STATs (signal transducers and activators of
transcription) are transcription factors that contain SH2 domains and
are activated by tyrosine phosphorylation, often in response to
cytokine stimulation. Recent evidence indicates that the transforming
tyrosine kinases encoded by the v-Src, v-Abl, and v-Fps oncogenes can
induce STAT activation, suggesting that their normal cellular homologs
may contribute to STAT activation under physiological conditions. In
this report, we provide direct evidence that c-Fes, the normal human
homolog of v-Fps, potently activates STAT3. Transient transfection of
human 293T cells with STAT3 and Fes resulted in strong stimulation of
STAT3 DNA binding activity. In contrast, only modest activation of
STAT5 by Fes was observed in this system, indicative of possible selectivity. To determine whether Fes-induced STAT3 activation is
dependent upon endogenous mammalian kinases, co-expression studies were
also performed in Sf-9 insect cells. Fes also induced a dramatic
increase in STAT3 DNA binding activity in this system, whereas no
activation of STAT5 was observed. As a positive control, both STAT3 and
STAT5 were shown to be activated by the Bcr-Abl tyrosine kinase in Sf-9
cells. Fes induced strong tyrosine phosphorylation of STAT3 in both
expression systems, consistent with the gel-shift results. Fes and
STAT3 have been independently linked to myeloid differentiation.
Results presented here suggest that these proteins may cooperate to
promote differentiation signaling in response to hematopoietic
cytokines.
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INTRODUCTION |
STATs (signal transducers and activators of transcription) are a
class of transcription factors with SH2 domains that are activated in
response to tyrosine phosphorylation. STATs are often activated by
members of the JAK family of protein-tyrosine kinases in response to
cytokine stimulation. The activation mechanism involves
SH2-dependent recruitment of the STATs to
tyrosine-phosphorylated cytokine receptors. The STATs are then
phosphorylated by receptor-associated JAKs, which induces dimerization
via reciprocal SH2-phosphotyrosine interaction. The STAT dimers then
enter the nucleus and bind specific DNA elements, leading to
transcriptional activation. The JAK-STAT pathway is the subject of many
recent comprehensive reviews (1-5).
In addition to JAKs, other classes of non-receptor protein tyrosine
kinases have recently been shown to induce STAT activation. For
example, STAT3 is constitutively activated in fibroblasts transformed
with v-Src (6, 7). Src forms a stable complex with STAT3, suggesting
that STAT3 activation may be direct (7). However, more recent work
shows that JAK1 and to some extent JAK2 are activated in
Src-transformed cells, suggesting that JAKs may contribute to STAT
activation by Src kinases (8). Another study has shown that STATs 1 and
5 are activated in fibroblasts transformed by v-Abl (9). In this case,
v-Abl was shown to form stable complexes with endogenous JAK kinases,
suggesting that STAT activation by v-Abl may involve JAKs as well.
Related studies with chronic myelogenous leukemia cell lines
transformed by Bcr-Abl show a similar pattern of STAT activation,
with some evidence for JAK involvement (10-12).
Recently, we observed that fibroblast transformation by v-Fps
correlates with potent activation of endogenous STAT3 (13). This
finding suggested that STAT3 may also be a substrate and effector
protein for c-Fes, the normal human homolog of v-Fps (14-16). The
c-Fes tyrosine kinase exhibits strong hematopoietic expression, is
activated in response to multiple cytokines (17-20), and has been
linked directly to the control of myeloid differentiation (21, 22).
Recent studies have shown that activation of STAT3 is necessary for
induction of myeloid differentiation in response to IL-6 (23, 24), one
of the cytokines known to activate c-Fes (19). Taken together, these
previous studies suggest that c-Fes may contribute to STAT activation
under physiological conditions. In this study, we provide evidence for
the activation of STAT3 by Fes in human cells as well as Sf-9 insect
cells. Fes potently activated STAT3 in both systems, suggesting that
STAT3 may be a direct substrate for Fes and may mediate Fes
differentiation signaling in myeloid cells.
