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(Received for publication, August 18,
1995; and in revised form, November 8, 1995) From the
Granulocyte-macrophage colony-stimulating factor (GM-CSF),
supports proliferation, differentiation, and functional activation of
hemopoietic cells by its interaction with a heterodimeric receptor.
Although GM-CSF receptor is devoid of tyrosine kinase enzymatic
activity, GM-CSF-induced peripheral blood polymorphonuclear leukocytes
(PMN) functional activation is mediated by the phosphorylation of a
large number of intracellular signaling molecules. We have previously
shown that JAK2 becomes tyrosine-phosphorylated in response to GM-CSF
in PMN. In the present study we demonstrate that also the signal
transducers and activators of transcription (STAT) family members STAT1
p91 and STAT3 p92 and the product of the c-fps/fes protooncogene become tyrosine-phosphorylated upon GM-CSF
stimulation and physically associated with both GM-CSF receptor Finally we demonstrate that GM-CSF induces a
DNA-binding complex that contains both p91 and p92. These results
identify a new signal transduction pathway activated by GM-CSF and
provide a mechanism for rapid activation of gene expression in
GM-CSF-stimulated PMN. Granulocyte-macrophage colony-stimulating factor (GM-CSF) ( All GM-CSF effects are mediated by a
heterodimeric receptor comprised of a ligand binding subunit, denoted
HEPG2 cells were maintained in RPMI 1640 medium supplemented with
10% bovine calf serum and serum-starved overnight before being treated
with rhIL-6 (30 ng/ml).
GM-CSF plays an important role in host defense by enhancing
the functional activities of mature leukocytes and, in particular,
neutrophils(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
The binding of GM-CSF to its heterodimeric receptor, which is devoid of
intrinsic kinase activity, leads to tyrosine phosphorylation of
cellular substrates(19) . It has been reported that, in growth
factor-dependent cell lines, JAK2 is constitutively associated with the
Figure 1:
JAK2 association with the
GM-CSF receptor
Figure 2:
Kinetic analysis of GM-CSF-induced JAK2
activation in human PMN. PMN were incubated in 1640 RPMI medium in the
absence or in the presence of GM-CSF (10 ng/ml) for the indicated time,
lysed, and immunoprecipitated with the anti-JAK2 antiserum. The filter
was probed with 4G10 anti-phosphotyrosine monoclonal antibody (upper panel) and reprobed with the antiserum against JAK2 (lower panel). The positions of the p130-JAK2 and the p90
phosphotyrosine proteins are indicated. IP,
immunoprecipitated; IB, immunoblotted; P-tyr,
phosphotyrosine.
A likely set of substrates for the JAKs is the family of
latent cytoplasmic transcription factors termed STATs(31) .
Ligand binding to several cytokine receptors induces tyrosine
phosphorylation of STAT family members that, subsequently, translocate
to the nucleus, bind to related DNA sequences, and promote
transcription(39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) .
The anti-phosphotyrosine blot of anti-JAK2 immunoprecipitates from
GM-CSF-treated PMN revealed, together with the phosphorylated p130
JAK2, a marked band of phosphotyrosine-containing protein(s) of
approximately 90 kDa (Fig. 2). It is known that among the STAT
proteins STAT1 and STAT3 exhibit a molecular mass of 91 and 92 kDa,
respectively. Therefore we sought to determine whether the JAK2
co-precipitating p90 phosphoprotein(s) included one or both STATs. It
has also been demonstrated that a p93 myeloid-specific protein, the
product of the protooncogene c-fps/fes, becomes phosphorylated
and associated to the GM-CSF receptor
Figure 3:
GM-CSF-induced tyrosine phosphorylation of
STATp91, STATp92, and p93
Figure 4:
p91, p92, and p93
Figure 5:
GM-CSF-induced JAK2 tyrosine
phosphorylation in human PMN. Cell lysates from unstimulated(-)
and IL-3- and GM-CSF-stimulated (10 ng/ml each for 5 min) (+) PMN
were immunoprecipitated with anti-JAK2 antiserum. The filter was probed
with 4G10 anti-phosphotyrosine monoclonal antibody (upper
panel) and reprobed with the antiserum against JAK2 (lower
panel). The positions of the p130-JAK2 and the p90 phosphotyrosine
proteins are indicated. IP, immunoprecipitated; IB,
immunoblotted; P-tyr,
phosphotyrosine.
Figure 6:
p91, p92, and p93
Tyrosine phosphorylation of various tyrosine kinases is
commonly associated with the activation of their catalytic
activity(53) . An in vitro kinase assay was performed
to examine whether phosphorylation of JAK2 and p93
Figure 7:
In vitro JAK2 and p93
PMN are terminally differentiated cells and do
not undergo proliferation; however, tyrosine phosphorylation of
intracellular substrates has been implicated in a number of functional
activities such as superoxide anion
production(54, 55, 56, 57, 58) ;
regulation of integrin surface expression, leading to adherence of PMN
to endothelial cells (59) ; regulation of microvascular
permeability, leading to migration of PMN into inflammatory
tissue(60, 61) ; and modulation of apoptotic
process(15) . The role of protein tyrosine phosphorylation in
physiological agonist-mediated or GM-CSF-mediated PMN activation is
further supported by the observation that PMN biological responses are
prevented by the addition of tyrosine kinase
inhibitors(15, 55, 56, 59, 62) .
