Constitutive Activation of JAK2 Confers Murine
Interleukin-3-independent Survival and Proliferation of BA/F3
Cells*
Chang-Bai
Liu
,
Tohru
Itoh
§,
Ken-ichi
Arai
¶, and
Sumiko
Watanabe
From the
Department of Molecular and Developmental
Biology, Institute of Medical Science, University of Tokyo and
¶ CREST, Japan Science and Technology Corp. (JST), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
 |
ABSTRACT |
The Janus tyrosine kinase 2 (JAK2) plays an
essential role of cytokine receptor signaling, including that of the
human granulocyte-macrophage colony-stimulating factor (GM-CSF)
receptor. We reported earlier that the activation of JAK2 is essential
for all the examined signals induced by human GM-CSF through the box1
region of
c, such as promotion of cell survival and proliferation.
To elucidate the role of JAK2 in cell survival and proliferation, we
generated an artificial activation system by constructing a chimeric
molecule (
/JAK2) consisting of
c extracellular and transmembrane
regions fused with JAK2, and we analyzed various signaling events in
interleukin-3-dependent mouse pro-B cell, BA/F3. The
/JAK2
was constitutively phosphorylated in the absence of
human GM-CSF and murine interleukin-3, and this led
to proliferation and cell survival. Western blot analysis showed that
STAT5, Shc, and SHP-2 were not phosphorylated in the cells, and the
consistent activation of
-casein and c-fos
promoters was not enhanced. In contrast, c-myc
transcription was constitutively activated. We propose that the
activation of
/JAK2 suffices for survival and proliferation and that
the activation of STAT5 and mitogen-activated protein kinase cascade is
not required for these activities in BA/F3 cells.
 |
INTRODUCTION |
The Janus tyrosine kinases (JAK) family has four known members,
JAK1, 2, 3 and Tyk2, that play an essential role in the signal transduction of cytokine receptors and interferon receptors (1). The
importance of their roles was first recognized in studies using mutant
cell lines defective in interferon signaling (2, 3). Subsequent studies
showed that JAKs were activated in response to a wide variety of
cytokines including granulocyte-macrophage colony-stimulating factor
(GM-CSF)1 and interleukin-3
(IL-3) (2, 4-6). Targeted disruption of JAKs in mice revealed the
essential roles of JAKs in cytokine signals in vivo. JAK2
knockout mice are embryonic lethal because of the absence of definitive
erythropoiesis (7, 8). The myeloid progenitors from the fetal liver of
JAK2-deficient mice failed to respond to erythropoietin, IL-3 and
GM-CSF; hence, the essential role of JAK2 in these cytokine signals
became evident.
The GM-CSF receptor (GM-CSFR) consists of an
subunit and a common
(
c) subunit, shared by the receptors for GM-CSF, IL-3 and IL-5
(9). We reported that although the addition of GM-CSF induces
oligomerization of
and
subunits, the
subunit is present as
a homodimer even before the addition of GM-CSF (10). It is also
reported that GM-CSF induced dimerization of
c (11).
c does not
contain intrinsic kinase activity; rather, GM-CSF activates JAK2
through the box1 region (4), and dominant negative JAK2 blocked all the
examined activities of human (h) GM-CSF in BA/F3 cells (5). In studies
done using a series of
c mutants, multiple signaling pathways were
induced by GM-CSFR, and all required the box1 region of
c (12-14).
One pathway is the activation of the Ras/Raf/mitogen-activated protein
kinase (MAPK) cascade followed by c-fos/c-jun
transcriptional activation. This pathway requires a membrane distal
region of
c containing tyrosine residues in addition to the box1
region. Phosphorylation of tyrosine residues of
c by JAK2 in
response to GM-CSF stimulation and the following recruitment of
SH2-containing effectors such as Shc, SHP-2 through association of SH2
domain, and phosphorylated tyrosine residues of
c has been proposed
(14-16). In contrast, cell survival and low level proliferation can be
induced through a
c mutant having no tyrosine residue or only the
membrane proximal region containing box1 and box2 regions. These
activities are abrogated by deletion of the box1 region or
co-expression of dominant negative JAK2, suggesting that cell survival
and low level proliferation require JAK2 activation but no cytoplasmic
region of
c. Nevertheless, only limited knowledge is available to
explain the promotion of proliferation and survival induced by GM-CSF,
because the precise mechanisms through which JAK2 transduces signals to
downstream pathways remain to be elucidated. One approach to resolve
such issues is to differentiate the activation of JAK2 from receptor phosphorylation-dependent signals. We generated a chimeric
protein consisting of the
c extracellular and transmembrane domains
and JAK2 designated as
/JAK2 and analyzed signaling in BA/F3 cells. Our evidence shows that
/JAK2 is phosphorylated constitutively in
BA/F3 cells, and the cells survive and proliferate without activation
of STAT5 or the MAPK cascade.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
RPMI 1640 was purchased from Nikken
BioMedical Laboratories Co. Ltd. Fetal calf serum was purchased from
Biocell Laboratories Co. Ltd. Recombinant mIL-3 produced in silkworm,
Bombyx mori was purified as described (17). Recombinant
hGM-CSF and G418 were kindly provided by Schering-Plough. The
anti-hGM-CSF receptor
chain (S-16, N-20), anti-JAK2 (HR-758, C-20),
anti-STAT5a (L-20), STAT5b (C-17), and anti-SHP-2 (C-18) antibodies
were from Santa Cruz Biotechnology (Santa Cruz, CA).
