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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4652-4662
Thrombopoietin Induces Association of Crkl With STAT5 But Not
STAT3 in Human Platelets
By
Katsutoshi Ozaki,
Atsushi Oda,
Hiroshi Wakao,
Jennifer Rhodes,
Brian J. Druker,
Akaru Ishida,
Masatoshi Wakui,
Shinichiro Okamoto,
Kayo Morita,
Makoto Handa,
Norio Komatsu,
Hideya Ohashi,
Atsushi Miyajima, and
Yasuo Ikeda
From the Division of Hematology, Department of Internal Medicine, and
Blood Center, Keio University, Tokyo, Japan; the Laboratory of Cellular
Biosynthesis, Institute of Molecular and Cellular Biosciences, Tokyo
University, Tokyo, Japan; the Division of Hematology and Medical
Oncology, Oregon Health Sciences University, Portland, OR; the Division
of Hematology, Department of Medicine, Jichi Medical School, Tochigi,
Japan; the Department of Pathophysiology and Therapeutics, Faculty of
Pharmacological Sciences, Hoshi University, Tokyo, Japan; and the
Pharmaceutical Research Laboratory, Kirin Brewery Co, Ltd, Takasaki,
Japan.
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ABSTRACT |
Crkl, a 39-kD SH2, SH3 domain-containing adapter protein, is
constitutively tyrosine phosphorylated in hematopoietic cells from
chronic myelogenous leukemia (CML) patients. We recently reported that
thrombopoietin induces tyrosine phosphorylation of Crkl in normal
platelets. In this study, we demonstrate that thrombopoietin induces
association of Crkl with a tyrosine phosphorylated 95- to 100-kD
protein in platelets and in UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line. With specific antibodies against STAT5, we
demonstrate that the 95- to 100-kD protein in Crkl immunoprecipitates is STAT5. This coimmunoprecipitation was specific in that Crkl immunoprecipitates do not contain STAT3, although STAT3 becomes tyrosine phosphorylated in thrombopoietin-stimulated platelets. The
coimmunoprecipitaion of Crkl with STAT5 was inhibited by the immunizing
peptide for Crkl antisera or phenyl phosphate (20 mmol/L). After
denaturing of Crkl immunoprecipitates, Crkl was still
immunoprecipitated by Crkl antisera. However, coimmunoprecipitation of
STAT5 was not observed. Coincident with STAT5 tyrosine phosphorylation, thrombopoietin induces activation of STAT5 DNA-binding activity as
demonstrated by electrophoretic mobility shift assays (EMSA). Using a
 -casein promoter STAT5 binding site as a probe, we have also
demonstrated that Crkl antisera supershift the STAT5-DNA complex,
suggesting that Crkl is a component of the complex in the nucleus.
Furthermore, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin also induce Crkl-STAT5 complex formation in responding cells in a
stimulation-dependent manner. In vitro, glutathione S-transferase
(GST)-Crkl bound to STAT5 inducibly through its SH2 domain. These
results indicate that thrombopoietin, IL-3, GM-CSF, and erythropoietin
commonly induce association of STAT5 and Crkl and that the complex
translocates to the nucleus and binds to DNA. Interestingly, such
association between STAT5 and Crkl was not observed in
cytokine-stimulated murine cells, suggesting an intriguing possibility
that components of the human STAT5-DNA complex may be different from
those of the murine counterpart.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN IS essential for
megakaryocytopoiesis/thrombocytopoiesis (reviewed in
Kaushansky1). One of the earliest biochemical events after
treatment of cells with thrombopoietin is the induction of tyrosine
phosphorylation. Using cell lines or platelets expressing
thrombopoietin receptors (c-Mpl), several groups have shown that
thrombopoietin treatment leads to tyrosine phosphorylation of Jak2,
Tyk2, Shc, c-Cbl, Vav, STAT proteins, and c-Mpl.2-17
Crkl is an SH2/SH3 domain-containing adapter protein that was shown to
be the major tyrosine-phosphorylated protein in chronic myelogenous
leukemia (CML) neutrophils.18-22 We recently reported that
thrombopoietin induces tyrosine phosphorylation of Crkl in normal
platelets and FDCP-hMpl5 cells, a cell line genetically engineered to
express human c-Mpl.17 Our data suggest that Crkl can be
phosphorylated in the absence of p210Bcr-Abl and that Crkl
may have a role in signaling by thrombopoietin. We have also shown that
Crkl is constitutively tyrosine phosphorylated in platelets from CML
patients.17
To further evaluate the role of Crkl in thrombopoietin signaling, we
examined Crkl immunoprecipitates for the presence of tyrosine-phosphorylated proteins. In this report, we demonstrate the
presence of a 95- to 100-kD tyrosine-phosphorylated protein in Crkl
immunoprecipitates after thrombopoietin treatment of platelets and
UT7/TPO cells, a thrombopoietin-dependent megakaryocytic cell line.