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EXPERIMENTAL PROCEDURES |
Cell Lines--
293T cells (25) were obtained from the American
Type Culture Collection (ATCC) and grown in Dulbecco's modified
Eagle's medium supplemented with 5% fetal bovine serum and 50 µg/ml
gentamicin. Sf-9 cells were obtained from the ATCC and grown in
Grace's insect cell medium supplemented with 10% fetal bovine serum
and 50 µg/ml gentamicin.
Expression of Fes and STAT Proteins in 293T Cells--
The
cDNAs encoding murine STAT3 and STAT5A were subcloned into the
respective expression vectors pCDNA3 and pCDNA3.1(
)
(Invitrogen). Construction of expression vectors for Fes and the
kinase-inactive Fes mutant K590E with C-terminal FLAG epitope tags and
calcium phosphate-mediated transfection of 293T cells is described
elsewhere (26).
Generation of Recombinant Baculoviruses and Expression of
Proteins in Sf-9 Insect Cells--
STAT3 and STAT5A cDNAs were
subcloned into the baculovirus transfer vectors pVL1392 and pVL1393,
respectively. The resulting constructs were used to make recombinant
baculoviruses by co-transfection with BaculoGold DNA (Pharmingen) using
the manufacturer's protocol. A cDNA encoding the p210 form of
Bcr-Abl (generous gift of Dr. Owen Witte, Howard Hughes Medical
Institute, UCLA) was used to make a recombinant baculovirus by the same
procedure. Construction of baculoviruses for the expression of
wild-type and kinase-defective Fes has been described elsewhere (26).
Baculovirus-expressed Fes proteins carry a C-terminal FLAG epitope tag
to allow for detection with the M2 monoclonal antibody. For protein
expression, Sf-9 cells were grown to 50% confluence on 60-mm tissue
culture plates and infected with recombinant baculoviruses for 1 h
at 27 °C. The virus was replaced with fresh medium, and the cells were incubated for 48 h prior to preparation of cytosolic
extracts.
Preparation of Cytosolic Extracts--
Cytosolic extracts were
made following a previously published protocol (6). Briefly, culture
plates of 293T or Sf-9 cells were washed twice with ice-cold PBS
followed by PBS containing 1 mM sodium orthovanadate and 5 mM sodium fluoride. The cells were rinsed with hypotonic
buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 1 mM EGTA, 20 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium
pyrophosphate, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and 25 µg/ml
aprotinin). The cells were then lysed in hypotonic buffer containing
0.2% Triton X-100 and clarified by microcentrifugation. The protein
concentration of the extract was determined using the Coomassie-plus
reagent (Pierce) and stored at
80 °C.
Electrophoretic Mobility Shift Assay--
The probes used for
STAT DNA binding included the sis-inducible element (SIE) (6) and the
mammary gland factor element (MGFE) (27). The sequences of the
SIE oligonucleotides are 5'-CTTCATTTCCCGTAAATCCCTAAAGCT-3' and
5'-AGCTTTAGGGATTTACGGGAAATGA-3'. The sequences of the MGFE oligonucleotides are 5'-AGATTTCTAGGAATTCAA-3' and
5'-GATTTGAATTCCTAGAAA-3'. Oligonucleotides (20 pmol) were
annealed in 10 µl of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) by heating to 70 °C and slowly cooling to room
temperature. The probes were labeled by combining 1 µl of annealed
oligonucleotide with 2 µl of Labeling Mix-dATP (Pharmacia Biotech
Inc.), 2 µl of [
-32P]dATP (10 mCi/ml; Amersham Life
Science, Inc.), and 1 µl of the Klenow fragment of DNA polymerase
(Life Technologies, Inc., 3.7 units/µl). The mixture was incubated at
room temperature for 30 min. and the unincorporated nucleotides were
removed by spermine precipitation (28). DNA binding reactions contained
40,000 cpm of labeled probe in a final volume of 20 µl and 2-6 µg
of cytosolic protein extract. In the case of the cytosolic extract from
Sf-9 cells, 0.5 µl of a 1.0 ml cytosolic extract from 6 × 106 cells was used in each reaction. The reactions were
incubated at 30 °C for 30 min and run on 5% polyacrylamide gels in
0.25× Tris borate-EDTA buffer. Gels were fixed with 10% acetic acid, 10% methanol, rinsed with water, dried, and exposed to a storage phosphor screen. Images were visualized using a Molecular Dynamics PhosphorImager.
