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RAPID COMMUNICATION
Thrombopoietin Induces Tyrosine Phosphorylation of Stat3 and Stat5 in
Human Blood Platelets
By Yoshitaka Miyakawa, Atsushi Oda, Brian J. Druker, Hiroshi Miyazaki, Makoto Handa, Hideya Ohashi,
and Yasuo lkeda
Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently,we and others have
shown that thrombopoietin induces rapid tyrosinephosphorylation of Jak2 and other proteins in human platelets
and BaF3 cells, genetically engineered to express c-Mpl, a
receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some ofthe effects of cytokines
or hematopoietic growth factors by recruitment and tyrosine phosphorylationof a variety of Stat (signal transducers
andactivatorsoftranscription)proteins.
Hence, we have
investigated whether Stat proteins are present in platelets
in
and, if so, whether they become tyrosine phosphorylated
response to thrombopoietin. We immunologically identified
Statl, Stat2, Stat3, and Stat5 in human platelet lysates.
Thrombopoietin induced tyrosine phosphorylationof Stat3
and Stat5 in these cells. Thrombopoietin also induced
tyrosine phosphorylationof Stat3 and Stat5 in FDCP-2 cells genetically engineeredto constitutively express humanc-Mpl.
Thus, ourdata indicatethat Stat3 and Stat5 may be involved
in signal transduction after ligand binding
to c-Mpl and that
this event may have a role in megakaryopoiesis/thrombopoiesis or possibly amature platelet function such as aggregation.
0 1996 by The American Societyof Hematology.
C
tein has been shown to be essential for homo- or heterodimerization, translocation into the nucleus, and formation
of DNA binding
Given the presence of functional thrombopoietin receptors on platelets and our previous
observation of Jak2 activation after stimulation with thrombopoietin, we examined the possibility that platelets may
have Stat proteins and, if so, whether they become tyrosine
phosphorylated after treatment with thrombopoietin. We also
examined whether Jak2 activation and Stat phosphorylation
correlated with parameters of platelet function, eg, aggregation.
LONING OF A cDNA for thrombopoietin has enabled
us to perform an extensive examination of the biologic
functions of this hematopoietic growth/maturation factor.
The results of these studies have indicated that thrombopoietin induces the growth and maturation of megakaryocytic
precursors.”” In support of this conclusion, mice lacking cMpl (a receptor for thrombopoietin) have significantly reduced numbers of circulating ~1atelets.l~
Despite extensive
knowledge of thrombopoietin function, the intracellular
events that occur after thrombopoietin binding to its receptor
are largely unknown.
It has been reported that, in BaF3 cells engineered to
express murine c-Mpl, thrombopoietin induces tyrosine
phosphorylation of several proteins, including c-Mpl itself,
Jak2, and Shc.14 We previously reported that treatment of
human platelets with thrombopoietin led to tyrosine phosphorylation of several intracellular proteins with relative molecular weights of 130, 95, 85, and 52 k D . 1 5 Using specific
antisera, we showed that Jak2 and Shc were two of the
proteins that were inducibly tyrosine phosphorylated in
platelets in response to thr~mbopoietin.’~
Furthermore, we
have preliminary data that suggest that treatment of platelets
with thrombopoietin (1 to 10 ng/mL) enhances aggregation
induced by various agonists such as epinephrine and ADP
(manuscript submitted). These data suggest that c-Mpl on
the surface of platelets is functional. Thus, thrombopoietin
appears to be similar to other cytokines such as granulocytemacrophage colony-stimulating factor (GM-CSF), interleukin-3 (E-3), and granulocyte colony-stimulating factor (GCSF), which are not only required for the proliferation and
maturation of progenitor cells but stimulate the functions of
mature hematopoietic cells.