Antiphosphotyrosine (4G10), anti-Shc, and anti-JAK2 antibodies were
purchased from Upstate Biotechnology, Inc. The antiphospho-Akt
(Ser-473) and anti-Akt antibodies were from New England BioLabs
(Beverly, MA). Phosphatidylinositol and phosphatidylserine were
purchased from Sigma. CIS cDNA and Bcl-xL cDNA used
as probes in the Northern blot analysis were gifts from Drs. A. Yoshimura (Kurume University, Japan) and Y. Tsujimoto (Osaka
University), respectively.
Plasmids Construction--
The JAK2 cDNA (pBSK-JAK2) was
kindly provided by Dr. J. Ihle (St. Jude Children's Research
Hospital). The
c cDNA was originally cloned into pME18S vector
containing SR
, as a promoter (18). The construction of pME-
/JAK2,
a chimera gene encoding
c extracellular and transmembrane regions
fused with the N terminus of the full-length JAK2 was done as follows.
Two fragments containing the coding region of JAK2, corresponding to
amino acid positions 4 to 455 and 456 to 1129, were isolated from
pBSK-JAK2 by digesting in HaeI/XhoI and
XhoI/NheI, respectively. The coding region of
c extracellular and transmembrane domains (corresponding to amino acid positions 1 to 455) was prepared by isolating the
XhoI/FspI fragment from pME18S-
c. The
XhoI/NheI fragment of JAK2 was inserted into the
expression vector pME18S using XhoI and SpeI sites, then the
XhoI/FspI fragment of
c and the
HaeI/XhoI fragment of JAK2 were inserted at the
XhoI site.
Cell Culture and Transfections--
COS7 cells were maintained
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. Transient
transfection of COS7 cells with plasmid was done using LipofectAMINETM
(Life Technologies, Inc.) according to the manufacturer's instruction.
A mIL-3-dependent pro-B cell line, BA/F3, was maintained in
RPMI 1640 medium containing 5% fetal calf serum, 0.25 ng/ml mIL-3, 50 units/ml penicillin, and 50 µg/ml streptomycin. To obtain stable
transfectants, BA/F3 cells or BAFGMR
, which stably express the
wild-type hGM-CSFR
subunit, were cotransfected with pME-
/JAK2
and the pKU-2Neo vector (containing a neomycin-resistant gene) by
electroporation, as described (18). After a 2-week selection using 1 mg/ml G418, drug-resistant clones were screened for surface expression
of
/JAK2 by FACS analysis (Becton Dickinson), and size of
/JAK2 was confirmed by Western blotting using an anti-JAK2 antibody.
DNA Fragmentation Assay by Agarose Gel--
Cells cultured with
or without mIL-3 and/or hGM-CSF for 12 h were lysed with TTE
buffer (0.5% Triton X-100, 5 mM Tris-HCl, pH7.4, 20 mM EDTA). Genomic DNA was extracted as described (19) and
was then suspended in 10 mM Tris, 1 mM EDTA, pH
8.0, containing 20 µg of DNase-free RNase. Equal amounts of nucleic
acids from each sample were separated through a 1.8% agarose gel then
the fragmented DNA was visualized after ethidium bromide staining.
Proliferation Assay--
Analysis of incorporation of
[3H]thymidine was done as described (5). Briefly, the
cells were seeded into a 96-well plate (1 × 104
cells/well) with various concentrations of mIL-3 or hGM-CSF as indicated, then the cells were cultured for 24 h, labeled with [3H]thymidine (1 µCi/well) for 4 h, then harvested
onto a glass fiber filter. Incorporation of [3H]thymidine
was measured using a filter counter (model 1450 MicroBetaTM, Wallac, Turku, Finland).
Immunoprecipitation and Western Blot
Analysis--
Immunoprecipitation and Western blotting were done as
described (5). Briefly, cells (1 × 107 cells/sample)
were lysed in 500 µl of lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride). Immunoprecipitation was
carried out with the indicated antibody and protein A-Sepharose beads
(Amersham Pharmacia Biotech). For Western blotting of total cell
lysates, 4 × 105 cell were lysed with 20 µl of
lysis buffer, mixed with a buffer containing 50 mM
Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 5 µg of bromphenol blue/ml, and boiled. Samples were separated through
10% SDS-polyacrylamide electrophoresis gels, and transferred to a
Immobilon polyvinylidene difluoride membrane (Millipore), and Western
blottings were performed using appropriate antibodies. The immuno
reactive bands were visualized by the enhanced chemiluminescence kit
(ECL, Amersham Pharmacia Biotech) according to the manufacturer's instruction.
