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INTRODUCTION |
The stem cell factor
(SCF)1 has been implicated in
a number of important developmental processes (1-3) and is a potent
co-stimulating protein that acts synergistically with hemopoietic
colony-stimulating factors, such as granulocyte-macrophage
colony-stimulating factor and interleukin-3 (IL-3), to promote
proliferation of early hemopoietic progenitors (1). SCF effects are
mediated by the presence, on the surface of its target cells, of the
transmembrane receptor c-Kit. SCF receptor belongs to the tyrosine
kinase receptor family, and in addition to the presence of an intrinsic
enzymatic activity it shares with other members of the family, such as
platelet-derived growth factor receptor (PDGFR) and the
colony-stimulating factor-1 (CSF-1) receptor, the presence of an
interkinase domain and consensus sequences for recognition of Src
homology 2 domains (SH2) (4-7). SCF binding to its receptor leads to
dimerization, transphosphorylation, and increased binding of
SH2-containing proteins such as phospholipase C-
,
phosphatidylinositol 3-kinase, Syp, and JAK2 (8-13).
The JAKs are non-transmembrane protein tyrosine kinases that are
rapidly tyrosine-phosphorylated upon ligand binding and play a critical
signaling function downstream of cytokine receptors (14). One such
function of JAKs is the activation of STAT proteins (signal transducers
and activators of transcription), latent cytoplasmic proteins that
undergo rapid tyrosine phosphorylation following cytokine stimulation
(15-17). Phosphorylation on a tyrosine immediately distal to the SH2
domain induces their homo- or heterodimerization through
phosphotyrosine-SH2 interactions. As a consequence they acquire DNA
binding activity, translocate into the nucleus, bind to specific
promoter elements, and control the expression of target genes. So far
seven different STAT proteins (STAT1, STAT2, STAT3, STAT4, STAT5A,
STAT5B, and STAT6) have been cloned (14-17). STAT1 has been originally
identified as a component of an interferon-activated transcriptional
factor that upon tyrosine phosphorylation and homodimerization binds to
the GAS sequences (18). STAT1 can also be activated by EGF (19) and
PDGF (20) to bind to the serum-inducible element (SIE) of c-Fos as
homodimers or heterodimers with another STAT member defined as STAT3
(19). A third STAT implicated in the receptor tyrosine kinase signaling
pathway, such as EGF (21), ErbB-1 (22), or PDGF (23), is STAT5. STAT5 was originally described as a prolactin-responsive transcription factor
in the mammary gland epithelium (24), and subsequently two different
but highly homologous STAT5 proteins were isolated and defined as
STAT5A and STAT5B (25). These transcriptional factors recognize a
specific palindromic sequence that is found in the
-casein promoter
(24) and also in a number of promoters of genes other than mammary
genes (25), suggesting that STAT5 may play a role other than the
induction of milk proteins. Consistent with this hypothesis is the
observation that a dominant negative variant of STAT5 was able to
inhibit IL-3-mediated cell proliferation (26), and a mutation of
tyrosine residues of the IL-2 receptor
chain, mediating STAT5
docking and activation, strongly reduced cell proliferation (27).
Moreover, a potential role of STAT5 in regulating the response of bone
marrow-derived mast cells to SCF has been reported (28).
In the present study we investigated the pattern of STAT protein
activation in response to SCF stimulation in growth
factor-dependent MO7e cell line and in transfected cells
expressing the wild type receptor or two different c-Kit mutants. We
found that SCF was able to induce tyrosine phosphorylation of STAT1
,
STAT5A, and STAT5B in cells expressing the wild type receptor and in
cells expressing a carboxyl-terminally deleted mutant (
CT). However, other STAT proteins, e.g. STAT2, STAT3, STAT4, and STAT6,
which are present in the human myeloid cell line MO7e, underwent no activation in response to SCF. Consistent with a role in regulation of
gene expression, the modified STAT1
, STAT5A, and STAT5B acquired the
ability to undergo dimerization and bind to specific DNA elements. Analysis of two mutants of c-Kit attributed a role to the
carboxyl-terminal region in activation of STAT5 proteins, whereas
STAT1
activation appears to be independent of this receptor domain.
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EXPERIMENTAL PROCEDURES |
Materials--
Iscove's modified Dulbecco's medium (Life
Technologies Inc.) supplemented with 5% bovine calf serum (HyClone,
Logan, UT) was the culture medium used throughout. RPMI 1640 medium
(Life Technologies, Inc.) was from HyClone (Logan, UT). SCF was gift
from Dr. S. Gillis (Immunex, Seattle, WA). Recombinant human IL-3 was
kindly provided by Sandoz Forschungsinstitut, Austria. The protein
A-Sepharose was purchased from Sigma. Nitrocellulose filters,
horseradish peroxidase-conjugated protein A, molecular weight markers,
[
-32P]dCTP, and chemiluminescence reagent (ECL) were
from Amersham Pharmacia Biotech. Poly(dI·dC):poly(dI·dC) was
obtained from Amersham Pharmacia Biotech (Uppsala, Sweden).
