(Received for publication, December 9, 1996, and in revised form, June 13, 1997)
From the Departments of Interleukin-9 receptor (IL-9R) complex consists
of a ligand-specific Interleukin 9 (IL-9)1 is
a T cell-derived multifunctional growth factor that exerts its effects
on activated T cells, B cells, mast cells, and hematopoietic
progenitors (1, 2). The in vitro biological functions of
IL-9 include its ability to stimulate proliferation of activated T
cells, to enhance production of immunoglobulin in B cells, and to
promote proliferation and differentiation of mast cells and
hematopoietic progenitors (3-7). The involvement of IL-9 in
lymphomagenesis has been suggested by in vivo studies (8) in
which a higher susceptibility to T cell lymphoma was observed in
transgenic mice expressing IL-9 constitutively and by in
vitro experiments (9) in which IL-9 has been shown to protect
mouse lymphoma cells from dexamethasone-induced apoptosis. The
functions of IL-9 are mediated by the IL-9 receptor (IL-9R), which
consists of a ligand-specific IL-9R The full-length cDNA for hIL-9R
Murine T cell clone, TS1,
was maintained in Click's medium (Irvine Scientific) supplemented with
10% fetal calf serum and 1 ng/ml mIL-9 (R & D Systems). For stable
expression of hIL-9R Transfectants expressing different
forms of hIL-9R Cells were starved
and stimulated with hIL-9 as described previously (18). Briefly, about
2 × 107 cells were starved for 8 h at 37 °C
in the absence of hIL-9 and serum, then cells were collected and
stimulated without or with hIL-9 (30 ng/ml) for 5 min. Cells were then
lysed in 0.5 ml of 1% Nonidet P-40 lysis buffer containing different
proteinase inhibitors (21). After centrifugation, clear cell lysates
were obtained and incubated with anti-JAK1, anti-JAK3, anti-IRS2, or
anti-STAT3 antibody (Upstate Biotechnology, Inc.) overnight at 4 °C.
Then protein A-agarose was added, and the mixture was rotated for
1 h. The immunoprecipitates were washed three times with lysis
buffer and separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted sequentially with
anti-phosphotyrosine (Upstate), anti-JAK1, anti-JAK3, anti-IRS-2, and
anti-STAT3 antibodies and finally detected with the ECL techniques
according to manufacturer instructions. For detection of FlagIL-9R
expression, total cell lysates were prepared from 2 × 107 cells and immunoprecipitated with 2 µg of anti-Flag
M2 antibody. The immunoprecipitates were separated by 10%
SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes,
and immunoblotted with anti-hIL-9R antibody (R & D Systems).
Northern blot analysis was performed
as described previously (22). For detection of primary response gene
expression, transfectants expressing different mutant forms of
hIL-9R Nuclear extracts were
prepared from unstimulated and stimulated TS1 cells as described
previously (24). A 32P-labeled oligonucleotide
corresponding to a high affinity dyed symmetrical acute-phase response
factor (APRF) binding site (5 To identify
structural elements critical for IL-9-mediated cell proliferation and
signal transduction in the cytoplasmic region of hIL-9R
To understand the role of
different regions of the cytoplasmic domain in hIL-9R
Accumulated evidence has revealed that molecular
cascades involved in cytokine-mediated cell growth are associated with
protein tyrosine phosphorylation. JAK1, JAK3, IRS-2, and STAT3 have
been demonstrated to be activated by IL-9 stimulation through tyrosine phosphorylation in our previous studies (17, 18). To examine the role
of hIL-9R
To further confirm the status of STAT3 activation by hIL-9 in different
hIL-9R
Activation of primary response genes,
which are signaling events downstream of JAK/STAT activation, has been
shown to play important roles in cell proliferation and
differentiation. hIL-9 has been demonstrated to induce c-Myc and JunB
expression in a human megakaryocytic leukemia cell line (19). To
understand the functional importance of the intracellular domains of
hIL-9R
In the present study, we have demonstrated the importance of the
cytoplasmic domain of hIL-9R In agreement with the study by Demoulin et al. (29), our
data also indicated that the BOX1 downstream region in hIL-9R Finally, we also investigated primary response gene expression in
hIL-9R-transfected cells. The importance of primary response genes as
potentially critical targets for cytokine-induced proliferation has
been extensively studied (40-42). Although our previous study has
shown that IL-9 could induce activation of junB and
c-myc in a human growth factor-dependent cell
line (19), the correlation between the activation of these two genes
and IL-9-mediated cell proliferation has never been demonstrated. Our
present study indicated that activation of c-myc upon
stimulation with IL-9 depends on the presence of both the
membrane-proximal and the BOX1 downstream regions in the cytoplasmic
domain of hIL-9R In conclusion, two important regions in the cytoplasmic domain of
hIL-9R We thank Steve Hulbert for his technical
assistance.
