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Steel Factor Induces Serine Phosphorylation of Stat3 in Human Growth
Factor-Dependent Myeloid Cell Lines
By Akihiko Gotoh, Hiroyuki Takahira, Charlie Mantel, Sara Litz-Jackson, H. Scott Boswell, and Hal E. Broxmeyer
Steel factor (SLF) acts synergistically with various hematopoietic growth factors that use the Jak-Stat pathways in
vivo and in vitro, although the contribution by SLF to this
pathway is unknown. We show here that SLF induces timeand dose-dependentphosphorylationof Stat3 in the human
growth factor-dependent cell lines M07e and TF-1. This
phosphorylation occurs exclusively on serine residues. Simultaneous stimulation with SLF plus other cytokines that
induce tyrosine phosphorylation of Stat3, such as interleukin-9 (IL-9) in M07e cells or IL-6 in TF-1 cells, resulted in
tyrosine phosphorylationand enhancedserine phosphorylation of Stat3. Serine phosphorylationalone did not promote
nuclear translocation or DNA binding activity to the sis-inducible element of Stat3. However, costimulation with SLF
plus IL-9 in M07e cells resulted in the nuclear translocation
of serine-hyperphosphorylatedStat3. Serine phosphorylation of Stat3 was also observed by the stimulation of cells
with granulocyte-macrophagecolony-stimulatingfactor and
IL-3, which do not induce tyrosine phosphorylationof Stat3.
These results suggest that SLF might modulate the JakStat3 pathway by serine phosphorylationand that the JakStat pathway may be differentially regulated by the combinational stimulation of two or more cytokines.
0 1996 by The American Society of Hematology.
S
proteins on a tyrosine immediately distal to the SH2 domain
induces their homo- or hetero-dimerization through phosphotyrosine-SH2 interactions. As a consequence, they translocate to the nucleus, bind to specific promoter elements, and
induce gene expression.'
Although SLF has been found to act synergistically with
various cytokines that use the Jak-Stat pathway, ie, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3),I0 erythropoietin (EPO)," IL-9,'* and
thrombopoietin (TPO),l3 the contribution by SLF to this
pathway has not been described. In the present study, we
show that SLF induces phosphorylation of Stat3 on serine
but not on tyrosine residues in human factor-dependent myeloid cell lines. Serine phosphorylation of Stat3 alone does
not promote nuclear translocation. However, costimulation
by SLF with other cytokines that induce tyrosine phosphorylation of Stat3 resulted in translocation of serine-hyperphosphorylated Stat3 to the nucleus.
TEEL FACTOR (SLF) is a potent costimulating cytokine
that acts synergistically with a number of cytokines to
stimulate growth of hematopoietic progenitor cells in vitro
and blood cell production in vivo.' c-kit, the receptor for
SLF, is a receptor-type protein tyrosine kinase (PTK) that is
a member of the platelet-derived growth factor receptor family. Binding of SLF to the c-kit-encoded surface protein
induces dimerization, followed by transphosphorylation and
subsequent PTK activation.' The resulting tyrosine-phosphorylated c-kit receptor binds SH2-containing proteins such
as phospholipase C-y (PLC-y), phosphatidylinositol 3-kinase, Syp, and HCP, which are in turn phosphorylated by
the c-kif PTK.'.'
The receptors for other cytokines and interferons of the
cytokine superfamily are composed of one to three polypeptides and are constitutively associated with members of the
Janus kinase (Jak) family of intracellular PTKs. Ligandbinding induces receptor oligomerization with concomitant
Jak activation.6 The PTK substrates that are most directly
implicated in signal transduction of the cytokine superfamily
are the Stat (the signal transducers and activators of transcription) proteins. To date, six Stats have been cloned and
characterized. These Stats possess an SH2 domain through
which they bind specific phosphotyrosine sites on the activated receptor.' Each subfamily of cytokine receptors regulates a unique set of Stat proteins in a fashion that is determined by the SH2-binding specificity of the receptor
phosphorylation sites.' Subsequent phosphorylation of Stat
From the Departments of Medicine and Microbiology/lmmunology and the Walther Oncology Center, Indiana University School
of Medicine, Indianapolis, IN.
Submitted November 29, 199.5; accepted March 7, 1996.
Supported by US Public Health Service Grants No. ROI HLS6416,
R37 CA 36464, ROI HL 54037, and ROI HL 49202 and by a project
in Grant No. POI HL 53586 from the National Cancer institute and
the National Institutes of Health to H.E.B.
Address reprint requests to Hal E. Broxmeyer, PhD, Walther
Oncology Center, Indiana University School of Medicine, 975 W
Walnut St, Indianapolis, IN 46202-5121.