Cells--
MO7e cells, maintained in Iscove's modified
Dulbecco's medium supplemented with 5% bovine calf serum and IL-3 (5 ng/ml), were washed twice, serum- and growth factor-starved in RPMI
1640 medium overnight, and incubated for 10 min at 37 °C without or
with IL-3 (10 ng/ml) (29) or SCF (20 ng/ml) (30). NIH-3T3 fibroblasts transfected and selected for overexpression of the human c-KIT cDNA
(HRS), NIH-3T3 fibroblasts transfected with a c-Kit mutant lacking the
kinase insert domain (
KI), and a carboxyl-terminal deletion mutant
of c-Kit (
CT) were cultured as described previously (9).
Antisera--
Polyclonal antibodies to p91 were produced in
rabbits by injection with a synthetic peptide corresponding to the
sequences of 84-96 residues of human transcription factor ISGR-3
(GenBankTM accession number M97935), RKSKRNLQDNFQEDC (31).
The 4G10 and PY20 anti-phosphotyrosine antibodies were obtained from
Upstate Biotechnology, Lake Placid, NY, and from Affinity Research
Products Ltd, Nottingham, UK, respectively. Anti-STAT5A (L-20),
anti-STAT5B (G-2), and anti-STAT5B (C-17), antisera raised against the
carboxyl terminus of STAT5 proteins, anti-STAT5B (N-20), raised against the amino terminus of STAT5B, and anti-STAT2, anti-STAT3, anti-STAT4, and anti-STAT6 antisera were purchased from Santa Cruz Biotechnology, Heidelberg, Germany. Anti-c-Kit antibodies (K44, Ab212, and anti-Kit-X) were obtained as described previously (9, 32).
Western Blot Analysis and Immunoprecipitation Studies--
The
MO7e cells, HRS,
CT-, and
KI-expressing cells, serum- and growth
factor-starved for 18 h at 37 °C, were supplemented for 4 h with phosphate-buffered saline (30% v/v), sodium orthovanadate 0.2 mmol/liter, EDTA 1 mmol/liter, and then incubated without or with SCF
(20 ng/ml) or IL-3 (10 ng/ml) at 37 °C for the indicated times. The
cells were then extracted with cold DIM buffer (50 mmol/liter Pipes, pH
6.8, 100 mmol/liter NaCl, 5 mmol/liter MgCl2, 300 mmol/liter sucrose, 5 mmol/liter EGTA, 2 mmol/liter sodium orthovanadate) plus 1% Triton X-100 and a mixture of protease inhibitors (1 mmol/liter phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.15 units/ml aprotinin, 1 µg/ml pepstatin A) for 20 min
at 4 °C, and centrifuged at 15,000 × g for 20 min.
The clarified supernatants were precleaned for 1 h with 50 µl of
protein A-Sepharose (3 mg/sample). The protein concentration of cell
lysates was determined by the Bradford's technique, and the protein
content of the samples was normalized to 250 mg/samples by appropriate
dilution with the cold DIM buffer. The samples were then adsorbed by
antisera coupled to protein A-Sepharose. Bound proteins were washed
several times in DIM buffer and eluted in boiling Laemmli buffer.
Thirty µl of eluted proteins were subjected to 8% SDS-PAGE. Proteins were then transferred electrophoretically to nitrocellulose; the filters were incubated with blocking solution (10% low fat milk or
bovine serum albumin in 20 mmol/liter Tris-HCl, pH 7.6, and 17 mmol/liter NaCl) for 1 h. Antisera were then added at the same solution, and the incubation was carried out for 2 h at room
temperature. For detection, the filters were washed three times (10 min
each wash) with phosphate-buffered saline, 0.05% Tween 20 and reacted for 1 h at room temperature with horseradish peroxidase-conjugated protein A. The enzyme was removed by washing as above. The filters were
reacted for 1 min with a chemiluminescence reagent (ECL) and exposed to
an autoradiography film for 1-15 min. To reprobe, nitrocellulose
filters were first stripped of antibody by 62 mmol/liter Tris-HCl, pH
6.7, 2% SDS, 100 mmol/liter
2- mercaptoethanol.