Medicine
(Hematology/Oncology) and
Biochemistry and Molecular Biology,
chain and IL-2R
chain. In this study, two
regions in the cytoplasmic domain of human IL-9R
were found to be
important for IL-9-mediated cell growth. A membrane-proximal region
that contains the BOX1 consensus sequence is required for IL-9-induced cell proliferation and tyrosine phosphorylation of Janus kinases (JAKs). Deletion of this region or internal deletion of the BOX1 motif
abrogated IL-9-induced cell proliferation and signal transduction. However, substitution of the Pro-X-Pro in the BOX1 motif
with Ala-X-Ala failed to abolish IL-9-induced cell
proliferation but decreased IL-9-mediated tyrosine phosphorylation of
JAK kinases, insulin receptor substrate-2, and signal transducer and
activator of transcription 3 (STAT3) and expression of c-myc
and junB. Another important region is downstream of
the BOX1 motif and contains a STAT3 binding motif YLPQ. Deletion of
this region significantly impaired IL-9-induced cell growth, activation
of JAK kinases, insulin receptor substrate-2, and STAT3 and expression
of early response genes. A point mutation changing YLPQ into YLPA
greatly reduced IL-9-induced activation of STAT3 and expression of
c-myc but did not affect cell proliferation. These results
suggest that cooperation or cross-talk of signaling molecules
associated with different domains of IL-9R
other than STAT3 is
essential for IL-9-mediated cell growth.
chain and IL-2 receptor (IL-2R)
chain. IL-2R
chain, normally referred to as the common
chain
(
c chain), is shared by receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (10-13). Some of the signaling pathways elicited by
these cytokines are quite similar, which probably explains in part the
functional redundancy of these cytokines.
chain (IL-9R
) is IL-9-specific and is responsible for IL-9
binding. The cDNAs encoding mouse and human IL-9R
have been
cloned (14-15). IL-9R
belongs to the hematopoietic receptor superfamily and has no intrinsic tyrosine kinase motif in its cytoplasmic region. Several homologous sequences, such as the BOX1
consensus sequence and a serine-rich region, which were demonstrated to
be important for IL-2R
function (16), are also present in the
cytoplasmic region of human IL-9R
(hIL-9R
). Recently, it has been
shown that several biochemical events including tyrosine phosphorylation of JAK kinases, activation of signal transducers and
activators of transcription (STAT) proteins, and expression of nuclear
proto-oncogenes are involved in IL-9-mediated signal transduction
(17-19). In the present study, a series of deletions and point
mutations were made in the cytoplasmic region of hIL-9R
. These
mutants were transfected into a mouse IL-9-dependent T cell line, TS1, to investigate the functions of different domains of hIL-9R
in IL-9-induced cell proliferation and activation of JAK/STAT and primary response genes.