It has been shown that, in PMN, GM-CSF causes a rapid tyrosine
phosphorylation of intracellular molecules including both 90- and
130-kDa proteins(15, 55, 62, 63) .
In agreement with these findings, the present study demonstrates that a
set of 90-kDa proteins namely STAT1, STAT3, and p93 It has been reported that treatment of cells with different
cytokines results in rapid STAT protein phosphorylation. Activated
STATs (one or more) form dimers that migrate in the nucleus and form
stable complexes with specific DNA sequences (response elements) and
stimulate transcription(31) . Three discrete complexes between
activated STAT proteins and DNA response elements have been
demonstrated upon EGF treatment(50) . These complexes seem to
be formed by STAT1 or STAT3 homodimers or by heterodimers between the
two STATs(50) . In contrast, in interferon-
Figure 8:
A, induction of DNA-binding activity by
GM-CSF and IL-6 in PMN and in HEPG2 cells. Nuclear extracts of
untreated(-) or 15-min IL-6-treated HEPG2 cells and
GM-CSF-treated PMN (+) were either treated (+) or
not(-) treated with a 50-fold excess of unlabeled oligonucleotide
(competitor) for 30 min before the addition of radiolabeled
oligonucleotide. The complexes were then resolved by nondenaturing
polyacrylamide gel electrophoresis. The DNA-binding complexes are
indicated. B, the GM-CSF-induced DNA-binding complex is
antigenically related to p91 and p92. Nuclear extracts from 15-min
GM-CSF-treated PMN or IL-6-treated HEPG2 cells were preincubated for 1
h at 4 °C with preimmune serum (PI), anti-p91 antiserum,
or anti-p92 antibodies before incubation with radiolabeled
oligonucleotide and separated on a nondenaturing polyacrylamide gel
electrophoresis. The IL-6- and GM-CSF-induced DNA-protein complexes and
the supershifted specie are indicated.
It has been shown that tyrosine-phosphorylated
proteins are involved in GM-CSF-mediated PMN functional activation and
c-fos gene transcription(55) . Moreover the role of
tyrosine kinases in controlling GM-CSF-induced c-fos gene
expression in PMN, has been demonstrated by the use of a tyrosine
kinase inhibitor(55) . Our finding that at least two of the
STAT proteins that become phosphorylated upon GM-CSF stimulation are
involved in the formation of a complex with the serum-inducible
elements of c-fos, supports the hypothesis that GM-CSF can
regulate the transcription of this gene via STAT1 and STAT3 activation. In conclusion, our study demonstrates that in PMN both STAT1 p91 and
STAT3 p92 and the myeloid-specific p93 The redundancy of growth factors inducing the same
DNA-responsive element to stimulate both cell proliferation and
functional activation raises the question of how their specificity can
be determined. The answer could be obtained by the identification of
more genes whose transcription can be activated by the binding of known
or unknown proteins.
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3562-3567
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
, STAT1 p91, and STAT3 p92 in
Polymorphonuclear Leukocytes (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
common subunit and JAK2. Moreover GM-CSF was able to induce JAK2 and
p93 catalytic activity. We also demonstrate that
the association of the GM-CSF receptor
common subunit with JAK2
is ligand-dependent.
)regulates proliferation and differentiation of hemopoietic
progenitor cells and functionally activates polymorphonuclear
leukocytes (PMN)(1) . In particular, GM-CSF exerts several
direct actions on neutrophils, including stimulation of changes in
surface expression of both chemotactic receptors and adherence
proteins(2, 3, 4, 5) , as well as
hydrogen peroxide production by neutrophils adhered to extracellular
matrix components(6, 7) . Moreover GM-CSF has indirect
effects on neutrophils, such as ``priming'' these cells for
enhanced responses to a number of physiologically relevant stimuli such
as ingestion of Staphylococcus aureus(8) ,
serum-opsonized particles(3) , antibody-dependent
cytotoxicity(3) , fMet-Leu-Phe (FMLP)-stimulated intracellular
calcium mobilization(9) , and oxyradical (2, 10) and platelet-activating factor (11) production as well as leukotriene
synthesis(12, 13, 14) . More recently it has
been reported that GM-CSF inhibits programmed cell death both in human
eosinophils and neutrophils (15) and that this effect is
mediated by tyrosine phosphorylation of intracellular
substrates(15) .