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.
0006-4971/96/8801-oO30$3.00/0
138
MATERIALS AND METHODS
Cells, cell culture, and cell proliferation assay. Human growth
factor-dependent myeloid cell lines, M07e and TF- I , were cultured
in RPMI 1640 supplemented with 20% fetal bovine serum (FBS)
and 100 U/mL GM-CSF. These cell lines have been described elseBefore cytokine stimulation. these cells were washed
twice, resuspended in RPMI 1640 supplemented with 0.5% bovine
serum albumin (BSA), and then incubated for 18 hours without
growth factors (factor starvation). ['Hlthymidine uptake was used
to measure cell proliferation by incubation of triplicate aliquots of
cells cultured in flat-bottom microtiter plates (1 X lo4 cellsll50 pL)
with or without cytokines for 72 hours at 37°C. Cells were labeled
with ['Hlthymidine (Amersham, Arlington Heights, IL) at 0.5 pCi/
well for the last 4 hours.
Cytokines, antibodies, and reagents. Recombinant human (rhu)
SLF, rhuIL-3, and rhuGM-CSF were kindly provided by Dr D.E.
Williams (Immunex Co, Seattle, WA). rhuIL-6 and rhuIL-9 were
obtained from R & D Systems (Minneapolis, MN). Anti-Stat2, Stat3,
Stad, and Stat6 monoclonal antibodies (MoAbs), anti-ISGF3 polyclonal Ab, and anti-phosphotyrosine MoAb (PY20) were purchased
from Transduction Laboratories (Lexington, KY). Anti-Stat3 and
Stat4 polyclonal Abs and synthetic double-strand oligonucleotide
corresponding to a binding site for sis-inducible factor (sis-inducible
element [SIE]) and its mutant in the DNA binding region were
from Santa Cmz Biotechnology Inc (Santa Cruz, CA). Agaroseconjugated antiphosphotyrosine (4G10) was obtained from Upstate
Biotechnology, Inc (Lake Placid, NY). Horseradish peroxidase
(HRP)-conjugated antimouse and antirabbit Ig and ['ZP]~rthoph~sphate were purchased from Amersham.
Blood, VOI 88. NO 1 (July 1). 1996 pp 138-145
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139
SERINE PHOSPHORYLATION OF Stat3 BY SLF
In vivo labeling with "P. After factor starvation, cells were
washed three times with Minimum Essential Medium without sodium phosphate (Sigma, St Louis, MO) and incubated for 45 minutes
in the same medium supplemented with 10% dialyzed FBS at a cell
density of 107/mL. [3ZP]orthophosphate(100 pCi/mL) was added
and cells were further incubated for 3 hours. After stimulation with
various cytokines, cells were washed with ice-cold phosphate-buffered saline and then lysed as described below.
Cell lysis and nuclear extract preparation. Cells were lysed in
lysis buffer as described previously." After 20 minutes on ice, insoluble fractions were removed by centrifugation at 14,000g for 20
minutes (whole cell lysate). Nuclear extract was prepared as described previously." Cells were washed with ice-cold buffer A (10
mmol/L HEPES, pH 7.9, 10 mmol/L KCl, 1.5 mmol/L MgCI2, 0.5
mmol/L dithiothreitol [DlT], 0.5 mmom phenylmethylsulfonyl
fluoride [PMSF]) and were resuspended in buffer A containing 0.1 %
Nonidet P-40 and incubated for 5 minutes on ice. The nuclear pellet
was pelleted by centrifugation. The supematant was centrifuged for
20 minutes at 14,OOOg and the resulting supernatant was saved as
cytoplasmic extracts. The nuclear pellets were rinsed with buffer A
and resuspended in buffer C (20 mmol/L HEPES, pH 7.9,420 mmoY
L NaCl, 1.5 mmol/L MgC12, 0.2 mmoVL EDTA, 0.5 mmol/L DTT,
0.5 mmol/L PMSF, 25% glycerol). After gently rocking for 15 minutes at 4"C, insoluble nuclear material was removed by centrifugation
at 14,OOOg for 15 minutes. The protein concentration of these cell
extracts was measured with a Bio-Rad protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded in each lane or
were used for immunoprecipitation and gel-mobility shift assay.
