
precursors expanded in vitro. This corroborates the recent study of Dai
et al33 and indicates that these studies are feasible.
Because these cells, unlike murine factor-dependent cell lines
genetically engineered to express erythropoietin receptors or murine
and human leukemic cell lines, regularly undergo full terminal
differentiation and become reticulocytes, we believe that these studies
are more likely to be reflective of in vivo situations, although the
presence of the artifacts arising from in vitro culture still cannot be
ruled out. When Drachman et al examined murine mature
megakaryocytes and established cell lines for proximate signal
transduction triggered by thrombopoietin, they found that there were
significant differences in these cells, suggesting that the results of
the studies using the established cell lines may not readily be
extended to primary cells.34 These considerations led us to
examine primary cultured human erythroid precursor cells for early
signaling induced by erythropoietin.
We found that Jak2 became tyrosine phosphorylated in a time- and
dose-dependent fashion in human erythroid cells, which was consistent
with previous reports.6-15 Erythropoietin-induced tyrosine phosphorylation of both STAT5A and STAT5B in human erythroid cells is
interesting, because only STAT5B became tyrosine phosphorylated in
chicken erythroid cells.15 We did not detect any increase in tyrosine phosphorylation of STAT1 and STAT3 in
erythropoietin-stimulated human erythroid cells. On the other hand,
Penta and Sawyer14 found that erythropoietin-induced STAT1
tyrosine phosphorylation and its activation in murine erythroid cells.
Further, in rat erythroid cells from fetal liver, STAT3 was tyrosine
phosphorylated on stimulation with erythropoietin.16 Thus,
these and our current data suggest the presence of species-specific
differences in signaling by erythropoietin, although it is formally
possible that different sensitivities used in detection of STAT5
activation may have contributed to the differences.
Further, our data suggest that erythropoietin and thrombopoietin may
induce tyrosine phosphorylation and activation of different sets of
STATs in human primary cells in spite of their similarity in amino acid
sequence,1-3,35,36 although such differences may be
obscured in the studies that used established cell
lines.8-11,36 We have previously reported that
thrombopoietin induces tyrosine phosphorylation of STAT3 and STAT5 in
thrombopoietin-stimulated human platelets.18 Drachman et
al reported that the same set of STATs was tyrosine
phosphorylated and activated in thrombopoietin-stimulated murine
megakaryocytes.34 Thus, it is possible that the differences in activated STATs by erythropoietin and thrombopoietin may be important in the lineage-specific developments of both erythrocytic and
megakaryocytic precursors.
The differences between the studies that used primary erythroids and
those that used cell lines are not limited to STAT proteins. Barber et
al have shown that Crkl becomes tyrosine phosphorylated and associated with c-Cbl on stimulation by
erythropoietin.37 However, we found that in primary
erythroids Crkl becomes associated with a 95-kD tyrosine phosphorylated
protein, which has been identified as STAT5. This coimmunoprecipitation
of Crkl and STAT5 is not likely caused by the cross reactivity of Crkl
antisera with STAT5 because of the following three reasons (Fig 5): (1)
Crkl antisera coimmunoprecipitated STAT5 mostly after stimulation of
cells with erythropoietin, suggesting that, if this is a cross
reactivity, the Crkl antisera must recognize only the activated form of
STAT5. (2) Crkl immunoprecipitation and STAT5 coimunoprecipitation were inhibited by Crkl immunizing peptides whose amino acid sequence is not
found in STAT5. (3) Phenylphosphate inhibited coimmunoprecipitation of
STAT5 without impairing the immunoprecipitation of Crkl, suggesting that Crkl-STAT5 interaction may be dependent on phosphotyrosine on
STAT5, because we did not detect tyrosine phosphorylation of Crkl. The
last supposition was supported by the results of in vitro binding of
GST-Crkl-SH2 to STAT5 from stimulated erythroid cells (Fig 5D),
suggesting that STAT5 could be binding to Crkl through its
phosphotyrosine-dependent interaction with the SH2 domain of Crkl.