Recently, a new class of transcription factors termed Stat
(signal transducers and activators of transcription) have been
identified.I6-l8They appear to have an essential role in signal
transduction elicited by cytokines, hematopoietic growth factors, interferons, growth hormone, prolactin, or epidermal
growth factor (EGF).I6-I8In all cases, the Stat proteins appear
to be dormant in resting cells. On stimulation of cells with
growth factor or cytokine, they become tyrosine phosphorylated, possibly as a consequence of the activation of a Jak
family tyrosine kinase. The phosphorylation of the Stat proBlood, Vol 87, No 2 (January 151, 1996: pp 439-446
MATERIALS AND METHODS
Materials. Prostaglandin El (PGE, ), epinephrine, aspirin, apyrase (type VIII), N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic
acid (HEPES), sodium dodecyl sulfate (SDS), 2-mercaptoethanol,
sodium orthovanadate, chicken egg albumin, protein A-Sepharose,
antimouse IgG-conjugated agarose, Triton X-100, and Tris (hydroxymethyl) aminomethane (Tris) were purchased from Sigma (St
Louis, MO). Polyvinylidene difluoride (PVDF) membrane (pore
size, 0.45 mm) was from Millipore Corp (Bedford, MA). SDSpolyacrylamide gel electrophoresis (SDS-PAGE) molecular standards were from Amersham (Arlington Heights, IL). Enhanced
Fromthe Division of Hematology, the Department of Internal
Medicine, and the Blood Center, Keio University, Tokyo, Japan:
the Division of Hematology and Medical Oncology, Oregon Health
Sciences University, Portland, OR: and the Pharmaceutical Research Laboratory, Kirin Brewery CO,Ltd, Maebashi, Japan.
Submitted June 30, 1995: accepted October 13, 1995.
Y.M. and A.O. equally contributed to this work andshould be
regarded as co-jirst authors.
Supported in part by Grants in Aid from The Ministry of Education, Science and Technology of Japan (Y.I. and A.O.) and The
Ryoichi Naito Foundation for Medical Research (A.0.).
Address reprint requests to Yasuo Ikeda, MD, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, 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” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8702-0051$3.00/0
439
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MIYAKAWA ET AL
440
chemiluminescence (ECL) reagents, including second antibodies,
were purchased from Amersham. Antiphosphotyrosine murine
monoclonal antibody (4G10) was used as described.”.’*.”’Anti-Jak2
rabbit polyclonal antibodies were from UBI (Lake Placid, NY). Specific anti-Statl, -Stat2, -Stat5, and -Stat6 antibodies and Rous sarcoma virus-transformed (RSV)-NIH-3T3 cell lysates, a positive con-
A
M.W.
1
2
3
4
5
6
1
2
3
4
M. W.
(kW
5
A
Stat5
. 200
-
. 97.4
(kW
69
Stat3 +
m
46
IP : anti-Stat3
WB: anti-PY
IP: Anti-PY (4G10)
WB: Anti-Stat5
IP : anti-PY
WB: anti-Jak2
LP : anti-Stat3
WB: anti-Stat3
M.W.
C
Stat3
-
+
.a
-
Stat5
1
+
2
-
-
(kW
200
91.4
69
97.4
69
WB: anti-Stat5
Fig 2. (AI Protein tyrosine phosphorylation of St& in platelets
stimulated by thrombopoietin(TPO 100 ng/mL). Platelets were lysed
in SDS and lysates were diluted10-fold. Tyrosine phosphorylated proteins were immunoprecipitated
with 4610. After boiling for5 minutes
in SDS-sample buffer, proteins were separated by SDS-PAGE and
transferred onto PVDF membranes. Stat5 was detected by a spacific
Fig 1. (A) Tyrosine phosphorylation of Stat3 in platelets stimuantibody. Lane 1, resting platelets. Lanes 2 through 5, 15 seconds, 1
lated by thrombopoietin (100 nglmL). Platelets were lysed by the
minute, 5 minutes, and 10 minutes after exposure t o thrombopoietin
addition of an equal amount of a buffer containing 2% Triton X-l00
(100 ng/mL). HC, heavy chain of IgG.(B) Protein tyrosine phosphorylabefore and after exposure t o thrombopoietin (100 ng/mL). Stat3 was
tion of Jak2 in platelets stimulated by thrombopoietin (TPO 100 ng/
immunoprecipitated with specific anti-Stat3 antisera. Immune commL). Platelets were lysed as in Fig 1. Tyrosine phosphorylated proteins
plexes were lysed with SDS-sample buffer. Proteins were separated
were immunoprecipitated with 4G10. After boiling for 5 minutes, proby 10% SDS-PAGE and transferred onto PVDF membranes. Immuteins wereseparated by SDS-PAGE and transferred onto PVDF memnoblots were probed with antiphosphotyrosine antibody and
bands
branes. J a M was detected by a specific antibody. Lanes arethe same
were visualized by chemiluminescence. Lane 1, resting platelets.
as in (A). IC)Anti-Stat5 recognized the same protein in lysates from
Lanes 2 through 6,15 seconds, 1 minute, 5 minutes, 10 minutes, and
platelets and RSV-3T3 cells. The same as
Fig 1C except that Stat5 was
30 minutes after exposure t o thrombopoietin (100 ng/mL). (B) The
same PVDF membrane used in (A) was stripped of the antibody and detected by a specific antibody. Lane 1, resting platelets 11.5 x lo’
cells/lane). Lane 2, RSV-3T3 cells (20 p g proteinllanel.
reprobed for Stat3. Bands were visualized by chemiluminescence.