Analysis of Phosphoinositide 3-Kinase (PI3-K) and Akt
Activities--
PI3-K activity associated with antiphosphotyrosine
antibody immunoprecipitates were assayed as follows. Cells (1.5 × 107/sample) were lysed and immunoprecipitated with
antiphosphotyrosine antibody (4G10) as described above except that the
lysis buffer contained 1% Nonidet P-40, 50 mM sodium
fluoride, 2 µg/ml leupeptin, and 1 µg/ml pepstatin A. Immunoprecipitates were washed four times with the lysis buffer, twice
with 0.5 M LiCl in 50 mM Tris-HCl, pH 7.5, and
twice with 100 mM NaCl in 50 mM Tris-HCl, pH
7.5. Lipid kinase assays were performed on the beads in a 50-µl
reaction mixture containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EGTA, 200 µM
adenosine, 0.1 mg/ml phosphatidylinositol, and 0.1 mg/ml phosphatidylserine. The kinase reaction was initiated by the addition of 10 µCi of [
-32P]ATP and MgCl2 to
final concentrations of 10 µM and 10 mM,
respectively. After incubation at 25 °C for 15 min, the reactions
were terminated by the addition of 125 µl of 1N HCl, and
the reaction mixtures were extracted with
CHCl3:CH3OH (2:1, v/v), spotted onto
oxalate-treated thin layer chromatography plates (Silica Gel 60, Merck), and developed using a solvent system of CHCl3,
CH3OH, H2O, 25% NH4OH (90:65:8:12, v/v/v/v). Phosphatidylinositol 3-phosphate was visualized by
autoradiography, and 32P incorporation was quantified using
a FUJI image analyzer (model BAS-2000). Activity of Akt was analyzed by
Western blotting of total cell lysate using antiphosphorylated-Akt antibody.
Luciferase Assay--
Cells (4.5 × 106
cells/sample) were transfected with 30 µg of
-casein
luciferase (20) or 3 µg of c-fos luciferase (18) plasmids
by electroporation as described (18). The cells were separated to three
groups and incubated with depletion medium for 6 h then stimulated
with mIL-3 (1 ng/ml), hGM-CSF (10 ng/ml), or left unstimulated for
another 6 h. The sample indicated as random culture was cultured
in mIL-3 (0.25 ng/ml)-containing media for 12 h immediately after
transfection. The cells were lysed with 20 µl of 250 mM
Tris-HCl, pH 7.4, and protein concentration was determined using the
BCA protein assay kit (Pierce). Luciferase activity was measured using
a luminometer (model LB9501; Berthold Lumat Co. Ltd., Japan) and a
luciferase assay substrate (Promega, Madison, WI).
Northern Blot Analysis--
Northern blots were performed with
mRNA prepared using the Fast Track 2.0 kit (Invitrogen, CA).
Briefly, 1 µg of mRNA was separated on a 1% agarose gel
containing 6% formaldehyde and transferred onto a nylon membrane
(Hybond-N, Amersham Pharmacia Biotech) by capillary blotting. The blots
were hybridized with cDNA probes (c-fos,
c-jun, c-myc, bcl-xL,
CIS, and glyceraldehyde-3-phosphate dehydrogenase genes)
labeled by the Ready-To-GoTM kit (Amersham Pharmacia
Biotech) using [
-32P]dCTP. The blotted membrane was
visualized using a FUJI image analyzer (model BAS-2000).
 |
RESULTS |
Construction and Expression of the
/JAK2--
To analyze the
direct signals from JAK2, we constructed a JAK2 chimera,
/JAK2, in
which the extracellular and transmembrane domains of
c were fused to
the N terminus of full-length JAK2. The total length of the
/JAK2 is
1580 amino acids (Fig. 1A). We
first examined the expression and size of
/JAK2 in COS7 cells. COS7
cells were harvested after 24 h of culture after transfection of
/JAK2 or wild-type JAK2 as a control, then subjected to
immunoprecipitation using anti-
c N terminus (S16) or anti-JAK2
antibodies followed by Western blotting using an anti-JAK2 antibody
(Upstate Biotechnology, Inc). A 170-180-kDa band that corresponds to
the expected size of
/JAK2 appeared (Fig. 1B). We next
prepared BA/F3 cells stably expressing
/JAK2 (BAF
/JAK2) and
/JAK2 together with the hGM-CSFR
subunit (BAF
,
/JAK2). The
expression of
/JAK2 in BA/F3 cells was confirmed by flow cytometry
assay (Fig. 1C) and Western blotting (Fig.
2).

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Fig. 1.
Construction and expression of
/JAK2 chimera. A, a schematic
diagram of /JAK2 chimera. The extracellular and transmembrane
regions of c are fused to the N terminus of full-length JAK2.
a.a., amino acid. B, transient expression of
/JAK2 in COS7 cells. The /JAK2 or wild-type JAK2 as a control was
expressed in COS7 cells, and expressed proteins were examined by
immunoprecipitation followed by Western blotting using an anti- c and
JAK2 antibodies. Sizes of /JAK2 (170-180 kDa) and JAK2 (120 kDa)
correspond to the predicted ones. C, surface expression of
/JAK2 in BA/F3 cells stably expressing /JAK2 (BAF /JAK2) or
/JAK2 and GM-CSFR subunit (BAF , /JAK2) was analyzed by flow
cytometry using an anti- c antibody. The shaded trace
indicates control staining using mouse IgG, and the open
trace indicates anti- c antibody staining.
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Fig. 2.