However, STAT5 is thought to dimerize when tyrosine phosphorylated
through its SH2 domain binding to the tyrosine phosphate of another
STAT protein.38,39 Possible explanations for this finding
include the fact that dimerization of STAT5 is required for binding to
Crkl or that erythropoietin treatment results in an altered subcellular
localization of Crkl or STAT5 that allows these proteins to interact.
Alternatively, another protein could be involved in mediating the
complex formation. Although more studies are necessary, our study
indicates that activated STAT5 complex is not simply made of dimerized
STAT5 in human erythroid cells and that the complex may include Crkl and possible other proteins as well. Taken together, our data suggest
that the results of the studies that used the primary erythroids may be
quite different from those obtained from the experiments that were
solely dependent on cell lines, giving a rationale for further
examinations of the signal transduction of primary human erythroid
precursors (Fig 5). Further, the importance of examinations of proximal
signal transduction was supported by the recent reports, suggesting
that abrogation of tyrosine phosphorylation may be related to the
increased erythropoiesis in polycythemic patients.33,40
| |
FOOTNOTES |
Submitted October 3, 1997;
accepted March 13, 1998.
A.O. and K.S. contributed equally to this work.
Supported in part by grants in aid from The Ministry of Education,
Science, Sports and Culture of Japan (A.O., K.S., and Y.I.), a research
grant for Idiopathic Disorders of Hematopoietic Organs Research
Committee from the Ministry of Health and Welfare of Japan (K.S.), the
Ryoichi Naito Foundation for Medical Research (A.O.), a research grant
for the Development of Advanced Therapeutics from Hokkaido University
School of medicine and the Sankyo Life Science Foundation (K.S.), and
research grants for Life Sciences and Medicine, Keio University Medical
Science Fund (A.O.).
Address reprint requests to Atsushi Oda, MD, PhD, Department of
Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Tokyo 160, Japan.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr C.I. Civin for the generous gift of monoclonal antibodies,
without which this work could not have been done. We are grateful to
Drs Hiroshi Wakao and Atsushi Miyajima for helpful discussions. We
thank Dr S.C. Clark, Dr Hiroshi Wakao, Genetics Institute, Kirin
Brewery Co, and Chugai Pharmaceutical Co for the generous gifts of
recombinant human growth factors; Dr Hisamaru Hirai for TF-1 cells; and
Taisho Pharmaceutical Co for providing chymopapain.
| |
REFERENCES |
1.
Krantz SB:
Erythropoietin.
Blood
77:419,
1991[Free Full Text]
2.
Koury MJ,
Bondurant MC:
The molecular mechanism of erythropoietin action.
Eur J Biochem
210:649,
1992[Medline]
[Order article via Infotrieve]
3.
Youssoufian H,
Longmore G,
Neuman D,
Yoshimura A,
Lodish HF:
Structure, function, and activation of the erythropoietin receptor.
Blood
81:2223,
1993[Free Full Text]
4.
Sayer ST,
Penta K:
Erythropoietin cell biology.
Hematol Oncol Clin North Am
8:895,
1994[Medline]
[Order article via Infotrieve]
5.
Sui X,
Tsuji K,
Tajima S,
Tanaka R,
Muraoka K,
Ebihara Y,
Ikebuchi K,
Yasukawa K,
Taga T,
Kishimoto T,
Nakahata T:
Erythropoietin-independent erythrocyte production: Signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.
J Exp Med
183:837,
1996[Abstract/Free Full Text]
6.
Witthuhn BA,
Quelle FW,
Silvennoinen O,
Yi T,
Tang B,
Miura O,
Ihle JN:
Jak2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
Cell
74:227,
1993[Medline]
[Order article via Infotrieve]
7.
Miura O,
Nakamura N,
Quelle FW,
Witthuman BA,
Ihle JN,
Aoki N:
Erythropoietin induces association of the Jak2 protein tyrosine kinase with the erythropoietin receptor in vivo.
Blood
84:1501,
1994[Abstract/Free Full Text]
8.
Wakao H,
Harada N,
Kitamura T,
Mui AL,
Miyajima A:
Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.