Lanes are the same as in (A). (ClAnti-Stat3 recognized the same
protein in lysates from platelets and RSV-3T3 cells. Platelets were
trol for the transcription factors, were
from
Transduction
lysed by the addition ofan equal amount of 2x SDS-sample buffer.
KY).
Anti-Stat.?,
anti-Stat4,
and
anti-Tyk2
Laboratories
(Lexington,
After boiling for 5 minutes, proteins were separated by SDS-PAGE
were from Santa Cruz (Santa Cruz, CA). Iscove’smodified Duland transferred onto PVDF membranes. Stat3 was detected by a
beco’s medium (IMDM) wasfrom GlBCO BRL (Gaithersburg,
specific antibody. Lane 1, resting platelets(1.5 x lO’cells/lanel. Lane
2, RSV-3T3 cells (20 p g protein/lane).
MD). Recombinant hirudin
kindly
was
provided
by Japan
Energy
WB : anti-Stat3
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441
PLATELETSANDSTATPROTEINS
M.W.
1
A
Stat3
+
2
F
"
3
4
-"-=W-"
S
"...
6
-~
l
(kDa)
D
97.4
Stat5
-
M.W.
L
1
+
2
3
#
4
5
6
69
(kDd
-
97.4
69
IP : anti-PY
WB: anti-Stat5
IP : anti-Stat3
WB: anti-PY
"
W_
..
..
1
R
Y
Stat3
2
3
4
+
5
6
-
M.W.
(kW
97.4
E
1
2
3
4
5
6
-
OdW
200
-
97.4
Ip : anti-Stat3
IP : anti-PY
WB: anti-Jak2
WB: anti-Stat3
1
C
Jak2
+
2
L
-
200
97.4
IP : anti-Jak2
WB: anti-PY
Fig 3. (A) Tyrosine phosphorylation of Stat3in FDCP-hMPL5 cells stimulated by thrombopoietin(100ng/mL). FDCP-hMPL5cells (lo' cells/
mL in PBS, pH 7.4)were lysed by the addition of an equal amount of a buffer containing 2% Triton X-l00before and after exposure t o
thrombopoietin (l00ng/mL). Stat3 was immunoprecipitated
with specific anti-Stat3 antisera. Immune complexes were lysed with SDS-sample
buffer. Tyrosine phosphorylation of Stat3 was detectedas described in Fig 1A. Lane 1, resting cells. Lanes 2 through 6, 1 minute, 5 minutes,
10 minutes, 30 minutes, and 60 minutes after exposure t o thrombopoietin (100ng/mL). (B) The same PVDF membrane used in (A) was stripped
of the antibody and reprobed with anti-Stat3
antisera. Bands were visualized by chemiluminescence. Lanes are the same as in (A). (C) Tyrosine
phosphorylation of JakZ in FDCP-hMPL5cells stimulated by thrombopoietin(100ng/mL). FDCP-hMPL5 cells (lo' cells/mL in PBS, pH 7.4)were
lysed by the addition of an equal amount of a buffer containing 2% Triton X-l00before and after exposure t o thrombopoietin (100ng/mLI.
Jak2 was immunoprecipitated andits tyrosine phosphorylation was detectedas described in Fig 1A. Lane 1, resting cells. Lanes 2 through 6,
l minute, 5 minutes, 10 minutes, 30 minutes, and 60 minutes after exposure t o thrombopoietin (100ng/mL). The same lysates as used in (A)
and (B) were used. Lanes are the same as in (A). (D) Tyrosine phosphorylation of Stat5 in FDCP-hMPL5 cells stimulated by thrombopoietin
(l00nglmL). Tyrosine phosphorylated proteins were immunoprecipitatedwith 4610.After boiling for5 minutes, proteins were separated by
SDS-PAGE and transferred onto PVDF membranes. Stat5 was detected by a specific antibody. Lane 1, resting cells. Lanes 2 through 6, 1
minute, 5 minutes, 10 minutes, 30 minutes, and 60 minutes after exposure t o thrombopoietin (100ng/mL). (E) Tyrosine phosphorylation of
Jak2 in FDCP-hMPL5 cells stimulated by thrombopoietin 1100 ng/mL). The same lysates as used in (D). JakZ was detected by a specific
antibody. Lanes are the same as in (D).