(16) , and of a transducing subunit designated as
(17) , which is also shared with interleukin-3 (IL-3) (17) and IL-5 receptor(18) . Although GM-CSF receptor
does not possess an intrinsic tyrosine kinase domain, several lines of
evidence indicate that signaling processes initiated by ligand binding
to the receptor induce activation of cellular tyrosine
kinases(19) . Studies on the biochemical interaction involved
in signaling from the GM-CSF receptor have demonstrated that a number
of transducing molecules such as
Shc(20, 21, 22) , Grb2(20) ,
Sos1(20) , Ras(23) , Raf-1 (24) , and
mitogen-activated protein kinase (25) become activated upon
GM-CSF stimulation. It has also been reported that a nonreceptor
tyrosine kinase, the c-fps/fes protooncogene product, is
phosphorylated in response to GM-CSF(26) . More recently the
receptor-associated protein JAK2 (27, 28) has been
reported to be rapidly phosphorylated upon GM-CSF receptor
activation(29, 30) . Recent data suggest that at least
two components of latent cytoplasmic proteins termed signal transducers
and activators of transcription (STATs)(31) , which become
activated upon ligand binding, are substrates of JAK family
members(32, 33, 34) . In order to
characterize the tyrosine-phosphorylated proteins involved in
GM-CSF-mediated PMN activation, we examined the role of two STAT
proteins, STAT1 (p91) and STAT3 (p92) and of
c-fps/fes protooncogene product
(p93) in this process. We demonstrate that, upon
GM-CSF stimulation, both STAT proteins and p93
become tyrosine-phosphorylated and physically associate with
GM-CSF receptor
common subunit as well as with JAK2. GM-CSF
stimulation was also able to induce p93 and JAK2
catalytic activity. Moreover we demonstrate that, as previously
reported for erythropoietin receptor (35) , JAK2 association
with the
common subunit is ligand-dependent. Finally we
demonstrate that the DNA-binding proteins p91 and p92 are early targets
of the GM-CSF-induced DNA-binding complex. These results identify a
signal transduction pathway that is activated in response to GM-CSF in
human PMN and provide evidence for the role of STAT proteins in
GM-CSF-mediated rapid modulation of gene expression in functionally
activated nonproliferating cells.
Reagents
Cells were cultured in RPMI 1640 medium
(Life Technologies, Inc.). Bovine calf serum was from Hyclone
Laboratories (Logan, UT). Gelatin was from Difco. rhGM-CSFs were kindly
provided by Sandoz Forschungsinstitut, Austria. rhIL-3 and rhIL-6 were
a gift from Genetic Institute (Cambridge, MA). The Sepharose-protein A
was purchased from Sigma. Nitrocellulose filters, horseradish
peroxidase-conjugated protein A, molecular weight markers,
[-P]dCTP,
[
-
P]ATP, and the chemiluminescence reagent
(ECL), were from Amersham Corp. Poly(dI-dC):poly(dI-dC) was obtained
from Pharmacia (Uppsala, Sweden).
Antisera
Polyclonal anti-IL-3/GM-CSF receptor
common subunit antiserum was obtained as described
previously(20, 30) . Polyclonal anti-c-fps/fes antibody was prepared from serum of a rabbit immunized against a
synthetic peptides, which was conjugated to keyhole limpet hemocyanin
by gluteraldehyde. The peptide sequences for c-fps/fes was
LLLQDDRHSTSSSEQEREGG (corresponding to amino acid residues
424-443)(26) . The specificity of the antisera was
demonstrated by the lack of the immunoprecipitation band in the
presence of saturating concentrations of the related peptides (data not
shown). Polyclonal antibodies to p91 were generated in rabbits that
were injected with a synthetic peptide whose sequence corresponds to
residues 84-96 of human transcription factor ISGR-3
(GenBank accession number M97935): RKSKRNLQDNFQEDC.
Peptide conjugation to a carrier protein, injection to rabbits and
bleeding were as described previously(36) .
Anti-phosphotyrosine antibody 4G10, anti-JAK1, and anti-JAK2 antisera
were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-STAT3
p92 monoclonal antibodies were obtained from Affinity Research Products
Ltd. (Nottingham, United Kingdom) and from Santa Cruz Biotechnology,
Inc. (Heidelberg, Germany).
Cells
Human neutrophils (PMN) were isolated from
venous blood of normal donors by gelatin sedimentation (2.5% gelatin in
PBS, pH 7.2, for 30 min at 37 °C) followed by Ficoll-Hypaque
gradient separation. Contaminating erythrocytes were removed by
hypotonic lysis, and the cells were resuspended RPMI 1640 medium at a
final concentration of 1 
10
cells/ml. The
percentage of neutrophils in cell preparation used in this study was
97% and cell viability as determined by trypan blue exclusion was 98%. Western Blot Analysis and Immunoprecipitation
Studies
Cells (4 10) were incubated with or
without GM-CSF (10 ng/ml) at 37 °C for 5 min, extracted with cold
DIM buffer (50 mmol/liter Pipes, pH 6.8, 100 mmol/liter NaCl, 5 mM MgCl
, 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 supernatant was precleaned for 1 h with
50 µl of Sepharose protein A (3 mg/sample). The precleared cell
lysates 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. Eluted proteins were subjected to 8%
SDS-polyacrylamide gel electrophoresis. Proteins were then transferred
electrophoretically to nitrocellulose; the filters were incubated with
blocking solution (10% low fat milk 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 B-mercaptoethanol.In Vitro Kinase Assay
Anti-JAK2 and
anti-p93 immunoprecipitates were washed and divided into
two equal parts. One part was resuspended in an equal volume of kinase
assay buffer (50 mmol/liter NaCl, 5 mmol/liter MgCl
, 5
mmol/liter MnCl, 0.1 mmol/liter
Na
VO
, 10 mmol/liter Hepes (pH 7.4) containing
[-
P]ATP (0.25 µCi/ml) for 30 min at
room temperature, washed, eluted with sample buffer for
SDS-polyacrylamide gel electrophoresis, separated on 8% gel, and
visualized by autoradiography; the other part was separated on 8% gel
and probed with anti-JAK2 or anti-p93
antisera.