Immunoprecipitation and Immunoblotting--
For
immunoprecipitation, clarified cytosolic extracts were incubated with 1 µg of anti-STAT3 or anti-STAT5 antibodies (Santa Cruz Biotechnology)
and 20 µl of protein G-Sepharose (50% slurry; Pharmacia) for 1 h at 4 °C. The immunoprecipitates were washed three times with 1 ml
of radioimmune precipitation buffer (26) followed by heating in
SDS-PAGE sample buffer. For immunoblotting, cytosolic extracts or
immunoprecipitated proteins were run on 8% polyacrylamide gels and
transferred to PVDF membranes. The membranes were incubated for 1 hour
with antibodies to Fes (M2 anti-FLAG antibody; Eastman-Kodak Co.),
phosphotyrosine (PY20; Transduction Laboratories), STAT3 (Santa Cruz)
or STAT5 (Transduction Laboratories) at 1 µg/ml in Tris-buffered
saline containing 0.05% Tween-20
(TBST)1 and 1.5% bovine
serum albumin. The membranes were then washed in TBST and probed with a
secondary antibody-alkaline phosphatase conjugate in TBST-bovine serum
albumin at the dilution recommended by the manufacturer (Southern
Biotechnology Associates). Following further washing with TBST, the
immunoreactive proteins were visualized colorimetrically using the
alkaline phosphatase substrate NBT/BCIP.
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RESULTS |
Activation of STAT3 by c-Fes in 293T Cells--
Recently we
observed that endogenous STAT3 is constitutively activated in
fibroblasts transformed by v-Fps, a retroviral homolog of c-Fes (13).
Given the relationships of c-Fes and STAT3 to myeloid differentiation
(21, 23, 24, 29), we decided to investigate whether STAT3 or other STAT
factors represent possible Fes substrates and effector molecules. To
determine whether STAT3 is activated by Fes, the two proteins were
expressed either alone or together in human 293T cells. Cytosolic
extracts were prepared and tested for the presence of active STAT3 in a
gel-shift assay with an SIE probe. As shown in Fig.
1, extracts from cells co-transfected with both Fes and STAT3 produced a dramatic shift in SIE mobility, indicative of STAT3 activation. Addition of a 100-fold molar excess of
unlabeled SIE completely blocked complex formation, providing evidence
of probe specificity.

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Fig. 1.
Activation of STAT3 by Fes in 293T
cells. Human 293T cells were transfected with STAT3, Fes, or a
kinase-inactive Fes mutant (Fes-KE) either alone or in the
combinations shown. Cytosolic extracts were prepared and tested for the
presence of active STAT3 by gel-shift analysis with an SIE probe.
Control lanes include incubation of the probe in the absence of extract
(SIE) and gel-shift assays of untransfected cell extracts
(293T). Duplicate gel-shift assays were conducted in the
presence of a 100-fold excess of unlabeled SIE to demonstrate probe
specificity (COMP). The shifted complex of activated STAT3
and the SIE probe is denoted by the arrow.
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To demonstrate that the activation of STAT3 requires the tyrosine
kinase activity of c-Fes, 293T cells were co-transfected with STAT3 and
a kinase-defective Fes mutant (Fes-K590E) (30). As shown in Fig. 1,
co-expression with the kinase-defective protein produced only low
background levels of STAT3·SIE complexes equivalent to those observed
when STAT3 was expressed alone. This result shows that stimulation of
STAT3 DNA binding activity requires an active Fes kinase domain and is
consistent with STAT3 tyrosine phosphorylation data shown below. We
also investigated the requirements for other structural features of
c-Fes in STAT3 activation using a series of Fes deletion and
autophosphorylation site mutants. Co-expression of STAT3 with a Fes
mutant lacking the unique N-terminal domain (amino acids 1-450) (26)
activated STAT3 to the same extent as wild-type c-Fes (data not shown).