This 95- to 100-kD protein was identified as STAT5. Interestingly, a
Crkl-STAT5 complex is present in the nucleus, as demonstrated by the
supershift of STAT-DNA complexes with Crkl antisera. Furthermore, we
demonstrate that the Crkl-STAT5 complex formation also occurs in cells
in response to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin, suggesting that
the complex is commonly involved in signaling pathways elicited by
various cytokines.
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MATERIALS AND METHODS |
Reagents.
Prostaglandin E1 (PGE1), aspirin, apyrase (type
VIII), N-2 hydroxyethylpiperazine-N -2-ethanesulfonic acid
(HEPES), sodium dodecyl sulfate (SDS), 2-mercaptoethanol, isopropyl
-D-thiogalactopyranoside (IPTG), sodium orthovanadate, chicken egg
albumin, protein A-Sepharose, Triton X-100, bovine serum albumin (BSA),
dithiothreitol (DTT), phenyl phosphate, and Tris (hydroxymethyl)
aminomethane (Tris) were purchased from Sigma (St Louis, MO).
Polyvinylidene difluoride (PVDF) membranes (pore size, 0.45 µm) were
from Millipore Corp (Bedford, MA). SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) molecular standards and
[ -32P]ATP were from Amersham (Arlington Heights, IL)
or Bio-Rad (Richmond, CA). Double-stranded Poly (dI-dC) was from
Pharmacia Biotech (Milwaukee, WI). The Jak2 immunizing peptide
(DSQRKLQFYEDKHQLPAPKC) was from Upstate Biotechnology Inc (Lake Placid,
NY). Enhanced chemiluminesence (ECL) reagents, including secondary
antibodies, were purchased from Amersham. The antiphosphotyrosine
murine monoclonal antibody (MoAb; 4G10) was used as
described.17 Crkl, STAT3, and c-Cbl antisera; an anti-GST
MoAb; and the Crkl immunizing peptide (amino acids 283-302 mapping at
the carboxy terminus of human Crkl) were from Santa Cruz (Santa Cruz,
CA). Iscove's modified Dulbeco's medium (IMDM), nitroblue tetrazolium
chloride (NBT), and 5-bromo-4 chloro-3-indolyl phosphate p-toluidine
salt (BCIP) were from GIBCO BRL (Gaithersburg, MD). For electrophoretic
mobility shift assay (EMSA), an anti-STAT5 antiserum, described
previously, was used.23 An anti-STAT5 MoAb was from
Transduction Laboratories (Lexington, KY). STAT5A and STAT5B antisera
were from R&D Systems (Minneapolis, MN). Recombinant thrombopoietin,
erythropoietin, IL-3, and GM-CSF were kindly provided by Kirin Brewery
Co Ltd (Maebashi, Japan).
Platelet preparation.
Blood from healthy volunteers and three CML patients was drawn by
venipuncture into 1/10 vol of 3.8% (wt/vol) trisodium citrate and
gently mixed. Before drawing blood, informed consent was obtained. The
three CML patients were BCR-ABL positive, as demonstrated by reverse
transcriptase-polymerase chain reaction (RT-PCR) on peripheral blood
samples. Platelet-rich plasma (PRP) was prepared by centrifuging the
whole blood at 200g for 20 minutes and aspirating PRP. PRP was
incubated with aspirin (2 mmol/L) for 30 minutes at room temperature.
PGE1 (1 µmol/L) was added from a stock solution in
absolute ethanol (1 mmol/L). The PRP was spun at 800g to form a
soft platelet pellet. The pellet was resuspended in 1 mL of a modified
HEPES-Tyrode buffer (129 mmol/L NaCl, 8.9 mmol/L NaHCO3, 0.8 mmol/L KH2PO4, 0.8 mmol/L
MgCl2, 5.6 mmol/L dextrose, and 10 mmol/L HEPES, pH 7.4)
containing apyrase (2 U/mL) and was washed twice with this buffer.
Platelets were resuspended in the same buffer at a concentration of 3 × 108 cells/mL with apyrase (2 U/mL) and 1 mmol/L
CaCl2 at 37°C. The contaminating white blood cells were
counted and were regularly less than 0.01% of platelets. If the
contaminating white blood cell count exceeded this value, the cell
suspension was repeatedly centrifuged at 200g for 5 minutes
until this value of the supernatant was less than 0.01%.
Gel electrophoresis and Western blotting.
Platelet stimulation was terminated by the addition of an equal volume
of 2× concentrated Laemmli's sample buffer (10% glycerol, 1%
SDS, 5% 2-mercaptoethanol, 50 mmol/L Tris-HCl [pH 6.8], 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-7.5% to 15% polyacrylamide gels
as described.24 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 1:3,000 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.