Immunoprecipitation and immunoblotting. Immunoprecipitation
and immunoblotting were performed as described previously.I6
Briefly, for immunoprecipitation, cell extracts were mixed with 1
pg of anti-Stat3 polyclonal Ab at 4°C for 2 hours. Antigen-Ab complexes were collected with protein A sepharose beads (Pharmacia
Biotech, Uppsala, Sweden). Immunoprecipitates were washed with
lysis buffer five to seven times before analysis. Immunoprecipitates
and cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore Corp, Bedford,
MA). Membranes were blocked in Tris-buffered saline containing
0.5% Tween 20 and 1% BSA for 1 hour at room temperature and
incubated with appropriate primary Abs for 2 hours. Blots were
visualized using HRP-conjugated secondary Abs and an enhancedchemiluminescent system (ECL; Amersham). To reprobe with another first Ab, membranes were incubated in stripping buffer (62.5
mmol/L Tris, pH 6.7, 100 mmol/L 2-mercaptoethanol, 2% SDS) for
30 minutes at 50°C, washed, and then used for further study.
Phosphoamino acid analysis. ["PI-labeled anti-Stat3 immunoprecipitates were separated by 7.5% SDS-PAGE, transferred to
PVDF membranes, and visualized by autoradiography. The region
corresponding to phosphorylated Stat3 on the membrane was excised
and hydrolyzed in 6 N HCl. Phosphoamino acid analysis was then
performed on nitrocellulose thin-layer chromatography plates (Merk,
Darmstadt, Germany) as described previously with 1 pg each of
standard phosphoamino acids in pH 3.5 electrophoresis buffer.'*
Electric gel mobility shift assay (EMSA). EMSA was performed
as described previously." A double-strand synthetic SIE consensus
oligonucleotide (5'-GTG CAT TTC CCG TAA ATC l T G TCT
ACA-3')'' was 5' end-labeled with [y3'P]ATP (Amersham) by polynucleotide T4 kinase (Boehringer Mannheim, Indianapolis, IN). Nuclear extract (5 pg) was incubated with 3 pg of poly(d1-dC) (Pharmacia Biotech) for 15 minutes on ice before adding 10,000 cpm of
radiolabeled probe in binding buffer (20 mmol/L HEPES, pH 7.9,
20 mmoVL KCl, 5 mmol/L MgCl', 5 mmol/L D l T , 10% glycerol).
For supershift assay, nuclear extract was preincubated with antiStat3 Ab or preimmune rabbit serum for 1 hour on ice. Competitions
were performed with either the unlabeled homologous probe or a
oligonucleotide mutated in the DNA binding region (5'-GTG CAT
CCA CCG TAA ATC l T G TCT ACA-3'). After binding reaction
for 10 minutes at room temparature, DNA-protein complexes were
separated by 5% naive polyacrylamide gel in 0 . 2 5 ~TBE buffer.
RESULTS
Tyrosine phosphorylation of Stat family proteins in factordependent cell lines. Tyrosine phosphorylation of Stat family proteins, presumably by Jak family tyrosine kinases, has
been shown to be essential for their DNA binding and transcriptional activation.' We first examined the effect of various hematopoietic growth factors on tyrosine phosphorylation of Stat family proteins in human factor-dependent cell
lines, M07e and TF-1. Factor-starved M07e cells were stimulated with 100 ng/mL SLF, 200 U/mL GM-CSF, 200 U/
mL IL-3, or 5 ng/mL IL-9 for 10 minutes at 37°C. Whole
cell lysates of cytokine-stimulated cells were immunoprecipitated with agarose-conjugated antiphosphotyrosine MoAb
and immunoprecipitates were separated by SDS-PAGE followed by immunoblotting with specific antibodies against
Stat family proteins. As shown in Fig lA, in M07e cells,
Stat5 was found to be tyrosine-phosphorylated in response
to GM-CSF, IL-3, and IL-9. Stat3 was tyrosine-phosphorylated by IL-9 (Fig 1B). Tyrosine phosphorylation of Stat3
reached a plateau at 5 ng/mL of IL-9 (data not shown). No
tyrosine phosphorylation of either Stat5 or Stat3 was detected after 100 ng/mL of SLF stimulation. Tyrosine phosphorylation of other Stat family proteins tested (ISGF3,
Stat2, Stat4, and Stat6) was not detectable or was very weak
after stimulation with IL-9, GM-CSF, IL-3, or SLF in these
conditions (data not shown).
In TF-1 cells, which respond to the proliferative stimulation effect of IL-3, IL-6, GM-CSF, and SLF, but not to IL9, tyrosine phosphorylation of Stat5 by IL-3 and GM-CSF
(Fig 1C) and tyrosine phosphorylation of Stat3 by IL-6 (Fig
1D) was observed. Maximal tyrosine phosphorylation of
Stat3 was obtained by 20 ng/mL of IL-6 (data not shown).