Preparation of Nuclear Extract and Gel Retardation
Assay
Nuclear extracts from untreated and GM-CSF-treated PMN and
untreated and IL-6-treated HEPG2 cells were prepared by Nonidet P40
lysis as described by Sadowski and Gilman(37) . The
oligonucleotides used were G GGG CAT TTC CCG TAA ATC and G GGG GAT TTA
CGG GAA ATG(38) . The annealed oligonucleotide was labeled by
filling in the overhanging ends with Klenow fragment in the presence of
[-P]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 NaMo0
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
incubated 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.
subunit(29, 30) . Moreover JAK2 has been shown
to be phosphorylated upon growth factor stimulation not only in
proliferating cells but also in PMN and eosinophils functionally
activated by GM-CSF (30) and IL-5(38) , respectively.
Moreover, a ligand-dependent association of JAK2 with erythropoietin
receptor has also been reported(35) . To further elucidate the
interaction between the subunit and JAK2 we performed
co-immunoprecipitation experiments in unstimulated and
GM-CSF-stimulated PMN. The results, reported in Fig. 1,
demonstrate that in PMN p130 JAK2 physically associates with the
subunit only upon GM-CSF stimulation, suggesting that, under
physiological conditions, the association between JAK2 and the receptor
may not be constitutive. Kinetic analysis of JAK2 activation, upon
GM-CSF stimulation, reported in Fig. 2, demonstrates a transient
JAK2 tyrosine phosphorylation peaking at 5 min and disappearing after
10 min.
common subunit in PMN. Cell lysates from
unstimulated(-) or GM-CSF-stimulated (10 ng/ml for 5 min)
(+) PMN were immunoprecipitated with anti- antiserum. The
cells were also immunoprecipitated with anti-JAK2 antiserum to indicate
the p130 JAK2 protein. The filter was probed with the anti-JAK2
antiserum (upper panel) and reprobed with the anti-
antiserum (lower panel). The p130 JAK2 is indicated. IP, immunoprecipitated; IB,
immunoblotted.
common subunit upon GM-CSF
stimulation in a growth factor-dependent cell line(26) .
Therefore, we tried to assess whether p93 was also
included in JAK2 co-precipitating p90 phosphoprotein(s). To test these
possibilities we first evaluated tyrosine phosphorylation of p91, p92,
and p93
upon GM-CSF stimulation. As shown in Fig. 3, both STAT proteins, p91 and p92, and p93
become phosphorylated after 5 min of GM-CSF treatment. In
addition, in the anti-p91 and -p92 immunoprecipitates together with the
marked band of approximately 90 kDa a faint band of approximately 130
kDa was detected. Taken together these results strongly suggest that at
least the two STAT proteins physically associated with JAK2. To confirm
this hypothesis, anti-JAK2 co-immunoprecipitates from unstimulated and
GM-CSF-stimulated PMN were divided into four aliquots, resolved by
SDS-polyacrylamide gel electrophoresis, and independently blotted with
the antibodies of interest. In the anti-phosphotyrosine immunoblot
reported in Fig. 4A a large band of approximately 90 kDa can
be detected only in GM-CSF-stimulated PMN. The anti-p91, anti-p92, and
anti-p93
immunoblots of anti-JAK2 immunoprecipitates from
untreated and GM-CSF-treated PMN are shown in Fig. 4, B, C, and D, respectively, demonstrating
that the two STAT proteins together with the p93
are
physically associated with JAK2 only upon GM-CSF stimulation. The
specificity of these results was further confirmed by the inability of
anti-JAK1 antiserum to co-immunoprecipitate these proteins (data not
shown). Moreover the correlation between the biochemical events induced
by GM-CSF stimulation and its biological effects on PMN was supported
by the observation that, upon IL-3 stimulation, neither functional
activation (data not shown) or protein tyrosine phosphorylation were
detected in anti-JAK2 immunoprecipitates (Fig. 5). The
observation that JAK2 physically associates with the
common
subunit as well as with p91 and p92 STATs and p93 implies
that the latter three proteins are also directly or indirectly, via
JAK2, associated with the
common. To evaluate this hypothesis
anti-beta co-immunoprecipitation experiments were performed. The
anti-phosphotyrosine immunoblot of anti- common immunoprecipitates
from unstimulated and GM-CSF-stimulated PMN, shown in Fig. 6A, demonstrates the presence of approximately 90-kDa
tyrosine-phosphorylated protein(s) only in GM-CSF-stimulated cells.
Moreover when aliquots of the same samples were resolved by
SDS-polyacrylamide gel electrophoresis and independently blotted with
the anti-p91 (Fig. 6B), anti-p92 (Fig. 6C),
and anti-p93 (Fig. 6D) antibodies, the
two STAT proteins together with the p93
were found to be
physically associated, upon ligand binding, with the
common
subunit.
in PMN.