CO (Tokyo, Japan). Human erythropoietin was from Kirin Brewery
CO, Ltd (Maebashi, Japan). Recombinant thrombopoietin was prepared as described.',x An expression plasmid containing the fulllengthhuman c-mpl cDNA driven by the long terminal repeat of
Moloney murine sarcoma virus was a kind gift from AmgenInc
(Thousand Oaks, CA)!
Plafelefpreparation. Human blood from healthy volunteers was
drawn by venipuncture into 1/10 volume of 3.8% (wt/vol) trisodium
citrate and gently mixed. Platelet-rich plasma (PRP) was prepared
by centrifuging the whole blood at 20013 for 20 minutes and aspirating PRP. PRP was incubated with aspirin (2 mmol/L) for 30 minutes
atroom temperature. PGE, ( 1 pmol/L) was added from a stock
solution in absolute ethanol (1 mmol/L). The PRP was spun at 8 0 0 ~
to form a soft platelet pellet. The pellet was resuspended in l mL
of a modified HEPES-Tyrode buffer (129 mmol/L NaCI, 8.9 mmol/
L NaHCOl, 0.8 mmol/L KH2P04, 0.8 mmol/L MgCI?, 5.6 mmol/L
dextrose, and 10 mmol/L HEPES, pH 7.4) also containing apyrase
(2 UlrnL) and washed twice. Platelets were resuspended in the same
buffer at a concentration of 3 X 10' cells/mL with apyrase (2 U/
mL) containing I mmol/L CaClz at37°C. To makehirudinPRP,
hirudin (final concentration, 1 0 0 U/mL) was mixed with wholeblood
instead of citrate.
Cell lines. Murine IL-%dependent myeloid cell lineFDCP-2"
(kindly provided by Dr Arinobu Tojo, Institute of Medical Science,
University of Tokyo, Tokyo, Japan) was maintained in IMDM containing 10% fetal calf serum (FCS) and I ng/mL murineIL-3. A
thrombopoietin-dependent cell line (FDCP-hMPL5) was established
by transforming FDCP-2 cells with an expression plasmid containing
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MIYAKAWA ET AL
442
A
Stat3
1
2
3
+
.
4
"....
I
-
M.W.
(kW
-
97.4
69
IP : anti-Stat3
WB: anti-PY
M.W.
1
B
stat5
2
+
(kW
-
-
91.4
69
P : anti-PY
WB: anti-Stat5
Fig 4. (AI Tyrosine phosphorylation of Stat3 in FDCP-hMPW cells
stimulated by thrombopoietin (100 nglmL) but not in FD-ER3 cells
treated with erythropoietin (15 UlmL). FD-ER3 or FDCP-hMPL5 cells
(lo' cellslmL in PBS, pH 7.4) were lysed by the addition of an equal
amount of a buffer containing 246 Triton X-l00 before and after exposure to erythropoietin (15 UlmL) or thrombopoietin (100 nglmL1.
Stat3 wasimmunoprecipitated with specific anti-Stat3 antisera. Immune complexes were lysed with SDS-sample buffer. Tyrosine phosphorylation of Stat3 was detected as described in Fig 1A. Lane1,
resting FD-ER3 cells. Lane 2,lO minutesafter exposure to erythropoietin (15 UlmL; FD-ER3 cells). Lane 3, resting FDCP-hMPL5 cells. Lane
4, 10 minutes after exposure to thrombopoietin (100 nglmL; FDCPhMPL5). The same PVDF membrane was stripped of the antibody
and reprobed with anti-Stat3 antisera and the sameamount of Stat3
was detected in each lane (data not shown). (B) Tyrosine phosphorylation of Stat5 inFD-ER3 stimulated by erythropoietin (15 UlmL).
Tyrosine phosphorylated proteins were immunoprecipitated with
4G10. After boiling for 5 minutes, proteins were separated by SDSPAGE and transferred onto PVDF membranes. Stat5 was detected
by a specific antibody. Lane 1, resting cells. Lane 2, 10 minutes after
exposure to erythropoietin (15 UlmLI.
the full-length human c-mpl cDNA driven by the long terminal
repeat of Moloney murine sarcoma virus?" This cell line expresses
human c-Mpl protein on its cell surface and can grow in the presence
of recombinant thrombopoietin in a dose-dependent manner. FDCP2 cells expressing murine erythropoietin receptors (FD-ER3 cells)
were established as described previously.2' Before treatment of the
cells with thrombopoietin or erythropoietin, they were incubated in
IMDM containing 10% FCS without exogenous IL-3 for 6 hours.