This result suggests that the Fes N-terminal region, while involved in
the recruitment of some Fes substrates (31, 32) is dispensable for
interaction with STAT3. However, deletion of the Fes SH2 domain or
mutagenesis of Tyr-713, the major Fes autophosphorylation site (26,
30), greatly diminished STAT3 activation (data not shown). The latter two mutations reduce Fes tyrosine kinase activity in vitro
(30), which may account for their reduced efficiency of STAT3
activation.
To determine whether Fes exhibits a preference for STAT3, Fes was
co-expressed with STAT5 in 293T cells. Cell lysates were tested for the
presence of activated STAT5 using the MGFE as a probe. As shown in Fig.
2, Fes stimulated STAT5 DNA binding
activity. However, activation of STAT5 by Fes was modest in comparison
to STAT3 (3-fold for STAT5 versus 35-fold for STAT3 relative
to background levels observed with the kinase-inactive Fes mutant).
These results suggest that Fes preferentially activates STAT3 over
STAT5 in this system. In addition, work with Sf-9 insect cells
described below suggests that STAT5 activation by Fes in mammalian
cells may be indirect, possibly involving an intermediate tyrosine
kinase.

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Fig. 2.
Activation of STAT5 by Fes in 293T
cells. Human 293T cells were transfected with STAT5A, Fes, or a
kinase-inactive Fes mutant (Fes-KE) either alone or in the
combinations shown. Cytosolic extracts were prepared and tested for the
presence of active STAT5 by gel-shift analysis with an MGFE probe.
Control lanes include incubation of the probe in the absence of extract (MGFE) and gel-shift assays of untransfected cell extracts
(293T). Duplicate gel-shift assays were conducted in the
presence of a 100-fold excess of unlabeled MGFE to demonstrate probe
specificity (COMP). The shifted STAT5-MGFE complex is
denoted by the arrow.
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Fes Induces Tyrosine Phosphorylation of STAT3 and STAT5 in 293T
Cells--
Tyrosine phosphorylation is required to induce STAT factor
dimerization, nuclear localization, and DNA binding (1, 2, 5). To
determine whether stimulation of STAT3 DNA binding activity by Fes
correlated with tyrosine phosphorylation, STAT3 was immunoprecipitated from transfected 293T cell lysates and analyzed by immunoblotting with
the anti-phosphotyrosine antibody, PY20. As shown in Fig. 3, STAT3 was strongly phosphorylated on
tyrosine following co-expression with Fes, consistent with the positive
gel-shift result. In contrast, control STAT3 immunoprecipitates from
cells expressing STAT3 alone or in the presence of the kinase-inactive
Fes mutant were devoid of tyrosine phosphorylation. Control immunoblots
show that equivalent amounts of STAT3 were immunoprecipitated in each
case and that equivalent levels of the wild-type and kinase-inactive
Fes proteins were expressed (Fig. 3). These results suggest that STAT3
is either a direct substrate for Fes, that Fes activates an endogenous
tyrosine kinase that in turn phosphorylates STAT3, or both. Data
described below with Sf-9 insect cells strongly support the hypothesis
that STAT3 is a direct substrate for Fes. However, the contribution of
an endogenous tyrosine kinase in mammalian cells cannot be ruled out.
Whatever the specific mechanism, these results demonstrate that c-Fes
can strongly activate STAT3 in a human cell context. This finding is
particularly interesting in light of recent data directly implicating
STAT3 in myeloid differentiation.

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Fig. 3.
Fes induces tyrosine phosphorylation of STAT3
and STAT5 in 293T cells. Human 293T cells were transfected with
STAT3, STAT5, Fes, or a kinase-inactive Fes mutant (Fes-KE)
either alone or in the combinations shown. Untransfected cells were
also included as an additional negative control (293T).
Cytosolic extracts were prepared and incubated with antibodies to
either STAT3 or STAT5. Immune complexes were precipitated with protein
G-Sepharose and immunoblotted with antibodies to phosphotyrosine
(PY20, top). To verify equivalent recovery of
STAT proteins, aliquots of the immunoprecipitates were also blotted
with the STAT antibodies (center). To verify the expression
of Fes and Fes-KE, the cytosolic extracts were immunoblotted with the
anti-FLAG antibody, M2 (bottom).