Immunoprecipitation.
Cells in suspensions (0.5 mL) were lysed by the addition of an equal
amount (0.5 mL) of lysis buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl 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 (40 µL of a 50% slurry) for 1 hour. Crkl antisera (2 µg/mL), STAT3 antisera (2 µg/mL), or the mixture of STAT5A and
STAT5B antisera (3 µL each) were then added and incubated for 2 to 3 hours on ice. Protein A-Sepharose (40 µL of a 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.
Cell lines.
UT7/TPO and UT7/EPO, sublines of UT7, have been previously
characterized.25 TF-1 cells were kindly provided by Dr
Hisamaru Hirai (Tokyo University, Tokyo, Japan).26
FDCP-hMpl5 cells were described previously.10,17 Cells were
maintained in IMDM containing 10% fetal calf serum (FCS) in the
presence of 10 ng/mL thrombopoietin (for UT7/TPO or FDCP-hMpl5), 10 U/mL erythropoietin (for UT7/TPO), or 1 ng/mL GM-CSF (for TF-1 and
UT7). Before treatment of the cells with various cytokines, they were
incubated in IMDM containing 10% FCS without cytokines for 18 hours.
Before stimulation with thrombopoietin, cells were washed twice with
phosphate-buffered saline (PBS; pH 7.4) and then resuspended in PBS at
2 × 107 cells/mL.
EMSA.
Nuclear extracts (40 µL, containing 10 to 20 µg protein) were
prepared from UT-7/TPO cells (2 × 107 cells) by lysis
followed by high salt extraction, as described previously.23 Two microliters of nuclear extracts was mixed with 20 µL of binding buffer [10 mmol/L Tris-HCl, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, 0.1% NP-40, 5% glycerol, 1 mg/mL BSA, and
2 mg/mL poly(dI-dC), pH 7.5] containing 50,000 cpm of an 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× TBE and detected by
autoradiography.
GST binding assays.
The GST-Crkl fusion proteins were generated as previously
described.21,27 GST-fusion constructs were transformed into
Escherichia coli DH-5 and protein expression was induced by
adding 0.5 mmol/L IPTG to exponentially growing cells. The GST-fusion
proteins were isolated from sonicated bacterial lysates using
glutathione sepharose beads. Coomassie-stained gels were used to
normalize for the expression of the various GST-fusion proteins.
Between 2.5 and 5 µg of GST-fusion proteins was 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%
2-mercaptoethanol). The beads were washed four times in PBS and
incubated for 4 hours with normalized cellular lysate of UT7 or TF-1
cells. The beads were washed three times with PBS and boiled in
SDS-sample buffer. Proteins were separated by SDS-PAGE and transferred
onto PVDF membranes for immunoblot analysis.
Isolation of platelet cytoskeleton.
The Triton X-100-insoluble cytoskeleton was isolated as described,
with the following modification.17 An equal amount of lysis
buffer was added to platelet suspensions to solubilize platelets. After
5 minutes on ice, the lysates were centrifuged at 10,000g. The
resulting pellet was washed twice in washing buffer. For
one-dimensional SDS electrophoresis, the Triton X-100-insoluble
residue was solubilized in SDS sample buffer. The supernatant was
diluted with an equal volume of 2× concentrated SDS sample
buffer.
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RESULTS |
As we have previously reported, Crkl becomes tyrosine phosphorylated
after thrombopoietin treatment of human platelets
(Fig 1A).17 Examination of phosphotyrosine immunoblots of the
Crkl immunoprecipitates from thrombopoietin-treated platelets
demonstrates the presence of a protein with a molecular weight of
approximately 95 to 100 kD (often seen as a doublet). Whole cell lysate
antiphosphotyrosine immunoblots show a protein of similar molecular
weight that is inducibly tyrosine phosphorylated.9 As seen
in Fig 1A, the association of this 95-to 100-kD tyrosine-phosphorylated
protein with Crkl occurs rapidly and is maximal 5 minutes after
treatment with thrombopoietin.
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| Fig 1.
(A and B) Crkl immunoprecipitates from
thrombopoietin-treated normal platelets contain a
tyrosine-phosphorylated 95- to 100-kD protein that is also recognized
by an anti-STAT5 MoAb. Platelets were lysed at the indicated times
after thrombopoietin treatment by the addition of an equal amount of a
buffer containing 2% Triton X-100 before and after exposure to
thrombopoietin (100 ng/mL). Crkl was immunoprecipitated with specific
Crkl antisera. Immune complexes were resuspended in SDS-sample buffer.
Proteins were separated by 7.5% to 15% SDS-PAGE and transferred onto
PVDF membranes. Immunoblots were probed with phosphotyrosine antibodies
(A) or a STAT5 MoAb (B) and bands were visualized by chemiluminescence.