After two washes, they were incubated in phosphate-buffered saline
(PBS; pH 7.4).
Gel electrophoresis and Western blotting to detect tyrosine phosphorylated proteins or Stat proteins. Platelet stimulation was terminated by the addition ofan equal volume of2X concentrated
Laemmli's sample buffer (10% glycerol, 1% SDS, 5% 2-mercaptoethanol, 50 mmol/L Tris-HCI [pH 6.81, and 0.002% bromophenol
blue),'' 10 mmol/L EGTA,and I mmol/L sodium orthovanadate.
After boiling at 95°C for 5 minutes, one-dimensional SDS-electrophoresis was performed on10% or 12% polyacrylamide gels as
described." Separated proteins were electrophoretically transferred
from the gel onto PVDF membranes 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, transfer membranes were incubated in TBST (Tris-buffered
saline [TBS], 10 mmol/L Tris, 150 mmol/L NaCI, 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 of1.0 & n L in TBST. The primary antibody
was removed and the blots were washed four times in TBST and
incubated with horseradish peroxidase-conjugated second antibody
diluted 1:3,000 in TBST. Blots were thenwashed four times in
TBST. Antibody reactions were detected with chemiluminescence
according to the manufacturer's instructions.
Immunoprecipitation. Cell stimulation was terminated by the addition of an equal amount of lysis buffer (15 mmol/L HEPES, 150
mmol/L NaCI, I mmol/L PMSF, 10 mmol/L EGTA, 1 mmol/L
sodium orthovanadate, 0.8 mg/mL leupeptin, 2% Triton X-100 [voll
wt], pH 7.4). After 20 minutes on ice, the lysates were centrifuged
at I0,OOOg (at 4°C) for 20 minutes. The supernatant was removed
and incubated with preimmune serum and protein A-Sepharose (40
pL of 50% slurry) for I hour. The desired polyclonal or monoclonal
antibodies were then added and incubated for 2 to 3 hours on ice.
Protein A-Sepharose or antimouse IgG-conjugated agarose (40 pL
of 50% slurry) was added and incubated for several hours. The
immune complexes were washed with 1 mL of cold washing buffer
(15 mmol/L HEPES, 150 mmol/L NaCI, 1 mmol/L PMSF, 10 mmoV
L EGTA, I mmol/L sodium orthovanadate, 0.8 mg/mL leupeptin,
1% Triton X-100 [vollwt], pH 7.4) three times and then lysed with
Laemmli's sample buffer. When immunoprecipitation of denatured
proteins was necessary, cells were lysed first in 1% SDS and boiled
for 5 minutes. The lysates were diluted IO-foldin the lysis buffer
that contains 1% Triton X-l 00 before immunoprecipitation.
Measurement of platelet aggregation. The measurement of
platelet aggregation was performed withan aggregometer (Hema
Tracer TM Model601; Niko Bio Science, Tokyo, Japan) with contin,O
O rpm).
uous stirring ( 1O
RESULTS
In preliminary experiments, we found that humanplatelets
express Statl, Stat2, Stat3, and Stat5 but not Stat4 or Stat6
by immunoblotting with specific antisera. A positive control
cell lysate (RSV-3T3) wasused to confirmthat the Stat
proteins were detected with the specific antisera and that the
proteins detected in platelets comigrated with the Stat proteins in the control lysates (Figs IC and 2C and data not
shown).
We next examined whether any of the Stat proteins became tyrosine phosphorylated after treatment of platelets
with thrombopoietin. Platelets treated with thrombopoietin
(100 ng/mL) for various times were lysed in a buffer containing 1% Triton X-100 and immunoprecipitated with specific Stat3 antisera. The same amount of a 95-kD protein
recognized by Stat3 antisera was immunoprecipitated from
all cellular lysates (Fig 1B). After treatment with thrombopoietin, this same protein was tyrosine phosphorylated (Fig
1A). Thus, platelets have Stat3 and itbecomes tyrosine phosphorylated after thrombopoietin treatment.
