©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-13 Signal Transduction in Lymphohemopoietic Cells
SIMILARITIES AND DIFFERENCES IN SIGNAL TRANSDUCTION WITH INTERLEUKIN-4 AND INSULIN (*)

Melanie J. Welham (§) , Leslie Learmonth , Heather Bone , John W. Schrader (1)

From the (1) Biomedical Research Centre Department of Medicine, 2222 Health Sciences Mall, University of British Columbia, Vancouver V6T 1Z3, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Interleukin-13 (IL-13) and interleukin-4 (IL-4) are related in structure and function and are thought to share a common receptor component. We have investigated the signal transduction pathways activated by these two growth factors, as well as insulin, in cell-lines and primary cells of lymphohemopoietic origin. All three factors induced the tyrosine phosphorylation of a protein of 170 kDa (p170), which coimmunoprecipitated with the p85 subunit of PI3`-kinase, via high affinity interactions mediated by the SH2 domains of p85. Antibodies raised against the entire insulin-receptor substrate-1 (IRS-1) protein immunoprecipitated p170 much less efficiently than they did IRS-1 from 3T3 cells. However, antibodies directed against the conserved pleckstrin homology domain of IRS-1 immunoprecipitated both p170 and IRS-1 with similar efficiency, suggesting they share structural similarities in this region. In lymphohemopoietic cells, IL-13, IL-4, and insulin failed to induce increased tyrosine phosphorylation of Shc, or its association with grb2, modification of Sos1, or activation of erk-1 and erk-2 mitogen-activated protein kinases, suggesting that p170 mediates downstream pathways distinct from those mediated by IRS-1. Both IL-13 and IL-4 induced low levels of tyrosine phosphorylation of Tyk-2 and Jak-1. IL-4 also activated the Jak-3-kinase, but, despite other similarities, IL-13 did not. Insulin failed to activate any of the known members of the Janus family of kinases. In that Jak-3 is reported to associate with the IL-2c chain, these data suggest that the IL-13 receptor does not utilize this subunit. However, both IL-13 and IL-4 induced tyrosine phosphorylation of the IL-4-140 kDa receptor chain, suggesting that this is a component of both receptors in these cells and accounts for the similarities in signaling pathways shared by IL-13 and IL-4.


INTRODUCTION

The cytokines IL¹(¹) -4 and IL-13 share structural and functional features and define a newly defined subfamily of cytokines (1) . Both IL-4 and IL-13 are produced by activated Th2 cells (2) and have a similar range of target cells, with the exception that only IL-4 affects T lymphocytes (1) . Both share important, specialized functions in promoting class switching in human B cells to IgE production (3, 4) and down-modulating proinflammatory events in human monocytes and murine macrophages, increasing production of IL-1 receptor antagonist, and decreasing expression of IL-1 and , tumor necrosis factor-, and IL-12 (reviewed in Ref. 1). Interestingly, the effects of IL-4 and IL-13 are not synergistic, suggesting that many of these overlapping functions are controlled by common signaling processes, potentially emanating from common receptor components (see below).

The IL-4 receptor is composed of at least two subunits. The first to be characterized was a 140-kDa subunit, termed IL-4R (5, 6, 7) . A second subunit of the IL-4 high affinity receptor has recently been shown to be the IL-2 chain, or c (8, 9) . Cells in which both IL-4R 140-kDa chain and c are expressed bind IL-4 with affinities two to three greater than those measured in cells expressing only the IL-4R 140-kDa chain (8, 9) . At least two regions of the cytoplasmic domain of the IL-4R 140-kDa molecule have been reported to be essential for transmitting signals important for mediating cell growth. These regions span residues 258-390 and 437-557 (10, 11, 12) .

The nature of the IL-13 receptor is unknown. Competitive binding studies on TF-1 cells, which bind both IL-4 and IL-13, have demonstrated that IL-13 can cross-compete with IL-4 for binding, leading to the suggestion that their receptors share a common component (13). The c subunit has been proposed as a likely candidate (8, 9) . However, it is also possible that the 140-kDa IL-4 receptor is the shared component (see below).

