(Received for publication, May 23, 1995)
From the
Interleukin (IL)-9 stimulates the proliferation of a variety of hematopoietic lineages through its interaction with a receptor of the cytokine receptor superfamily. In the studies presented here, we have begun to characterize the downstream signaling pathways activated by IL-9. In addition to the activation of JAK1 and JAK3 tyrosine kinases, IL-9, unlike most hematopoietic cytokines but similar to IL-4, induces the tyrosine phosphorylation of a 170-kDa protein that is related to the insulin receptor substrate-1 (IRS-1). We further demonstrate for the first time that IRS-1 is not only associated with JAK1 but also tyrosine phosphorylated and functionally involved in IL-9 signaling in TS1 lymphocytes transfected with the murine IRS-1 cDNA. Cotransfection studies and in vitro experiments directly demonstrate that JAK1, JAK2, or JAK3 is capable of tyrosine phosphorylating IRS-1, suggesting a functional role for these kinases in vivo. Lastly, we demonstrate that IL-9 induces the tyrosine phosphorylation of Stat3 and in this regard differs from IL-4, which triggers tyrosine phosphorylation of Stat6. Taken together, these results strongly suggest that IL-9 and IL-4 utilize common and unique signaling pathways via inducing the similar and distinct tyrosine-phosphorylated proteins.
IL-9 ()is a T cell-derived lymphokine with growth
promoting activity for certain murine T helper clones(1) . The
cDNAs encoding murine and human IL-9 have been
cloned(2, 3) . Subsequently, it was found that IL-9
has a variety of biological activities including T cell and mast cell
growth-promoting activity(4, 5) , B cell
differentiation activity(6) , and stimulatory activity toward
erythroid and myeloid precursors(7, 8) . Recently, the
possible involvement of IL-9 in T cell tumorigenesis has been
suggested(9) . These results suggest that IL-9 is a
multifunctional T cell cytokine that may play an important role in
immunohematopoiesis.
IL-9 receptor belongs to the hematopoietin receptor superfamily(10) . Although there is no intrinsic tyrosine kinase motif in the IL-9 receptor, IL-9 has been shown to stimulate protein tyrosine phosphorylation in D10G4.1 T lymphocytes(11) . Recently, it has been demonstrated that JAK1 and JAK3 tyrosine kinases are activated following IL-9 stimulation(11, 12, 13) . Activation of JAK tyrosine kinases has been linked to tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat)(14, 15) . However, it is not clear whether JAK kinases can directly phosphorylate other proteins involved in IL-9 signaling. Here we show for the first time that insulin receptor substrate-1 (IRS-1) and IRS-1-related IL-4-induced phosphorylated substrate (4PS), which play essential roles in insulin- and IL-4-mediated signal transduction(16, 17, 18, 19) , are not only rapidly tyrosine-phosphorylated but also functionally involved in IL-9 signal transduction. Furthermore, we provide evidence that activated JAK1, JAK2, and JAK3 tyrosine kinases induced by either IL-9 stimulation or overexpression in COS-1 cells can phosphorylate IRS-1 on tyrosine residues both in vitro and in vivo. Our results also indicate that IL-9 induces tyrosine phosphorylation of Stat3, whereas IL-4 activates Stat6. These results strongly suggest that IL-9 and IL-4 can trigger common and unique signaling pathways via inducing the same and distinct tyrosine-phosphorylated proteins.