Thus, SLF had no effect on the tyrosine phosphorylation of
Stat family proteins in TF-1 cells (Fig 1C and D) or in M07e
cells (Fig 1A and B). Regarding GM-CSF, IL-3, and IL-6,
these results confirm reports by others,'0.11.2'
while these data
show that IL-9 uses both Stat3 and Stat5 as downstream
signaling pathways, probably as a result of activation of Jak
family tyrosine kinases (Jakl, Jak2, and Tyk2) in M07e
cells.
Slower mobility of Stat3 in SDS-PAGE by SLF is caused
by hyperphosphorylation. Immunoblotting of whole cell
lysates with anti-Stat5 MoAb showed mobility shift of Stat5
in response t o tyrosine phosphorylation in M07e cells (Fig
2A). SLF did not modify mobility of Stat5 in SDS-PAGE
(Fig 2A). Although SLF also did not induce tyrosine phosphorylation of Stat3 (Fig IB), slightly slower mobility of
Stat3 in SDS-PAGE was observed in M07e cells treated
with SLF compared with control medium-treated cells (Fig
2B). It has been reported by others that IL-6 and epidermal
growth factor (EGF) induce two tyrosine-phosphorylated
forms of Stat3 and that the slower band is serine phosphorylated.22,23
Unlike the results found in those previous reports,
Stat3 in cells treated with SLF did not form doublet bands,
but rather formed one slower band (Fig 2B). To investigate
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GOTOH ET
140
A
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-stat5
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Blot:
if this slower mobility was caused by phosphorylation of
Stat3 on amino acids other than tyrosine, M07e cells were
labeled with ['2P]orthophosphate and then stimulated with
various cytokines. As shown in Fig 3, immunoprecipitation
with anti-Stat3 Ab of ["PI-labeled cell lysates showed that
117
-
89
-
a-.rrrm--Y?n
Blot
+stat5
a - Stat 5
117 -
89 -
Blot
-!Bat3
a-Stat3
Fig 2. Mobility change of Stat5 and Stat3 after stimulation of
M07e cells. Factor-starved M07e cells were stimulated with the indicated cytokines for 10 minutes at 37°C. Whole cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Stat5 MoAb
(A) and anti-Stat3 MoAb (B). Concentrations of cytokines were the
same as in Fia 1. Similar results were obtained in five indeDendent
experiments.
I
c - 3
":!i
f g 2
kDa
117
Blot:
AL
u
- a,-,
.1
Fig 1. Tyrosine phosphorylation of Stat5 and Stat3 in M07e
and TF-1 cells. Factor-starved
M07e (A and Bl and TF-1 I C and
D) cells were stimulated with indicated cytokines for 10 minutes
at 37°C. Cells were lysed in lysis
buffer and immunoprecipitated
with agarose-conjugated antiphosphotyrosine MoAb. Immunoprecipitates were separated
by 7.546 SDS-PAGE and immunobloted with anti-Stat5 MoAb
IA and C) and anti-Stat3 MoAb
IB and D). The concentrations of
cytokines used were as follows:
100 nglmL SLF, 200 UlmL GMCSF, 200 UlmL IL-3.20 nglmL IL6, and 5 ng/mL IL-9. These are
representative results of three
independent studies.
SLF can induce time- and dose-dependent phosphorylation
of Stat3.
Effect of SLF alone and in combination on phosphorylation ofStat3. Proliferative effects of SLF and cytokines that
induced tyrosine phosphorylation of Stat3 were examined by
['Hlthymidine uptake. It was confirmed that SLF and IL-9
in M07e cells (Fig 4A) and SLF and IL-6 in TF-I cells (Fig
4B) had significant proliferative effects at the concentrations
used in this study. Moreover, simultaneous stimulation with
SLF plus these cytokines induced synergistic proliferation
(Fig 4).
We next determined the nature of SLF-induced Stat3
phosphorylation in more detail. "P-labeled factor-starved
M07e cells were stimulated with cytokines, and Stat3 immunoprecipitates were analyzed by immunoblotting and autoradiography. Medium-treated cells showed basal phosphorylation of Stat3 (Fig 5C, lane 2) as compared with
immunoprecipitation with preimmune serum (Fig 5C, lane
I). Simultaneous stimulation with SLF plus IL-9, which induced synergistic proliferation of M07e cells (Fig 4A), resulted in enhanced phosphorylation of Stat3 (Fig 5C, lane
5 ) . Enhancement of overall phosphorylation was 3.5-fold by
SLF, 2.5-fold by IL-9, and 4.2-fold by SLF plus IL-9 compared with medium-treated cells. In SLF plus IL-9-treated
cells, tyrosine-phosphorylated Stat3 appeared as a single
slightly slower migrating band in SDS-PAGE (Fig 5B, lane
5), whereas that of IL-9-treated cells appeared as a doublet
band (Fig 5B, lane 4). To define amino acid residues phosphorylated by the stimulation with SLF, phosphoamino acid
analysis was used. Regions corresponding to the mobility of
Stat3 of the membrane were cut and hydrolyzed at the same
time. Afterwards, all of the hydrolyzed amino acids were
loaded in each lane and were separated on the same TLC
plate. Figure 5D shows that SLF induces phosphorylation of
Stat3 exclusively on serine residues. IL-9 induces tyrosine
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SERINE PHOSPHORYLATION OF Stat3 BY SLF
141
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117 89-
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Blot:
90
mln.
.-CSm3
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60
a-Stat3
-
a Stat 3
Fig 3. Time- and dose-dependent phosphorylationof Stat3 by SLF. Factor-starved M07e cells were labeled with ["Plotthophosphate. Cells
were stimulated with 100 ng/mL SLF for the indicated period of time (A) or stimulated with the indicated concentrationof SLF for 7 minutes
(B) at 37°C and then lysed in lysis buffer. These whole cell lysates were immunoprecipitatedwith anti-Stat3 Ab. One half of each immunoprecipitate was separated by 7.5% SDS-PAGE and blots were visualized by autoradiography.The rest of immunoprecipitateswere separated by 7.5%
SDS-PAGE and immunoblotted with anti-Stat3 MoAb (left panels). Relative phosphorylationof Stat3 was determined by the densitometric
analysis with autoradiography and normalized by relative Stat3 amount in these immunoprecipitates (right panels). Similar results were
obtained from t w o separate experiments.
phosphorylation and, to a lesser extent than does SLF, serine
phosphorylation (Fig 5D). Densitometric analysis showed
that serine phosphorylation was approximately 3.5 times
higher after stimulation with 100 ng/mL of SLF-treated than
in nontreated cells. Notably, simultaneous stimulation with
SLF plus IL-9 induced phosphorylation of Stat3 on both
serine and tyrosine residues (Fig 5D). Serine phosphorylation of Stat3 by the stimulation with SLF plus IL-9 was
approximately 1.7 times higher than that of IL-9 alone as
determined by densitometric analysis.
Phosphorylation of Stat3 was also detected after stimulation with GM-CSF and IL-3 (Fig 5C, lanes 6 and 7), which
were confirmed not to induce tyrosine phosphorylation of
Stat3 (Fig 5B, lanes 6 and 7). Using densitometric analysis,
phosphorylation of Stat3 was 3.5-fold and 2.7-fold enhanced,
respectively, in GM-CSF- and IL-3-treated cells compared
with medium-treated cells. GM-CSF and IL-3, which induced tyrosine phosphorylation of Stat5 but not Stat3, induced serine phosphorylation of Stat3 (Fig 5D).
To confirm that serine phosphorylation of Stat3 by SLF
is induced not only in M07e cells but also in other myeloid
cells, TF- 1 cells were labeled with [."PI-orthophosphate and
stimulated with SLF, IL-6, SLF plus IL-6, and GM-CSF.
Phosphorylation of Stat3 was also shown in TF-I cells by
the stimulation with SLF and GM-CSF (Fig 6C, lanes 3 and
6) that did not induce tyrosine phosphorylation of Stat3 (Fig
6B, lanes 3 and 6). Simultaneous stimulation with IL-6 plus
SLF that induced synergistic proliferation in TF-I cells (Fig
4B) resulted in enhanced overall phosphorylation (Fig 6C,
lane 5) and serine phosphorylation (Fig 6D) of Stat3 compared with that with IL-6 alone (Fig 6C, lane 4 and Fig
6D). After stimulation of cells with SLF plus IL-6, tyrosinephosphorylated Stat3 migrated as a slower single band in
SDS-PAGE (Fig 6B, lane 5) compared with cells stimulated
with IL-6 alone (Fig 6B, lane 4). Also, stimulation with 100
nmol/L 12-0-tetradecanoyl phorbol- 13-acetate(TPA) for 10
minutes induced serine phosphorylation but not tyrosine
phosphorylation of Stat3 both in M07e and TF-I cells (data
not shown).