Plasmid Construction
was provided by Dr. Ming-Shi Chang at Amgen (Thousand Oaks, CA). The
hIL-9R
cDNA fragments containing different C-terminal
truncations of the cytoplasmic region were generated by polymerase
chain reaction. The full-length and C-terminal-truncated cDNAs for
hIL-9R
were subcloned into blunt-ended XbaI site in
pRc/CMV vector (Invitrogen). Site-directed mutations in the BOX1 and
STAT3 binding motifs (BOXm and STAT3 m) within the cytoplasmic domain
of hIL-9R
were generated using the Altered Sites II in
vitro mutagenesis systems (Promega). Internal deletion of the BOX1
motif (between amino acids 298 and 315) in hIL-9R
(BOXd) was
performed using the polymerase chain reaction method as described
previously (20). To construct pFlagIL-9R plasmids, an
NdeI-XbaI restricted fragment from pFlag-CMV-1
expression vector (Eastman Kodak Co.) that contained part of the CMV
promoter and the coding sequence for preprotrypsin leader peptide and
flag epitope tag was subcloned into various pRc/CMV-IL-9R constructs to
replace NdeI-XbaI-restricted DNA fragments. The
resulting plasmids were designated as pFlagIL-9R W, pFlagIL-9R D1,
pFlagIL-9R D2, pFlagIL-9R D3, pFlagIL-9R D4, pFlagIL-9R BOXm,
pFlagIL-9R STAT3 m, and pFlagIL-9R BOXd (see Fig. 1). All plasmid
constructs were confirmed by DNA sequencing.
Fig. 1.
Schematic diagram of wild-type, C-terminal
truncations, internal deletion, and point mutations of hIL-9R.
The construction of pFlagIL-9R plasmids containing wild-type
(W) and mutated hIL-9R
was described under "Materials
and Methods." Flag peptide, transmembrane domain (TM),
BOX1 motif (BOX1), serine-rich region (SR), and
point mutation within BOX1 motif (BOXm) or STAT3 binding
site (STAT3 m) are as indicated. The numbers given at the C
terminus of each protein represent relevant positions of amino acids at
the truncated end (according to the numbering system in published hIL-9
cDNA sequence; Ref. 14). CMV and bovine growth hormone poly(A)
represent CMV promoter and polyadenylation signal from bovine growth
hormone present in the vector. aa, amino acid(s).
[View Larger Version of this Image (15K GIF file)]
, TS1 cells in exponential growth phase were
suspended at 2 × 107 cells/ml in culture medium. 0.3 ml of these cells was transferred into a 0.4-cm cuvette with 30 µg of
pFlagIL-9R DNAs. Following a single 250-V/960-microfarad pulse with
Bio-Rad Gene Pulser, each sample was cultured in 5 ml of medium with
mIL-9 (1 ng/ml) at 37 °C. Stable transfectants were isolated by
selection in 1 mg/ml G418 for 3 weeks. Cells expressing high levels of
FlagIL-9R proteins were further enriched by Fluorescence-activated Cell Sorter using an anti-Flag M2 monoclonal antibody (Kodak) and goat anti-mouse IgG1-fluorescein isothiocyanate (Southern Biotechnology Associates, Inc.).
were washed three times, seeded in triplicates at
5 × 103 cells/200 µl of culture medium/well either
without growth factors or supplemented with various concentrations of
hIL-9. After incubation at 37 °C for 3 days, 1 µCi of
[3H]thymidine (Amersham Life Science, Inc., specific
activity 5 Ci/mmol) was added per well and further incubated for 4 h at 37 °C before harvest. The incorporated
[3H]thymidine was determined using a liquid scintillation
counter (Beckman Instruments, LS 6000IC).
were starved and stimulated with hIL-9 (30 ng/ml) for 1 h. Total RNAs were isolated from cells using a guanidinium
isothiocyanate method (23). 5 µg of total RNA was used for Northern
blot and hybridized with 32P-labeled c-myc,
junB, c-jun, and c-fos cDNAs. The
mouse glyceraldehyde-3-phosphate dehydrogenase probe was hybridized to
the same filters to ensure equal loading in each lane. The expression
of different pFlagIL-9R constructs in TS1 transfectants was determined
using the hIL-9R
cDNA as the probe.