Phosphorylation of the
/JAK2 in BA/F3 cells. The BAFGMR (BA/F3 cells
expressing wild-type hGM-CSFR), BAF /JAK2, and BAF , /JAK2 cells
were depleted of mIL-3 for 6 h, stimulated with mIL-3 or hGM-CSF
for 10 min, and subjected to immunoprecipitation using an anti-JAK2
antibody followed by Western blotting. /JAK2 and endogenous
wild-type JAK2 are indicated by arrows.
anti-PTyr, antibody against phosphorylated Tyr.
|
|
Phosphorylation of
/JAK2 in BA/F3--
To examine the activity
of
/JAK2, we first examined tyrosine phosphorylation of
/JAK2 in
BA/F3 cells by immunoprecipitation followed by Western blotting. We
used BA/F3 cells expressing wild-type hGM-CSFR (BAFGMR) or parental
BA/F3 cells as a control in the following experiments. Fig. 2 shows
Western blotting patterns obtained with either an antiphosphotyrosine
antibody (4G10) or anti-JAK2 antibodies of anti-JAK2 immunoprecipitants
of BAFGMR, BAF
/JAK2, or BAF
,
/JAK2 cells. The anti-JAK2
antibody immunoprecipitated
/JAK2 as well as the endogenous JAK2,
and
/JAK2 was phosphorylated even in the absence of mIL-3 or hGM-CSF
in both BAF
/JAK2 cells and BAF
,
/JAK2 cells. In contrast,
endogenous JAK2 was phosphorylated only after mIL-3 stimulation.
hGM-CSF stimulation induced phosphorylation of endogenous JAK2 in
BAFGMR cells but not in BAF
/JAK2 or BAF
,
/JAK2 cells.
Therefore,
/JAK2 is constitutively phosphorylated in BA/F3 cells
regardless of the presence of hGM-CSFR
subunit or hGM-CSF stimulation. In addition, transphosphorylation to endogenous JAK2 from
/JAK2 did not occur.
BA/F3 Cells Expressing
/JAK2 Survived and Proliferated in the
Absence of mIL-3--
Because there was a constitutive phosphorylation
of
/JAK2, we next examined survival and proliferation of the
BAF
/JAK2 and BAF
,
/JAK2 cells. Fig.
3A shows DNA fragmentation
patterns of BAFGMR, BAF
/JAK2 and BAF
,
/JAK2 cultured in the
absence or presence of mIL-3 or hGM-CSF. As expected, the DNA from
BAFGMR cells cultured in growth factor-free media for 12 h was
fragmented. When BAFGMR cells were cultured either in mIL-3 or hGM-CSF,
no fragmentation was observed. In the case of BAF
/JAK2 and
BAF
,
/JAK2 cells, only a residual ladder DNA was observed.

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Fig. 3.
Cell proliferation and survival of BA/F3 cell
expressing /JAK2. Cell survival and
proliferation were analyzed by DNA fragmentation (A),
[3H]thymidine incorporation (B), or trypan
blue dye exclusion (C) assays. The BAFGMR, BAF /JAK2, and
BAF , /JAK2 cells were incubated for 12 h (A),
24 h (B), or the indicated days in the presence of
mIL-3 (closed circle) or hGM-CSF (open circle) or
the absence of factor (open squares) (C).
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|
We next analyzed the proliferation of BAF
/JAK2 and BAF
,
/JAK2
cells by [3H]thymidine incorporation analysis. As shown
in Fig. 3B, in BAFGMR, incorporation of
[3H]thymidine was not evident in the absence of factor,
and incorporation was stimulated in the presence of mIL-3 or hGM-CSF in
a dose-dependent manner. Both BAF
/JAK2 and
BAF
,
/JAK2 cells had a significant level of
[3H]thymidine incorporation in the absence of mIL-3 or
hGM-CSF. When cells were cultured in mIL-3-containing media, increased [3H]thymidine incorporation occurred in a mIL-3
dose-dependent manner. In contrast, hGM-CSF did not
stimulate [3H]thymidine incorporation in BAF
/JAK2 or
in BAF
,
/JAK2 cells. These results indicate that the expression of
/JAK2 supports DNA replication in BA/F3 cell in addition to cell
survival. It should be noted that the level of proliferation was less
than that observed with mIL-3 stimulation.
We then examined long term proliferation of these cells by examining
cell number and viability by making use of trypan blue dye exclusion
(Fig. 3C). As expected, the BAFGMR cells all died within one
day after mIL-3 depletion (left upper panel). In contrast, more than 70% of BAF
/JAK2 cells and 90% of BAF
,
/JAK2 cells survived without mIL-3 for at least 4 days. The cell number of BAF
/JAK2 and BAF
,
/JAK2 cells constantly increased, but the growth rate was slower than that of the cells cultured in mIL-3. Furthermore, we were able to maintain both BAF
/JAK2 and
BAF
,
/JAK2 cells in medium containing no mIL-3 for more than four
months (data not shown).
Neither PI3-K nor Akt Was Activated in BAF
/JAK2 or
BAF
,
/JAK2 Cells--
A role for PI3-K followed by Akt activation
in cell survival was reported in various cells. Because we found that
both PI3-K and Akt are activated by mIL-3 or hGM-CSF stimulation in
BAFGMR cells,2 we examined
whether these kinases are activated in BAF
/JAK2 and BAF
,
/JAK2
cells. PI3-K activity co-immunoprecipitated with antiphosphotyrosine
antibody was determined by in vitro kinase assay. As shown
in Fig. 4A, residual kinase
activity was observed after depletion of mIL-3 for 5 h, and
activity was induced by mIL-3 stimulation in all the three cells.
Random culture samples showed almost the same level of activity as that
of the depleted sample. The addition of hGM-CSF into BAF
,
/JAK2
cells did not affect the PI3-K activity. Akt is assumed to be
downstream of PI3-K, and we next analyzed Akt activity by Western
blotting using antiphospho-Akt antibody (Fig. 4B).