The asterik in (A) indicates the tyrosine-phosphorylated 95- to 100-kD
protein. (C) The same PVDF membrane used in (B) was stripped of the
antibody and reprobed with Crkl antisera. Bands were visualized using
alkaline phosphatase-conjugated second antibody and NBT/BCIP. (D) Crkl
immunoprecipitates do not contain STAT3. Platelets were lysed as in (A)
before and after stimulation with thrombopoietin (100 ng/mL for 10 minutes). Crkl or STAT3 was immunoprecipitated with specific antisera
as indicated. The membranes were probed with STAT3 antisera (top
panel), Crkl antisera (middle panel), or 4G10 (bottom panel). Bands
were visualized by ECL. (E) Thombopoietin-induced tyrosine
phosphorylation of STAT5 in platelets. Platelets were lysed as in (A)
before and after stimulation with thrombopoietin (100 ng/mL for 10 minutes). STAT5 was immunoprecipitated with specific antisera (3 µL
of anti-STAT5A and STAT5B each). The membranes were probed with 4G10
(upper panel) or an anti-STAT5 MoAb (lower panel). Bands were
visualized by ECL. (F) Association of STAT5 with the Triton
X-100-insoluble residue. Platelets were lysed with Triton X-100-EGTA
buffer before or after stimulation with thrombin (1 U/mL), with or
without stirring. Lysates were separated by high-speed centrifugation
into soluble and insoluble residues. Proteins from each fraction were
separated by 10% SDS-PAGE and immunoblotted with an anti-STAT5 MoAb
(upper panel) or Crkl antiserum (lower pane). (Left lane) Triton
X-100-soluble residue of resting cells (1.5 × 107
cells); (middle lane) Triton X-100-insoluble residue of resting cells
(3.0 × 107cells); (right lane) Triton X-100-insoluble
residue from 3.0 × 107 cells, prepared 10 minutes after
exposure to thrombin (1 U/mL) with stirring.
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We and others have previously demonstrated that STAT3 and STAT5 become
tyrosine phosphorylated after thrombopoietin treatment of platelets or
various cell lines expressing c-Mpl.3,10,15,25 As expected,
STAT3 and STAT5 were tyrosine phosphorylated in
thrombopoietin-stimulated platelets (Fig 1D and E). Although STAT3
became tyrosine phosphorylated in thrombopoietin-stimulated platelets,
Crkl immunoprecipitates did not contain STAT3 and vice versa (Fig 1D).
However, Crkl immunoprecipitates from thrombopoietin-stimulated
platelets did contain STAT5 as detected by either STAT5 MoAbs or
polyclonal antibodies (Fig 1B and data not shown). The proteins
detected by the STAT5 antibodies are often a doublet that may reflect
STAT5A and STAT5B. No reactivity was seen with either STAT1 or Vav
antisera (data not shown). We have previously shown that Crkl is
incorporated into platelet cytoskleton during platelet aggregation (Oda
et al17 and Fig 1F, lower panel). Accordingly, we
postulated that STAT5, the function of which has been unknown in
platelets, may be incorporated into the platelet cytoskeleton during
platelet aggregation. STAT5 also became incorporated into the
cytoskeletal pellet (Fig 1F, lower panel), suggesting that STAT5 may
have a role in the reorganization of the cytoskeleton during platelet
aggregation.
Because Crkl is constitutively tyrosine phosphorylated in platelets
from CML patients, we next asked whether Crkl tyrosine phosphorylation
is sufficient for an association with STAT5
(Fig 2). Before and after stimulation by
thrombopoietin (100 ng/mL for 1 minute), Crkl was tyrosine
phosphorylated. However, Crkl immunoprecipitates only contained STAT5
after stimulation with thrombopoietin (Fig 2), suggesting that Crkl
tyrosine phosphorylation in CML platelets is not sufficient for
association of Crkl with STAT5.
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| Fig 2.
Crkl immunoprecipitates from thrombopoietin (100 ng/mL
for 1 minute) -treated platelets of CML patients contain a
tyrosine-phosphorylated 95- to 100-kD protein that is also recognized
by an anti-STAT5 MoAb. The same methods as in Fig 1 were used, except
that platelets from CML patients were analyzed. Results are
representative of three experiments from three different donors. ( )
Resting CML platelets; (+) thrombopoietin-treated CML platelets.