Similar experiments were performed to examine whether
Stat5 was inducibly tyrosine phosphorylated in platelets. Because the anti-Stat5 antibody used does not work for immunoprecipitations, tyrosine phosphorylated proteins were immunoprecipitated, from denatured cellular lysates,with a
specific antiphosphotyrosine antibody (4G10). Wefound
that,in
the antiphosphotyrosine immunoprecipitates, increased amounts of a 95-kD protein were recognized by an
anti-Stat5 antibody after stimulation of platelets with throm-
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443
PLATELETS AND STATPROTEINS
-
Stat3 +
97.4
69
4
6
IP: Anti-Stat3
W:
Anti-PY
B
Fig 5. (A) Thrombopoietininduced tyrosine phosphorylationof Stat3 in platelets in a dose-dependent
fashion. Platelets were lysed by the addition of an
equal amount of a buffer containing2% Triton X-l00
before and after exposure to thrombopoietin (0.1 to
100 ng/mL). Tyrosine phosphorylationof Stat3 was
detected as described in Fig 1A. (B) The priming of
epinephrineinduced platelet aggregation in hirudinPRP. Aspirin-pretreated hirudin-PRP was incubated
with thrombopoietin (10 nglmL)for 5 minutes. Epinephrine (1 prnollL)
was then added (the upper line).
In the lower line, thrombopoietin (10 nglmL) was
added after stimulation with epinephrine (1 pmollL)
for 1 minute. Aggregation was measured turbimetrically.
.-OmC
l:
TPO
’l-
bopoietin (Fig 2A). Similarly, we examined whether Stat1
and Stat2 were tyrosine phosphorylated and we could find
no evidence for tyrosine phosphorylation of either protein
before or after stimulation of platelets with thrombopoietin
(data not shown).
Jak2 has been reported to phosphorylate Stat5 on tyrosine
in vitro and possibly in ~ i v o . ” . ’ Consistent
~~’~
with our previous observations, increased amounts of Jak2 were detected
in antiphosphotyrosine immunoprecipitates after stimulation
of platelets with thrombopoietin (Fig 2B). Tyrosine phosphorylation of Jak2 occurred over a similar time course to
that of Stat3 and Stat5 (compare Fig IA and Fig 2A and B).
To further confirm the link between thrombopoietin stimulation and Stat3/Stat5, we examined whether Stat3 and Stat5
were tyrosine phosphorylated in FDCP-2 cells expressing
humanc-Mpl (FDCP-hMPL5). FDCP-hMPL5 cells were
treated with thrombopoietin (100 ng/mL) for various times
and were lysed in a buffer containing I % Triton X- 100. The
same amount of a 95-kD protein recognized by Stat3 antisera
was immunoprecipitated from all cellular lysates (Fig 3B).
This same protein became tyrosine phosphorylated after
treatment with thrombopoietin (Fig 3A). We also found that
lmin
I
I
lag time
Jak2 was tyrosine phosphorylated after treatment with
thrombopoietin (Fig 3C), which corroborates the data obtained in platelets and BaF3 cells transfected with murine cMpI.l4.’’ Furthermore, tyrosine phosphorylation of Jak2 occurred along with that of Stat3 (compare Fig 3A and C). We
also found that 4G10 immunoprecipitates contained increased amounts of Stat5 and Jak2 from lysates of FDCPhMPL5 cells (Fig 3D and E) after thrombopoietin stimulation. As in platelets, we did not detect any tyrosine phosphorylation of Stat 1 or Stat2 either before or after stimulation of
FDCP-hMPL5 cells with thrombopoietin (data not shown).
Because of the structural similarity between thrombopoietin
and erythropoietin, we also examined whether Stat3 and
Stat5 were tyrosine phosphorylated in FDCP-2 cells expressing erythropoietin receptors (FD-ER3 cells). Erythropoietin
induced tyrosine phosphorylation of Stat5 but not of Stat3
in these cells (Fig 4A and B).
We have found that treatment of platelets with thrombopoietin enhances aggregation induced by various agonists
such as shear stress, epinephrine, and ADP (manuscript submitted). The effect was observed at 1 ng/mL of thrombopoietin and reached a maximum at IO ng/mL. Figure 5A shows
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MIYAKAWA ET AL
444
M.W.
(kW
A
1
Tyk2 +D
2
3
4
5
2
._;
-
200
-
97.4
LP : anti-Tyk2
WB: anti-PY
M.W.