Preparation of Nuclear Extract and Gel Retardation
Assay--
Nuclear extracts from untreated and SCF-treated cells were
prepared by Nonidet P-40 lysis as described by Sadowski and Gilman (33). The oligonucleotides used, corresponding to the
prolactin-inducible element (PIE) of the
-casein promoter, were G
GGG GGA CTT CTT GGA ATT AAG GGA and G GGG TCC CTT AAT TCC AAG AAG TCC
(24) and corresponding to the serum-inducible element of c-Fos (SIE)
were G GGG CAT TTC CCG TAA ATC and G GGG GAT TTA CGG GAA ATG (34). The
annealed oligonucleotide was labeled by filling in the over-hanging ends with Klenow fragment in the presence of
[
-32P]dCTP. Gel retardation reactions were performed
in 13 mmol/liter Hepes, pH 7.6, 80 mmol/liter NaCl, 3 mmol/liter NaF, 3 mmol/liter NaMoO4, 1 mmol/liter dithiothreitol, 0.15 mmol/liter EDTA, 0.15 mmol/liter EGTA, and 8% glycerol (including
contribution from the nuclear extract) and contained 75 µg/ml
poly(dI·dC):poly(dI·dC) approximately 0.3 ng of radiolabeled probe,
and 5-10 µg of protein. Reactions were carried out at room
temperature for 40 min and then resolved on 4% polyacrylamide gels
containing 0.25× TBE (1× TBE is 89 mmol/liter Tris borate, 1 mmol/liter EDTA, pH 8) and 5% glycerol. Gels were run at 4 °C in
0.25× TBE at 20 V/cm, dried, and autoradiographed. Oligonucleotide
competition was performed by preincubating nuclear extracts with the
competitor oligonucleotide (50 fold excess) and
poly(dI·dC):poly(dI·dC) for 30 min at room temperature before the
addition of labeled probe. Gel mobility shift assays were done with
nuclear extract that had been reacted for 1 h at 4 °C with the
indicated antibodies.
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RESULTS |
c-Kit Expression and Tyrosine Phosphorylation in Human Myeloid
Cells Expressing Wild Type or c-Kit Mutants--
To evaluate
SCF-induced signaling we used the following cell types: the myeloid
growth factor MO7e cell line that survives and proliferates in response
to different growth factor, including SCF (30), NIH-3T3 fibroblasts
transfected and selected for overexpression of the human c-Kit cDNA
(HRS) (9), and two previously described mutants of c-Kit (9) as
follows: NIH-3T3 fibroblasts transfected with a c-Kit mutant lacking
the kinase insert domain (
KI), and a carboxyl-terminal deletion
mutant of c-Kit (
CT). Three antibodies to c-Kit were used for
immunoprecipitation or Western blotting. These are the anti-Kit-X
antiserum raised against the extracellular domain of c-Kit, the Ab212
rabbit antiserum directed to the most carboxyl-terminal 14-amino
acid-long sequence of c-Kit, and monoclonal antibody K44 recognizing
the second immunoglobulin-like domain of the human c-KIT. As is evident
from Fig. 1A, anti-Kit-X was able to detect the full-length receptor in the MO7e, HRS, and
CT
cells, as well as the lower molecular weight mutant of c-Kit protein in
the
KI cells. By contrast, the Ab212 antiserum was unable to detect
the c-Kit mutant lacking the carboxyl terminus (Fig. 1A).
When the four cell lines were stimulated with SCF and c-Kit proteins
analyzed for tyrosine phosphorylation, we observed increased
phosphorylation of the wild type and
CT c-Kit proteins (Fig.
1B). However, no SCF-induced tyrosine phosphorylation of the
KI mutant was detectable, in agreement with a previous report that
characterized this mutant as a catalytically inactive form of c-Kit
(9).

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Fig. 1.
Expression and tyrosine phosphorylation of
wild type and mutant Kit proteins. A, expression of
wild type and mutant Kit proteins. Cells were lysed and Kit proteins
immunoprecipitated with an anti-Kit antibody (K44). The
immunocomplexes were subjected to SDS-PAGE and immunoblotted with the
anti-Kit-X. MO7e and CT cell lysates were also immunoprecipitated
with the K44 antibody and immunoblotted with the Ab 212 antiserum (right panel). B, SCF-mediated tyrosine
phosphorylation of Kit proteins. Cell lysates prepared from
unstimulated ( ) or SCF-stimulated (+) cell lines were subjected to
immunoprecipitation (IP) with K44 antibody. Proteins were
electrophoretically transferred to nitrocellulose filters which were
then immunoblotted (IB) with an anti-phosphotyrosine
(P-Tyr) antibody and reprobed with the anti-Kit-x antiserum.
Note that the KI mutant undergoes no tyrosine phosphorylation in
response to SCF. The positions of the various Kit proteins are
indicated. The arrows correspond to the less glycosylated
Kit precursor.