Translocation of hyper-phosphoylated Stat3 to the nucleus. For transcriptional activity of Stat3, translocation
from the cytoplasm to the nucleus is a crucial event. In this
context, we examined the localization of Stat3. Cytoplasmic
and nuclear extracts were prepared from M07e cells stimulated with SLF andor IL-9. Immunoblotting of nuclear extracts with anti-Stat3 MoAb showed translocation of Stat3
to the nucleus in response to IL-9 and IL-9 plus SLF in
M07e cells (Fig 7A). SLF alone had no effect on this translocation of Stat3, and GM-CSF did not induce translocation
(Fig 7A). Stat3 was observed in the cytoplasm and was
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GOTOH ET AL
142
[3H ] thymidine incorporation
x 103cpm
A
none
P
M07e
I
SLF
IL-9
translocated Stat3 showed that Stat3 in the nucleus was phosphorylated both on serine and tyrosine residues (data not
shown), and the ratio of "P incorporated into serine/tyrosine
was increased from 5.3 to 8.6 by simultaneous stimulation
of cells with IL-9 plus SLF (Fig 7E).
Activation of DNA binding activih containing Stat3 in
response to IL-9 or simultaneous stimulation with IL-9 plus
SLF. Stat3 is known to bind SIE DNA consensus site upon
a~tivation.'.'~Therefore, SIE binding activity was assessed
by EMSA with nuclear extracts of SLF and/or IL-9-treated
M07e cells. As shown in Fig 8, IL-9 activated a specific
DNA-binding protein (Fig 8, lane 3) whose binding could
be competed with excess unlabeled probe itself but not by
the unlabeled probe mutated in the DNA binding region (Fig
8, lanes 5 and 7). This DNA binding activity was not observed in nontreated cells or after SLF treatment alone (Fig
x 103cpm
B
5
10
15
20
none
1
2
3
mot
SLF
B
117 2
11-6
ea1
SLF+IL-6
2
3
117-
6
7
4
5
CSt.13
6
7
-
n P3r
Blot
c k l h
5
--
k h
-
Fig 4. Synergistic proliferation of M07e and TF-1 cells by the stimulation with SLF plus cytokines that induce Stat3 tyrosine phosphorylation. Factor-starved M07e (A) or TF-1 (B) cells were plated at 1 x
10' per well in RPMl 1640 containing 596 FBS with indicated cytokines. The concentrations of cytokines used were as follows: 100 n g l
mL SLF, 5 ng/mL IL-9, and 20 nglmL IL-6. Cells were labeled with
I'Hlthymidine for the last 4 hours of 72 hours of culture and then
the amount of incorporated ['Hlthymidine was calculated using a
scintillation counter. Data are the mean of triplicate culture SD. P
values were measured using the unpaired Student's t-test. " P < .0005
v control medium; * * P < .005 v control medium; # P < .0001 for
greater than additive effects.
4
a-Slat3
p
891
2
3
4
5
6
7
Autoradlogmphy
D
P-p-Thr
serine-phosphorylated after stimulation of cells with SLF
(data not shown). These results suggest that serine phosphorylation occurs in the cytoplasm and phosphorylation on tyrosine but not on serine residues is crucial for nuclear translocation of Stat3.
Although the protein amount and tyrosine phosphorylation
state of nuclear Stat3 were almost equal between IL-9 stimulation and IL-9 plus SLF stimulation (Fig 7B and C), overall
phosphorylation was enhanced approximately 1.6-fold by
stimulation with IL-9 plus SLF (Fig 7D). Furthermore, nuclear Stat3 formed a doublet band in SDS-PAGE after stimulation of the cells with IL-9 alone, whereas Stat3 formed a
single slower migrating band after stimulation with IL-9 plus
SLF (Fig 7B and C). Phosphoamino acid analysis of nuclear-
P - V
None
SLF
IL9
SLF
+IL9
IL3
GM
Fig 5. Phosphorylation and phosphoamino acid analysis of Stat3
in M07e cells. Factor-starved M07e cells labeled with "P were stimulated with SLF I100 nglmL), IL-9 (5 nglmLI, SLF + IL-9, GM-CSF (200
UlmL), or IL-3 (200 UlmL) for 10 minutes at 37°C. Anti-Stat3 immunoprecipitates or an immunoprecipitate with preimmune rabbit serum
as a mock control were separated by 7.5% SDS-PAGE and immunoblotted with anti-Stat3 MoAb (A) or with antiphosphotyrosine
MoAb (B) and visualized by autoradiography IC). Lane 1, mock; lane
2, control medium; lane 3, SLF; lane 4, IL-9; lane 5, SLF + IL-9; lane
6, GM-CSF; lane 7, IL-3. The corresponding region of Stat3 in the
membrane was excised and phosphoamino acid analysis was performed (D) as described in the Materials and Methods. These are
representative results of three independent experiments.
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143
SERINE PHOSPHORYLATION OF Stat3 BY SLF
myeloid cell lines. It has been reported by others that the
PMSP sequence, conserved in the C-terminal region of
Stat la, Stat3, and Stat4, is similar to the MAP kinase recognition consensus sites.'? We have previously reported that
MAP kinase is activated after stimulation with SLF and GMCSF but not with IL-9.'"'' Moreover, it has recently been
reported by others that dominant negative MAP kinase inhibits interferon-D-induced transcription in human fibroblasts." These lines of evidence suggest that MAP kinase
might play a role in the serine phosphorylation of Stat3 by
SLF. The serine-threonine kinase cascade activated by SLF
and GM-CSF involves not only MAP kinase but also raf-l
and other serine kinases that are downstream of MAP kinase
and protein kinase C that might be activated as a result of
PLC--y activation.'"*' In the future, it should be determined
which serine kinase is responsible for serine phosphorylation
-
Blot:
u aiai J
kUa
11789
1
2
3
111-
--
80-
.
kDa
-
4
5
6
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.
I
-
A
1
2
3
4
5
kDa
6
z":
2
2 2 2 5
Autoradlagraphy
-.~
117-
89-
4-stat3
-
Blot:
a Stat 3
P-*
p-Thr
P-TLr
SLF
None
IL6
SLF
GM
+IL6
Fig 6. Phosphorylation and phosphoamino acid analysis of Stat3
in TF-1 cells. Factor-starved TF-1 cells labeled with 32Pwere stimulated with SLF (100 nglmLI, 11-6 (20 nglmLI, SLF + IL-6, and GM-CSF
(200 UlmL) for 10 minutes at 37°C. Anti-Stat3 immunoprecipitates
were separated by 7.5% SDS-PAGE and immunoblotted with antiStat3 MoAb (A) or with antiphosphotyrosine MoAb (B) and visualized
by autoradiography (C). Lane 1, mock; lane 2, control medium; lane
3, SLF; lane 4, IL-6; lane 5, SLF IL-6; lane 6, GM-CSF. The corresponding region of Stat3 was excised and subjected t o phosphoamino acid
analysis 1D). Similar results were obtained from three independent
experiments.
+
D
E
-
Autoradiography
11-9
8, lanes 1 and 2). Simultaneous stimulation of cells with IL9 and SLF induced an SIE binding activity that comigrated
in EMSA with that induced by IL-9 alone (Fig 8, lanes 4
and 8). These DNA-protein complexes were shown to contain Stat3 by performing a supershift with an anti-Stat3 Ab
that resulted in the retarded migration of the complexes (Fig
8, lanes 9 and IO).
DISCUSSION
The role of tyrosine phosphorylation for Stat family proteins are well delineated. We describe here for the first time
that cytokines that do not induce tyrosine phosphorylation
of Stat3, such as SLF, GM-CSF, and IL-3, can induce serine
phosphorylation of Stat3 in human growth factor-dependent
SLF+IL-B
Fig 7. Phosphorylation of nuclear translocated Stat3 by the simultaneous stimulation with SLF and IL-9. Factor-starved M07e cells
were labeled with "P and stimulated with SLF (100 ng/mL), IL-9 15
nglmL), SLF + IL-9, and GM-CSF (200 UlmLl for 10 minutes at 37°C.
Nuclear extracts were prepared as described in the Materials and
Methods and then separated by 7.5% SDS-PAGE followed by immunoblotting with anti-Stat3 MoAb (A). Anti-Stat3 immunoprecipitates
of nuclear extracts stimulated with IL-9 alone or SLF + IL-9 were
separated by 7.5% SDS-PAGE and immunoblotted with anti-Stat3
MoAb (B)or immunoblotted with antiphosphoryrosine MoAb (C) and
visualized by autoradiography (D1. These results are representative
of four independent experiments. The corresponding region of Stat3
was excised and subjected t o phosphoamino acid analysis, and then
corresponding regions of tyrosine phosphorylation and serine phosphorylation were quantitated by densitometry. The ratio of 32Pincorporated serineltyrosine was calculated. Pvalue was measured using
the unpaired Student's t-test. *P < .01 (El.
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GOTOH ET AL
144
Antibody:
=
= 5 5
d W S l
Competitor:
SLF
Treatment: IL-Q
- + - + - + - + - + - +
+ + + + + + + + + +
- -
ss+
b
freeprobe+
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
Fig 8. EMSA analysis of SIE binding activity in M07e cells. Factorstarved M07e cells (1 x 1O8/samplel were stimulated with control
medium (lane 11, SLF (100 nglmL, lane 21, IL-9 (5nglmL, lanes 3, 5,
7,9, and 111, and SLF + 11-9 (lanes 4,6,8, 10, and 121 for 10 minutes
at 37°C. Nuclear extracts were prepared and EMSA was performed
as described in the Materials and Methods using a double-strand
syntheticoligonucleotidecorrespondingto SIE (5‘-GTG CAT TTC CCG
TAA ATC l T G TCT ACA-3‘1. Competitions were performed with excess (100x1 unlabeled homologous oligonucleotide (lanes 5 and 6)
or mutated oligonucleotide (5’-GTG CAT CCA CCG TAA ATC l T G TCT
ACA-3’; lanes 7 and 8 ) . Supershifts were performed with anti-Stat3
Ab (lanes 9 and 101 or preimmune rabbit serum (lanes 11 and 12).