Consistent with the results of PI3-K, almost no activity was observed
from depleted samples of any of the cells. Activity was induced by the
addition of mIL-3, and random-cultured samples showed very weak
activity in comparison to the depleted cells. Taken together, these
results indicate that no constitutive activation of the PI3-K pathway
in BAF
/JAK2 and BAF
,
/JAK2 cells exists in the absence of
factors. We also examined the effect of wortmannin, a PI3-K-specific
inhibitor. The addition of wortmannin up to 200 nM, which
completely suppressed the GM-CSF-induced activation of PI3-K and Akt in
BAFGMR cells, did not show any inhibitory effect on factor-independent
proliferation of BAF
/JAK2 and BAF
,
/JAK2 cells (data not
shown), further eliminating the possible involvement of this pathway
within these cells.

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Fig. 4.
Analysis of PI3-K and Akt. PI3-K
activity was analyzed by immunoprecipitation followed by in
vitro kinase assay (A), and Akt activity was analyzed
by Western blotting of total cell lysate using antiphospho-Akt antibody
(B). A, the cells were depleted of mIL-3 and
stimulated either by mIL-3 or hGM-CSF for 1 min. The sample indicated
as random culture is a sample cultured continuously in mIL-3 media.
Immunoprecipitation using antiphosphotyrosine antibody (4G10) and then
lipid kinase assay were done as described under "Experimental
Procedures." Lipid products were separated by TLC.
PIP indicates phosphatidylinositol phosphate. B,
the upper panel shows the blotting pattern of
antiphospho-Akt, and the lower panel shows the
blotting pattern using anti-Akt antibody of the same membrane.
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STAT5, Shc, or SHP-2 Was Not Phosphorylated in BAF
/JAK2 and
BAF
,
/JAK2 Cells--
To examine whether cellular proteins are
phosphorylated in BAF
/JAK2 and BAF
,
/JAK2 cells, we did Western
blot analysis of total cell lysates using antiphosphotyrosine antibody
(Fig. 5A). Cells were depleted
of mIL-3 for 6 h and restimulated by mIL-3 or hGM-CSF for 10 min.
In all the cells, the addition of mIL-3 induced tyrosine
phosphorylation of various cellular proteins. Random-cultured cells in
the presence of mIL-3 showed significant tyrosine phosphorylation in
every cell lines. In contrast, although BAF
/JAK2 and BAF
,
/JAK2
cells grew in the absence of factors, no significant
tyrosine-phosphorylated band was observed.

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Fig. 5.
Tyrosine phosphorylation of cellular proteins
in BAF /JAK2,
BAF , /JAK2, and
parental BA/F3 cells. BAF /JAK2, BAF , /JAK2, and BA/F3
cells were depleted of mIL-3 and stimulated either by mIL-3 or hGM-CSF.
The sample indicated as a random culture is a sample cultured
continuously in mIL-3 media. A, total cell lysates were
subjected to Western blotting using antiphosphotyrosine antibody
(4G10). B, immunoprecipitation using the indicated
antibodies followed by Western blotting were done as described under
"Experimental Procedures." In the bottom panel, the
arrow indicates SHP-2.
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We previously found that STAT5, Shc, and SHP-2 are
tyrosine-phosphorylated in response to hGM-CSF in BA/F3 cells
expressing hGM-CSFR, and this phosphorylation requires the tyrosine
residues of
c (14, 15). We analyzed the tyrosine phosphorylation of these molecules in both BAF
/JAK2 and BAF
,
/JAK2 cells by
immunoprecipitation followed by Western blotting. When BA/F3 cells were
depleted of mIL-3 for 5 h, no residual phosphorylation of STAT5
was observed, and the STAT5 was tyrosine-phosphorylated in response to
mIL-3, in accord with our previous reports (Fig. 5B). In
BAF
/JAK2 and BAF
,
/JAK2 cells, no phosphorylation of STAT5 was
observed with samples of factor-depleted cells. The addition of hGM-CSF
induced little tyrosine phosphorylation of STAT5 in BAF
,
/JAK2
cells. Because tyrosine phosphorylation of STAT5 is transient, it is possible that phosphorylation of STAT5 is not observed with
unsynchronized cells. We examined the state of tyrosine phosphorylation
of STAT5 from random-cultured BA/F3 cells. STAT5 is weakly but
significantly tyrosine-phosphorylated within the sample of
random-cultured cells, thus indicating that
/JAK2 cannot
phosphorylate STAT5 in the absence of mIL-3 in BAF
/JAK2 and
BAF
,
/JAK2 cells. Although both BAF
/JAK2 and BAF
,
/JAK2
cells grew without mIL-3, tyrosine phosphorylation of STAT5 was never observed.
Similarly, we analyzed the tyrosine phosphorylation of Shc and SHP-2 by
immunoprecipitation followed by Western blotting under the same
conditions used for STAT5. Both Shc and SHP-2 were phosphorylated by
mIL-3 stimulation in all the cells, and weak phosphorylation was
observed in the random-cultured BA/F3 cells. In contrast, in
mIL-3-depleted BAF
/JAK2 and BAF
,
/JAK2 cells, neither Shc nor
SHP-2 was phosphorylated. A positive role for the activation of Shc and
SHP-2 in the MAPK pathway was proposed (15). The results suggested that
neither STAT5 nor MAPK pathway is required for survival and
proliferation of BA/F3 cells in this system.