Untreated(-) and GM-CSF-treated (for 5 min with 10 ng/ml of
GM-CSF) (+) PMN were immunoprecipitated with anti-p91 (A)
anti-p93
(B), and anti-p92 (C)
antibodies. The filters were probed with 4G10 anti-phosphotyrosine
monoclonal antibody (upper panels) and reprobed with anti-p91 (A), anti-p93
(B), and
anti-p91 (C) antibodies (lower panels). The position
of p91, p93
, and p92 are indicated. IP,
immunoprecipitated; IB, immunoblotted; P-tyr,
phosphotyrosine.
are physically associated with JAK2. Unstimulated(-)
and GM-CSF-stimulated (+) PMN were lysed and immunoprecipitated
with anti-JAK2 antiserum. The immunoprecipitates were divided into four
aliquots that were resolved in SDS-polyacrylamide gel and blotted
independently. A, the filter was probed with 4G10
anti-phosphotyrosine monoclonal antibody (upper panel) and
reprobed with anti-JAK2 antiserum (lower panel). B,
the filter was probed with anti-p91 antiserum. The cells were also
immunoprecipitated with anti-p91 antiserum to indicate the migration of
the p91 protein. C, the filter was probed with anti-p92
antibody. The cells were also immunoprecipitated with anti-p92
antiserum to indicate the migration of the p92 protein. D, the
filter was probed with anti-p93
antiserum. The
cells were also immunoprecipitated with anti-p93
antiserum to indicate the migration of the p93
protein. The position of p91, p92, and p93
are indicated. IP, immunoprecipitated; IB,
immunoblotted; P-tyr,
phosphotyrosine.
physically associate with the GM-CSF receptor
common
subunit. Unstimulated(-) and GM-CSF-stimulated (+) PMN were
lysed and immunoprecipitated with anti-GM-CSF receptor common
subunit antiserum. The immunoprecipitates were divided into four
aliquots that were resolved in SDS-polyacrylamide gel and blotted
independently. A, the filter was probed with 4G10
anti-phosphotyrosine monoclonal antibody (upper panel) and
reprobed with anti-GM-CSF receptor common subunit antiserum (lower panel). B, the filter was probed with anti-p91
antiserum. The cells were also immunoprecipitated with anti-p91
antiserum to indicate the migration of the p91 protein. C, the
filter was probed with anti-p92 antibody. The cells were also
immunoprecipitated with anti-p92 antiserum to indicate the migration of
the p92 protein. D, the filter was probed with
anti-p93 antiserum. The cells were also
immunoprecipitated with anti-p93
antiserum to
indicate the migration of the p93
protein. The
positions of p91, p92, and p93
are indicated. IP, immunoprecipitated; IB, immunoblotted; P-tyr, phosphotyrosine.
correlates with their intrinsic kinase activity. As shown in Fig. 7, anti-JAK2 (panel A) and anti-p93
(panel B) immunoprecipitates from GM-CSF-stimulated, but
not from IL-3-stimulated, PMN have a detectable in vitro kinase activity.
kinase activity following IL-3 and GM-CSF stimulation in
human PMN. Anti-JAK2 (panel A) and anti-p93
(panel B) immunoprecipitates from
unstimulated(-) and IL-3- and GM-CSF-stimulated (10 ng/ml each
for 5 min) (+) PMN were washed and divided in two aliquots. The
first one was resuspended in the kinase assay buffer containing
[
-
P]ATP for 30 min at room temperature,
washed and eluted with sample buffer for SDS-polyacrylamide gel
electrophoresis, separated on 8% gel, and detected by autoradiography (upper panels). The second one was separated on 8% gel and
probed with anti-JAK2 and anti-p93
antisera,
respectively (lower panels). The p130-JAK2 and the
p93
proteins are indicated. IP,
immunoprecipitated; IB,
immunoblotted.
and a
130-kDa protein, identified as JAK2, become phosphorylated upon GM-CSF
stimulation. Therefore, it is reasonable to assume that at least some
PMN functional activities may be regulated by the JAK/STAT signaling
pathway.
-(44) ,
IL-6-(50) , and GM-CSF-treated cells(51) , only one
complex can be detected containing either the STAT1 (45) or the
STAT3 homodimers(51) . The rapid tyrosine phosphorylation of
p91 and p92 observed in GM-CSF-stimulated PMN led us to evaluate, by
gel retardation assay, the formation of DNA-protein complexes in
nuclear extract of untreated and treated cells. As shown in Fig. 8A, both in IL-6-treated HEPG2 cells and in
GM-CSF-treated PMN a DNA-protein complex appears. Moreover when the
same nuclear extracts were incubated with an excess of unlabeled
oligonucleotide, both the IL-6- and GM-CSF-induced complexes are
competed (Fig. 8A) demonstrating its sequence specificity.
Moreover it is also clear that the DNA-binding complex observed in
IL-6-stimulated HEPG2 cells shows a slower migration than that observed
in GM-CSF-stimulated PMN. It has been reported that in HEPG2 cells IL-6
induces only the formation of a major complex, defined also as SIF-A (40, 45) , corresponding to the complex containing p92
homodimers(50) . In contrast, EGF-activated proteins have been
shown to form three complexes, designated as SIF-A, SIF-B, and
SIF-C(50) , with the serum-inducible element of c-fos (in its mutated, hyperactive form)(40, 45) .
Therefore, it is possible that the faster migrating complex observed in
GM-CSF-stimulated PMN contains either STAT1
STAT3 heterodimers
and/or STAT1 homodimers. We thus tested the GM-CSF-induced complex for
reactivity with anti-STAT1 p91 and anti-STAT3 p92 antibodies. As shown
in Fig. 8B, when anti-p91 and anti-p92 antisera were
added to GM-CSF-treated nuclear extract, a new band, which was not
present in the binding reaction with preimmune serum, appeared in the
upper part of the gel, thus demonstrating the formation of a
supershifted species. A supershifted species appears also when nuclear
extract from IL-6-stimulated HEPG2 cells was preincubated with anti-p92
antiserum (Fig. 8B) The presence of the supershifted
complex observed both in anti-p91 and anti-p92-pretreated nuclear
extract suggests that, in PMN, GM-CSF can rapidly modulate gene
expression by the induction of a DNA-binding complex containing p91 and
p92 heterodimer.
become
phosphorylated upon GM-CSF stimulation and are co-immunoprecipitated by
anti-JAK2 and anti-
common subunit antibodies, and that GM-CSF
induces the formation of a DNA-protein complex containing both p91 and
p92.