EMBO J
14:2527,
1995[Medline]
[Order article via Infotrieve]
8.
Sawyer ST,
Penta K:
Association of Jak2 and STAT5 with erythropoietin receptors.
J Biol Chem
271:32430,
1996[Abstract/Free Full Text]
9.
Quell FW,
Wang D,
Nosaka T,
Thierfelder WE,
Stravopodis D,
Weinstein Y,
Ihle JN:
Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for mitogenic response.
Mol Cell Biol
16:1622,
1996[Abstract]
10.
Ohashi T,
Masuda M,
Ruscetti SK:
Induction of sequence-specific DNA-binding factors by erythropoietin and the spleen focus-forming virus.
Blood
85:1454,
1995[Abstract/Free Full Text]
11.
Klingmuller U,
Bergelson S,
Hsio JG,
Lodish HF:
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Proc Natl Acad Sci USA
93:8324,
1997[Abstract/Free Full Text]
12.
Darnell J Jr:
The JAK-STAT pathway: Summary of initial studies and recent advances.
Recent Prog Horm Res
51:391,
1996
13.
Ihle JN:
Janus kinases in cytokine signaling.
Philos Trans R Soc Lond B Biol Sci
351:159,
1996[Medline]
[Order article via Infotrieve]
14.
Penta K,
Sawyer ST:
Erythropoietin induces the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1 and STAT5 in erythroid cells.
J Biol Chem
270:31282,
1995[Abstract/Free Full Text]
15.
Mellitzer G,
Wesseley O,
Decker T,
Meinke A,
Hayman MJ,
Beug H:
Activation of STAT5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.
Proc Natl Acad Sci USA
93:9600,
1996[Abstract/Free Full Text]
16. (abstr, suppl)
Chreitien S,
Verdier F,
Varlet P,
Gobert S,
Devermy E,
Billat C,
Gisselbrecht,
Mayyeux P,
Lacombe C:
Identification of STAT proteins activated by erythropoietin in normal erythroid progenitors and in murine and human erythroleukemia.
Blood
88:54a,
1996
17.
Miyakawa Y,
Oda A,
Druker BJ,
Kato T,
Miyazaki H,
Handa M,
Ikeda Y:
Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets.
Blood
86:23,
1995[Abstract/Free Full Text]
18.
Miyakawa Y,
Oda A,
Druker BJ,
Miyazaki H,
Handa M,
Ohashi H,
Ikeda Y:
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets.
Blood
87:439,
1996[Abstract/Free Full Text]
19.
Oda A,
Miyakawa Y,
Druker BJ,
Ishida A,
Ozaki K,
Ohashi H,
Wakui M,
Handa M,
Watanabe K,
Okamoto S,
Ikeda Y:
Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin.
Blood
88:4304,
1996[Abstract/Free Full Text]
20.
Wakao H,
Gouilleux F,
Groner B:
Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response.
EMBO J
13:2182,
1994[Medline]
[Order article via Infotrieve]
21.
Sato N,
Sawada K,
Takahashi TA,
Mogi Y,
Asano S,
Koike T,
Sekiguchi S:
A time course study for optimal harvest of peripheral blood progenitor cells by granulocyte colony stimulating factor in healthy volunteers.
Exp Heamatol
22:973,
1994[Medline]
[Order article via Infotrieve]
22.
Sawada K,
Krantz SB,
Dai CH,
Koury ST,
Horn ST,
Glick AD,
Civin CI:
Purification of human blood burst forming units erythroid and demonstration of the evolution of erythropoietin receptors.
J Cell Physiol
142:219,
1993
23.
Yamaguchi M,
Sawada K,
Sato N,
Koizumi K,
Sekiguchi S,
Koike T:
A rapid nylon fiber syringe system to deplete CD14+ cells for positive selection of human blood CD34+ cells. Use of immunomagnetic microspheres.
Bone Marrow Transplant
19:373,
1997[Medline]
[Order article via Infotrieve]
24.