(kW
B
1
2
3
4
5
-
200
97.4
IP : anti-Tyk2
WB: anti-Tyk2
Fig 6. (A) Tyrosine phosphorylation ofTyk2 in platelets stimulated
by thrombopoietin(100 nglmL1. Platelets were lysed by the addition
of an equal amount of a buffer containing 2% Triton X-l00 before
and afterexposure t o thrombopoietin (100 ng/mL). Tyk2 was immunoprecipitated with specific anti-Tyk2 antisera. Immune complexes
were lysed with SDS-sample buffer. Proteins were separated by 10%
SDS-PAGE and transferred onto PVDF membranes. lmmunoblots
were probed with antiphosphotyrosine antibody and bands were
visualized by chemiluminescence. Lane 1, resting platelets. Lanes 2
through 5, 15 seconds, 1 minute, 5 minutes, and 30 minutes after
exposure t o thrombopoietin (100 ng/mL). (B1 The same PVDF memof the antibody and reprobed forTyk2.
brane used in (A) was stripped
Bands were visualized by chemiluminescence. Lanes are the same
as in (A).
that tyrosine phosphorylation of Stat3 also occurs at I ng/
mL and reached a maximum at 10 ng/mL. We found that
epinephrine, which does not induce platelet aggregation in
hirudin-PRP,” does so in the presence of thrombopoietin
(manuscript submitted). Figure SB shows that thrombopoietin ( I O ng/mL) did not induce aggregation or shape change.
Subsequent addition of epinephrine induced aggregation
within 15 seconds. When the order of the agonists was reversed, there was a significant lag time (the lower line).
However, aggregation was still observed within 1 minute
after the addition of thrombopoietin, suggesting that the
priming of epinephrine-induced platelet aggregation occurred within 1 minute.
Finally, we examined whether other Jak family kinases
other than Jak2 may also be tyrosine phosphorylated after
treatment of platelets with thrombopoietin. We found that
thrombopoietin induced tyrosine phosphorylation of Tyk2 in
platelets and FDCP-hMPL5 cells (Figs 6 and 7). Under the
same conditions, we did not detect any tyrosine phosphorylation of Jakl and Jak3 (data not shown).
DISCUSSION
This report shows the presence of Stat proteins and thrombopoietin-induced tyrosine phosphorylation of Stat3 and
Stat5 in human platelets. Both were also tyrosine phosphorylated in FDCP-hMPL5 expressing human c-Mpl, a receptor
for thrombopoietin. The tyrosine phosphorylation of Stat
proteins induced by thrombopoietin was apparently limited
to Stat3 and Stat5. Our data do not rule out the possibility
that other unknown Stat-like proteins are also tyrosine phosphorylated in platelets or in FDCP-hMPL5 or that Statl or
Stat2 could be phosphorylated below the level of detection
in our systems. Interestingly, Gurney et al” showed that Stat
1 and 3 were activated, as measured by binding to a sisinducible element (SIE), in CMK megakaryoblastoid cells
stimulated with thrombopoietin. Tyrosine phosphorylation
of Statl wasnotmeasured
directly by these investigators
and further studies will be required to explore the possibility
that a variety of Stat heterodimers are formed after thrombopoietin stimulation.
In a study by Goullieux et al,”’ it was also reported that
phosphorylation of tyrosine at position 697 was essential for
DNA binding activity of Stat5 and that Stat5 was tyrosine
phosphorylated by Jak2 in vitro. Our data show that tyrosine
phosphorylation of Jak2 occurred along with that of Stat3
or Stat5 Because phosphorylation of Jak2 is known to be
accompanied by enhanced kinase activity,’”’’.’” it is likely
that Jak2 may at least partially contribute to tyrosine phosM.W.
(kW
A
l
2
3
4
5
Tyk2 +
-
200
97.4
IP : anti-Tyk2
WB: anti-PY
M.W.
(kW
B
1
2
3
4
Tyk2 +
5
-
-
200
97.4
IP : anti-Tyk2
WB: anti-Tyk2
Fig 7. (AI Tyrosine phosphorylation of Tyk2 in FDCP-hMPL5 cells
stimulated by thrombopoietin (100 nglmL1. FDCP-hMPL5 cells (10’
cells/mL in PBS, pH 7.41 were lysed by the addition of an equal
amount of a buffer containing2% Triton X-l00 before and after exposure t o thrombopoietin (100 ng/mL). Tyk2 was immunoprecipitated
with specific anti-Tyk2 antisera. Immune complexes were lysed with
SDS-sample buffer. Tyrosine phosphorylation of Tyk2 was detected
as described in Fig 1A. Lane 1, resting cells. Lanes 2 through 5, 1
minute, 5 minutes, 10 minutes, and 30 minutes after exposure t o
thrombopoietin (100 ng/mLI. (B] The same PVDF membrane used in
(A) was stripped of the antibody and reprobed with anti-Tyk2 antisera. Bands were visualized by chemiluminescence. Lanes are the
same as in (Al.