IL-4 is unusual in that it fails to activate many of the pathways activated by other growth factors. IL-4 fails to activate p21(14, 15, 16) , induce tyrosine phosphorylation of Shc (17) , activate erk-1 and erk-2 kinases (16, 18) , or activate Raf-1 (19) . IL-4 also fails to induce tyrosine phosphorylation of p120 GAP (15, 20) , phospholipase C1 (20) , or SH-PTP2 (21) . In addition, in some, but not all cells, IL-4 fails to activate protein kinase C (22) , induce Ca mobilization (22, 23) , or activate phospholipase C (22, 23, reviewed in Ref. 24). However, IL-4 does activate PI3`-kinase (20, 25, 26) . The p85 subunit of PI3`-kinase has been shown to bind to the major substrate of IL-4-induced tyrosine protein kinases that has been termed 4PS (20, 25, 27) . 4PS is a 170-kDa protein, which is reported to weakly react with antibodies directed against the major insulin receptor substrate, IRS-1, suggesting some limited homology of 4PS/p170 to IRS-1 (27) . In the factor-dependent hemopoietic cell line 32D, IL-4 failed to induce tyrosine phosphorylation of 4PS and also failed to promote proliferation. However, after transfection of 32D with IRS-1, IL-4 stimulated increased tyrosine phosphorylation of IRS-1 and cell proliferation (28) . Recently, a region of the IL-4R 140-kDa subunit containing a motif shared with the insulin receptor has been shown to be important for mediating IRS-1 phosphorylation and cell growth of 32D cells (29) . This region maps to that previously shown to be important for IL-4 receptor mediated mitogenesis in Ba/F3 cells (10, 11, 12) . The c-fes kinase has been reported to associate with the IL-4 receptor (30) , although this kinase was not activated by stimulation with IL-4. It has also recently been shown that IL-4 activates Jak-1 and Jak-3 in T cells (31, 32) . Jak-1 may interact directly with the 140-kDa subunit of the IL-4 receptor (33) , and Jak-3 has been shown to associate with the IL-2c subunit (34, 35) . Activation of the Jak family of kinases is likely to be related to the ability of IL-4 to activate the Stat molecules termed NF-IL-4 (36) and Stat-IL-4 (37) or the recently cloned IL-4-Stat (38) .

Little is currently known about IL-13 signal transduction. Two pieces of evidence point to similarities with IL-4 signal transduction, one being the cross-competition studies, pointing to a shared receptor component (13) , and the second being the report that IL-13 activates NF-IL-4 (39) . We have characterized some of the early events which occur following treatment of cells with IL-13 and compared them to signals triggered by IL-4 and insulin in cell lines and primary cultures of lymphohemopoietic cells.


MATERIALS AND METHODS

Cell Culture

Cells were cultured in humidified incubators at 37 °C, 5% CO (v/v) in either RPMI 1640 medium or Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.), 20 µM 2-mercaptoethanol, 100 units of penicillin/streptomycin, and 2 mM glutamine. FDMAC11/4.5 (FD-5) is a variant of FDMAC11 which will grow in response to IL-4, as well as IL-3, granulocyte macrophage-colony stimulating factor, and colony stimulating factor (16) and was maintained in 3% (v/v) IL-4-conditioned medium derived from the AgX63/OMIL-4 cells (40) , which were a gift of Dr. F. Melchers (Basel Institute of Immunology, Basel, Switzerland). TF-1 is a human erythroleukemia cell line (41) and was maintained in RPMI supplemented with 3% (v/v) gibbon IL-3-conditioned medium derived from AgX63/gIL-3 cells. TF-1 cells respond to IL-3, IL-5, granulocyte macrophage-colony stimulating factor, Epo, IL-4, IL-13, and insulin (13, 41) . B9 is a murine plasmacytoma cell line and was grown in Dulbecco's modified Eagle's medium supplemented with 2% (v/v) of a 10-fold concentrated stock of conditioned medium from Swiss 3T3 cells as a source of IL-6 (42) . These cells respond to IL-13 and IL-4 (1) . (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (MTT) assays were performed as described previously (43) . CT.4S, HDK-1, and D10 were grown as described previously (16, 44, 45) . Populations of primary murine ConA-activated splenic T cells and murine lipopolysaccharide-(LPS) activated splenic B cells were prepared as described previously (46, 47) .

Cell Stimulation and Growth Factors

Stimulation of cells with different growth factors was carried out as described previously (17). Synthetic forms of murine IL-3, IL-13, IL-4, and human IL-4 were kindly provided by Dr. Ian Clark-Lewis (The Biomedical Research Centre, Vancouver, British Columbia). mIL-13 has been demonstrated to bind equally well to cells of both human and murine origin (13) . Porcine insulin was purchased from Sigma. mIL-2 was purchased from Genzyme. Gibbon IL-3 expressed in AgX63 cells is fully bioactive on human cells and was used as a source of IL-3 for studies with TF-1 cells. Cells were stimulated for a length of time and with a particular concentration of growth factor which had been previously determined to induce maximal levels of tyrosine phosphorylation. These were as follows, mIL-3 at 10 µg/ml (17) , mIL-13 at 20 µg/ml, mIL-4 at 20 µg/ml (16) , mIL-2 at 100 units/ml (46) , hIL-4 at 20 µg/ml, and insulin at 5 µg/ml. All stimulations were carried out for 10 min, except for those with insulin, which were carried out for 2 min. In some cases time course analyses were performed to examine the kinetics of tyrosine phosphorylation and are indicated in the appropriate figures. Cell pellets were lysed in immunoprecipitation buffer (IP buffer: 50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 10 mM sodium fluoride, 40 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml soyabean trypsin inhibitor, 10 µg/ml leupeptin, 0.7 µg/ml pepstatin).