Sawada K,
Krantz SB,
Dai CH,
Sato N,
Ieko M,
Sakurama S,
Yasukouchi T,
Nakagawa S:
Transitional change of colony stimulating factor requirements for erythroid progenitors.
J Cell Physiol
149:1,
1991[Medline]
[Order article via Infotrieve]
25.
Sawada K,
Krantz SB,
Dessypris EN,
Koury ST,
Sawyer ST:
Human colony forming units erythroid do not require accessory cells, but do require direct interaction with insulin like growth factor I and/or insulin for erythroid development.
J Clin Invest
83:1701,
1989
26.
McLeod DL,
Shreeve MM,
Axelrad AA:
Improved plasma culture system for production of erythrocytic colonies in vitro. Quantitative assay method for CFU-E.
Blood
44:517,
1974[Abstract/Free Full Text]
27.
Sawada K,
Krantz SB,
Kans JS,
Dessypris EN,
Sawyer S,
Glick AD,
Civin CI:
Purification of human erythroid colony forming units and demonstration of specific binding of erythropoietin.
J Clin Invest
80:357,
1987
28.
Sawada K,
Sato N,
Notoya A,
Tarumi T,
Hirayama S,
Takano H,
Koizumi K,
Yasukouchi T,
Yamaguchi M,
Koike T:
Proliferation and differentiation of myelodysplastic CD34+ cells: Phenotypic subpopulations of marrow CD34+ cells.
Blood
85:194,
1995[Abstract/Free Full Text]
29.
Laemmli UK:
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680,
1970[Medline]
[Order article via Infotrieve]
30.
Kitamura T,
Tange T,
Terasawa T,
Chiba S,
Kuwaki T,
Miyagawa K,
Piao YF,
Miyazono K,
Urabe A,
Takaku F:
Establishment and characterization of a unique human cell line that proliferates dependent on GM-CSF, IL-3, or erythropoietin.
J Cell Physiol
140:323,
1989[Medline]
[Order article via Infotrieve]
31.
Oda T,
Heaney C,
Hagopian JR,
Okuda K,
Griffin JD,
Druker BJ:
Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia.
J Biol Chem
269:22925,
1994[Abstract/Free Full Text]
32.
Heaney C,
Kolibaba K,
Bhat A,
Oda T,
Ohno S,
Fanning S,
Druker BJ:
Direct binding of CRKL to BCR-ABL is not required for BCR-ABL transformation.
Blood
89:297,
1997[Abstract/Free Full Text]
33.
Dai CH,
Krantz SB,
Sawyer ST:
Polycythemia vera. V. Enhanced proliferation and phosphorylation due to vanadate are diminished in polycythemia vera erythroid progenitor cells: A possible defect of phosphatase activity in polycythemia vera.
Blood
89:3574,
1997[Abstract/Free Full Text]
34.
Drachman JG,
Sabath DF,
Fox NE,
Kaushansy K:
Thrombopoietin signal transduction in purified murine megakaryocytes.
Blood
89:483,
1997[Abstract/Free Full Text]
35.
Kaushansky K:
Thrombopoietin: The primary regulator of platelet production.
Blood
86:419,
1995[Free Full Text]
36.
Nagata Y,
Todokoro K:
Interleukin 3 activates not only Jak2 and STAT5, but also Tyk2, STAT1, and STAT3.
Biochem Biophys Res Commun
221:785,
1996[Medline]
[Order article via Infotrieve]
37.
Barber DL,
Mason JM,
Fukazawa T,
Reedquist KA,
Druker BJ,
Band H,
D'Andrea AD:
Erythropoietin and interleukin-3 activate tyrosine phosphorylation of CBL and association with CRK adaptor proteins.
Blood
89:3166,
1997[Abstract/Free Full Text]
38.
Schindler C,
Darnell JE Jr:
Transcriptional responses to polypeptide ligands: The JAK-STAT pathway.
Annu Rev Biochem
64:621,
1995[Medline]
[Order article via Infotrieve]
39.
Ihle JN:
Signaling by the cytokine receptor superfamily in normal and transformed hematopoietic cells.