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We also investigated whether Fes could induce tyrosine phosphorylation
of STAT5 in 293T cells using the same approach. As shown in Fig. 3,
STAT5 immunoprecipitates from 293T cells co-transfected with STAT5 and
Fes showed evidence of tyrosine phosphorylation. This finding is in
good agreement with the positive gel-shift result shown in Fig. 2. As
with STAT3, tyrosine phosphorylation was not observed when STAT5 was
expressed alone or in the presence of the kinase-inactive Fes mutant,
K590E. Control blots show equivalent levels of STAT5 in the
immunoprecipitates and equal levels of wild-type and kinase-defective
Fes in the cell lysates (Fig. 3). These results show that Fes can
induce the tyrosine phosphorylation and activation of STAT5 in
mammalian cells. Recent studies suggest that STAT5, like STAT3, may
contribute to hematopoietic differentiation (see Discussion).
Activation of STAT3 but Not STAT5 by Fes in Sf-9 Insect
Cells--
Experiments conducted in 293T cells clearly demonstrate the
ability of Fes to induce STAT3 DNA binding activity (Fig. 1). One
possible mechanism of activation involves direct phosphorylation of
STAT3 by Fes, leading to dimerization and DNA binding. Alternatively, it is possible that Fes may mediate its effects on STAT3 indirectly by
activating endogenous JAKs or other tyrosine kinases, which in turn
mediate STAT activation. The latter mechanism may contribute to STAT
activation by both v-Abl and v-Src (8, 9). To address the issue of STAT
activation by Fes more directly, we utilized the baculovirus/Sf-9 cell
system to circumvent the issue of endogenous mammalian tyrosine
kinases. For these experiments, Sf-9 insect cells were infected with a
recombinant STAT3 baculovirus either alone or in combination with
wild-type or kinase-inactive Fes baculoviruses. Cytosolic extracts were
examined for the presence of activated STAT3 by gel-shift assay with
the SIE probe. As shown in Fig. 4,
co-expression with Fes induced potent activation of STAT3 in Sf-9
cells, consistent with the result observed in 293T cells (Fig. 1). The
Fes-induced SIE·STAT3 complex was fully competed with a 100-fold
excess of unlabeled SIE, indicative of binding specificity. In
contrast, the kinase-inactive mutant of Fes produced no detectable
increase in STAT3 activation above background. These results strongly
suggest that Fes is capable of directly activating STAT3 without the
requirement for a member of the JAK family or another intermediate
tyrosine kinase.

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Fig. 4.
Activation of STAT3 by Fes in Sf-9
cells. STAT3, STAT5, Fes, or a kinase-inactive Fes mutant
(Fes-KE) were expressed in Sf-9 insect cells either alone or
in the combinations shown. Cytosolic extracts were prepared and tested
for the presence of active STAT3 by gel-shift analysis with an SIE
probe. Control lanes include incubation of the probe in the absence of
extract (SIE) and gel-shift assays of extracts from cells
infected with a wild-type baculovirus (AcMNPV). Duplicate
gel-shift assays were conducted in the presence of a 100-fold excess of
unlabeled SIE to demonstrate probe specificity (COMP). The
shifted complex of activated STAT3 and the SIE probe is denoted by the
arrow.
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The effect of Fes on STAT5 activation was also investigated in Sf-9
insect cells using the gel-shift assay and MGFE probe. As shown in Fig.
5, no detectable STAT5 DNA binding
activity was observed in the presence of Fes. This result is in
contrast to the tyrosine phosphorylation and activation of STAT5 by Fes
observed in 293T cells (Figs. 2 and 3). Taken together, these results
suggest that activation of STAT5 by Fes in 293T cells is indirect,
possibly involving activation of an endogenous tyrosine kinase by Fes
which in turn activates STAT5.

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Fig. 5.
Co-expression with Fes fails to activate
STAT5 in Sf-9 cells. STAT3, STAT5, Fes, or a kinase-inactive Fes
mutant (Fes-KE) were expressed in Sf-9 insect cells either
alone or in the combinations shown. Cytosolic extracts were prepared
and tested for the presence of active STAT5 by gel-shift analysis with
an MGFE probe. Control lanes include incubation of the probe in the
absence of extract (MGFE) and gel-shift assays of extracts
from cells infected with a wild-type baculovirus (AcMNPV).
Duplicate gel-shift assays were conducted in the presence of a 100-fold
excess of unlabeled SIE to demonstrate probe specificity
(COMP). Note that activated STAT3 (Fes + STAT3)
does not produce a gel-shift with the MGFE, indicative of probe
specificity.
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To control for the expression of Fes and STAT proteins in the Sf-9 cell
experiments, immunoblots were performed on the cell extracts used for
the gel-shift assays. As shown in Fig. 6,
Fes and STAT proteins were all strongly expressed in the infected cells. In addition, immunoblots of the same extracts with an
anti-phosphotyrosine antibody revealed potent tyrosine phosphorylation
of STAT3 in the presence of Fes, consistent with the strong signal
observed in the gel-shift assay (Fig. 4). Also visible in this
experiment is autophosphorylated Fes. On the other hand, no tyrosine
phosphorylation of STAT5 was detected, despite the presence of active
Fes. This result is consistent with the negative gel-shift result with
extracts from cells co-infected with Fes and STAT5 (Fig. 5).

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Fig. 6.
Fes phosphorylates STAT3 but not STAT5 in
Sf-9 cells. Immunoblots were performed on the cytosolic extracts
from the Sf-9 insect cells infected with the Fes and STAT baculoviruses used for the gel-shift assays shown in Figs. 4 and 5. The
anti-phosphotyrosine antibody, PY20, detects Fes autophosphorylation
(upper arrow, top panel) and tyrosine
phosphorylation of STAT3 when co-expressed with Fes (lower
arrow, top panel). Immunoblots performed with antibodies against Fes, STAT3, and STAT5 confirm the expression of
these proteins (bottom three panels).
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An alternative explanation for the negative gel-shift result with Fes
and STAT5 in Sf-9 cells is that the baculovirus-expressed STAT5 protein
may be resistant to activation in the insect cell context. To rule out
this possibility, STAT5 was co-expressed with the p210 form of Bcr-Abl,
the constitutively activated protein-tyrosine kinase associated with
chronic myelogenous leukemia (33). Several recent reports have shown
that Bcr-Abl induces the activation of STAT5 as well as STATs 1 and 3 (10-12). Using the gel-shift assay with the MGFE probe, we observed
that STAT5 was readily activated by Bcr-Abl in Sf-9 cells, whereas no
detectable activation occurred with Fes (Fig.
7). This result indicates that
baculovirus-expressed STAT5 can bind to DNA following phosphorylation
by the appropriate tyrosine kinase. We also observed potent activation
of STAT3 by Bcr-Abl in Sf-9 cells (Fig. 7). Our observation of
Bcr-Abl-induced STAT activation in Sf-9 cells is consistent with
previous work by Ilaria and Van Etten (12), which suggests that STATs
are directly activated by Bcr-Abl and do not require JAKs or other endogenous kinases.

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Fig. 7.
Activation of STAT3 and STAT5 by Bcr-Abl in
Sf-9 insect cells. STAT3, STAT5, Fes or the p210 form of Bcr-Abl
were expressed in Sf-9 insect cells either alone or in the combinations
shown. Cytosolic extracts were prepared and tested for the presence of active STAT3 (top) or STAT5 (bottom) by gel-shift
analysis with the SIE or MGFE probes, respectively. Duplicate gel-shift
assays were conducted in the presence of a 100-fold excess of unlabeled probe to demonstrate probe specificity (COMP).