)
We thank Prof. G. Borreani for carefully reading this
manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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  N. N. Bewry, R. R. Nair, M. F. Emmons, D. Boulware, J. Pinilla-Ibarz, and L. A. Hazlehurst Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance Mol. Cancer Ther., October 1, 2008; 7(10): 3169 - 3175. [Abstract] [Full Text] [PDF]
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 E. Voisset, S. Lopez, P. Dubreuil, and P. De Sepulveda The tyrosine kinase FES is an essential effector of KITD816V proliferation signal Blood, October 1, 2007; 110(7): 2593 - 2599. [Abstract] [Full Text] [PDF]
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 P. M.-C. Dang, C. Elbim, J.-C. Marie, M. Chiandotto, M.-A. Gougerot-Pocidalo, and J. El-Benna Anti-inflammatory effect of interleukin-10 on human neutrophil respiratory burst involves inhibition of GM-CSF-induced p47PHOX phosphorylation through a decrease in ERK1/2 activity FASEB J, July 1, 2006; 20(9): 1504 - 1506. [Abstract] [Full Text] [PDF]
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 C. F. Fortin, A. Larbi, O. Lesur, N. Douziech, and T. Fulop Jr. Impairment of SHP-1 down-regulation in the lipid rafts of human neutrophils under GM-CSF stimulation contributes to their age-related, altered functions J. Leukoc. Biol., May 1, 2006; 79(5): 1061 - 1072. [Abstract] [Full Text] [PDF]
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 P. Boutet, D. Boulanger, L. Gillet, A. Vanderplasschen, R. Closset, F. Bureau, and P. Lekeux Delayed Neutrophil Apoptosis in Bovine Subclinical Mastitis J Dairy Sci, December 1, 2004; 87(12): 4104 - 4114. [Abstract] [Full Text] [PDF]
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 T. J. Warby, S. M. Crowe, and A. Jaworowski Human Immunodeficiency Virus Type 1 Infection Inhibits Granulocyte-Macrophage Colony-Stimulating Factor-Induced Activation of STAT5A in Human Monocyte-Derived Macrophages J. Virol., December 1, 2003; 77(23): 12630 - 12638. [Abstract] [Full Text] [PDF]
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 T. M. C. Hornell, G. W. Beresford, A. Bushey, J. M. Boss, and E. D. Mellins Regulation of the Class II MHC Pathway in Primary Human Monocytes by Granulocyte-Macrophage Colony-Stimulating Factor J. Immunol., September 1, 2003; 171(5): 2374 - 2383. [Abstract] [Full Text] [PDF]
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S. Faderl, D. Harris, Q. Van, H. M. Kantarjian, M. Talpaz, and Z. Estrov Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces antiapoptotic and proapoptotic signals in acute myeloid leukemia Blood, July 15, 2003; 102(2): 630 - 637. [Abstract] [Full Text] [PDF]
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 J. Kim, Y. Ogata, and R. A. Feldman Fes Tyrosine Kinase Promotes Survival and Terminal Granulocyte Differentiation of Factor-dependent Myeloid Progenitors (32D) and Activates Lineage-specific Transcription Factors J. Biol. Chem., April 18, 2003; 278(17): 14978 - 14984. [Abstract] [Full Text] [PDF]
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A. S. Cowburn, K. A. Cadwallader, B. J. Reed, N. Farahi, and E. R. Chilvers Role of PI3-kinase-dependent Bad phosphorylation and altered transcription in cytokine-mediated neutrophil survival Blood, September 18, 2002; 100(7): 2607 - 2616. [Abstract] [Full Text] [PDF]
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 R. A. Zirngibl, Y. Senis, and P. A. Greer Enhanced Endotoxin Sensitivity in Fps/Fes-Null Mice with Minimal Defects in Hematopoietic Homeostasis Mol. Cell. Biol., April 15, 2002; 22(8): 2472 - 2486. [Abstract] [Full Text] [PDF]
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H. Nagase, M. Miyamasu, M. Yamaguchi, M. Imanishi, N. H. Tsuno, K. Matsushima, K. Yamamoto, Y. Morita, and K. Hirai Cytokine-mediated regulation of CXCR4 expression in human neutrophils J. Leukoc. Biol., April 1, 2002; 71(4): 711 - 717. [Abstract] [Full Text] [PDF]
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J. Kim and R. A. Feldman Activated Fes Protein Tyrosine Kinase Induces Terminal Macrophage Differentiation of Myeloid Progenitors (U937 Cells) and Activation of the Transcription Factor PU.1 Mol. Cell. Biol., March 15, 2002; 22(6): 1903 - 1918. [Abstract] [Full Text] [PDF]
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E. Nijhuis, J.-W. J Lammers, L. Koenderman, and P. J. Coffer Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning J. Leukoc. Biol., January 1, 2002; 71(1): 115 - 124. [Abstract] [Full Text] [PDF]
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 R K Turlej, L Fievez, C F Sandersen, S Dogne, N Kirschvink, P Lekeux, and F Bureau Enhanced survival of lung granulocytes in an animal model of asthma: evidence for a role of GM-CSF activated STAT5 signalling pathway Thorax, September 1, 2001; 56(9): 696 - 702. [Abstract] [Full Text] [PDF]
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 S. Doublier, V. Ruotsalainen, G. Salvidio, E. Lupia, L. Biancone, P. G. Conaldi, P. Reponen, K. Tryggvason, and G. Camussi Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome Am. J. Pathol., May 1, 2001; 158(5): 1723 - 1731. [Abstract] [Full Text] [PDF]
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 S. Bhattacharya, B. A. Stout, M. E. Bates, P. J. Bertics, and J. S. Malter Granulocyte Macrophage Colony-Stimulating Factor and Interleukin-5 Activate STAT5 and Induce CIS1 mRNA in Human Peripheral Blood Eosinophils Am. J. Respir. Cell Mol. Biol., March 1, 2001; 24(3): 312 - 316. [Abstract] [Full Text] [PDF]
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 M. F. Brizzi, L. Formato, P. Dentelli, A. Rosso, M. Pavan, G. Garbarino, M. Pegoraro, G. Camussi, and L. Pegoraro Interleukin-3 Stimulates Migration and Proliferation of Vascular Smooth Muscle Cells : A Potential Role in Atherogenesis Circulation, January 30, 2001; 103(4): 549 - 554. [Abstract] [Full Text] [PDF]
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 J. A. Rogers, H. Y. Cheng, and T. E. Smithgall Src Homology 2 Domain Substitution Modulates the Kinase and Transforming Activities of the Fes Protein-Tyrosine Kinase Cell Growth Differ., November 1, 2000; 11(11): 581 - 592. [Abstract] [Full Text]
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J. Tu, N. Karasavvas, M. L. Heaney, J. C. Vera, and D. W. Golde Molecular characterization of a granulocyte macrophage-colony-stimulating factor receptor alpha subunit-associated protein, GRAP Blood, August 1, 2000; 96(3): 794 - 799. [Abstract] [Full Text] [PDF]
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E. Rollet-Labelle, C. Gilbert, and P. H. Naccache Modulation of Human Neutrophil Responses to CD32 Cross-Linking by Serine/Threonine Phosphatase Inhibitors: Cross-Talk Between Serine/Threonine and Tyrosine Phosphorylation J. Immunol., January 15, 2000; 164(2): 1020 - 1028. [Abstract] [Full Text] [PDF]
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Y. Senis, R. Zirngibl, J. McVeigh, A. Haman, T. Hoang, and P. A. Greer Targeted Disruption of the Murine fps/fes Proto-Oncogene Reveals that Fps/Fes Kinase Activity Is Dispensable for Hematopoiesis Mol. Cell. Biol., November 1, 1999; 19(11): 7436 - 7446. [Abstract] [Full Text] [PDF]
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M. A. Cassatella, S. Gasperini, C. Bovolenta, F. Calzetti, M. Vollebregt, P. Scapini, M. Marchi, R. Suzuki, A. Suzuki, and A. Yoshimura Interleukin-10 (IL-10) Selectively Enhances CIS3/SOCS3 mRNA Expression in Human Neutrophils: Evidence for an IL-10-Induced Pathway That Is Independent of STAT Protein Activation Blood, October 15, 1999; 94(8): 2880 - 2889. [Abstract] [Full Text] [PDF]
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P. Dentelli, L. D. Sorbo, A. Rosso, A. Molinar, G. Garbarino, G. Camussi, L. Pegoraro, and M. F. Brizzi Human IL-3 Stimulates Endothelial Cell Motility and Promotes In Vivo New Vessel Formation J. Immunol., August 15, 1999; 163(4): 2151 - 2159. [Abstract] [Full Text] [PDF]
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A. W. B. Craig, R. Zirngibl, and P. Greer Disruption of Coiled-coil Domains in Fer Protein-tyrosine Kinase Abolishes Trimerization but Not Kinase Activation J. Biol. Chem., July 9, 1999; 274(28): 19934 - 19942. [Abstract] [Full Text] [PDF]
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M. F. Brizzi, P. Dentelli, A. Rosso, Y. Yarden, and L. Pegoraro STAT Protein Recruitment and Activation in c-Kit Deletion Mutants J. Biol. Chem., June 11, 1999; 274(24): 16965 - 16972. [Abstract] [Full Text] [PDF]
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 M. F. Brizzi, E. Battaglia, G. Montrucchio, P. Dentelli, L. Del Sorbo, G. Garbarino, L. Pegoraro, and G. Camussi Thrombopoietin Stimulates Endothelial Cell Motility and Neoangiogenesis by a Platelet-Activating Factor–Dependent Mechanism Circ. Res., April 16, 1999; 84(7): 785 - 796. [Abstract] [Full Text] [PDF]
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A. Al-Shami and P. H. Naccache Granulocyte-Macrophage Colony-stimulating Factor-activated Signaling Pathways in Human Neutrophils. INVOLVEMENT OF Jak2 IN THE STIMULATION OF PHOSPHATIDYLINOSITOL 3-KINASE J. Biol. Chem., February 26, 1999; 274(9): 5333 - 5338. [Abstract] [Full Text] [PDF]