Adv Cancer Res
68:23,
1996[Medline]
[Order article via Infotrieve]
40.
Sui X,
Krantz SB,
Zhao Z:
Identification of increased tyrosine phosphatase activity in polycythemia vera erythroid progenitor cells.
Blood
90:651,
1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. Saito, M. Hirokawa, K. Inaba, H. Fukaya, Y. Kawabata, A. Komatsuda, J. Yamashita, and K. Sawada
Phagocytosis of codeveloping megakaryocytic progenitors by dendritic cells in culture with thrombopoietin and tumor necrosis factor-{alpha} and its possible role in hemophagocytic syndrome
Blood,
February 15, 2006;
107(4):
1366 - 1374.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Ruscher, D. Freyer, M. Karsch, N. Isaev, D. Megow, B. Sawitzki, J. Priller, U. Dirnagl, and A. Meisel
Erythropoietin Is a Paracrine Mediator of Ischemic Tolerance in the Brain: Evidence from an In Vitro Model
J. Neurosci.,
December 1, 2002;
22(23):
10291 - 10301.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Osawa, T. Yamaguchi, Y. Nakamura, S. Kaneko, M. Onodera, K.-i. Sawada, A. Jegalian, H. Wu, H. Nakauchi, and A. Iwama
Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis
Blood,
September 26, 2002;
100(8):
2769 - 2777.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M.-S. Dai, N. Chevallier, S. Stone, M. C. Heinrich, M. McConnell, T. Reuter, H. E. Broxmeyer, J. D. Licht, L. Lu, and M. E. Hoatlin
The Effects of the Fanconi Anemia Zinc Finger (FAZF) on Cell Cycle, Apoptosis, and Proliferation Are Differentiation Stage-specific
J. Biol. Chem.,
July 12, 2002;
277(29):
26327 - 26334.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Imrie, R. Esmail, R. M. Meyer, and and the Members of the Hematology Disease Site Gro
The Role of High-Dose Chemotherapy and Stem-Cell Transplantation in Patients with Multiple Myeloma: A Practice Guideline of the Cancer Care Ontario Practice Guidelines Initiative
Ann Intern Med,
April 16, 2002;
136(8):
619 - 629.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. D. Bunting, H. L. Bradley, T. S. Hawley, R. Moriggl, B. P. Sorrentino, and J. N. Ihle
Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5
Blood,
January 15, 2002;
99(2):
479 - 487.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Oda, H. D. Ochs, L. A. Lasky, S. Spencer, K. Ozaki, M. Fujihara, M. Handa, K. Ikebuchi, and H. Ikeda
CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk
Blood,
May 1, 2001;
97(9):
2633 - 2639.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
Y. Kashii, M. Uchida, K. Kirito, M. Tanaka, K. Nishijima, M. Toshima, T. Ando, K. Koizumi, T. Endoh, K.-i. Sawada, et al.
A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction
Blood,
August 1, 2000;
96(3):
941 - 949.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Choi, K. Muta, A. Wickrema, S. B. Krantz, J. Nishimura, and H. Nawata
Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin
Blood,
June 15, 2000;
95(12):
3742 - 3749.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Haseyama, K.-i. Sawada, A. Oda, K. Koizumi, H. Takano, T. Tarumi, M. Nishio, M. Handa, Y. Ikeda, and T. Koike
Phosphatidylinositol 3-Kinase Is Involved in the Protection of Primary Cultured Human Erythroid Precursor Cells From Apoptosis
Blood,
September 1, 1999;
94(5):
1568 - 1577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Silva, A. Benito, C. Sanz, F. Prosper, D. Ekhterae, G. Nunez, and J. L. Fernandez-Luna
Erythropoietin Can Induce the Expression of Bcl-xL through Stat5 in Erythropoietin-dependent Progenitor Cell Lines
J. Biol. Chem.,
August 6, 1999;
274(32):
22165 - 22169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Du, Y. M. Alsayed, F. Xin, S. J. Ackerman, and L. C. Platanias
Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils
J. Biol. Chem.,
October 13, 2000;
275(42):
33167 - 33175.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract