Granulocyte-macrophage colony-stimulating factor (GM-CSF) (
)is a cytokine that stimulates proliferation and
differentiation of various hematopoietic cells(1) . IL-3
exhibits biological activities similar to those of GM-CSF. Receptors of
IL-3 and GM-CSF consist of
and
subunits, both of which are
members of a cytokine receptor superfamily(2) . The
subunit is specific for each cytokine, and the
subunit is shared
by IL-3 and GM-CSF in addition to IL-5(3) . IL-3 and GM-CSF
induce early response genes and cell proliferation in both
hematopoietic cells and fibroblasts(4) . With IL-3 or GM-CSF
stimulation, phosphorylation of tyrosine residues of several
cytoplasmic proteins and the
subunit itself
occurs(5, 6, 7, 8) . Because the
cytokine receptor family, including IL-3R or GMR, does not contain a
kinase domain or kinase activity in the receptor itself, cellular
tyrosine kinase may be involved. Phosphorylation or activation of
tyrosine kinases such as src family tyrosine kinases and janus
kinase (JAK) 2 by IL-3 or GM-CSF stimulation has been
reported(9, 10, 11, 12, 13, 14) but
with no direct evidence of the involvement of these tyrosine kinases(s)
in IL-3 and GM-CSF activities, the exact roles of these kinases have
remained unknown.
The JAK family kinase consists of JAK1, JAK2,
JAK3, and Tyk2 in mammalian species(15) , but what role they
have in hematopoiesis remained to be determined. Interestingly, the
dominant mutation of Drosophila homolog, hop gene (hopscotch
) resulted in
hematopoietic defects(16) . Much attention has been directed to
JAK family kinases because their functions in interferon signals were
recognized(17) . Studies with interferon receptor signals
revealed that JAK family kinases are involved in interferon-specific
gene expression in cooperation with STAT
proteins(18, 19, 20) . Subsequent studies on
IL-6 and MGF signaling revealed that the JAK-STAT system plays a role
in cytokine-specific gene expression(21, 22) .
However, it is unclear whether or not JAK is involved in activities
shared by many cytokines, for example induction of cell proliferation
or activation of immediate response genes.
JAK2 is phosphorylated or
activated by many cytokines including IL-3 and
GM-CSF(9, 10, 23) , and association of JAK2
with the common
subunit of IL-3R and GMR was noted(24) .
We reported that two distinct signaling pathways function in hGMR
signaling, one for activation of c-myc promoter/proliferation
and the other for activation of c-fos/c-jun promoters(25, 26) . The membrane-proximal region
of hGMR
containing box1 and box2 motifs conserved among members of
the cytokine receptor family is essential for both signaling pathways,
and in addition, the membrane-distal region is required for activation
of c-fos/c-jun promoters. A role for tyrosine kinases
in these two signals has been considered, as deduced from studies using
kinase inhibitors(26) . Genistein and herbimycin almost
completely suppressed activation of the c-myc promoter and
cell proliferation, whereas herbimycin only partially suppressed
activation of c-fos/c-jun promoters and genistein did
not suppress the induction of c-jun mRNA; it even augmented
activation of the c-fos promoter. These findings suggest an
essential role for genistein/herbimycin sensitive kinase(s) in cell
proliferation and in c-myc mRNA induction. However, whether or
not tyrosine kinase is involved in activation of
c-fos/c-jun genes remained to be clarified. Because
the membrane-proximal region of hGMR
is required for
phosphorylation of JAK2(10, 27) , we asked whether or
not JAK2 is involved in activation of both signaling pathways. An
approach using dominant negative JAK2 indicated a role for JAK2 in
erythropoietin-induced proliferation and the partial requirement of
JAK2 in IL-3-induced cell proliferation(28) , but the role of
JAK2 in signaling pathway leading to activation of c-fos promoter is not known. In the present work, we attempted to
determine whether or not JAK2 is involved in hGMR signals using BA/F3
cells. We found that JAK2, which is activated through the box1 region
of hGMR
, plays essential roles in both signaling pathways.