-CCTTCCCGGAATTC-3
) was used as the probe
(25). For the gel shift assay, 10 µg of nuclear proteins were
incubated with 2 µg of poly[d(I·C)] before the addition of 2 × 105 cpm of probe. After incubation at room temperature
for 30 min, the reactions were analyzed with a 5% native
polyacrylamide gel. For the gel supershift assay, nuclear extracts were
preincubated with 2 µg of anti-Stat3 antibody or the same amount of
preimmune IgG at 4 °C for 2 h and then incubated with the probe
for 30 min at room temperature. In oligonucleotide competition
experiments, a 100-fold molar excess of various competitors were
preincubated with nuclear extracts for 10 min before the binding
reactions. The sequences of c-sis-inducible element (SIE), AP-1, and
Oct-1 competitors are: SIE, 5
-GTCGACATTTCCCGTAAATCTTGT-3
(11, 26); AP-1, 5
-CGCTTGATGAGTCAGCCGGAA-3
(Promega); Oct-1,
5
-TGTCGAATGCAAATCACTAGAA-3
(Santa Cruz Biotechnology).
Expression of Mutant hIL-9R Proteins
, a series of
mutants with deletions or point mutations in hIL-9R
were constructed
and stably transfected into TS1 cells. TS1 is a mouse
IL-9-dependent T cell line that does not respond to hIL-9
stimulation. It has been shown previously that transfection of
hIL-9R
cDNA into TS1 cells conferred responsiveness of these cells to hIL-9 (14). A schematic diagram of mutant receptor constructs
used in the present study is shown in Fig.
1. To determine the expression of
different pFlagIL-9R constructs in transfected TS1 cells, Northern blot
and immunoprecipitation analysis were performed to detect hIL-9R
mRNAs and proteins in different transfectants. As shown in Fig.
2, A and B, the
parental TS1 cells did not express hIL-9R
transcripts and proteins.
Cells transfected with different pFlagIL-9R constructs expressed
various sizes of hIL-9R
mRNAs and proteins. It was noticed that
two different sizes of hIL-9R
proteins were expressed in each
transfectant, which may represent different glycosylated forms of
hIL-9R
. Cell surface expression of FlagIL-9R proteins was further
confirmed by flow cytometric analysis. As shown in Fig. 2C,
cells transfected with pRc/CMV vector alone (negative control) showed a
similar fluorescence intensity to untransfected cells, whereas
transfectants expressing various pFlagIL-9R cDNAs had a higher
relative fluorescence intensity than control cells. The percentage of
positive cells was very similar among different transfectants.
Fig. 2.
Expression of different pFlagIL-9R
constructs in TS1 cells. A, northern blot analysis. 5 µg
of total RNA from untransfected TS1 cells (C) and
transfected TS1 cells with different pFlagIL-9R constructs were
size-fractionated by gel electrophoresis, transferred onto a
nitrocellulose membrane, and hybridized with a 32P-labeled
hIL-9R
cDNA probe. B, immunoprecipitation and Western blot analysis. Total lysates from about 2 × 107 cells
of different transfectants were immunoprecipitated with anti-Flag2
antibody, transferred to a PVDF membrane, and immunoblotted with
anti-hIL-9R antibody. Kd, kilodalton; IP,
immunoprecipitation analysis. C, flow cytometric analysis.
Surface expression of FlagIL-9R proteins was demonstrated by flow
cytometric analysis. Various TS1 transfectants were incubated
sequentially with anti-Flag M2 and anti-mouse-IgG1-fluorescein
isothiocyanate (FITC) antibodies. Dashed lines
represent fluorescence intensity of untransfected TS1 cells;
solid lines represent fluorescence intensity shifts with
transfected cells. Control, cells transfected with vector alone.
[View Larger Version of this Image (59K GIF file)]
Play Important
Roles in IL-9-induced Cell Growth
for
IL-9-mediated cell growth, TS1 cells transfected with different
hIL-9R
cDNAs were tested for their proliferative response to
various concentrations of hIL-9. As shown in Fig.