Figure 1:
Protein tyrosine
phosphorylation and activation of JAK1 and JAK3 kinases induced by IL-9
in TS1 cells. After factor starvation, TS1 cells (2 10
cells in 0.5 ml of serum-free medium) were stimulated with 50
ng/ml of IL-9 or IL-4 for 5 min (A and D) or for the
indicated periods of time (B and C). Cells were lysed
with 1% Nonidet P-40 lysis buffer. A, tyrosine phosphorylation
of cellular proteins induced by IL-9 and IL-4. Equal amounts of total
cell lysates (4
10
cells/lane) were separated by
SDS-PAGE (7.5%), and proteins were transferred to PVDF membranes. B, kinetics of tyrosine phosphorylation of JAK1 and JAK3
kinases induced by IL-9. Cell lysates (2
10
cells/sample) were incubated with 1 µl of JAK1 or JAK3
antisera for 2 h at 4 °C. The immune complexes were precipitated by
the addition of protein A-agarose beads and washed with lysis buffer as
indicated under ``Materials and Methods.'' The
immunoprecipitates were separated by SDS-PAGE (7.5%) and transferred to
PVDF membranes. The membranes from A and B were
immunoblotted with the indicated antibodies. C, JAK1 and JAK3 in vitro kinase activity induced by IL-9. The
immunoprecipitates with JAK1 or JAK3 antisera as described in B were washed five times with lysis buffer and once with kinase
buffer. The immune complex kinase assays were performed by adding
[
-
P]ATP as described under ``Materials
and Methods.'' The products of kinase reactions were resolved by
SDS-PAGE (7.5%) and transferred to PVDF membranes. The kinase activity
was detected by autoradiography, and the membranes were then
immunoblotted with JAK1 or JAK3 antisera as indicated. D,
tyrosine phosphorylation of Stat proteins induced by IL-9 and IL-4. The
Stat proteins were immunoprecipitated with specific Stat antibodies as
indicated and separated by SDS-PAGE (7%). The proteins were then
transferred to PVDF membranes and immunoblotted with the indicated
antibodies. PTyr, Tyr(P). IP,
immunoprecipitation.
We also examined
whether IL-9 can induce tyrosine phosphorylation of known Stats. The
results in Fig. 1D demonstrated that Stat3 but not
Stat1, Stat2, Stat4, Stat5, or Stat6 is tyrosine-phosphorylated
following IL-9 stimulation in TS1 cells. In contrast, IL-4 specifically
induced Stat6 tyrosine phosphorylation in these cells (Fig. 1D). The results demonstrated that IL-4 and IL-9
utilize distinct Stats despite sharing IL-2 receptor chain and
activating the same JAK tyrosine kinases(11, 13) ,
implying that signaling specificity for each cytokine may be achieved
by activating distinct Stat.
Figure 2:
Tyrosine phosphorylation of the IRS-1 and
IRS-1-related 4PS induced by IL-9 and IL-4 in TS1 cells. A,
tyrosine phosphorylation of IRS-1-related 4PS induced by IL-9 and IL-4.
Treatment and stimulation of cells with IL-9 and IL-4 were the same as
described in the legend to Fig. 1. Cell lysates (2
10
cells/sample) were incubated with 10 µg of
anti-IRS-1 antibodies raised against the intact IRS-1 protein overnight
at 4 °C. B, comparison of cell proliferation in TS1 cells
transfected with vector alone (TS1) or with murine IRS-1 cDNA (TS1-IRS-1) following IL-9 or IL-4 stimulation. Cells (200
cells/0.2 ml/well in triplicate) were incubated in 96-well plates for 3
days with different concentrations of IL-9 or IL-4 as indicated. The
number of cells in each well was counted by cell counter at the end of
culture. The results represent one of three different individual
experiments. C, association of IRS-1 with JAK1 kinase. TS1
cells transfected with IRS-1 cDNA were starved and stimulated with IL-9
or IL-4 as described in the legend to Fig. 1. Cells (2
10
cells/sample) were lysed, and IRS-1 was
immunoprecipitated with 3 µg of antibodies for the C terminus of
IRS-1 for 2 h at 4 °C. The immune complexes from A and C were precipitated by the addition of protein A-agarose beads
as described under ``Materials and Methods.'' The
immunoprecipitates were separated by SDS-PAGE (7.5%) and transferred to
PVDF membranes. The membranes were sequentially immunoblotted with
antibodies for Tyr(P) (PTyr), intact IRS-1 (A), or C
terminus of IRS-1 (C) and JAK1 or JAK3 (C) as
indicated. IP,
immunoprecipitation.