MATERIALS AND METHODS
Chemicals, Media, and Cytokines
Fetal calf serum
was from Biocell laboratories Co. Ltd. RPMI 1640 and Dulbecco's
modified Eagle's medium were from Nikken BioMedical Laboratories
Co. Ltd. Recombinant hGM-CSF was kindly provided by Schering-Plough.
mIL-3 produced by silkworm, Bombyx mori, was purified as
described elsewhere(29) . Genistein was from Wako Pure Chemical
Industries, Ltd. G418 was a gift from Schering-Plough.
Plasmids and Genes
JAK2 cDNA (pBSK-JAK2) was
kindly provided by Dr. J. Ihle (St. Jude Children's Research
Hospital). Construction of the plasmid containing JAK2 under the
control of the SR
promoter was as follows. The coding region of
JAK2 was isolated from pBSK-JAK2 at NotI and SalI
sites. The insert, which was blunt-ended using the Klenow fragment and
attached to the NotI linker, was subcloned into pME18S at the NotI site. Dominant negative JAK2 (
JAK2) was constructed
as follows.
Jak2 that lacks the C terminus kinase domain was
isolated at NotI and AvrII(2724) sites, and the NotI site was blunt-ended by the Klenow fragment before AvrII digestion. The fragment was inserted at blunt-ended EcoRI site and intact SpeI site of pME18S.
hIL-2R
and hIL-2R
under the SR
promoter were kind gifts
from Dr. K. Sugamura (Tohoku University, Japan). Dominant negative ras
gene (MMTV promoter-N17 Ras; pMT64AA) was kindly provided by Dr. G. M.
Cooper (Harvard Medical School). To construct a plasmid containing N17
Ras under the control of the SR
promoter, the coding region of N17
Ras was isolated from pMT64AA at BamHI sites. The insert was
blunt-ended using the Klenow fragment and was subcloned into pME18S at
blunt-ended XhoI sites. Construction of hGMR
mutants
box1 (lacking amino acid positions 458-465) and
box2
(lacking amino acid positions 518-530) are described
elsewhere(50) .
Cell Lines and Culture Methods
A mIL-3-dependent
proB cell line, BA/F3 was maintained in RPMI 1640 medium containing 10%
fetal calf serum, 1 ng/ml mIL-3, 100 units/ml penicillin, and 100
µg/ml streptomycin. Various BA/F3 cell clones expressing hGMR
and hGMR
(BA/FGMR) or hGMR
mutants (BA/F
box1,
BA/F
box2, BA/F589, BA/F 544) were grown in the same type of medium
but supplemented with 500 µg/ml G418.
Immunoprecipitation, SDS-Polyacrylamide Gel
Electrophoresis, and Western Blotting
BA/F3 cells (1
10
cells/sample) were harvested, washed with
phosphate-buffered saline containing 1 mM sodium
orthovanadate, and lysed for 30 min in 500 µl of ice-cold lysis
buffer (0.5% Nonidet P-40 (for BA/F3), 20 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride). Nuclei and
cell debris were removed by centrifugation for 5 min at 4 °C in a
microfuge. Protein A-Sepharose (Pharmacia Biotech Inc.) or protein
G-Sepharose (Pharmacia), 30 µl of 50% slurry equilibrated in
phosphate-buffered saline, and antibodies were added to the supernatant
followed by rotation for 2 h at 4 °C. The protein A or G bound
immunoprecipitates were washed four times with lysis buffer and eluted
in 2
SDS loading buffer by boiling. Samples were separated on
7.5% SDS-polyacrylamide gels, transferred electrophoretically to
Immobilon(TM) polyvinylidene difluoride membrane (Millipore, MA).
Membranes were blocked with 4% bovine serum albumin (Fraction V) in
TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween
20) for 1 h, washed in TBST, incubated with the relevant primary
antibody for 1 h, and then washed in TBST. Protein antibody complexes
were detected and visualized with horseradish peroxidase-coupled
secondary antibodies (anti-rabbit or anti-mouse, as appropriate,
Amersham Corp.) using a chemiluminescence system (ECL(TM), Amersham
Corp.). To reprobe immunoblots, the filters were incubated in 62.5
mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS
at 50 °C for 30 min. Antibodies used for immunoprecipitation were
polyclonal rabbit antiserum against PTP 1D (sc-280, Santa Crutz, CA),
JAK2 (HR-758, c-20, Santa Crutz, CA), Shc (Upstate Biotechnology,
Inc.), monoclonal rat anti-hGMR
(5A5). Antibodies used for
immunoblotting were polyclonal rabbit antiserum against PTP 1D
(sc-280), JAK2 (Upstate Biotechnology, Inc.) and hGMR
(MBL,
Nagoya, Japan) and monoclonal mouse anti-phosphotyrosine (Tyr(P))
antibody 4G10 (Upstate Biotechnology, Inc.).