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STAT1
Tyrosine Phosphorylation and Coupling to c-Kit in
SCF-treated Cells--
It has been previously shown that STAT1
, as
well as STAT3, becomes tyrosine-phosphorylated upon activation of
certain growth factor receptors (14-17). To evaluate the ability of
SCF to induce STAT1
phosphorylation on tyrosine residues, we treated
cells with SCF, immunoprecipitated STAT1
, and analyzed its state of phosphorylation by using the anti-phosphotyrosine antibody. The results
presented in Fig. 2A
demonstrate that SCF was able to induce tyrosine phosphorylation of
STAT1
in MO7e, HRS, and
CT cells but not in the
KI-expressing
cells, consistent with the defect of c-Kit in these cells. Moreover,
kinetic analysis of STAT1
phosphorylation in MO7e cells revealed
that its tyrosine phosphorylation occurred already after 5 min of SCF
stimulation, but it disappeared after 15 min of ligand stimulation
(data not shown). Importantly, a 145-kilodalton protein corresponding
to the full-length c-Kit was detectably co-immunoprecipitated with STAT1
from lysates of SCF-stimulated MO7e and HRS cells, and in the
CT mutant was also detectable upon long film exposure, but no
association with the
KI mutant was evident (Fig. 2A). Nevertheless, physical association of the
CT mutant, as well as the
wild type c-Kit, with STAT1
was evident from a reciprocal co-immunoprecipitation assay in which anti-c-Kit immunoprecipitates were analyzed by using an anti-STAT1
antibody (Fig. 2B).
In conclusion, both wild type and a carboxyl-terminally deleted c-Kit
proteins are able to increase tyrosine phosphorylation of and
physically associate with the STAT1
protein, but the
kinase-defective
KI mutant is inactive in STAT1
coupling.

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Fig. 2.
SCF-induced tyrosine phosphorylation of
STAT1 and physical association with Kit.
A, tyrosine phosphorylation of STAT1 . Unstimulated ( )
and SCF-stimulated (+) cells were lysed, and cell extracts were
subjected to immunoprecipitation (IP) with an anti-STAT1
antiserum. Following SDS-PAGE proteins were electrophoretically
transferred to nitrocellulose filter, and the filter was immunoblotted
(IB) with an anti-phosphotyrosine antibody (upper
panel) and reprobed with an anti-STAT1 antiserum (lower
panel). The location of the STAT1 protein is indicated.
B, STAT1 physically associates with the activated c-Kit
receptor. The indicated cell lines were lysed, and their whole extracts
were subjected to immunoprecipitation with the K44 antibody. Proteins
were electrophoretically transferred to nitrocellulose filter, and the
filter was immunoblotted with an anti-STAT1 antiserum (upper
panels), stripped, and reprobed with the anti-Kit-X antiserum
(lower panel). MO7e total cell lysate (TCL) was
used to indicate the location of STAT1 . The positions of p145
protein and STAT1 are indicated.
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SCF Induces the Formation of a Protein Complex Containing STAT1
on the SIE--
It is known that STATs can bind to a specific DNA
element in the c-Fos promoter, located distally to the serum response
element (SRE) and known as the c-sis-inducible element (SIE)
(19). SIE binds to STATs other than STAT1 and STAT3 rather weakly, but
it displays high affinity for homo- and heterodimers of STAT1 and STAT3
(19). Therefore, we addressed the ability of the SCF-activated STAT1
to bind to the SIE. To this end we incubated nuclear extract from
SCF-treated cells with a radiolabeled SIE probe and analyzed complex
formation by using non-denaturing gel electrophoresis. The results
presented in Fig. 3A indicate
that SCF stimulation induced SIE binding activity in MO7e cells but not
in cells expressing a kinase-defective
KI mutant of c-Kit. That
STAT1
is present in the SCF-induced DNA-protein complex was evident
from a supershift analysis in which an antibody to STAT1
specifically caused a mobility shift of the SIE-containing complex
(Fig. 3B). Similarly, a carboxyl-terminal deletion mutant of
c-Kit was able to mediate formation of an SIE·STAT1
complex (Fig.
3B, right panel), implying that the distal part
of the intracellular domain of c-Kit is not essential for coupling to
and activation of STAT1
.

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Fig. 3.
SCF-induced formation of the SIE complex in
wild type Kit- and CT-expressing cells but not
in KI-expressing cells. A, SIE
complex formation. Nuclear extracts were prepared from the indicated
untreated ( ) or SCF-treated (+) cells in the presence or in the
absence of unlabeled oligonucleotide (competitor).
B, the SCF-induced SIE-binding complex is antigenically
related to STAT1 . Nuclear extracts from SCF-treated cells were
preincubated with a preimmune serum (PI) or with an
anti-STAT1 antiserum. The DNA-protein complexes were then resolved
by nondenaturing polyacrylamide gel electrophoresis. The SCF-induced
complexes and the supershifted species are indicated by
arrows (lower and upper arrows,
respectively).
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STAT5A and STAT5B Become Tyrosine-phosphorylated upon SCF
Stimulation and Form a Stable Complex with Kit, Possibly through Its
Carboxyl-terminal Region--
To extend the analysis of the
interaction between Kit and STAT proteins, we tested tyrosine
phosphorylation of and physical association with other members of the
STAT family. Although STAT2, STAT3, STAT4, and STAT6 are expressed in
MO7e cells, we were unable to detect their interaction with a
ligand-activated c-Kit (data not shown). However, two other STAT
proteins, STAT5A and STAT5B, were found to be coupled to c-Kit upon
ligand binding. These STAT5 proteins were originally reported to
undergo activation in response to prolactin in the mammary gland (24),
but subsequently a large body of evidence supporting STAT5 activation
in response to many cytokines and growth factors has accumulated
(14-17). For example, stimulation of MO7e cells with IL-3 resulted in
phosphorylation of STAT5A and STAT5B (Fig.
4, A and B).
Likewise SCF-mediated tyrosine phosphorylation of STAT5A and STAT5B was
detectable in both MO7e and HRS cells (Fig. 4, A and
B). Similarly analysis of cells expressing a
carboxyl-terminally deleted mutant of c-Kit (
CT), by immunoblotting
with anti-phosphotyrosine antibody, revealed that this mutant retained
the ability to phosphorylate both proteins (Fig. 4C).
However, in experiments that are not presented, no tyrosine
phosphorylation of these proteins was detectable in fibroblasts expressing the kinase-defective
KI mutant of c-Kit. Moreover, in
lysates from IL-3- and SCF-stimulated cells, additional phosphorylated bands of unknown tyrosine-phosphorylated substrates were also detected
(Fig. 4, A and B). Kinetic analysis of STAT5
protein activation in
CT-expressing cells (Fig. 4C)
revealed that their tyrosine phosphorylation was rapid and transient,
occurring already after 1 min of SCF stimulation and disappearing by 10 min of ligand stimulation. By contrast, a less transient (still present
after 15 min of ligand stimulation) tyrosine phosphorylation of STAT5A (Fig. 4D) and STAT5B (data not shown) was detectable in
SCF-stimulated MO7e cells.

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Fig. 4.
Wild type and carboxyl-terminally deleted Kit
proteins mediate SCF-induced tyrosine phosphorylation of STAT5A and
STAT5B. A and B, STAT5A and STAT5B
phosphorylation by wild type Kit. The indicated cells were either
unstimulated ( ) or stimulated (+) with IL-3 or SCF. Cell lysates were
prepared and subjected to immunoprecipitation (IP) with an
anti-STAT5A (A) or an anti-STAT5B (B) antiserum.
C, STAT5A and STAT5B phosphorylation by a
carboxyl-terminally deleted Kit protein. CT-expressing cells were
stimulated with SCF for the indicated time intervals. Cell lysates were
prepared and subjected to immunoprecipitation with an anti-STAT5A or an
anti-STAT5B antiserum. D, kinetics of STAT5A phosphorylation
in MO7e cells. MO7e were incubated in the absence or in the presence of
SCF for the indicated times, lysed, and immunoprecipitated with an
anti-STAT5A antiserum. Proteins were electrophoretically transferred to
nitrocellulose filters, and the filters were immunoblotted
(IB) with an anti-phosphotyrosine (P-tyr)
antibody (upper panels) and reprobed with the indicated
antiserum (lower panels). The positions of STAT5A and STAT5B
proteins were indicated. The positions of unknown
tyrosine-phosphorylated substrate are indicated by the
arrows.
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It has been shown that upon ligand stimulation the activated STAT
proteins tightly interact, through their SH2 domains, with specific
phosphotyrosine residues of activated receptors (16). To evaluate the
ability of the activated wild type and a carboxyl-terminally deleted
c-Kit to form a stable complex with STAT5 proteins, we performed
co-immunoprecipitation experiments whose results are shown in Fig.
5, A and B.
Evidently, STAT5A and STAT5B could be immunoprecipitated with the wild
type form of c-Kit from cell extracts of SCF-stimulated MO7e cells
(Fig. 5, A and B). However, despite the ability
of the
CT mutant to mediate tyrosine phosphorylation of STAT5
proteins, our co-immunoprecipitation experiments failed to detect
complex formation between STAT5 proteins and this mutant form of c-Kit
(Fig. 5, C-E).

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Fig. 5.