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UT7/TPO cells are a recently characterized megakaryocytic cell line
that is thrombopoietin-dependent.25 Treatment of UT7/TPO cells with thrombopoietin resulted in a similar induction of tyrosine phosphorylation of Crkl and association of Crkl with STAT5 as seen in
platelets (Fig 3). To examine the
specificity of the Crkl-STAT5 interaction, several experiments were
performed. As seen in Fig 4A, the Crkl
immunizing peptide but not a Jak2 peptide inhibited the Crkl
immunoprecipitation and STAT5 coimmunoprecipitation. Crkl
immunoprecipitates from UT7/TPO were next divided, and half of the
beads were boiled in lysis buffer plus 2% SDS for 5 minutes. The
denatured immunoprecipitates were diluted into lysis buffer containing
1% Triton X-100 to lower the concentration of SDS to 0.5%. Crkl was
again immunoprecipitated (2nd IP). Before and after denaturing of Crkl
immunoprecipitates, Crkl was immunoprecipitated by Crkl antisera (Fig
4B, lower panel). However, no STAT5 was seen in the denatured Crkl
immunoprecipitates (Fig 4B, upper panel). To see whether tyrosine
phosphorylation is involved in Crkl and STAT5 association, Crkl was
immunoprecipiated in the presence and absence of phenyl phosphate (Fig
4C). Crkl was immunoprecipitated before and after stimulation of
UT7/TPO cells, irrespective of the presence of phenyl phosphate (20 mmol/L; upper and lower panels). However, STAT5 coimmunoprecipitation
after cell stimulation was inhibited by phenyl phosphate, suggesting
that protein tyrosine phosphorylation is involved in the Crkl-STAT5
interaction.
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| Fig 3.
(A, B, and C) Crkl immunoprecipitates from
thrombopoietin-treated UT7/TPO cells contain a tyrosine-phosphorylated
95- to 100-kD protein that is also recognized by a STAT5 MoAb. UT7/TPO
cells were lysed at the indicated times by the addition of an equal
amount of a buffer containing 2% Triton X-100 before and after
exposure to thrombopoietin (100 ng/mL). Tyrosine-phosphorylated
proteins (A), STAT5 (B), and Crkl (C) in the Crkl immunoprecipitates
were detected as described in Fig 1.
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| Fig 4.
(A) Crkl and STAT5 immunoprecipitation was inhibited by
the Crkl immunizing peptide. (Upper and lower panels) Crkl
immunoprecipitation was performed as in Fig 3A and C, except that
immunoprecipitation was performed in the presence of the Jak2 (control;
left 2 lanes) or the Crkl immunizing peptides (right 2 lanes). (B)
STAT5 was not immunoprecipitated by Crkl antisera after denaturing of
Crkl immunoprecipitates. The left two lanes are same as in (A; 1st IP).
After denaturing of Crkl immunoprecipitates and dilution into lysis
buffer containing 0.5% SDS and 1% Triton X-100, Crkl was again
immunoprecipitated (2nd IP; the right 2 lanes). Crkl and STAT5 were
detected as in Fig 2A and C. (C) Crkl and STAT5 coimmunoprecipitation
was inhibited by phenyl phosphate (20 mmol/L). (Upper and lower panels)
Crkl immunoprecipitation was performed as in Fig 3A and C, except that
immunoprecipitation was performed in the presence (right 2 lanes) or
absence (left 2 lanes) of phenyl phosphate (20 mmol/L).
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STAT5 is thought to be tyrosine phosphorylated in the cytoplasm and
translocates to the nucleus when cells are stimulated with various
factors, including prolactin, GM-CSF, IL-3, epidermal growth factor
(EGF), erythropoietin, and thrombopoietin25,28-31 (reviewed
in Darnell32 and Ihle33). After tyrosine
phosphorylation, STAT5 dimerizes and translocates to the nucleus, where
it activates or represses transcription.
Binding of STAT5 to a radiolabeled probe from the -casein promoter
was analyzed by EMSA (Fig 5). Nuclear
extracts were prepared from UT7/TPO cells before and after stimulation
with thrombopoietin (100 ng/mL for 20 minutes). Thrombopoietin induced
the electrophoretic shift of the end-labeled probe in UT7/TPO cells
(Fig 5, Shifts). STAT5 but not STAT3 antisera supershifted the
DNA-protein complex and bound to the labeled probe (Fig 5, Super Shift
2, lanes 4 and 6), indicating that a STAT5 or a STAT5-like factor was
activated by thrombopoietin. Crkl antisera also supershifted a portion
of the same DNA-protein complex induced by thrombopoietin, indicating that Crkl is a component of the DNA-STAT5 complex (Super Shift 1, lane
5). In contrast, STAT3 antiserum had no effect (lane 6), indicating
that the effects of STAT5 and Crkl antisera are specific.
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| Fig 5.
Thrombopoietin activates STAT5 in UT7/TPO cells. Growth
factor-deprived UT-7/TPO cells were stimulated with 100 ng/mL
thrombopoietin and nuclear extracts were prepared. Nuclear extracts
were subject to EMSA using a -casein probe. Lane 1, nuclear extracts
from unstimulated cells; lane 2, nuclear extracts from
thrombopoietin-stimulated cells; lane 3, the same as in lane 2 in the
presence of 50-fold excess cold probe; lane 4, the same as in lane 2, except that the nuclear extracts were incubated with STAT5 antisera (2 µg/mL) before EMSA; lane 5, + Crkl antisera (2 µg/mL); lane 6, + STAT3 antisera (2 µg/mL). Shift, probe complex in the absence of
the antibodies; Super Shift 1, the supershifted band in the presence of
STAT5 antisera; Super Shift 2, the supershifted band in the presence of
Crkl antisera.
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We also tested the possibility that IL-3, GM-CSF, and erythropoietin,
which are known to induce activation STAT5 in various cells, may induce
the Crkl-STAT5 complex formation. As shown in Fig 6, Crkl-STAT5 complex formation was
also induced in IL-3- or GM-CSF-stimulated TF-1 cells (Fig 6A) or in
erythropoietin-stimulated UT7/EPO cells (Fig 6B). Similar results were
obtained in IL-3- or GM-CSF-stimulated UT7 cells (data not shown). In
each set of experiments, the presence of Crkl-STAT5 complex formation
was also confirmed by EMSA as in Fig 5 (data not shown).
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| Fig 6.
GM-CSF, IL-3, and erythropoietin induce the association
of Crkl with STAT5. (A) (Upper panel) TF-1 cells were lysed before
(pre) and after exposure to GM-CSF or IL-3 (100 ng/mL for 5 minutes).
Crkl immunoprecipitates were resuspended in SDS-sample buffer. Proteins
were separated by 10% SDS-PAGE and transferred onto PVDF membranes.
Immunoblots were probed with an anti-STAT5 MoAb and bands were
visualized by chemiluminescence. (Lower panel) The same PVDF membrane
used in (A) was stripped of the antibody and reprobed with anti-Crkl
antisera. The arrows indicated the relative position of STAT5, heavy
chain of IgG, and Crkl. GM, GM-CSF. (B) Association of Crkl with STAT5
in UT7/EPO cells stimulated by erythropoietin. The same as in Fig 4A,
except that UT7/EPO cells were stimulated by erythropoietin (10 U/mL
for 10 minutes). EPO, erythropoietin.
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The interaction of Crkl and STAT5 was next examined using bacterially
expressed Crkl (Fig 7). Lysates from
starved () TF-1 cells or cells stimulated with 100 ng/mL of
GM-CSF for 10 minutes (+) were incubated with various GST-Crkl
constructs. Crkl constructs include full-length Crkl, full-length Crkl
with a mutation of Arg to Lys in the SH2 domain FLVRES sequence (K39),
the SH2 domain, and the N-terminal SH3 domain (SH3n) of Crkl. Bound
proteins were boiled in 1% SDS and separated by SDS-PAGE, transferred
to PVDF membranes, and immunoblotted with STAT5 antisera. Full-length Crkl or the SH2 domain demonstrated inducible binding to STAT5 from
starved cells, with a significant increase using lysates from
GM-CSF-stimulated cells. No association with STAT5 was seen using the
SH3n or K39 Crkl constructs. No binding was seen to GST or beads.
Similar data were obtained using lysates from GM-CSF-treated UT7 cells
or thrombopoietin-stimulated platelets (data not shown).
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| Fig 7.
Binding of STAT5 to bacterially expressed Crkl. Lysates
from starved () TF-1 cells or cells stimulated with 100 ng/mL of
GM-CSF for 10 minutes (+) were analyzed for binding to GST or various
GST-Crkl constructs. Crkl constructs include full-length Crkl,
full-length Crkl with a mutation of Arg to Lys in the SH2 domain FLVRES
sequence (K39), the SH2 domain, and the N-terminal SH3 domain (SH3n) of
Crkl. Bound proteins were separated by SDS-PAGE, transferred to PVDF
membranes, and immunoblotted with STAT5 antisera.
|
|
Finally, we also examined whether Crkl-STAT5 association may be
observed in murine FDCP-hMpl5 cells in response to thrombopoietin. As
we have reported, thrombopoietin (100 ng/mL) induced tyrosine phosphorylation of Crkl (Fig 8A).
Thrombopoietin also induced coimmunoprecipitation with Crkl of 120-kD
tyrosine-phosphorylated protein indicated by the asterik instead of 95- to 100-kD proteins. Crkl was immunoprecipitated similarly from each
sample (Fig 8B). The Crkl immunoprecipitate contains a protein reactive
with anti-cCbl (Fig 8C), suggesting that the 120-kD protein contains
c-Cbl. Using the same samples, we have never seen any
coimunoprecipitation of STAT5 using the same Crkl antiserum (data not
shown), despite the fact that we regularly observe
thrombopoietin-induced tyrosine phosphorylation of STAT5.10
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| Fig 8.
Crkl and c-Cbl were coimmunoprecipitated from the lysates
from FDCP-hMpl5 cells stimulated by thrombopoietin. (A and B) Crkl
immunoprecipitates from thrombopoietin-treated FDCP-hMpl5 cells contain
a tyrosine phosphorylated 120-kD protein (*). FDCP-hMpl5 cells were
lysed at the indicated times by the addition of an equal amount of a
buffer containing 2% Triton X-100 before and after exposure to
thrombopoietin (100 ng/mL). Tyrosine-phosphorylated proteins (A) and
Crkl (B) in the Crkl immunoprecipitates were detected as described in
Fig 1. Lane 1, resting FDCP-hMpl5 cells; lanes 2 through 5, the lysates
from FDCP-hMpl5 cells 1, 5, 10, and 30 minutes after stimulation with
thrombopoietin. (C) Crkl immunoprecipitate from
thrombopoietin-stimulated FDCP-hMpl5 cells contains c-Cbl. FDCP-hMpl5
cells were lysed by the addition of an equal amount of a buffer
containing 2% Triton X-100 before and 10 minutes after exposure to
thrombopoietin (100 ng/mL). c-Cbl (upper panel) and Crkl (lower panel)
in the Crkl immunoprecipitates were detected as described in Fig 1.
|
|
| |
DISCUSSION |
In this study, we have confirmed and extended our previous observations
that Crkl becomes tyrosine phosphorylated upon thrombopoietin stimulation of cells (platelets and UT7/TPO cells). Crkl
immunoprecipitates from platelets or UT7/TPO, treated with
thrombopoietin, contained a prominent tyrosine-phosphorylated protein
of 95 to 100 kD. We have identified this protein as STAT5. Furthermore,
our results suggest that Crkl is also present in the nucleus with STAT
5 and is a component of a STAT5-DNA complex in cells stimulated with thrombopoietin (Fig 5).
The association of Crkl with STAT5 occurs within 1 minute after
stimulation with thrombopoietin, when Crkl tyrosine phosphorylation in
platelets is minimal (Fig 1). Thus, Crkl tyrosine phosphorylation may
not be required for its association with STAT5. In support of this,
constitutive tyrosine phosphorylation of Crkl in CML platelets was
readily detected, and Crkl immunoprecipitates from lysates of resting
CML platelets did not contain STAT5 (Fig 2). However, thrombopoietin
(100 ng/mL) -induced treatment resulted in Crkl-STAT5 association
without changing the degree of tyrosine phosphorylation of Crkl. In
cells transformed by v-Crk, the v-Crk protein is not tyrosine
phosphorylated, because it lacks the major site of Crk tyrosine
phosphorylation.34 However, Crk
immunoprecipitates from v-crk-transformed cells contain numerous
tyrosine-phosphorylated proteins.35 Crk II has been shown
to bind to a variety of tyrosine-phosphorylated proteins, such as
c-Cbl, paxillin, and p130cas.34-36 In the case of c-Cbl,
this association is dependent on the tyrosine phosphorylation of c-Cbl
after EGF receptor activation or T-cell receptor engagement and does
not require tyrosine phosphorylation of Crk II.34,36 Thus,
Crkl may be capable of binding to STAT5 without becoming tyrosine
phosphorylated.
Because phenyl phosphate inhibited Crkl-STAT5 association, protein
tyrosine phosphorylation may be required for Crkl-STAT5 association.