To determine whether STAT5 and MAPK pathways are functionally defective
in BAF
/JAK2 and BAF
,
/JAK2 cells, we further analyzed the
activities of
-casein and c-fos promoters,
assumed to be targets of STAT5 and the MAPK cascade, respectively (21).
A transient transfection analysis was made using
-casein
or c-fos promoters fused to the luciferase-coding region.
Luciferase activity of random-cultured cells, factor-depleted cells,
and factor restimulated cells was examined. As shown in Fig.
6A, the
-casein
luciferase activity was induced about 5-fold in all the cells after
depletion and readdition of mIL-3. The
-casein luciferase
activity of random-cultured BA/F3 cells showed higher luciferase
activity than was observed in mIL-3 restimulated cells. In contrast,
hGM-CSF stimulation did not induce the activity of
-casein luciferase in BAF
/JAK2 and BAF
,
/JAK2
cells. These results are consistent with those observed in experiments
examining tyrosine phosphorylation of STAT5. We suggest that
constitutively activated
/JAK2 does not activate STAT5 in
BAF
/JAK2 and BAF
,
/JAK2 cells. Similarly, c-fos
luciferase activity in unstimulated BAF
/JAK2 and BAF
,
/JAK2 cells was significantly lower than that observed in cells subjected to
mIL-3 stimulation (Fig. 6B). In contrast, the
c-fos luciferase activity of random-cultured BA/F3 cells was
higher than that in cells stimulated with mIL-3.

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|
Fig. 6.
Induction of gene expression in
BAF /JAK2,
BAF , /JAK2, and
parental BA/F3 cells. A and B, examination
of promoter activities of -casein and c-fos
using transient transfection analysis. The -casein
promoter luciferase (A) or c-fos promoter
luciferase (B) plasmids were transiently transfected to
BA/F3, BAF /JAK2, and BAF , /JAK2 cells, and luciferase
activities of indicated conditions were examined. Values are expressed
as relative to the values obtained by mIL-3-stimulated samples.
C, Northern blot analysis of c-fos,
c-jun, CIS,
bcl-xL, and
c-myc. The cells were depleted for 6 h and stimulated
for 30 min (c-fos, c-jun, CIS) or
3 h (bcl-xL, c-myc)
by mIL-3 or hGM-CSF. random culture indicates samples
cultured continuously in mIL-3. G3PDH,
glyceraldehyde-3-phosphate dehydrogenase.
|
|
c-myc but Not c-fos, c-jun, CIS, and bcl-xL Was
Constitutively Activated in BAF
/JAK2 and BAF
,
/JAK2
Cells--
We previously found that immediate early genes,
c-fos, c-jun, bcl-xL, and
c-myc are activated either by mIL-3 or hGM-CSF stimulation in BA/F3 cells. For c-fos and c-jun gene
activation, the membrane distal region of
c is required in addition
to the membrane proximal region. In contrast, c-myc
activation requires only the membrane proximal region, and this
requirement is the same as that required for cell proliferation. To
analyze whether these genes are activated or not in BAF
/JAK2 and
BAF
,
/JAK2 cells, we performed Northern blot analysis.
Factor-depleted cells were restimulated for 30 min for
c-fos, c-jun, and CIS analysis and for
3 h for c-myc and bcl-xL
analysis. As shown in Fig. 6C, by 6 h of mIL-3
depletion, levels of c-fos, c-jun,
bcl-xL, and CIS fell to background
levels, and mIL-3-induced these genes in all the three cells. In
contrast, even after mIL-3 depletion, the level of c-myc
expression remained significantly higher in BAF
/JAK2 and
BAF
,
/JAK2 cells than in the parental BA/F3 cells. These results
indicate that c-myc transcription is selectively activated
by the constitutively active
/JAK2 molecule.
 |
DISCUSSION |
In the present study, we found that activation of JAK2 using a
chimeric
/JAK2 supports long term survival and proliferation of
BA/F3 cells in the absence of mIL-3. Chimeric JAK2 is
tyrosine-phosphorylated constitutively in BA/F3 cells regardless of the
presence of hGM-CSFR
subunit or hGM-CSF. Similar to the model
proposed for the receptor-type tyrosine kinase (22), JAKs are assumed
to be activated by homodimer formation followed by transphosphorylation
by JAKs themselves (2, 3). Thus, constitutive tyrosine phosphorylation
of
/JAK2 in the present work may occur through constitutive dimer
formation of the
/JAK2. We reported that hGM-CSF receptor
subunit forms a dimer even in the absence of hGM-CSF in BA/F3 cells and
that the cytoplasmic region of
c is not required for this dimer
formation (10). Although attempts to show the presence of a dimer form of
/JAK2 using a chemical cross-linker were not successful (data not
shown), we speculate that the chimeric
/JAK2 forms a dimer through
transmembrane and extracellular regions of
c.
Analyses using mutant receptors as well as mutant JAKs showed an
essential role of JAK2 in the IL-3 and GM-CSF signaling pathway (5).
