©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-9 Induces Tyrosine Phosphorylation of Insulin Receptor Substrate-1 via JAK Tyrosine Kinases (*)

(Received for publication, May 23, 1995)

Tinggui Yin (1) Susanne R. Keller (3) Frederick W. Quelle (4) Bruce A. Witthuhn (4) Monica Lik-Shing Tsang (5) Gustav E. Lienhard (3) James N. Ihle (4) Yu-Chung Yang(§) (2)

From the  (1)Walther Oncology Center and the Departments of Medicine (Hematology/Oncology) and (2)Biochemistry/Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, the (3)Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, the (4)Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, and (5)R & D Systems, Minneapolis, Minnesota 55413

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

IL-9 (^1)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.


MATERIALS AND METHODS

Reagents and Cytokines

Antibodies for JAK1, JAK2, JAK3, TyK2, Stat2, IRS-1 (polyclonal antibodies against the intact molecule and the C terminus of the protein), and phosphotyrosine (Tyr(P), 4G10) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Antibodies for the N terminus of IRS-1 and Stat1 were purchased from Santa Cruz (Santa Cruz, CA). Antibodies for Stat3, Stat4, Stat5, and Stat6 were produced as described(20) . ECL detection kit was purchased from Amersham Corp. Recombinant murine IL-9 and IL-4 were from R & D Systems (Minneapolis, MN). DOTAP transfection reagents were purchased from Boehringer Mannheim.

Cell Culture and Cytokine Stimulation

IL-9-dependent murine T cell line (TS1) was maintained in Click's medium (Irvine Scientific) plus 10% fetal bovine serum in the presence of 0.1 ng/ml murine IL-9(1) . For experiments, TS1 cells were washed three times with serum-free medium, starved for 7 h in the absence of IL-9, and washed once every 2 h with serum-free medium during starvation. Cells (2 10^7 cells/ml) were stimulated with IL-9 (50 ng/ml) or IL-4 (50 ng/ml) for the indicated periods of time and lysed with 1% Nonidet P-40 lysis buffer as described(21) .

Immunoprecipitation and Immunoblotting

Anti-JAK1, JAK2, JAK3, TyK2, Stat, and IRS-1 immunoprecipitations were done as described (20, 21, 22) with minor modifications (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM EDTA, and 0.5% Nonidet P-40 lysis buffer for coimmunoprecipitation). Briefly, cell lysates (2 10^7 cells/sample) were incubated with 1 (JAK1, JAK2, JAK3, or TyK2) or 2 µl (Stat3, Stat4, Stat5, or Stat6) of antisera and 1 µg of Stat1, 20 µg of Stat2, or 3 µg of IRS-1 (C terminus) antibodies for 2 h at 4 °C or 10 µg of anti-IRS-1 antibodies against the intact IRS-1 overnight at 4 °C with rotation, and then protein A-agarose beads were added for an additional 1 h. The precipitates were washed five times with lysis buffer and proteins were dissociated from protein A-agarose beads by boiling for 5 min with SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE (7.5%) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were immunoblotted with antibodies for Tyr(P) (0.1 µg/ml), JAK1 (1:500), JAK2 (1:1000), JAK3 (1:1000), Stat1 (2 µg/ml), Stat2 (5 µg), Stat3/Stat4/Stat5/Stat6 (1:2000), or IRS-1 (2 µg/ml for the C terminus and 10 µg/ml for the intact molecule) as indicated under ``Results'' and ``Discussion.''

Expression Vectors and Transfections

The cDNA-encoding murine IRS-1(23) , JAK1, JAK2, and JAK3 (24, 25, 26) kinases were subcloned into the mammalian expression vectors pRc/CMV (Invitrogen). TS1 cells were transfected with IRS-1 cDNA or vector alone by electroporation and selected with G418 (1 mg/ml). The G418-resistant cells were further cloned by limiting dilution, and 40 clones were picked for screening IRS-1 expression by immunoblotting. Two clones that expressed high levels of IRS-1 were used for experiments. For cotransfection, subconfluent COS-1 cells in 100-mm culture dishes were transfected with various combinations of IRS-1, JAK1, JAK2, or JAK3 expression constructs by the DOTAP method according to the manufacturer's instructions. Cells were lysed with lysis buffer as described above following transfection for 48 h, and proteins were immunoprecipitated with the indicated antibodies.