Transfection and CAT/Luciferase Assay in BA/F3
Cells
BA/FGMR cells were transfected either with 5 µg of
SRE-CAT reporter plasmid(30) , 15 µg of pmycPCAT or 3
µg of c-fos-luciferase, and 10 µg of control vector,
JAK2, or
JAK2 plasmids. c-fos-luciferase plasmid contains
a 0.4-kilobase pair c-fos promoter upstream of the initiation
site and luciferase coding region(4) . In some cases, receptor
plasmids, 2 µg each of hGMR
and hGMR
, were co-transfected
with these genes to BA/F3 cells(4) . Transfection was done by
electroporation as described elsewhere(25) . Briefly, cells
suspended in 0.2 ml of Opti MEM (3
10
cells) were
transferred to a cuvette (0.4-cm electrode gap, Bio-Rad) and mixed with
DNA. Cells were electroshocked using 960 microfarads at 200 V using a
Gene Pulser electroporation apparatus (Bio-Rad). After 30 min of
incubation at room temperature, cells were divided into three portions
and transferred to 6-well plates in mIL-3-depleted medium. After 10 h
of restimulation with 5 ng/ml of mIL-3, hGM-CSF, or hIL-2, cells were
harvested and lysed by three cycles of freezing and thawing in liquid
N
. Each sample containing approximately 20 µg of total
protein was subjected to luminescence assay or CAT assay. For
luciferase assay, the substrate was automatically injected into the
sample in the luminometer (model LE9501; Berthold Lumat Co. Ltd.), and
luminescence of 30 s was counted and expressed as arbitrary units. CAT
assay was done by diffusion analysis(31) . Protein
concentration was estimated using the BCA protein assay reagent
(Pierce) according to the manufacturer's instructions.
Replication Assay Using Plasmid Containing Polyoma (Py)
Replication Origin
Activity to induce DNA replication by mIL-3
or hGM-CSF in BA/F3 cells was analyzed by replication of the
transfected plasmid containing the Py replication origin, using an
assay involving DpnI analysis, as described
elsewhere(32) . Briefly, plasmids (1 µg of indicated
template, 10 µg of RSV-LTag, and 10 µg of control vector, JAK2,
or
JAK2) were introduced into semiconfluent BA/F3 cells by the
DEAE dextran method. After factor depletion for 5 h, cells were
stimulated with 5 ng/ml of either mIL-3 or hGM-CSF. After incubation
for an additional 24 h, cells were harvested, and low molecular weight
DNA was isolated by the Hirt extraction method(33) . Ten µl
of extracted DNA solution was digested with HindIII, which
linearizes template plasmid, and DpnI. DpnI digests
only the methylated or hemimethylated recognition site of DNA, and
newly synthesized DNA is resistant to DpnI digestion. Southern
blotting of digested DNA were done using with HindIII-digested
pPyOICAT DNA as a probe. Blots were washed and exposed to an imaging
plate for 15 min and visualized using a FUJI Image Analyzer (model
BAS-2000).
Incorporation of
[
H]Thymidine
Plasmids of hGMR
(5
µg), hGMR
(5 µg), and either vector or
JAK2 (15
µg) were transfected to BA/F3 cells (10
/sample) by
electroporation. Cells were cultured for 8 h and seeded into 96-well
plates (10
viable cells/well) with various concentrations
of hGM-CSF. After 48 h of culture, [
H]thymidine
(1 µCi/well) was added, and cells were harvested after 4 h of
incubation. [
H]Thymidine incorporation was
measured using a liquid scintillation spectrophotometer.
RESULTS
Phosphorylation of JAK2 by hGM-CSF Requires box1 Motif
but Not box2 Motif of hGMR
in BA/FGMR Cells
It had been
reported that the membrane-proximal region of hGMR
is required for
phosphorylation of JAK2(10, 27) . This region contains
both box1 and box2 motifs, which are conserved among several cytokine
receptors, including gp130 and erythropoietin
receptor(2, 34) . We first examined the requirement of
these motifs in activating JAK2 kinase using hGMR
mutants that
lacked either box1 or box2 motif (
box1 and 
box2),
as schematically shown in Fig. 1. BA/F3 cells expressing wild
type hGMR
and the wild type hGMR
, 
box1, or

box2 (BA/FGMR, BA/F
box1, BA/F
box2, respectively)
were depleted of mIL-3 for 5 h and restimulated with 5 ng/ml of either
mIL-3 or hGM-CSF. After 5 min of incubation, immunoprecipitation of
JAK2 was performed followed by Western blotting using monoclonal
anti-Tyr(P) antibody 4G10. JAK2 was phosphorylated in response to
either mIL-3 or hGM-CSF stimulation. Deletion of 8 amino acids at the
box1 region resulted in a complete loss of hGM-CSF-induced JAK2
phosphorylation (Fig. 2A). On the other hand, deletion
of the box2 region had no effect on JAK2 phosphorylation. As indicated
in the bottom panel of Fig. 2A, the box1 motif
is essential, whereas the box2 motif is not required for activation of
the c-fos promoter by hGM-CSF.
Figure 1:
Schematic drawing of various hGMR
mutants.
Figure 2:
IL-3/GM-CSF induced phosphorylation of
JAK2 in BA/FGMR cells. A, BA/F3 cells expressing wild type
hGMR
and wild type hGMR
, 
box1, or 
box2
were depleted mIL-3 for 5 h and restimulated with either mIL-3 (5
ng/ml) or hGM-CSF (5 ng/ml) for 5 min. Cells were harvested and
immunoprecipitated with anti-JAK2 antibody. The immunoprecipitant was
electrophoresed and subjected to Western blot analysis with anti-Tyr(P)
(4G10) or anti-JAK2 antibodies. Bands were visualized using a
chemiluminescence system (ECL(TM)). For c-fos-luciferase
assay, 2 µg of c-fos-luciferase plasmid was transfected,
and luciferase activities induced by either mIL-3 or hGM-CSF were
analyzed as described under ``Materials and Methods.'' The
values given at the bottom of the figure are the averages of
three samples. B, effect of tyrosine kinase inhibitor
genistein on the hGM-CSF-induced phosphorylation of JAK2. Factor
depleted BA/FGMR cells were treated with the indicated concentrations
of genistein for 30 min and then stimulated with 5 ng/ml either mIL-3
(data not shown) or hGM-CSF for 5 min. JAK2 protein were
immunoprecipitated and analyzed by Western blotting using anti-Tyr(P)
or anti-JAK2 antibodies.
Tyrosine Kinase Inhibitor Genistein Did Not Inhibit
hGM-CSF-induced Phosphorylation of JAK2
In previous work, we
noted that genistein completely suppressed cell proliferation or
activation of c-myc gene in response to IL-3/GM-CSF. In
contrast, activation of the c-fos gene was not suppressed;
rather it was augmented by genistein(26) . We next examined the
effect of genistein on the phosphorylation of JAK2. After depletion of
mIL-3 for 5 h, BA/FGMR cells were treated with various concentrations
of genistein for 30 min before GM-CSF stimulation. The cells were then
stimulated with hGM-CSF (5 ng/ml) and harvested after 5 min of
incubation. As shown in Fig. 2B, hGM-CSF induced
phosphorylation of JAK2 even in the presence of 10 µg/ml of
genistein, a concentration that completely suppressed hGM-CSF-induced
proliferation(26) . At 20 µg/ml genistein, the level of
phosphorylation decreased but the level of protein was also reduced,
suggesting that genistein inhibited protein synthesis rather than
tyrosine phosphorylation. Essentially the same results were obtained in
response to mIL-3 stimulation (data not shown). Lack of any appreciable
inhibition of JAK2 phosphorylation by genistein is consistent with the
notion that JAK2 is involved in hGM-CSF-induced activation of the
c-fos promoter, which is not inhibited by genistein.
Dominant Negative JAK2 (
JAK2) Suppressed c-myc Gene
Activation and DNA Replication Induced by mIL-3/hGM-CSF
To
examine the involvement of JAK2 in mIL-3 and hGM-CSF signals, we
constructed mutant JAK2 lacking kinase activity. Kinase domain at the C
terminus (nucleotide position 2714) was deleted from JAK2 (
JAK2)
based on an earlier report(28) . Similar to the construct
described by Wojchowsky and co-workers(28) , our
JAK2
construct inhibits autophosphorylation of wild type JAK2, in a dominant
negative manner in the COS7 cells. We also examined the effects of
JAK2 on activation and autophosphorylation of JAK1 or JAK3 in COS7
cells and found that they were not inhibited by the co-expression of
JAK2 (data not shown). We next examined the effect of
JAK2 on
hGM-CSF-induced JAK2 activation in BA/F cells by transient transfection
assay. Plasmids carrying hGMR
and
subunits were
co-transfected with or without
JAK2. After stimulation of BA/F3
cells by hGM-CSF for 5 min, immunoprecipitation of JAK2 followed by
Western blotting was performed. As shown in Fig. 3, JAK2 was
phosphorylated through transiently expressed hGMR in BA/F3 cells, and
this phosphorylation was suppressed by the co-expression of
JAK2. Fig. 3(lower panel) shows the pattern of anti-JAK2
antibody blotting, and bands indicated by the upper arrow have
a molecular weight corresponding to the wild type. The bands indicated
by the lower arrow have a molecular weight that corresponding
to the
JAK2. We next examined the effect of
JAK2 on mIL-3 or
hGM-CSF activities in BA/FGMR cells. We previously reported that DNA
replication and c-myc activation are mediated through the
membrane-proximal region of hGMR
. A kinase-negative JAK2 mutant
used by Wojchowsky and co-workers (28) partially inhibited the
mIL-3-induced proliferation of DAER cells. We first examined the effect
of
JAK2 on c-myc gene activation using c-myc reporter plasmid (pmyPCAT)(25) , which contains the
2.6-kilobase pair fragment of the c-myc promoter fused to the
CAT coding region. In this construct, the mycI site (E2F recognition
site) was seen to be a major site responding to mIL-3 or hGM-CSF
signals(25) . Fig. 4A shows that co-expression
of
JAK2 completely suppressed mIL-3- or the hGM-CSF-induced
c-myc-CAT activity. We previously described T
antigen-dependent replication of Py origin in BA/FGMR cells as a model
system for initiation of DNA replication(32) . As shown in Fig. 4B,
JAK2 suppressed Py origin-dependent DNA
replication induced by mIL-3 or hGM-CSF in BA/F3GMR. We also examined
effect of
JAK2 on thymidine incorporation promoted by hGM-CSF via
transiently expressed hGMR in BA/F3 cells. We found that co-expression
of
JAK2 suppressed hGM-CSF-induced
[
H]thymidine incorporation almost completely even
in the presence of excess amounts of hGM-CSF (Fig. 4C).
Figure 3:
Effect of dominant negative JAK2
(
JAK2) on hGM-CSF-induced JAK2 phosphorylation in BA/F3 cells
transiently expressing hGMR. hGMR
and
subunit plasmids were
transfected to BA/F3 cells with either vector control or
JAK2
plasmids. Cells were cultured overnight with mIL-3 containing medium
and depleted for 5 h. Immunoprecipitation was done with anti-JAK2
antibody after stimulation by hGM-CSF for 5 min. The upper panel shows blotting pattern with anti-Tyr(P) (4G10), and the lower
panel shows blotting pattern with anti-JAK2 (Upstate
Biotechnology, Inc.). The blot was visualized using a chemiluminescent
system as described under ``Materials and
Methods.''
Figure 4:
Characterization of
JAK2 in BA/FGMR
cells. A, effects of
JAK2 on hGM-CSF-induced c-myc activation in BA/FGMR cells were analyzed by transient assay of
c-myc promoter CAT. pmycPCAT plasmid was transfected with
either control vector or
JAK2, and CAT activities induced by mIL-3 (hatched bars) or hGM-CSF (shaded bars) were analyzed
by diffusion assay as described under ``Materials and
Methods.'' B, mIL-3- or hGM-CSF-induced Py
origin-dependent DNA replication in the presence or the absence of
JAK2 in BA/FGMR cells were analyzed as described under
``Materials and Methods.'' The arrows indicate DpnI-resistant replicated plasmid and DpnI-sensitive
unreplicated transfected plasmids. C,
[
H]thymidine incorporation into BA/F3 cell
transiently expressing hGMR was analyzed in the presence or the absence
of
JAK2 as described under ``Materials and Methods.''
The values are the average of duplicate samples. The standard deviation
is shown as error bars in the
figure.
JAK2 Completely Suppressed Activation of the c-fos
Gene Induced by mIL-3/hGM-CSF but Not by hIL-2
We next
determined the effect of
JAK2 on activation of the c-fos gene, which is mediated through the box1 region and the more
membrane-distal region of hGMR
. c-fos-luciferase,
hGMR
, and hGMR
plasmids were co-transfected with control
vector, JAK2, or
JAK2 into BA/F3 cells, and luciferase activities
induced by mIL-3 or hGM-CSF were analyzed. As shown in Fig. 5A, hGM-CSF stimulated c-fos-luciferase
activity in BA/F3 cells as noted previously(4) , and
co-expression of
JAK2 completely abolished the hGM-CSF-induced
c-fos activation. Essentially the same results were obtained
with mIL-3 stimulation (data not shown). IL-2 activated JAK1 and JAK3
but not JAK2 (35, 36) and, in response to IL-2,
IL-2R
and IL-2R
transduced signals to activate the c-fos promoter even in the absence of IL-2R
(37) . To
determine whether or not the observed effect of
JAK2 is specific
to IL-3/GM-CSF signals, we examined the effect of
JAK2 on
hIL-2-induced c-fos-luciferase activity. hIL-2R
and
hIL-2R
were transiently expressed in BA/F3 cells together with the
c-fos-luciferase plasmid in the presence or the absence of
JAK2. In contrast to a complete suppression of hGM-CSF-induced
c-fos-luciferase activity, the same activity induced by hIL-2
was only partially inhibited by
JAK2 (Fig. 5A). It
should be noted, however, that wild type JAK2 co-transfected with
hIL-2R
and hIL-2R
also partially suppressed the
c-fos-luciferase activity induced by hIL-2 (data not shown).
On the other hand, dominant negative Ras (N17-Ras) almost completely
suppressed both hIL-2-induced and hGM-CSF-induced
c-fos-luciferase activity. Taken together, these results
suggest that suppression of c-fos-luciferase activity by
JAK2 is specific to the mIL-3- or hGM-CSF-dependent pathway.
Figure 5:
Effects of
JAK2 on hGM-CSF-induced
c-fos activation signaling pathway in BA/FGMR cells. A, effect of
JAK2 on c-fos-luciferase activity
induced by hGM-CSF or hIL-2. hGMR
and hGMR
or hIL-2R
and
hIL-2R
plasmids were co-transfected with control vector,
JAK2, or dominant negative Ras(N17) and c-fos-luciferase
activities induced by either hGM-CSF (hatched bars) or IL-2 (shaded bars) were analyzed. B, plasmid containing
three tandem SRE sites fused to the CAT coding region were transfected
with or without
JAK2, and mIL-3- (hatched bars) or
hGM-CSF-induced (shaded bars) CAT activities were analyzed by
diffusion assay as described in the legend to Fig. 4. C, effect of
JAK2 on transiently expressed hGMR-dependent
Shc and PTP 1D phosphorylation. Plasmids encoding hGMR
and
hGMR
(10 µg each) were transfected to BA/F3 cells (2
10
cells/sample) with either 20 µg of control vector or
JAK2and cultured overnight with medium containing 1 ng/ml of
mIL-3. After 5 h of mIL-3 depletion, cells were restimulated with 5
ng/ml of hGM-CSF and harvested after 5 min of incubation.
Immunoprecipitation with either anti-Shc or PTP 1D was done and
followed by Western blotting.
The c-fos promoter contains SRE and SIE sites, and the
latter is known to carry the GAS sequence(38) . Deletion
analysis of c-fos promoter suggested that both SRE and SIE
sites are essential for c-fos activation by IL-3/GM-CSF in
BA/FGMR cells (data not shown). It should be noted that the SRE site
also plays an important role for activation of egr1 by GM-CSF. (
)To examine whether or not
JAK2 exerts its effect
through the SRE site, we next carried out similar experiments using
SRE-CAT(30) . As shown in Fig. 5B,
JAK2
inhibited CAT activity of SRE induced by mIL-3/hGM-CSF.
JAK2 Suppressed Shc and PTP 1D Activities Induced by
mIL-3/hGM-CSF Stimulation
To analyze the role of Jak in
activation of SRE or c-fos, we next examined the effect of
JAK2 on signal transducing molecules known to be involved in
activation of the c-fos promoter. SH2 containing tyrosine
phosphatase PTP 1D (also called Syp, SH-PTP2, and PTP2C) (39) and adaptor molecule Shc (40) are well documented
constituents of the Grb2-Sos cascade(41) , and they are
phosphorylated upon mIL-3 or hGM-CSF stimulation(42) . To
determine if JAK2 is involved in the phosphorylation of Shc or PTP 1D
induced by hGM-CSF stimulation, hGMR
and hGMR
were
transiently transfected into BA/F3 cells, and immunoprecipitation was
done with either anti-Shc or PTP 1D antibodies. As shown in Fig. 5C, phosphorylation of Shc or PTP 1D through
transiently expressed hGMR was evident but was abolished with the
co-expression of
JAK2. It appears that
JAK2 interferes with
signaling event(s) upstream of Shc or PTP 1D activation, thereby
indicating that JAK2 plays an essential role in activation of both
signaling molecules.
Phosphorylation of hGMR
Induced by hGM-CSF Is
Abolished by
JAK2
The hGMR
is tyrosine-phosphorylated
in response to hGM-CSF stimulation, yet the nature of the tyrosine
kinase involved is unknown. To determine whether or not JAK2
participates in hGMR
phosphorylation, we examined the effect of
JAK2. hGMR
with control vector or with
JAK2 were
transiently transfected into BA/FGM
cells and cultured overnight.
After depletion of mIL-3 for 5 h, cells were stimulated with hGM-CSF (5
ng/ml) for 5 min, and immunoprecipitation was done with anti-hGMR
antibody (Fig. 6). Transiently reconstituted hGMR was
phosphorylated by hGM-CSF stimulation, and this phosphorylation was not
observed when
JAK2 was present. These results indicated that JAK2
is involved in ligand-induced phosphorylation of hGMR
.
Figure 6:
Phosphorylation of hGMR
in the
absence or the presence of
JAK2. Plasmids encoding hGMR
(10
µg) were transfected to BA/FGMR
cells (2
10
cells/sample) with 20 µg of either control vector or
JAK2 and cultured overnight. After 5 h of mIL-3 depletion, cells
were restimulated with 5 ng/ml of hGM-CSF and harvested after 5 min of
incubation. Immunoprecipitation with anti-hGMR
was done and
followed by Western blotting.
DISCUSSION
JAK2 Is Apparently the Primary Kinase Regulating
IL-3/GM-CSF Signals
We obtained evidence that in BA/FGMR cells,
JAK2 is involved in activities controlled by hGM-CSF, including cell
proliferation and activation of c-myc and c-fos promoters. We previously described two signaling pathways of hGMR,
one for activation of c-fos/c-jun genes and the other
for activation of c-myc gene/cell proliferation. We also
reported that these two signaling pathways differ in sensitivity to
tyrosine kinase inhibitor genistein, where c-fos gene
activation is hardly inhibited by genistein(25) . Other
investigators reported that gp130-dependent JAK2 activation is
suppressed by genistein(43) . If this is also the case for
IL-3/GM-CSF systems, JAK2 is unlikely to be involved in
IL-3/GM-CSF-dependent activation of c-fos promoter. However,
as noted in the present work, JAK2 activation in BA/F3 cells is
insensitive to genistein, an observation that raises the possibility
that JAK2 has a role in c-fos promoter activation. This view
was more directly supported by experiments using dominant negative
JAK2.