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445
PLATELETS AND STAT PROTEINS
phorylation of the Stat proteins in our system. We have
found that, consistent with the report of Rodriguez-Linares
et alZ7TyM and Jakl are also present in human blood platelets and that TyM becomes tyrosine phosphorylated after
stimulation of platelets and FDCP-hMPL5 cells with thrombopoietin (Figs 6 and 7). Thus, Tyk2 may also contribute to
tyrosine phosphorylation of the Stat proteins in our system.
Recently, Sattler et a12* also reported that thrombopoietin
induces tyrosine phosphorylation of Tyk2 in Mo7e cells and
BaF3 cells, which were genetically engineered to express cMpl, although Tortolani et a129did not detect increased tyrosine phosphorylation of Tyk2 in thrombopoietin-treated
Mo7e cells. The reason for the different observations is unknown.
Involvement of both Stat3 and Stat5 in signaling induced
by thrombopoietin is interesting in view of the recent reports
of activation and tyrosine phosphorylation of Stat5 but not
of Stat3 induced by erythropoietin (Fig 4),growth hormone,
and GM-CSF.3@35
Indeed, Stahl et a132determined that the
YXXQ motif in gp130 of IL-6 receptor, which is required
for tyrosine phosphorylation and activation of Stat3, is absent
in the cytoplasmic domain of erythropoietin or GM-CSF
receptor. This motif is present in the c-Mpl cDNA used in
our transfection studies? Given that the signal transduction
pathways used by these cytokines overlap significantly, the
involvement of specific combinations of Jak family members
and Stat proteins may contribute to the unique effects of
each cytokine.
Previously, we showed that thrombin and thrombopoietin
induced distinctively different patterns of protein tyrosine
phosphorylation in platelets.” Although Jak2 may be tyrosine phosphorylated and activated by a few fold in thrombinstimulated platelets:’ we did not find measurable increase
in tyrosine phosphorylation of Stat3 or Stat5 proteins after
thrombin treatment of platelets (data not shown). Thus, the
thrombin receptor and c-Mpl appear to use differing signaling pathways and tyrosine phosphorylation of the Stat proteins seems to be unique to the thrombopoietin receptor activation in platelets.
One of the most interesting findings in thisstudyisthe
presence of Stat proteins in human platelets because they are
devoid of nuclei. One possibility is that they are simply remnants from megakaryocytes. However, this would be consistent with a role
for Stat proteins in
megakaryopoiesis/thrombopoiesis.Anothermoreintriguinghypothesis
is thatStat
proteins may haverolesinplateletactivation.Indeed,
we
found that thrombopoietin-induced tyrosine phosphorylation
of Stat3 was observed at concentrations of thrombopoietin of
1 ng/mL and reached a maximum at 10 ng/mL (Fig 5A). In
separate experiments, we found that thrombopoietin primed
platelet aggregation induced by various agonists in a similar
dose range (manuscript submitted). Furthermore, the priming
of epinephrine-induced platelet aggregation and tyrosine phosphorylation of Stat3 and Stat5 occurred within 1 minute after
stimulation of platelets with thrombopoietin (compare Figs
IA, 2A, and 5B). Because serum levels of thrombopoietin
may reach 10 n g / d inthrombocytopenicpatients,36 these
data suggest that tyrosine phosphorylation of Stat3 and Stat5
and priming of platelet aggregation by thrombopoietin occurred in a similar time framework and in a dose range that
can be achieved in pathophysiologic states. Because thrombopoietin may enhance the activation of platelets, it is possible
that Stat proteins, perhaps through their SH2 domains, could
be an element in this enhancement. Most importantly, these
data show that thrombopoietin is similar to other cytokines,
such as GM-CSF, IL-3, and G-CSF, that
are not only required
for the proliferation and maturation of progenitor cells but
stimulate the functions of mature hematopoietic cells.
ACKNOWLEDGMENT
We thank Dr Arinobu Tojo, Japan Energy CO, and Amgen Inc
for kindly providing reagents or cells. We thank Akihiko Shimosaka
for his continual encouragement and helpful comments.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1996 87: 439-446
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5
in human blood platelets
Y Miyakawa, A Oda, BJ Druker, H Miyazaki, M Handa, H Ohashi and Y Ikeda
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