Because IRS-1-related 4PS cDNA and its specific antibody are not available at the present time and IRS-1 and IRS-1-related 4PS share many biological features(16, 18, 19) , we decided to transfect IRS-1 cDNA into IRS-1-negative TS1 cells to further determine whether IRS proteins are indeed involved in IL-9 signaling pathways. The results in Fig. 2B showed that the overexpression of IRS-1 in TS1 cells significantly enhances the sensitivity of TS1 cells to IL-9 and IL-4 stimulation (3-5-fold lower dosage required to achieve the same level of cell proliferation when compared with vector-transfected TS1 cells). Furthermore, we demonstrated that transfected IRS-1 is rapidly tyrosine-phosphorylated and preferentially associated with JAK1 kinase following IL-9 or IL-4 stimulation (Fig. 2C). These results strongly implicate that IRS-1 may be phosphorylated by JAK1 kinase and is functionally involved in IL-9 signaling pathways in TS1 cells.
Figure 3: Tyrosine phosphorylation of IRS-1 by JAK1, JAK2, and JAK3 kinases. A and B, tyrosine phosphorylation and association of IRS-1 with JAK1, JAK2, or JAK3 kinase in COS-1 cells. IRS-1 cDNA (4 µg) was cotransfected into COS-1 cells with 6 µg of JAK1, JAK2, or JAK3 cDNAs as indicated. A, IRS-1 was immunoprecipitated with 4 µg of IRS-1 antibodies for 2 h at 4 °C after transfection for 48 h. B, JAK1, JAK2 and JAK3 were immunoprecipitated from the same cell lysates as in A with 2 µl of JAK1, JAK2, or JAK3 antisera as indicated. The immunoprecipitates were separated by SDS-PAGE and transferred to the PVDF membranes. The membranes were immunoblotted with the indicated antibodies. C and D, in vitro tyrosine phosphorylation of IRS-1 by activated JAK1 and JAK3 kinases. The immunoprecipitated IRS-1 proteins from COS-1 cells transfected with IRS-1 cDNA were mixed with the immunoprecipitated JAK1 or JAK3 kinase from either TS1 cells (C) stimulated without (JAK-TS1) or with IL-9 (JAK-TS1-IL-9) or COS-1 cells (D) transfected with JAK1 (JAK1-COS-1) or JAK3 cDNA (JAK3-COS-1) as indicated. The kinase reactions were initiated by the addition of ATP as described under ``Materials and Methods.'' The reaction mixtures were separated by SDS-PAGE (7.5%) and transferred to the PVDF membranes. The membranes were immunoblotted by ECL with anti-Tyr(P), anti-IRS-1, or anti-JAK1 and JAK3 antibodies as indicated in the figure. PTyr, Tyr(P).
We have shown that IL-9 and IL-4 induced common and unique
tyrosine-phosphorylated proteins in TS1 cells. IL-9 stimulated tyrosine
phosphorylation of Stat 3, whereas IL-4 activated Stat6. It is noted
that the cytoplasmic domain of IL-9 but not that of IL-2 or IL-4
receptor ligand binding subunit contains the motif YLPQ, which has been
shown to be a binding site for Stat3 in IL-6 signal transducer, gp130 (29) . These results suggest that unique motif(s) presented in
the cytoplasmic domain of cytokine receptors can determine specific
Stat activation. We previously showed that IL-9 but not IL-4 induces
tyrosine phosphorylation of Stat1 in D10G4.1 lymphocytes(11) .