STAT5A and STAT5B physically associate with
the activated wild type protein but not with the carboxyl-terminally
deleted CT mutant. Unstimulated ( ) or
SCF-stimulated (+) MO7e (A and B) and
CT-expressing cells (C-E) were lysed, and their extracts
were subjected to immunoprecipitation (IP) with the K44
antibody. The immunoprecipitates were divided into 2 or 3 aliquots that
were subjected to SDS-PAGE. Proteins were electrophoretically
transferred to nitrocellulose filters, and the filters were separately
immunoblotted (IB) with an anti-STAT5A antiserum
(A and D), an anti-STAT5B antiserum (B
and E), or with the anti-phosphotyrosine (P-Tyr)
antibody (C) as indicated. The arrows correspond
to the less glycosylated Kit precursor. TCL , total cell
lysate.
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The Carboxyl-terminally Deleted c-Kit Shows an Impaired PIE Complex
Formation in Response to SCF--
STAT5 proteins can interact with the
-casein promoter region known as the prolactin-inducible element
(PIE) (25). Therefore, to evaluate the ability of the activated STAT5
protein to bind to the PIE, nuclear extract from SCF-stimulated MO7e,
HRS, and
CT cells were incubated with a radiolabeled PIE probe, and
complex formation was analyzed by non-denaturing gel electrophoresis. The results shown in Fig. 6A
indicate that SCF stimulation leads to the formation of a PIE complex
in MO7e and in HRS cells but not in cells expressing the
carboxyl-terminally deleted c-Kit. That the SCF-induced DNA-protein
complex contains both STAT5 proteins was evident from supershift
experiments in which antibodies to STAT5A and STAT5B were able to
induce a mobility shift of the PIE complex (Fig. 6B).

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Fig. 6.
SCF induces the formation of a PIE-binding
complex in MO7e and HRS cells but not in
CT-expressing cells. A, PIE complex
formation. Nuclear extracts were prepared from the indicated untreated
( ) or SCF-treated (+) cells in the presence or in the absence of
unlabeled oligonucleotide (competitor). B, the
SCF-induced PIE-binding complex is antigenically related to STAT5A and
STAT5B. Nuclear extracts from SCF-treated MO7e and HRS cells were
preincubated with a preimmune serum (PI) or with an
anti-STAT5A or an anti-STAT5B antiserum. The DNA-protein complexes and
the supershifted species (lower and upper arrows,
respectively) were then resolved by nondenaturing polyacrylamide gel
electrophoresis.
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SCF Stimulation Leads to the Formation of Different STAT
Dimers--
Receptor activation leads to the recruitment of STAT
monomers which then homo- or heterodimerize and migrate to the nucleus to activate gene transcription (14-17). To extend the analysis of STAT
protein activation upon SCF stimulation, we evaluate STAT dimer
formation in MO7e and in
CT cells. Co-immunoprecipitation experiments, whose results are shown in Fig.
7, were performed using
non-cross-reacting antisera to STAT5A and STAT5B. Heterodimer formation
between STAT5A and STAT5B was clearly detectable in MO7e cells.
However, despite the ability of the
CT receptor mutant to induce
tyrosine phosphorylation of STAT5, reciprocal co-immunoprecipitation assays were unable to detect dimer formation between STAT5A and STAT5B
in SCF-stimulated
CT cells (Fig. 7, right panel).

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Fig. 7.
SCF induces heterodimerization of STAT5A and
STAT5B proteins in MO7e cells. Cells were unstimulated ( ) or
stimulated (+) with SCF, lysed, and STAT5 proteins immunoprecipitated
(IP). Following SDS-PAGE, proteins were electrophoretically
transferred to nitrocellulose filters, and the filters were
immunoblotted (IB) with the reciprocal anti-STAT5, as
indicated (upper panels). The filters were then reprobed
with the other anti-STAT5 antibody as indicated (lower
panels). Total cell lysates (TCL) from MO7e cells were
used to localize the STAT5A and STAT5B proteins.
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It has been reported that upon activation of CSF-1 and PDGF receptors
tyrosine phosphorylation of STAT5 and STAT3 proteins can interact to
form heterodimers (35). Thus, we addressed the ability of SCF-activated
STAT1
and STAT5 proteins to form stable complex. To this end we
performed reciprocal co-immunoprecipitation experiments in MO7e and in
CT-expressing cells. As is evident from the anti-STAT1
immunoblot
presented in Fig. 8A, both
STAT5 proteins can heterodimerize with STAT1
in SCF-stimulated MO7e cells. By contrast, analysis of cells expressing a carboxyl-terminally deleted mutant of c-Kit (Fig. 8B) detected no STAT1
/STAT5
heterodimers. The DNA sequence specificity of the STAT1
/STAT5
heterodimeric complexes in MO7e cells was evaluated by electrophoretic
mobility shift assay, using PIE and SIE elements as probes. As shown in Fig. 8C, the apparent mobility of SCF-mediated PIE complex
could be altered by preincubating nuclear extracts with an antiserum to
STAT1
. By contrast, no supershifted species was detectable upon
adding either an antiserum to STAT5A or an antiserum to STAT5B to
SCF-treated nuclear extracts incubated with the SIE probe. In
conclusion, the PIE element binds to STAT1
/STAT5 complex, but the
SIE element cannot bind to such heterodimer in SCF-stimulated cells.