The JAK2 knock-out mice revealed the essential role of JAK2 for IL-3
signals in vivo (7, 8). Our initial idea to isolate the
activity of JAK2 was based on the finding that there are several
activities of hGM-CSF that require JAK2 activation but not the
C-terminal region of
c. During mutation analysis of
c, we found
distinct signaling pathways of hGM-CSFR (13). The signaling pathway
that requires the receptor C terminus region was initiated by
phosphorylation of
c tyrosine residues, and then a cascade of
signaling molecules such as the MAPK pathway by protein-protein
interactions followed. STAT activation also requires tyrosine residues,
because it is assumed that STAT binds to phosphorylated tyrosine
through its SH2 domain. On the other hand, there are signaling pathways
that require only JAK2 activation through the box1 region but not the
receptor tyrosine residues, leading to cell survival, cell
proliferation, and c-myc activation. The only event that is
constitutively activated in BAF
/JAK2 and BAF
,
/JAK2 cells was
c-myc transcription. Taken together, an important role for
c-myc in cell survival and proliferation is strongly
suggested. In the present work, the activation of JAK2 occurred without
involvement of the cytoplasmic region of
c.
/JAK2 transduces
signals for cell survival and weak proliferation. We previously
revealed that a mutant
c lacking all the tyrosines that cannot
activate STAT5 or the MAPK cascade is still capable of maintaining cell
survival and weak but significant proliferation. In addition, we
recently found that PI3-K and Akt activation induced by IL-3 or GM-CSF
does not play an essential role in cell survival in BA/F3
cells,2 which is consistent with the current observation
that no induction of PI3-K and Akt occurs in BAF
/JAK2 and
BAF
,
/JAK2. Taken together, it is strongly suggested that
activation of the MAPK and PI3-K cascades as well as STAT5 by cytokines
is not essential for survival and proliferation of BA/F3 cells.
We constructed and analyzed another type of chimeric JAK2 using Gyrase
B as an artificial dimerizer (23, 24). With this fusion protein of
Gyrase B and JAK2 (GyrB/JAK2), which is assumed to be inducibly
dimerized by binding to the chemical compound coumermycin, we obtained
different results from
/JAK2. The addition of coumermycin results in
activation of this chimeric JAK2 molecule and induces phosphorylation
of STAT5 but does not support long term survival, proliferation, and
MAPK cascade activation of BA/F3 cells. It is of interest that
phosphorylation of STAT5 and proliferation are antiparallel events in
GyrB/JAK2 and
/JAK2. This finding supports the notion that STAT5 may
not be involved in signaling for proliferation or survival of BA/F3
cells. The different activities of these chimeras may be explained by
differences in subcellular localization of these molecules, because
GyrB/JAK2 may exclusively locate in cytoplasm, and
/JAK2 is the
transmembrane protein. There are other examples of JAK2 fusions with
transmembrane molecules such as the EGF receptor and the erythropoietin
receptor (25, 26). In both cases, the chimeras can mediate
proliferative signaling without activating the MAPK cascade. Finally,
naturally occurring TEL-JAK2 fusion proteins, as a result of
chromosomal translocations, have been found via patients with acute
lymphoblastic leukemia and chronic myelogenous leukemia and are assumed
to cause malignancy within hematopoietic cells (27, 28). TEL-JAK2
exhibits constitutive tyrosine kinase activity, and its expression
confers mIL-3-independent proliferation to BA/F3 cells, yet the
molecular pathways that mediate the signals from this molecule remain
to be clarified. Further studies using our
/JAK2 chimera may help
gain an insight into the mechanism of JAK2 signaling in hematopoiesis
as well as in leukemogenesis.
 |
ACKNOWLEDGEMENTS |
Authors grateful to Yutaka Aoki and Yukitaka
Izawa for excellent technical assistance and Marty Dahl and Mariko
Ohara for critical reading of the manuscript.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
A recipient of a research fellowship from the Japan Society for the
Promotion of Science for Young Scientists.
To whom correspondence should be addressed. Tel.:
81-3-5449-5660; Fax: 81-3-5449-5424; E-mail:
sumiko{at}ims.u-tokyo.ac.jp.
2
R. Liu, T. Itoh, and S. Watanabe, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
GM-CSF, granulocyte-macrophage colony-stimulating factor;
GM-CSFR, GM-CSF
receptor;
IL-3, interleukin-3;
MAPK, mitogen-activated protein kinase;
hGM-CSF, human GM-CSF;
mIL-3, murine IL-3;
PI3-K, phosphoinositide
3-kinase;
CIS, cytokine-inducible SH2-containing protein.
 |
REFERENCES |
-
Ihle, J. N.,
Witthuhn, B. A.,
Quelle, F. W.,
Yamamoto, K.,
Thierfelder, W. E.,
Kreider, B.,
and Silvennoinen, O.
(1994)
Trends Biochem. Sci.
19,
222-227[CrossRef][Medline]
[Order article via Infotrieve]
-
Ihle, J.,
and Kerr, I. M.
(1995)
Trends Genet.
11,
69-74[CrossRef][Medline]
[Order article via Infotrieve]
-
Watanabe, S.,
and Arai, K.
(1996)
Curr. Opin. Gen. Dev.
6,
587-596[CrossRef][Medline]
[Order article via Infotrieve]
-
Quelle, F. W.,
Sato, N.,
Witthuhn, B. A.,
Inhorn, R. C.,
Eder, M.,
Miyajima, A.,
Griffin, J.,
and Ihle, J. N.
(1994)
Mol. Cell. Biol.