In M07E megakaryocytic leukemia cells, Stat1 and 88-kDa associated
protein are activated by IL-9(12) . These results implicate
that activation of Stat by IL-9 may bear cell type specificity. Taken
together, although IL-4 and IL-9 share the IL-2 receptor
chain(13) , induce tyrosine phosphorylation of IRS-1/4PS, and
activate the same JAK1 and JAK3 kinases, the activation of distinct
Stat and other tyrosine-phosphorylated substrates suggests that the
specificity for IL-9 and IL-4 signaling could be achieved through the
unique tyrosine-phosphorylated proteins. For example, dysregulated
expression of IL-9 but not IL-4 can induce T cell
transformation(29, 30) , and IL-9 has erythroid
progenitor stimulating activity(8) , whereas IL-4 lacks this
function. On the other hand, the common signaling pathways triggered by
IL-4 and IL-9 may explain some of the overlapping activities of IL-4
and IL-9 such as stimulation of T cell proliferation as well as
induction of B cell differentiation(5, 31) .
It has
been demonstrated that the IRS-1 or IRS-1-related 4PS is essential for
signal transduction mediated by IL-4, insulin, and insulin-like growth
factor(27) . IRS-1 has also been shown to be involved in growth
hormone-mediated signaling(32) . We demonstrated here that
IRS-1 is not only tyrosine-phosphorylated but also functionally
involved in IL-9 signaling in TS1 cells. We further showed that IRS-1
is associated with JAK1 kinase following IL-9 or IL-4 stimulation,
implying that IRS-1 may be phosphorylated by JAK1 kinase in
vivo. It has been suggested that activation of JAK3 but not JAK1
may be critical in IL-4-mediated proliferative signaling(33) .
Although JAK3 is also activated by IL-9 in TS1 cells, we were unable to
show an association between JAK3 and IRS-1 using current extraction
procedures. It is possible that JAK3 may form a complex with IRS-1 in vivo; however, the association is disrupted during
extraction. Further studies using a two-hybrid system will be required
to verify such interactions. The conserved sequence motif
(PLXNPXYXSXSD) (27) , which interacts with IRS-1 or IRS-1-related 4PS in IL-4,
insulin, and insulin-like growth factor receptors, is not present in
the IL-9 receptor(10) . Therefore, the mechanisms by which IL-9
induces tyrosine phosphorylation of the IRS-1 and IRS-1-related 4PS
require further investigation.
It has been shown that Stat proteins are physiological substrates for JAK tyrosine kinases that can be activated by a variety of cytokines (14, 15) . We showed here that IRS-1 is tyrosine-phosphorylated and associated with JAK1 in TS1 cells and both JAK1 and JAK2 are coimmunoprecipitated with tyrosine-phosphorylated IRS-1 in COS-1 cotransfection experiments, suggesting that IRS-1 is a substrate for JAK1 and JAK2 kinases. Cotransfection of JAK3 with IRS-1 also resulted in tyrosine phosphorylation of IRS-1, although we failed to show the direct association between JAK3 and IRS-1. In vitro experiments showed that activated JAK3 kinase is capable of phosphorylating IRS-1 on tyrosine residues, implying that IRS-1 is also a substrate for JAK3 kinase. It is well known that IRS-1 and IRS-1-related 4PS are substrates for insulin and insulin-like growth factor receptor tyrosine kinases and play essential roles in insulin and insulin-like growth factor signaling pathways(17, 18, 19, 34) . It has been identified that the NPXY motif present in insulin receptor cytoplasmic domain mediates interaction between IRS-1 and insulin receptor tyrosine kinase(35) . Although the mechanisms by which JAK kinases interact with IRS-1 require further investigation because the NPXY motif is absent in JAK kinases, the demonstration for the involvement of IRS-1 and IRS-1-related 4PS in IL-9 signal transduction suggests that IL-9 shares some common signaling pathways with those of IL-4, insulin, and insulin-like growth factor. However, it remains to be answered whether JAK kinases and insulin/insulin-like growth factor receptor tyrosine kinases can induce the same sites of tyrosine phosphorylation in IRS-1 and IRS-1-related 4PS and whether the functions of tyrosine-phosphorylated IRS-1 and IRS-1-related 4PS induced by these different tyrosine kinases are the same in vivo.