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Fig. 8.
The carboxyl-terminal region of c-Kit is
essential for STAT5/STAT1 heterodimer
formation, which binds specifically to the PIE. MO7e
(A) and CT-expressing cells (B) were
unstimulated ( ) or stimulated (+) with SCF, lysed, and the indicated
STAT proteins immunoprecipitated (IP) with the respective
antiserum. Proteins were electrophoretically transferred to
nitrocellulose filters that were immunoblotted (IB) with an
anti-STAT1 antiserum (upper panels) and reprobed with an
anti-STAT5A or an anti-STAT5B antiserum (lower panels).
Total cell lysates (TCL) from CT-expressing cells were
used to indicate the migration of STAT1 . C,
STAT1 /STAT5 heterodimers bind to PIE but not to the SIE sequence.
Nuclear extracts from SCF-treated MO7e cells were preincubated with the
indicated antibodies (PI, preimmune serum). Then,
radiolabeled oligonucleotides corresponding to the PIE or the SIE
sequences, as indicated, were added. The formed DNA-protein complexes
and the supershifted species are indicated by arrows
(lower and upper arrows, respectively).
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 |
DISCUSSION |
In the present study we investigated the ability of SCF to induce
physical association with and tyrosine phosphorylation of STAT
proteins. Wild type and two different deletion mutants of c-Kit were
examined. We found that, among different STAT proteins, only STAT1
,
STAT5A, and STAT5B were activated in response to SCF stimulation both
in human myeloid MO7e cells and in c-Kit-transfected fibroblasts.
Moreover, the results obtained with a mutant, lacking the kinase insert
domain, demonstrated that ligand stimulation failed to induce STAT
tyrosine phosphorylation and physical association with the mutated
c-Kit. In the cytokine receptor family ligand stimulation induces rapid
activation of JAK proteins leading to STAT tyrosine phosphorylation
(14-17). By contrast, in tyrosine kinase receptors, the role of JAK
proteins in STAT signaling is not yet clearly defined. Consistent with
a JAK-independent pathway, a catalytically inactive form of PDGFR is
unable to stimulate STAT1 and STAT3 activation in response to PDGF
stimulation (36). Likewise, our finding that the
KI mutant of c-Kit,
which displayed reduction in autophosphorylation in vitro
(9), cannot signal to STAT (Fig. 2) or to JAK2 upon SCF treatment (data
not shown), supports the possibility that STAT proteins may represent
direct substrates of receptor tyrosine kinases, and that, indeed, a
full functional enzymatic activity of the receptor is required for coupling to and activate STATs. Consistent with this hypothesis are the
observations that STAT1 can directly interact with EGF (37) and PDGF
(20) receptors and that these receptors can phosphorylate STAT1
in vitro (20, 38). Moreover, the absence of any JAKs does
not affect the ability of EGF or PDGF to phosphorylate STAT1 (20, 36,
39). Furthermore, the non-receptor tyrosine kinase, Src, is associated
with STAT3 in vivo and in vitro, and it can
phosphorylate STAT3 in vitro (40, 41). However, our observation does not rule out the possibility that the kinase insert
domain, which is known to selectively interact with the p85 regulatory
subunit of phosphatidylinositol 3-kinase (9), also contains
STAT-binding sites. It has been recently shown that activation of STAT5
by PDGF is dependent on phosphorylation sites in PDGFR
juxtamembrane and kinase insert domains (23). Comparing the sequences
surrounding receptor's tyrosine residues mediating STAT docking and
activation, it appears that a consensus YVDP or YLDP is required for
binding of the STAT5 SH2 domain (20, 42). A similar consensus sequence
does not surround tyrosine residue 721 and 730 contained in the kinase
insert domain of c-Kit (4, 9), suggesting that reduction of the
catalytic activity of the receptor, rather than loss of tyrosine
residues, may account for the inability of the
KI mutant of c-Kit to
interact with and activate STAT proteins.