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J. M. Stephens, S. J. Lumpkin, and J. B. Fishman Activation of Signal Transducers and Activators of Transcription 1 and 3 by Leukemia Inhibitory Factor, Oncostatin-M, and Interferon-gamma in Adipocytes J. Biol. Chem., November 20, 1998; 273(47): 31408 - 31416. [Abstract] [Full Text] [PDF]
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B. J. Jenkins, T. J. Blake, and T. J. Gonda Saturation Mutagenesis of the beta Subunit of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor Shows Clustering of Constitutive Mutations, Activation of ERK MAP Kinase and STAT Pathways, and Differential beta Subunit Tyrosine Phosphorylation Blood, September 15, 1998; 92(6): 1989 - 2002. [Abstract] [Full Text] [PDF]
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 T. Adachi and R. Alam The mechanism of IL-5 signal transduction Am J Physiol Cell Physiol, September 1, 1998; 275(3): C623 - C633. [Abstract] [Full Text] [PDF]
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S. E. Doyle and J. C. Gasson Characterization of the Role of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor alpha Subunit in the Activation of JAK2 and STAT5 Blood, August 1, 1998; 92(3): 867 - 876. [Abstract] [Full Text] [PDF]
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F. Guidez, A. C. Li, A. Horvai, J. S. Welch, and C. K. Glass Differential Utilization of Ras Signaling Pathways by Macrophage Colony-Stimulating Factor (CSF) and Granulocyte-Macrophage CSF Receptors during Macrophage Differentiation Mol. Cell. Biol., July 1, 1998; 18(7): 3851 - 3861. [Abstract] [Full Text]
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J. Li and T. E. Smithgall Fibroblast Transformation by Fps/Fes Tyrosine Kinases Requires Ras, Rac, and Cdc42 and Induces Extracellular Signal-regulated and c-Jun N-terminal Kinase Activation J. Biol. Chem., May 29, 1998; 273(22): 13828 - 13834. [Abstract] [Full Text] [PDF]
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K. L. Nelson, J. A. Rogers, T. L. Bowman, R. Jove, and T. E. Smithgall Activation of STAT3 by the c-Fes Protein-tyrosine Kinase J. Biol. Chem., March 20, 1998; 273(12): 7072 - 7077. [Abstract] [Full Text] [PDF]
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 T. E. Smithgall Signal Transduction Pathways Regulating Hematopoietic Differentiation Pharmacol. Rev., March 1, 1998; 50(1): 1 - 20. [Abstract] [Full Text] [PDF]
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C. Bovolenta, S. Gasperini, P. P. McDonald, and M. A. Cassatella High Affinity Receptor for IgG (Fc{gamma}RI/CD64) Gene and STAT Protein Binding to the IFN-{gamma} Response Region (GRR) Are Regulated Differentially in Human Neutrophils and Monocytes by IL-10 J. Immunol., January 15, 1998; 160(2): 911 - 919. [Abstract] [Full Text] [PDF]
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A. Al-Shami, W. Mahanna, and P. H. Naccache Granulocyte-Macrophage Colony-stimulating Factor-activated Signaling Pathways in Human Neutrophils. SELECTIVE ACTIVATION OF Jak2, Stat3, AND Stat5B J. Biol. Chem., January 9, 1998; 273(2): 1058 - 1063. [Abstract] [Full Text] [PDF]
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T. Matsuguchi, Y. Zhao, M. B. Lilly, and A. S. Kraft The Cytoplasmic Domain of Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Receptor alpha Subunit Is Essential for Both GM-CSF-mediated Growth and Differentiation J. Biol. Chem., July 11, 1997; 272(28): 17450 - 17459. [Abstract] [Full Text] [PDF]
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H. Chin, H. Wakao, A. Miyajima, R. Kamiyama, N. Miyasaka, and O. Miura Erythropoietin Induces Tyrosine Phosphorylation of the Interleukin-3 Receptor beta Subunit (beta IL3 ) and Recruitment of Stat5 to Possible Stat5-Docking Sites in beta IL3 Blood, June 15, 1997; 89(12): 4327 - 4336. [Abstract] [Full Text] [PDF]
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R. Soldi, L. Primo, M. F. Brizzi, F. Sanavio, M. Aglietta, N. Polentarutti, L. Pegoraro, A. Mantovani, and F. Bussolino Activation of JAK2 in Human Vascular Endothelial Cells by Granulocyte-Macrophage Colony-Stimulating Factor Blood, February 1, 1997; 89(3): 863 - 872. [Abstract] [Full Text] [PDF]
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A. Al-Shami, S. G. Bourgoin, and P. H. Naccache Granulocyte-Macrophage Colony-Stimulating Factor-Activated Signaling Pathways in Human Neutrophils. I. Tyrosine Phosphorylation-Dependent Stimulation of Phosphatidylinositol 3-Kinase and Inhibition by Phorbol Esters Blood, February 1, 1997; 89(3): 1035 - 1044. [Abstract] [Full Text] [PDF]
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 K. Yates, G. Crooks, and J. Gasson Analysis of Fes kinase activity in myeloid cell growth and differentiation Stem Cells, November 1, 1996; 14(6): 714 - 724. [Abstract]
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