3, cells transfected with vector alone
did not grow in the presence of hIL-9; in fact, >95% of cells were
dead before the addition of [3H]thymidine. TS1 cells
transfected with wild-type hIL-9R
(pFlagIL-9R W) showed a strong
response to hIL-9 induction, with cell proliferation detected even at a
very low concentration of hIL-9. This suggests that the FlagIL-9R
fusion protein functioned like the natural form of hIL-9R
in
response to hIL-9 stimulation in TS1 cells. Transfectants harboring
pFlagIL-9R D1 and pFlagIL-9R D2 (D1 and D2) showed a proliferative
response comparable to wild-type transfectants, suggesting that distal
C terminus and the serine-rich region of hIL-9R
are dispensable for
IL-9-induced cell proliferation. This is in part different from
IL-2R
in which the serine-rich region has been shown to be critical
for IL-2-induced cell growth (27). Two regions in the cytoplasmic
domain of hIL-9R
were implicated to be critical for IL-9-mediated
cell proliferation in the present study. A membrane-proximal region
between amino acids 295 and 338 including a BOX1 consensus sequence,
was shown to be essential for IL-9-induced cell proliferation. The
proliferative response to IL-9 was abrogated when this region was
further truncated (D4) or if the BOX1 motif in this region was deleted
(BOXd), indicating that the BOX1 consensus sequence in hIL-9R
is
required for IL-9-mediated cell growth. However, unlike other receptor
systems, mutation of the Pro-X-Pro motif in BOX1 consensus
sequence of hIL-9R
(BOXm) did not abolish but slightly reduced
IL-9-mediated cell growth. The second important region is located
downstream of the BOX1 consensus sequence between amino acids 338 and
422. This region contains a STAT3 binding site and was found to play a
crucial role in promoting IL-9-induced cell proliferation. When this
region was truncated (D3), cells only proliferated at a higher
concentration of hIL-9 (1 ng/ml). Since STAT 3 has been shown to be the
only STAT protein activated by IL-9 in TS1 cells (18), we also mutated STAT3 binding site within the hIL-9R
to evaluate the role of STAT3
in IL-9-mediated cell growth. Interestingly, point mutation at the
STAT3 binding site from YLPQ to YLPA (STAT3 m transfectants) did not
significantly affect IL-9-induced cell proliferation, although a
similar point mutation (from YMPQ to YMPA) has been shown to abolish
tyrosine phosphorylation of STAT3 induced by gp130 family of cytokines
(28). We also noticed that wild type, D1, D2, and STAT3 m transfectants
had a higher incidence of becoming factor-independent compared with D3,
D4, BOXm, and BOXd transfectants, implicating that overexpression of
specific domains of IL-9R
may result in transformation of TS1
cells.
Fig. 3.
hIL-9-dependent DNA synthesis of
TS1 transfectants expressing hIL-9R. TS1 cells stably
transfected with different pFlagIL-9R constructs or vector alone were
cultured with different concentrations of hIL-9, and incorporation of
[3H]thymidine into cells was measured. The data shown
here represent the mean values in a typical experiment with triplicates
from three independent experiments.
[View Larger Version of this Image (34K GIF file)]
Abrogates Tyrosine Phosphorylation of JAK Kinases, IRS-2, and STAT3
Induced by hIL-9
in IL-9-induced growth-promoting signals, the status of
hIL-9-induced tyrosine phosphorylation of JAK kinases, IRS-2, or STAT3
in TS1 cells expressing wild-type and mutant hIL-9R
s were tested. As
illustrated in Fig. 4, tyrosine
phosphorylation of JAK1, JAK3, IRS-2, and STAT3 was comparable in
wild-type hIL-9R
transfectants (W) and in parental TS1
cells stimulated with hIL-9 and mIL-9, respectively, indicating that
transfected hIL-9R
transduces similar signals as its murine
counterpart following hIL-9 stimulation. Deletion of the C terminus and
the serine-rich region of hIL-9R
(D1 and D2) failed to change
hIL-9-induced tyrosine phosphorylation of JAK kinases, IRS-2, and
STAT3. The BOX1 consensus sequence in the membrane-proximal region was
shown to be absolutely required for IL-9-induced signaling, since
truncation (D4) or internal deletion (BOXd) of this region completely
abolished IL-9-induced tyrosine phosphorylation of JAK kinases, IRS-2,
and STAT3. Furthermore, point mutation of Pro-X-Pro motif
into Ala-X-Ala (BOXm) within the BOX1 consensus sequence
also decreased IL-9-induced tyrosine phosphorylation of JAK kinases,
IRS-2, and STAT3. It was also found that tyrosine phosphorylation of
JAK kinases and IRS-2 was greatly decreased when the BOX1 downstream
region of hIL-9R
was truncated (D3). This observation is consistent
with a recent report, in which the BOX1 downstream region of hIL-9R
was found to be absolutely required for IL-9-induced JAK1 activation in
BW 5147 cells (29). This region was also found to be indispensable for activation of STAT3, since truncation of this region eliminated IL-9-induced tyrosine phosphorylation of STAT3 (D3). Point mutation of
the STAT3 binding motif from YLPQ to YLPA in this region (Stat3 m)
greatly decreased IL-9-induced tyrosine phosphorylation of STAT3,
suggesting activation of STAT3 is most likely through this YLPQ
motif.
Fig. 4.
Tyrosine phosphorylation of JAK1, JAK3,
IRS-2, and STAT3 in TS1 transfectants expressing hIL-9R. Cells
were starved in serum-free medium for 8 h. 2 × 107 cells were not stimulated or stimulated with hIL-9 (30 ng/ml) for 5 min and immunoprecipitated with 1.5 µg of anti-JAK1,
JAK3, or STAT3 or 4 µg of anti-IRS-2 antibody at 4 °C overnight.
The immunoprecipitates were separated by 7.5% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were
immunoblotted with anti-phosphotyrosine (P-Tyr), anti-JAK1, anti-JAK3, anti-IRS-2, and anti-STAT3.
[View Larger Version of this Image (45K GIF file)]
transfectants, gel shift and gel supershift assays were
performed to detect specific DNA-protein complexes following hIL-9
stimulation. As indicated in Fig.
5A, the nuclear extracts from
wild-type transfectants stimulated by hIL-9 specifically bind to the
APRF sequence that has a high affinity binding site for STAT3 proteins
(25). This binding activity was induced within 1 min after hIL-9
stimulation and persisted for at least 15 min. The competition
experiments (Fig. 5B) showed that the APRF binding activity
induced by IL-9 could be completely blocked with 100-fold excess of
cold APRF or SIE competitor but not with 100-fold excess of AP-1 or
Oct-1 oligonucleotide. It was further shown by the gel supershift assay
(Fig. 5B) that most of the IL-9-induced APRF-protein complexes can be specifically supershifted by anti-STAT3 antibody, indicating that the major proteins in IL-9-induced APRF-protein complexes are STAT3 or STAT3-related molecules. IL-9-induced APRF binding activities were also examined in different hIL-9R transfectants (Fig. 5C); it was found that the induction of APRF binding
activities correlated with STAT3 tyrosine phosphorylation in these
transfectants following IL-9 stimulation.
Fig. 5.
Activation of STAT3 by hIL-9 in TS1
transfectants expressing wild-type or mutant hIL-9R. A,
nuclear extracts were prepared from wild-type hIL-9R transfectants
treated with hIL-9 for the indicated period of time, and the APRF DNA
binding activity was analyzed by the gel shift assay. B, gel
shift and gel supershift assays were performed with nuclear extracts
from wild-type hIL-9R
transfectant stimulated by hIL-9 for 5 min in
the presence of 100-fold excess of unlabeled competitors or 2 µg of
pre-immune IgG or anti-STAT3 antibody. Comp., competitor.
C, gel shift assays were performed with nuclear extracts
from TS1 cells stimulated by mIL-9 for 5 min (as a positive control) or
from different hIL-9R
transfectants stimulated by hIL-9 for 5 min.