The positions of the specific complex and the supershifted complex
(SSI are indicated. Similar results were obtained in three independent experiments.
of Stat3 for a further understanding of Stat-mediated signaling. However, the fact that TPA can induce serine phosphorylation of Stat3 (data not shown) suggests that, at least in
part, activation of protein kinase C might be involved in this
process.
The biological significance of phosphorylation on serine
residues of Stat family proteins remains to be defined. However, recent reports have begun to clarify the importance of
serine phosphorylation of Stats. Serine phosphorylation is
reported to be required to form complexes of DNA with Stat3
homo-dimers.22Our data showed that serine phosphorylation
alone by SLF did not induce nuclear translocation or DNA
binding activity of Stat3 (Figs 7A and 8). However, simultaneous stimulation of the cells with SLF plus other cytokines
that induce tyrosine phosphorylation of Stat3 resulted in the
translocation of the serine-hyperphosphorylatedStat3 to the
nucleus (Fig 7). This suggest that SLF might modulate JakStat pathway. Because GM-CSF and IL-3 also induced serine
but not tyrosine phosphorylation of Stat3 (Figs 5 and 6).
Jak-Stat pathway could be modulated by the hematopoietic
progenitor cells that express particular sets of the cytokine
receptors in the presence of multiple cytokines. EMSA using
an SIE oligonucleotide as probe showed that IL-9 and IL-9
plus SLF induced a DNA binding activity that containing
Stat3 in M07e cells (Fig 8). Although these results did not
clarify the role of serine hyperphosphorylation of Stat3, it
is possible that transcriptional activity of serine-phosphorylated Stat3 is augmented without affecting its DNA binding
or that IL-9 plus SLF can induce distinct DNA binding activity other than SIE.
Mechanisms underlying the synergism of SLF and other
cytokines remain largely unknown, although a number of
possible candidate intracellular events have been implicated.?7’?clConsidering SLF synergy, our observation is potentially important. First, increased phosphorylation of Stat3
by SLF plus IL-9 in M07e cells (Fig SC, lane 5 ) and by
SLF and 1L-6 in TF-I cells (Fig 6C. lane 5 ) corresponds to
increased mitogenesis at the cellular level (Fig 4). Second,
translocation of serine-hyperphosphorylated Stat3 to the nucleus can be achieved only by the simultaneous stimulation
of SLF and other cytokines that use Stat3 but not by SLF
alone (Fig 7). It is noteworthy that others have reported
recently that maximal activation of transcription by Stat la
and Stat3 requires both tyrosine and serine phosphorylation.” Thus, it is highly possible that concomitant stimulation with SLF and other cytokines that use Stat3 can induce
a higher transcriptional activation status of Stat3 in hematopoietic progenitor cells. It has been reported by others that
dominant negative StatS inhibits IL-3-dependent cell
growth3”and that StatS antisense oligodeoxynucleotides inhibit proliferation followed by erythroid differentiation in
Friend virus-transformed murine erythroleukemia cells.”
Therefore, it is likely that the Jak-Stat3 pathway might also
contribute to some proliferation process(es). In addition, in
U937 monocytic cells, during differentiation induced by interferon-y, Stat1 is reported to be phosphorylated on serine
residues but not on tyrosine residues.” This finding suggests
that serine phosphorylation of Stats might play a role in the
differentiation process in some cell types.
The contribution of Jak-Stat pathways to the functional
response of hematopoietic progenitor cells requires further
investigation. However, taken together with these previous
reports and our data presented here, serine phosphorylation
of Stat3 by SLF and other cytokines, especially through
simultaneous stimulation with hematopoietic growth factors
that induce tyrosine phosphorylation of Stat3, might be involved in the regulation of proliferation and/or differentiation of myeloid stem and progenitor cells.
ACKNOWLEDGMENT
We thank Rebecca Miller for her assistance in preparation of this
manuscript.
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1996 88: 138-145
Steel factor induces serine phosphorylation of Stat3 in human growth
factor-dependent myeloid cell lines
A Gotoh, H Takahira, C Mantel, S Litz-Jackson, HS Boswell and HE Broxmeyer
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