JAK2 inhibited the activation of c-fos and
c-myc promoters as well as cell proliferation. Complete
inhibition of cell proliferation by genistein suggests that in addition
to JAK2, other kinase(s) sensitive to genistein probably play(s) an
essential role in the pathway downstream of JAK2. In fact,
proliferation induced by mitogenic factors such as EGF and fetal calf
serum were also inhibited by genistein(25) . These results
suggest that tyrosine kinase, which is sensitive to genistein, has role
in the common pathway responding to various mitogenic factors.
Activation of JAK2 Depends on the hGMR
box1 Region
Essential for All Known GM-CSF Activities
box1 and box2 motifs
in hGMR
are conserved among cytokine receptors(34) . We
showed that the region including box1 is essential for
hGM-CSF-dependent activation of JAK2 and the c-fos promoter,
whereas box2 is not required for these activities. Similar analyses
using the same constructs revealed that these regions affect DNA
replication and egr1 promoter activation in a manner similar
to that related to JAK2 activation.
The level of
hGM-CSF-induced proliferation is significantly reduced by the removal
of the box2 region of hGMR
, thereby implying an enhancing role for
box2 for cell proliferation. A close correlation between the activation
of JAK2, cell proliferation, and c-fos and c-myc promoters, all of which depend on the box1 region, is consistent
with the notion that JAK2 is the primary kinase regulating GM-CSF
signals. It should be noted that in COS7 cells, overexpression of JAK2
resulted in phosphorylation of hGMR
in a ligand-independent and
box1-independent manner (data not shown). It is tempting to speculate
that box1 facilitates establishment of an effective link between
extracellular signals and intracellular events resulting in
ligand-dependent activation of JAK2 in BA/F3 cells. Other receptors
such as gp130 and erythropoietin receptor also require the box1
motif(44, 45, 46) .
JAK2 Induces Phosphorylation of hGMR
and Activates a
Signaling Pathway Leading to Activation of c-fos Promoter
Our
observation that the SRE sequence, which lacks the putative STAT
recognition site, is suppressed by
JAK2 is in keeping with the
notion that JAK2 regulates the STAT-independent pathway involving
c-fos promoter activation. Deletion analysis suggests that the
SRE site is essential and the SIE site has an enhancing activity for
IL-3/GM-CSF-induced c-fos promoter activation (data not
shown). IL-3 or GM-CSF activated STAT5 and
STAT6(47, 48, 49) , and dominant negative
STAT5 partially suppressed endogenous c-fos gene activation in
response to mIL-3 in BA/F3 cels. (
)It is possible that the
SIE site containing the GAS sequence is the target of STAT activation.
Taken together, it is tempting to speculate that c-fos activation is regulated by STAT-dependent and -independent
mechanisms via SIE and SRE sites, respectively, and that JAK2 plays an
essential role in activation of both pathways.Interestingly,
JAK2 suppresses hGM-CSF-dependent phosphorylation of hGMR
in
BA/F3 cells. To explain the primary role of JAK2 in hGM-CSF-dependent
activation of c-fos promoter, we considered that JAK2
phosphorylates hGMR
itself. We further speculate that Shc or PTP1D
interacts with phosphorylated tyrosine residue(s) of hGMR
and
transduces signals downstream. Our finding that
JAK2 inhibited
activation of PTP1D and Shc supports this view.
In contrast to
activation of the c-fos promoter, the hGMR
region
containing phosphorylatable tyrosine residues is not required for
activation of the c-myc promoter and cell proliferation.
Dominant negative STAT5 did not affect c-myc activation by
mIL-3 in BA/F3 cells.
This means that even if box1 and JAK2
are essential for activation of c-myc promoter/cell
proliferation, the mechanism of JAK2 action differs from that related
to activation of the c-fos promoter. Further work is under way
to clarify the role of JAK2 in activation of cell proliferation and
c-myc promoter.