[View Larger Version of this Image (75K GIF file)]
in the expression of primary response genes and to correlate
expression of these genes with cell growth, we examined expression of
c-myc, junB, c-jun, and c-fos
in response to hIL-9 by Northern blot analysis. It was observed
that the expression of c-fos and c-jun mRNA
was not affected by hIL-9 treatment (data not shown), whereas the expression of c-myc and junB mRNAs was
up-regulated in wild-type, D1, and D2 transfectants upon stimulation
with hIL-9 (Fig. 6). In contrast,
c-myc and junB were not induced in D4, D3, and
BOXd transfectants, suggesting the membrane-proximal region and the adjacent downstream region of the cytoplasmic domain of hIL-9R
are
indispensable for the activation of c-myc and
junB. In BOXm transfectants, induction of both
c-myc and junB genes by hIL-9 was greatly reduced
compared with wild-type transfectants, indicating downstream signaling
molecules associated with the Pro-X-Pro motif may affect the
expression of c-myc and junB. Interestingly, in STAT3 m transfectants, induction of c-myc was greatly
diminished, whereas the expression of junB was not
significantly affected, suggesting that activation of c-myc
and junB by hIL-9 is mediated, at least in part, by
different mechanisms.
Fig. 6.
Induction of early response gene in TS1
transfectants expressing hIL-9R. Cells were starved in
serum-free medium for 8 h and were either not stimulated or
stimulated with hIL-9 (30 ng/ml) for 1 h. 5 µg of total RNAs
from unstimulated and stimulated cells were prepared and separated
through a 1% formaldehyde-agarose gel and transferred onto a
nitrocellulose membrane. The same membrane was sequentially hybridized
with different 32P-labeled c-Myc, JunB, c-Jun, c-Fos, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probes. W, wild type.
[View Larger Version of this Image (61K GIF file)]
in IL-9-mediated cell proliferation and
signal transduction. We identified two regions, a membrane-proximal region containing BOX1 consensus sequence and the adjacent downstream region containing a STAT3 binding site, as required for IL-9-mediated signal transduction. In our study, the membrane-proximal region including 47 amino acids of the cytoplasmic domain of hIL-9R
(D3)
was shown to support IL-9-induced cell proliferation and tyrosine
phosphorylation of JAK kinases even though the response of this mutant
to IL-9 was greatly reduced compared with wild type hIL-9R
. This
observation is different from a recent report by Demoulin et
al. (29) in which a mouse IL-3-dependent cell line,
Ba/F3, transfected with truncated hIL-9R
plasmid encoding 115 amino
acids of the cytoplasmic region containing the BOX1 motif no longer
responds to hIL-9. The difference between the two studies could be due
to the difference in the cell types used in each of these experiments.
The function of the membrane-proximal region in hIL-9R
is most
likely to be mediated by the BOX1 motif, which is also found in the
membrane-proximal region of many cytokine receptors and has been
demonstrated to be important for JAK1/JAK2 activation and cell growth
in several cytokine receptor systems (30-34). In our study, truncation
or internal deletion of the BOX1 motif in the membrane-proximal region
of hIL-9R
totally abolished hIL-9-induced cell proliferation and
tyrosine phosphorylation of JAK1 and JAK3, suggesting the BOX1
consensus sequence in hIL-9R
plays a preeminent role in IL-9-induced
activation of JAK kinases and cell growth. Since mutation of the
conserved Pro-X-Pro motif in the BOX1 consensus sequence
inactivated the receptor and JAK1 activation in certain receptor
systems (30, 35, 36), we also introduced point mutations
(Pro-X-Pro to Ala-X-Ala) in the BOX1 consensus
sequence of hIL-9R
(BOXm). To our surprise, these mutations did not
totally abolish IL-9-mediated cell growth, but decreased tyrosine
phosphorylation of JAK1 and JAK3. Although activation of JAK3 has been
demonstrated to be mainly associated with IL-2R
chain (37, 38),
tyrosine phosphorylation of JAK3 was shown to be affected by mutations
in the membrane-proximal and the BOX1 downstream regions of hIL-9R
in our study. Recently, a membrane-proximal region of human IL-2R
was shown to be critical for JAK3 activation (35), raising the
possibility that JAK3 may bind to receptor subunits other than IL-2R
chain for its activation. In this study, we also investigated
tyrosine phosphorylation of IRS-2 by IL-9 in various transfectants. It
appears that tyrosine phosphorylation of IRS-2 correlated with tyrosine
phosphorylation of JAK kinases, which is consistent with our earlier
observation that JAK kinases are responsible for IL-9-induced tyrosine
phosphorylation of IRS proteins (18).