In Vitro Kinase Assay

Anti-JAK1 and JAK3 antibody immunoprecipitates were washed five times with lysis buffer and once with kinase buffer. The in vitro kinase assay was performed and visualized as described(21, 22) . For in vitro tyrosine phosphorylation of IRS-1, JAK1 or JAK3 kinase was immunoprecipitated and washed with kinase buffer minus ATP and then mixed with immunoprecipitated IRS-1. The kinase reactions were initiated by adding 100 µM of ATP and incubated for 30 min at 30 °C as described previously (21, 22, 27) and indicated under ``Results.''


RESULTS

Characterization of Tyrosine-phosphorylated Proteins Induced by IL-9 in TS1 Lymphocytes

As shown in Fig. 1A, IL-9 induced multiple tyrosine-phosphorylated proteins with apparent molecular masses of 170, 145, 130, 125, 97, 89, 67, 64, and 40 kDa in TS1 cells. We have identified the 170-, 130-, and 125-kDa tyrosine-phosphorylated proteins as the IRS-1-related 4PS protein, JAK1, and JAK3 tyrosine kinases, respectively (Fig. 1, B and C, and 2A). Furthermore, JAK1 and JAK3 kinases were activated according to in vitro autophosphorylation reactions following IL-9 stimulation (Fig. 1C). JAK2 and TyK2 kinases, however, were not activated by IL-9 in TS1 cells (data not shown). The 89-kDa tyrosine-phosphorylated protein is likely to be Stat3 (see below). The 40-kDa tyrosine-phosphorylated protein is unlikely to be mitogen-activated protein kinases, because its molecular size is different from those of mitogen-activated protein kinases and IL-9 did not induce mitogen-activated protein kinase tyrosine phosphorylation in TS1 cells (data not shown), suggesting that IL-9, similar to IL-4 but unlike most cytokines, may not utilize mitogen-activated protein kinase signaling cascades. The identity of the 67- and 64-kDa tyrosine-phosphorylated proteins requires further investigation. In comparison with IL-9, IL-4 induced the common (170-, 145-, 130-, 125-, 67-, and 64-kDa) and distinct (86- and 72-kDa) tyrosine-phosphorylated proteins in TS1 cells (Fig. 1A).


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 10^7 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 10^5 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 10^7 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.

Identification of Tyrosine Phosphorylation of the IRS-1 and IRS-1-related 4PS Induced by IL-9 in TS1 Cells

As shown in Fig. 1A, IL-9 induced tyrosine phosphorylation of a 170-kDa protein. IL-4 also induced tyrosine phosphorylation of a 170-kDa protein in TS1 cells. It has been shown that the 170-kDa tyrosine-phosphorylated protein induced by IL-4 in a variety of cell lines is the IRS-1 related 4PS protein(16, 18, 19) . To test whether the 170-kDa tyrosine-phosphorylated protein in IL-9 signaling is 4PS, immunoprecipitation experiments with anti-intact IRS-1 antibodies were performed. The results demonstrated that the 170-kDa tyrosine-phosphorylated protein induced by IL-9 or IL-4 in TS1 cells can be precipitated with antibodies for intact IRS-1, although efficiency seems low when compared with total cell lysates (Fig. 1A and Fig. 2A). Normal rabbit IgG did not precipitate the 170-kDa tyrosine-phosphorylated protein (data not shown). Furthermore, antibodies against the C or the N terminus of the IRS-1 protein did not immunoprecipitate the 170-kDa tyrosine-phosphorylated protein induced by IL-9 or IL-4 in TS1 cells (data not shown). These results suggest that TS1 cells do not express IRS-1 and that the 170-kDa tyrosine-phosphorylated protein induced by IL-9 or IL-4 in TS1 cells shares some antigenic epitopes with IRS-1. Based on the characteristics of this protein, it is most likely that the 170-kDa tyrosine-phosphorylated protein induced by IL-9 in TS1 cells is the IRS-1-related 4PS(19) .