Tight binding of phosphotyrosine residues of the receptor to the SH2
domain of STAT proteins seems to play a dominant role in STAT signaling
(16). Requirement of intracellular phosphotyrosine residues in
mediating STAT5 docking and activation has been reported for the GH
receptor (43). Similarly, PRL-induced activation of STAT5 was abolished
in fibroblasts expressing PRL receptor mutants lacking all
intracellular tyrosines (44). Our results demonstrate that following
SCF stimulation, STAT1
and STAT5 proteins, phosphorylated on
tyrosine, physically associate with the full-length but not with the
kinase-defective receptor indicating that a catalytically active
receptor, able to provide phosphorylated docking site for STAT, is also
required in the tyrosine kinase receptor family. Furthermore, evidence
for alternative mechanisms of STAT receptor interaction has been
reported for GH. In particular, GH receptor mutants devoid of all
cytoplasmic sites for tyrosine phosphorylation can nonetheless support
STAT1 and STAT3 activation when GH is provided (45). In contrast, STAT5
activation relies on the presence of different tyrosine-phosphorylated
residues in the cytoplasmic tail of the GH receptor (46), indicating
that a redundancy in tyrosine residue requirement may be employed in
GH-mediated STAT5 signal (46). Likewise, three different
tyrosine-phosphorylated residues are required for maximal STAT5
activation in response to PRL (44), suggesting that full STAT5-mediated
gene transcription depends on several tyrosine-phosphorylated docking
sites. Therefore, the lack of tyrosine residues in the cytoplasmic tail
of the
CT mutant can explain why the SCF-activated COOH-terminally
deleted c-Kit is unable to recruit STAT5s but still retains the ability to transiently phosphorylate these proteins. However, other models can
account for our observation as follows: JAK2 itself, which is activated
upon SCF stimulation (12, 13), may function as a bridge, bringing STAT5
proteins to the receptor through its tyrosine residues (47). We were
unable to detect any JAK2-STAT5 interaction (data not shown). However,
we cannot rule out the possibility that a weak molecular interaction
may account for this result. Alternatively, non-receptor tyrosine
kinases, other than JAK2, may be implicated in STAT tyrosine
phosphorylation and/or STAT-receptor interaction. Nevertheless,
independently of the exact mechanism, the ability of the
CT mutant
to signal to STAT1
but not to STAT5 proteins suggests that the
recruitment of STAT1
and STAT5 depends on different intracellular
c-Kit domains. This interpretation is in agreement with the model in
which STAT5 but not STAT1 activation requires the box 3 region of the
cytoplasmic tail of GH receptor (46).
Accumulating evidence suggests that tyrosine-phosphorylated STATs
undergo intermolecular dimerization through their SH2 domain and
conserved phosphotyrosine present in the COOH-terminal of the SH2
domain (48). This dimerization is required for subsequent release of
STAT from the receptor complex and for translocation to the nucleus for
DNA binding (15). Our result demonstrates that in the
CT mutant,
which fails to recruit the phosphorylated STAT5 proteins, no dimer and
PIE complex can be formed, supporting the possibility that, as
described previously for STAT1
(49), a specific receptor interaction
may also dictate dimerization and DNA binding activity of STAT5 proteins.
It has been reported that STAT5 proteins, besides forming homo- and
heterodimers, may also contribute to the formation of heterodimers with
other STAT proteins (33). In response to CSF-1 and PDGF stimulation,
STAT5/STAT3 heterodimers apparently bind to the c-Fos promoter region,
whereas STAT5 homodimers are inactive. On the other hand, binding to
the
-casein promoter occurs as homodimers or STAT5A/STAT5B
heterodimers (35). Similarly, we found that SCF stimulation leads to
the formation of heterodimers containing STAT5A or STAT5B together with
STAT1
. The analysis of the DNA binding activity of these
heterodimeric complexes revealed that the sequence corresponding to the
-casein promoter region (PIE), but not that corresponding to the
c-Fos promoter region (SIE), could be specifically recognized by these
STAT5s/STAT1
heterodimeric complexes. Although the biological
significance of these different dimers is thus far unclear, the
observation that the heterodimeric complexes formed by STAT5 proteins
and STAT1
were detected in cells expressing the full-length
receptor, but not in
CT-expressing cells, supports the possibility
also that the rate of dephosphorylation of the already activated
molecules is crucial for dimer formation and final transcriptional
output from activated STATs.
The results of the present study demonstrate that different regions of
c-Kit are involved in STAT1
- and STAT5-receptor interaction and that
the COOH-terminal region of c-Kit is essential for full activation of
STAT5A and STAT5B but not of STAT1
. Moreover, we demonstrate that
upon SCF stimulation STAT1
can also form heterodimers with STAT5A or
STAT5B, and the complex formed specifically binds to the PIE sequence.
Better understanding of the role played by various activated STATs may
provide new insights into the signaling pathways required to achieve
divergent biological effects in response to the same growth factor.