plays
an important role in IL-9-mediated cell growth. Deletion of this region
not only greatly reduced hIL-9-stimulated cell proliferation and
tyrosine phosphorylation of JAK kinases and IRS-2 but also abrogated
tyrosine phosphorylation of STAT3. A STAT3 binding site (YLPQ) was
found within this region. YXXQ has been shown to be
important for tyrosine phosphorylation of STAT3 in gp130 family
receptors (28), and the important role of the tyrosine residue within
YLPQ motif in hIL-9R
has recently been reported (29). Since the
tyrosine residue within this motif was shown to play a key role in
IL-9-mediated cell growth and activation of STAT1
, STAT3, and STAT5,
STAT factors were suggested to be involved in IL-9-mediated cell
proliferation. However, in our study, a point mutation in this STAT3
binding site from YLPQ to YLPA did not significantly affect
IL-9-induced cell proliferation but greatly reduced tyrosine
phosphorylation and activation of STAT3, suggesting that STAT3 may not
be the major signaling molecule in IL-9-induced cell growth in TS1
cells. In the gp130 system, a recent report suggested that STAT3 may be
involved in cell differentiation and growth arrest mediated by IL-6
(39).
, suggesting that IL-9-induced expression of
c-myc may require JAK kinases and signals associated with
the BOX1 downstream region. This is different from IL-2R
, in which
the serine-rich region is important for the activation of c-myc
(27). In conjunction with the functional study, c-myc
is not likely to play an important role in IL-9-mediated cell
proliferation in TS1 cells, since the induction of c-myc by
IL-9 was greatly impaired in STAT3 m and BOXm transfectants that can
proliferate in response to hIL-9. Interestingly, tyrosine phosphorylation and activation of STAT3 by IL-9 were also decreased in
both STAT3 m and BOXm transfectants, suggesting that STAT3 may be
involved in IL-9-mediated activation of c-myc. hIL-9-induced junB activation appears to be attributed to both regions of
hIL-9R
and correlates with IL-9-induced cell proliferation. Although STAT3 was recently shown to be involved in the transcriptional regulation of the junB promoter through an IL-6 response
element (43, 44), the induction of junB by IL-9 was not
significantly decreased in STAT3 m transfectants, implicating that
additional transcription factors may be induced by IL-9 in the
activation of junB.
have been demonstrated to play important roles in IL-9-induced cell growth and signal transduction. A membrane-proximal region between amino acids 295 and 338 containing the BOX1 motif is
absolutely essential for IL-9-induced cell proliferation and tyrosine
phosphorylation of JAK kinases and a BOX1 downstream sequence between
amino acids 338 and 422 including a YLPQ motif is critical for
IL-9-induced cell growth, activation of STAT3, and up-regulation of
c-myc and junB gene expression. Since both of
these two regions are important for optimal cellular proliferation induced by IL-9, we suggest that the reconstitution of a fully functional hIL-9R requires the cooperation or cross-talk between different signaling transducers associated with hIL-9R cytoplasmic domain, even though activation of distinctive signaling molecules may
be mediated by individual cytoplasmic regions of hIL-9R.
*
This work was supported in part by United States Public
Health Service Grants RO1HL48819 and RO1DK 50570 (to Y.-C. Y.).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.
**
To whom correspondence should be addressed. Tel.: 317-274-7512;
Fax: 317-274-7592; E-mail: yu-chung_yang{at}iucc.iupui.edu.
1
The abbreviations used are: IL, interleukin; R,
receptor; h, human; m, murine; JAK, Janus kinase; STAT, signal
transducer and activator of transcription; IRS-2, insulin receptor
substrate-2; SIE, c-sis-inducible element; APRF, acute-phase response
factor; CMV, cytomegalovirus; PVDF, polyvinylidene difluoride.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.