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 10^7 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 10^7 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.

Demonstration for Tyrosine Phosphorylation of IRS-1 by JAK Tyrosine Kinases

We have demonstrated that IL-9 induces tyrosine phosphorylation of IRS-1 and IRS-1-related 4PS in TS1 T cells. It is important to identify possible tyrosine kinases responsible for tyrosine phosphorylation of IRS-1 and IRS-1-related 4PS in IL-9 signaling pathways. Because IL-9 activates JAK1 and JAK3 tyrosine kinases, we hypothesize that JAK1 and/or JAK3 may be tyrosine kinases capable of phosphorylating IRS-1 and IRS-1-related 4PS. It is known that overexpression of JAK kinases resulted in kinase autophosphorylation and activation (28) and that cotransfection of JAK kinase expression constructs with plasmids containing Stat cDNAs can result in tyrosine phosphorylation and activation of Stats(14, 20, 28) . To directly test our hypothesis, we subcloned IRS-1, JAK1, JAK2, and JAK3 cDNAs into mammalian cell expression vector pRc/CMV and performed cotransfection experiments with various combinations of IRS-1, JAK1, JAK2, or JAK3 cDNA plasmids in COS-1 cells. The results in Fig. 3, A and B, clearly indicated that cotransfection of IRS-1 plasmid with JAK1, JAK2, or JAK3 plasmids resulted in not only tyrosine phosphorylation and activation of JAK kinases but also tyrosine phosphorylation of IRS-1. Fig. 3A shows that JAK1 and JAK2 are specifically coimmunoprecipitated with IRS-1 because anti-IRS-1 antibodies did not precipitate any JAK kinases in COS-1 cells transfected with JAKs alone. (^2)The association of IRS-1 with JAK3 is too weak to be detected in these experiments. The efficiency of coprecipitation of IRS-1 with anti-JAK antibodies seems extremely low and is under detection (data not shown). The results from COS-1 cell transfection experiments strongly implicate that IRS-1 is a substrate for JAK kinases. Furthermore, in vitro experiments with immunoprecipitated JAK1 or JAK3 kinase activated by IL-9 in TS1 cells or overexpression in COS-1 cells showed that activated JAK kinases can phosphorylate IRS-1 on tyrosine residues (Fig. 3, C and D). Based on our cotransfection experiments in COS-1 cells and in vitro experiments, we conclude that JAK1, JAK2, and JAK3 kinases can directly interact with IRS-1 both in vitro and in vivo and that JAK 1 and JAK3 kinases are responsible for tyrosine phosphorylation of IRS-1 in IL-9 and IL-4 signaling pathways in T 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).




DISCUSSION

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 (PLX(4)NPXYXSXSD) (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.


FOOTNOTES

*
This work was supported in part by Grant IRG-161 (to T. Y.) from the American Cancer Society, by Grants R01HL48819, R01DK43105 (to Y. C. Y.), RO1DK42816 (to G. E. L.), P30CA21765, and PO1HL53745 (to J. N. I.) from the National Institutes of Health, and by funds from the American Lebanese Syrian Associated Charities (to J. N. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Scholar of the Leukemia Society of America. To whom correspondence should be addressed: Walther Oncology Center, Indiana University School of Medicine, IB 501, 975 West Walnut St., Indianapolis, IN 46202.

(^1)
The abbreviations used are: IL, interleukin; IRS-1, insulin receptor substrate-1; 4PS, IL-4-induced phosphorylated substrate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

(^2)
T. Yin, S. R. Keller, F. W. Quelle, B. A. Witthuhn, M. L.-S. Tsang, G. E. Lienhard, J. N. Ihle, and Y.-C. Yang, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. J. Van Snick at the Ludwig Institute for Cancer Research for providing the TS1 lymphocytes.


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