Subcellular Fractionations

Cytosol (C) and crude membrane (M) fractions were prepared using sonication buffer (150 mM NaCl) or homogenization buffer (0 mM NaCl) as described previously (17) . Briefly, cells were disrupted on ice by 4 2-s pulses using a Sonics and Materials ``Vibra Cell'' (Danbury, CT), or by 100 strokes of a tight fitting Dounce homogenizer. Similar results were obtained irrespective of the mechanical procedure used. Nuclei and intact cells were removed by centrifugation for 15 s, at 4 °C, in a microcentrifuge at full speed. The supernatant was then subjected to centrifugation at 150,000 g at 4 °C for 20 min. The resulting supernatant (S150) was designated the cytosol and the pellet (P150) the crude membrane. The pellet was solubilized in the appropriate buffer containing 1% Nonidet P-40 and 0.5% sodium deoxycholate and recentrifuged to remove remaining insoluble material. The cytosolic fraction was adjusted to contain 1% Nonidet P-40.

Immunoprecipitations

Extracts were precleared with 25 µl of a 50% (v/v) slurry of protein A-Sepharose beads (Pharmacia) for 15 min, at 4 °C on a rotator prior to addition of the appropriate antibody. The following antibodies were purchased from UBI, Lake Placid, NY with the amounts used per IP stated in parentheses: polyclonal rabbit antibodies to either the p85 subunit of PI3`kinase (2 µl), Jak-1 (06-272, 3 µl), Jak-2 (2 µl), Jak-3 (5 µl), Shc (2 µl), or IRS-1 (2 µl). A polyclonal antibody recognizing Tyk-2 was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA) and 2 µg/sample used for immunoprecipitations. Anti-human Jak-3 antibodies (31) were a kind gift of Dr. J. O'Shea (National Cancer Institute, Frederick, MD); 3 µl were used per sample. Monoclonal anti-IL-4 receptor antibody (M2) was a kind gift of Immunex Corporation (Seattle, WA); 5 µg were used per sample. Polyclonal rabbit antibodies to the pleckstrin homology domain of IRS-1 were raised against a GST fusion protein comprising amino acids 1-124 of murine IRS-1 fused to GST²(²) ; 10 µl of antibody were used per sample. After addition of antibody, the samples were vortexed and incubated on ice for 30 min to 1 h. 40 µl of a 50% (v/v) slurry of either protein A-Sepharose beads (for rabbit antibodies) or protein G-Sepharose beads (for mouse monoclonal antibodies) were added to each IP and samples incubated at 4 °C on a rotator for 1 h. Immunoprecipitates were washed three times with IP buffer at 4 °C. Bound proteins were eluted by boiling in SDS-PAGE sample buffer containing 2-mercaptoethanol. Aliquots of cell extracts were removed both prior to (``Pre IP'') and following (``Post IP'') immunoprecipitation and were diluted with the same SDS-PAGE sample buffer as the immunoprecipitates.

GST Fusion Proteins

The construction, expression, and purification of full-length human grb2 and p85 PI3`-kinase SH2 domain GST fusion proteins have been previously described (17, 48) . 2 µg of either the PI3`-kinase amino-terminal SH2 (N-SH2) or carboxyl-terminal SH2 (C-SH2) fused to GST or 5 µg of the full-length human grb2-GST were used per precipitation.

Kinase Assays

Assays were performed as described previously (16). Anti erk-1 and anti erk-2 antibodies conjugated to beads were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and 30 µl of a 50% (v/v) slurry was used per immunoprecipitation.

SDS-PAGE and Immunoblotting

SDS-PAGE and immunoblotting were carried out as described previously (17, 49) . Primary antibodies were used at the following concentrations: the antiphosphotyrosine monoclonal antibody 4G10 at 0.1 µg/ml, the polyclonal anti-Shc antibody at 0.25 µg/ml, polyclonal anti-Sos1 at 1:4000, anti-PI3`-kinase antibody at 1:4000, anti-Jak-1 and Jak-3 antibodies at 1:2000, and anti-Tyk-2 antibody at 0.2 µg/ml. Both goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated antibodies (Dako, Dimension Laboratories, Missisauga, Ontario) were used at a concentration of 0.05 µg/ml. Immunoblots were developed using the ECL system (Amersham). Kodak X-AR 5 film was used for detection of ECL signals. Blots were stripped completely of antibodies by incubation at 55 °C for 60 min with stripping solution (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 100 mM 2-mercaptoethanol). After extensive washing, blots were reblocked prior to reprobing. The results observed in different experiments were independent of the sequence of antibodies used for probing the immunoblots.