Because tyrosine phosphorylation of Crkl does not seem to be required
for Crkl-STAT5 association as discussed above and the association
occurs apparently after tyrosine phosphorylation of STAT5, it is likely
that STAT5 tyrosine phosphorylation may be involved in the Crkl-STAT5
complex formation. Furthermore, the binding of Crkl to STAT5 occurs
rapidly upon stimulation of the cells, suggesting that Crkl binds to
STAT5 directly after tyrosine phosphorylation of STAT5. STAT5 could be
binding to Crkl through a phosphotyrosine-dependent interaction of
STAT5 with the SH2 domain of Crkl. The GST fusion protein data confirms
such interaction of Crkl and STAT5. In these experiments, STAT5 binds to the SH2 domain of Crkl after stimulation of cells with cytokines. These data are consistent with the observed inducible
coimmunoprecipitation of Crkl and STAT5 and suggest a
phosphotyrosine-dependent interaction of STAT5 with the SH2 domain of
Crkl. Such a possibility seemed to be unlikely, because STAT5 is
thought to dimerize when tyrosine phosphorylated through its SH2 domain
binding to the tyrosine phosphate of another STAT protein (reviewed in
Darnell32 and Ihle33). This model suggests
that, when STAT5 binds to DNA, its SH2 domain and tyrosine phosphate
would not be available for binding to another SH2 domain-containing
protein. Because STAT proteins are believed to be tyrosine
phosphorylated at only one site, this suggests that Crkl must displace
a STAT protein by binding through its SH2 domain to the
tyrosine-phosphorylated residue of a STAT protein. A recent report
suggests that STAT1 or STAT2 requires only a tyrosine-phosphorylated
STAT protein, which binds to an SH2 domain of the other STAT protein
for homodimerization or heterodimerization of the STAT
complex and binding of the complex to DNA.37
Thus, our data suggest that this may also be true for the STAT5 dimers
and that one phosphotyrosine residue in a STAT5 dimer-DNA complex may
be available for binding to SH2 domain of other molecules such as Crkl
without disrupting the complex. Interestingly, this is not the case for
murine STAT5, because as we did not see any association of Crkl with
STAT5 in FDCP-hMpl5 cells stimulated by thrombopoietin (Fig 8). Because
amino acid sequences of both STAT5 and Crkl are well conserved in both
species, we initially suspected that the lack of coimmunoprecipitation was merely dilutional. However, even when we used large number of cells
(108 cells/mL) for immunoprecipitation studies, we did not
observe the coimmunoprecipitation (data not shown). Furthermore, we saw the coimmunoprecipitation of Crkl with c-Cbl (Fig 8), a process mediated by the binding of Crkl-SH2 domain to c-Cbl.38
Furthermore, we did not detect any Crkl-STAT5 association in murine 32D
cells stimulated by GM-CSF (data not shown). Thus, our data indicate that the lack of coimmunoprecipitation may not be a technical artifact,
suggesting an intriguing possibility that human and murine STAT5-DNA
complexes are different, because only the former contains Crkl.
STAT5 proteins are also known to be tyrosine phosphorylated in a
variety of situations, including cellular stimulation with GM-CSF,
IL-3, erythropoietin, and thrombopoietin.28-33 We have shown that Crkl is also associated with STAT5 under these conditions (Fig 6). These data suggest that the complex formation of STAT5 and
Crkl may be solely dependent on activation of STAT5. Several reports
have suggested that, in BCR/ABL-transformed cells, STAT5 is
constitutively tyrosine phosphorylated and activated.39-42
The molecular mechanisms of STAT5 activation are unknown, but one of
the possible mechanisms is tyrosine phosphorylation of STAT5 directly
by the BCR/ABL kinase.41 However, no direct association of
STAT5 and the BCR/ABL kinase has been demonstrated.41 As expected, we also found that, in BCR/ABL-positive K562 cells, Crkl-STAT5 complex is constitutively formed (data not
shown). As we have shown that Crkl-STAT5 formation is
mediated by Crkl-SH2 domain, N-teminal SH3 domain of Crkl is available
for the binding for BCR-ABL chimeric protein.38 Thus, it is
possible that Crkl may bridge STAT5 and the BCR/ABL protein so that the
kinase can phosphorylate STAT5.
In UT7 cell backgrounds, STAT5 seems to be dispensable for cell
proliferation.43 Furthermore, Crkl-STAT5 complex formation is also observed in platelets that do not proliferate. As we have demonstrated that STAT5 become incorporated into platelet cytoskleton, it is possible that Crkl-STAT5 complex may have a role that is not
directly related to STAT5 functions as a transcription factor. Such a
view is not heretical, because inactive STAT1 and STAT2 may mediate
interferon-induced apoptosis (reviewed in
Scinder44).
In summary, we have found the novel physical interaction of Crkl-STAT5
in human but not in murine hematopoietic cells, suggesting that the
components of the Crkl-STAT5 complex could be different in the cells
from different species, and we have further clarified a part of the
mechanisms of the Crkl-STAT5 association. Despite the extensive
investigations, the pathophysiologic role of Crkl in blood cells is
still obscure.38 Because Crkl is an adapter protein, it is
critical to determine a potentially important ligand for Crkl. Our
study indicates that STAT5 is a novel ligand for Crkl.
| |
ACKNOWLEDGMENT |
The authors thank Kirin Brewery Co Ltd for kindly providing recombinant
cytokines. We also thank Dr Hisamaru Hirai for TF-1 cells.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted August 12, 1998.
K.O. and A.O. contributed equally to this work and should be regarded
as cofirst authors.
Supported in part by Grants in Aid from The Ministry of Education,
Science and Technology of Japan (A.O., A.M., and Y.I.), Torey Research
Foundation (A.M.), New Energy and Industrial Technology Development
Organization (A.M.), The Ryoichi Naito Foundation for Medical Research
(1994 and 1996; A.O.), and Research Grants for Life Sciences and
Medicine, Keio University Medical Science Fund (A.O.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Atsushi Oda, MD, PhD, Hokkaido Red Cross
Blood Center, Yamanote 2-2, Nishi-ku, Sapporo 063-0002, Japan.
| |
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Abstract
Introduction