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DISCUSSION |
Data presented in this report demonstrate for the first time that
STAT3 can be activated by the c-Fes tyrosine kinase. Our approach was
to compare STAT activation in human versus insect cell
backgrounds to address the issue of whether endogenous mammalian tyrosine kinases play a role in the activation mechanism. Our observations that Fes strongly activates STAT3 in both human and insect
cells suggest that Fes is sufficient to activate STAT3 without a
requirement for JAKs or other tyrosine kinase intermediates. However,
the possible contribution of other tyrosine kinases to the activation
mechanism in mammalian cells cannot be formally ruled out. These
findings with STAT3 are consistent with our recent work showing that
endogenous STAT3 activation occurs in fibroblasts transformed by v-Fps,
the retroviral homolog of c-Fes (13). Taken together, these data
suggest that v-Fps may directly induce endogenous STAT3 activation as
well. In contrast to STAT3, however, we observed that Fes was unable to
activate STAT5 in insect cells, despite some degree of activation in
mammalian cells. These findings suggest that Fes activates STAT5 via an
indirect mechanism, possibly via endogenous tyrosine kinases. Such a
phenomenon has been reported previously for the activation of STATs by
both v-Abl and Src kinases (8, 9).
Potent activation of STAT3 by Fes in transfected human cells suggests
that such an event may also be induced by Fes in hematopoietic cells,
an important site of Fes expression (14-16). Fes has been linked to
the induction of myeloid differentiation in both normal and leukemic
cell lines (21, 22, 34). Two recent studies have strongly implicated
STAT3 as a key component of myeloid differentiation as well. Both of
these studies utilized dominant-negative mutants of STAT3 (23, 24).
These mutants were produced by substitution of the STAT3 tyrosine
phosphorylation site with phenylalanine or by mutation of residues
critical for DNA binding. When these mutants were introduced into M1
myeloid leukemia cells, the cells no longer responded to IL-6 with
growth arrest and terminal differentiation. Interestingly, IL-6 is one
of the cytokines reported to activate Fes (19). Taken together, these
results suggest that Fes may contribute to the IL-6-mediated activation
of STAT3 and the subsequent differentiation response.
Recent studies of GM-CSF-treated neutrophils also suggest a connection
between Fes and STAT3 activation (35). In these experiments, GM-CSF was
shown to induce the formation of a multiprotein complex consisting of
Fes, JAK2, STAT1, STAT3, and the
-subunit of the GM-CSF receptor.
However, this previous study did not address the possible contribution
of Fes to STAT activation. Data presented here clearly demonstrate the
tyrosine phosphorylation of STAT3 by Fes and induction of its DNA
binding activity, strongly suggesting that this activation mechanism
may be direct. The GM-CSF receptor may serve as a docking site to bring
Fes and STAT3 into close proximity to allow the phosphorylation event
to occur. Such an interaction may be initiated by JAK2, which could
create binding sites for the SH2 domains of Fes and STAT3 on the
receptor
-subunit. Further experiments will be required to address
the possible contribution of Fes to STAT activation in response to
cytokine treatment. However, a definitive demonstration that Fes is
involved in endogenous STAT3 activation by GM-CSF, IL-6, or other
cytokines may be difficult because of the multiple non-receptor
tyrosine kinases activated in response to cytokine treatment.
Although our results in Sf-9 insect cells indicate that STAT5 is not a
direct substrate for Fes, co-expression of these two proteins in human
cells induced both STAT5 tyrosine phosphorylation and DNA binding
activity. These results suggest that Fes may indirectly induce STAT5
activation under physiological conditions. Like STAT3, STAT5 may also
contribute to hematopoietic differentiation in some cases. Recent
studies have established a strong correlation between STAT5 activation
and differentiation of myeloid leukemia cell lines (36). In these
experiments, induction of monocytic differentiation in human U937 cells
with phorbol ester, retinoic acid, or 1
,25-dihydroxy-vitamin
D3 induced strong DNA binding activity of STAT5. Similar
results were observed following chemically induced differentiation of
HL-60 promyelocytic leukemia cells. In the same study, STAT5 activation
was observed in primary cultures of chicken myeloblasts following
differentiation to macrophages with chicken myelomonocytic growth
factor. Another recent study has shown that induction of megakaryocytic
differentiation by thrombopoietin correlates with activation of STAT5.
This study also identified the cyclin-dependent kinase
inhibitor p21WAF1/Cip1 as a possible target gene for STAT5
in the differentiation response (37). Further work will be required to
determine whether Fes can stimulate similar STAT5 responses as part of
a differentiation signaling pathway in hematopoietic cells.