14,
4335-4341[Abstract]
-
Watanabe, S.,
Itoh, T.,
and Arai, K.
(1996)
J. Biol. Chem.
271,
12681-12686[Abstract/Free Full Text]
-
Rao, P.,
and Mufson, R. A.
(1995)
J. Biol. Chem.
270,
6886-6893[Abstract/Free Full Text]
-
Parganas, E.,
Wang, D.,
Stravopodis, D.,
Topham, D. J.,
Marine, J.-C.,
Teglund, S.,
Vanin, E. F.,
Bodner, S.,
Colamonici, O. R.,
van Deursen, J. M.,
Grosveld, G.,
and Ihle, J. N.
(1998)
Cell
93,
385-395[Medline]
[Order article via Infotrieve]
-
Neubauer, H.,
Cumano, A.,
Muller, M.,
Wu, H.,
Huffstadt, U.,
and Pfeffer, K.
(1998)
Cell
93,
397-409[Medline]
[Order article via Infotrieve]
-
Miyajima, A.,
Mui, A. L.-F.,
Ogorochi, T.,
and Sakamaki, K.
(1993)
Blood
82,
1960-1974[Medline]
[Order article via Infotrieve]
-
Muto, A.,
Watanabe, S.,
Miyajima, A.,
Yokota, T.,
and Arai, K.
(1996)
J. Exp. Med.
183,
1911-1916[Abstract]
-
Lopez, A. F.,
Vadas, M. V.,
Woodcock, J. M.,
Milton, S. E.,
Lewis, A.,
Elliott, M. J.,
Gillis, D.,
Ireland, R., E.,
Olwell, E.,
and Park, L. S.
(1991)
J. Biol. Chem.
266,
24741-24747[Abstract/Free Full Text]
-
Sakamaki, K.,
Miyajima, I.,
Kitamura, T.,
and Miyajima, A.
(1992)
EMBO J.
11,
3541-3550[Abstract]
-
Watanabe, S.,
Muto, A.,
Yokota, T.,
Miyajima, A.,
and Arai, K.
(1993)
Mol. Biol. Cell
4,
983-992[Abstract]
-
Itoh, T.,
Muto, A.,
Watanabe, S.,
Miyajima, A.,
Yokota, T.,
and Arai, K.
(1996)
J. Biol. Chem.
271,
7587-7592[Abstract/Free Full Text]
-
Itoh, T.,
Liu, R.,
Yokota, T.,
Arai, K.,
and Watanabe, S.
(1998)
Mol. Cell. Biol.
18,
742-752[Abstract/Free Full Text]
-
Okuda, K.,
Smith, L.,
Griffin, J.,
and Foster, R.
(1997)
Blood
90,
4759-4766[Abstract/Free Full Text]
-
Miyajima, A.,
Schreurs, J.,
Otsu, K.,
Kondo, A.,
Arai, K.,
and Maeda, S.
(1987)
Gene
58,
273-281[Medline]
[Order article via Infotrieve]
-
Watanabe, S.,
Mui, A. L.-F.,
Muto, A.,
Chen, J. X.,
Hayashida, K.,
Miyajima, A.,
and Arai, K.
(1993)
Mol. Cell. Biol.
13,
1440-1448[Abstract]
-
Taglialatela, G.,
Gegg, M.,
Perez-Polo, J. R.,
Williams, L. R.,
and Rose, G. M.
(1996)
Neuroreport
7,
977-980[Medline]
[Order article via Infotrieve]
-
Mui, A. L.-F.,
Wakao, H.,
O'Farrell, A.-M.,
Harada, N.,
and Miyajima, A.
(1995)
EMBO J.
14,
1166-1175[Abstract]
-
Wakao, H.,
Gouilleux, F.,
and Groner, B.
(1994)
EMBO J.
13,
2182-2191[Abstract]
-
Ullich, A.,
and Schlessinger, J.
(1990)
Cell
61,
203-212[Medline]
[Order article via Infotrieve]
-
Farrar, M. A.,
Alberola-Ila, J.,
and Perlmutter, R. M.
(1996)
Nature
383,
178-181[CrossRef][Medline]
[Order article via Infotrieve]
-
Mohi, M. G.,
Arai, K.,
and Watanabe, S.
(1998)
Mol. Biol. Cell
9,
3299-3308[Abstract/Free Full Text]
-
Nakamura, N.,
Chin, H.,
Miyasaka, N.,
and Miura, O.
(1996)
J. Biol. Chem.
271,
19483-19488[Abstract/Free Full Text]
-
Joneja, B.,
and Wojchowski, D. M.
(1997)
J. Biol. Chem.
272,
11176-11184[Abstract/Free Full Text]
-
Lacronique, V.,
Boureux, A.,
Della Valle, V.,
Poirel, H.,
Quang, C. T.,
Mauchauffe, M.,
Berthou, C.,
Lessard, M.,
Berger, R.,
Ghysdael, J.,
and Bernard, O. A.
(1997)
Science
278,
1309-1312[Abstract/Free Full Text]
-
Peeters, P.,
Raynaud, S. D.,
Cools, J.,
Wlodarska, I.,
Grosgeorge, J.,
Philip, P.,
Monpoux, F.,
Van Rompaey, L.,
Baens, M.,
Van den Berghe, H.,
and Marynen, P.
(1997)
Blood
90,
2535-2540[Abstract/Free Full Text]
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