RESULTS

Induction of Tyrosine Phosphorylation in Lymphohemopoietic Cells in Response to IL-13, IL-4, and Insulin

We examined the responsiveness of a number of cell lines of lymphohemopoietic origin to IL-13, IL-4, and insulin. In each case the ability of the individual factors to induce tyrosine phosphorylation of proteins was assessed by immunoblotting with antiphosphotyrosine antibodies, and the results are shown in Fig. 1 . In both TF-1 (Fig. 1A) and B9 cells (Fig. 1C), IL-13, IL-4, and insulin all induced the tyrosine phosphorylation of a major species of 170 kDa. This protein is similar in molecular mass to the protein termed 4PS, which had been shown by others to be the major substrate which becomes phosphorylated on tyrosine residues following IL-4 and insulin treatment of murine myeloid cells (20, 25, 27, 28, 29) . In biological response MTT assays, both TF-1 and B9 responded to IL-13 and IL-4 (data not shown). Interestingly, both IL-4 and IL-13 induced tyrosine phosphorylation of a 112-kDa protein in TF-1 cells. In addition, IL-4, but not IL-13, was also capable of inducing tyrosine phosphorylation of a protein of 115 kDa in TF-1 (see below). Treatment of the myeloid line FD-5 with either IL-4 or insulin, but not IL-13, induced p170 tyrosine phosphorylation (Fig. 1B). Treatment of two T cell lines, HDK-1 (of Th1 phenotype, Fig. 1D) and D10 (of Th2 phenotype, Fig. 1E) with either IL-4 or insulin (but not IL-2) induced tyrosine phosphorylation of a 170-kDa protein. In both HDK-1 and D10, IL-4 also induced phosphorylation of a protein of 135-140 kDa, likely to be the IL-4 receptor. Importantly, treatment with IL-4 of the T cell line CT.4S, which is absolutely dependent on the presence of IL-4 for its continued proliferation, did not induce detectable tyrosine phosphorylation of p170 (Fig. 1F), although both IL-4 and IL-2 induced tyrosine phosphorylation of a 115-kDa protein.


Figure 1: Induction of tyrosine phosphorylation by IL-13, IL-4, and insulin in lymphohemopoietic cells. All cells were factor deprived for 16 h prior to stimulations, with the exception of B9 cells. All samples were separated by SDS-PAGE using 7.5% acrylamide gels and immunoblotting performed with 4G10 antiphosphotyrosine antibodies. Samples from control untreated cells are labeled C in each case. The position of molecular mass standards are shown and expressed in kDa. The positions of p170, p140, p115, and p112 are also indicated. A, TF-1 cells were treated for 10 min with either IL-3 (3), hIL-4 (4), or mIL-13 (13) or for 2 min with insulin (I). B, FDMAC11/4.5 (FD-5) cells were treated for 2 min with insulin (I) or for 10 min with either mIL-4 (4) or mIL-13 (13). C, B9 cells were treated for 2 min with insulin (I) or for 10 min with either mIL-4 (4) or mIL-13 (13). D-F, time course analyses were performed to examine kinetics of tyrosine phosphorylation. Cells were treated with factor for the indicated periods of time. D, HDK-1 cells were treated with insulin (INS), IL-2, or IL-4. E, D10 cells were treated with insulin (I), IL-2 (2), or IL-4. F, CT.4S cells were treated with either IL-4 or IL-2.



It was important to investigate the effects of IL-13, IL-4, and insulin not only on cell lines, but also on primary cells. Therefore, we generated populations of murine ConA-activated splenic T cells or LPS-activated splenic B cells, stimulated them with IL-2, IL-4, IL-13, or insulin, and subjected cell extracts to SDS-PAGE and immunoblotting with 4G10. As shown in Fig. 2, IL-4 and insulin both induced tyrosine phosphorylation of a 170-kDa protein in primary populations of T cells (Fig. 2A and data not shown) and B cells (Fig. 2B). IL-13 failed to stimulate p170 tyrosine phosphorylation in populations of T cells (data not shown) or B cells (Fig. 2B), although it has been reported to be active on human B cells (1) .


Figure 2: Induction of tyrosine phosphorylation in primary T and B lymphocytes. A, primary murine ConA-activated CD8-depleted splenic T cells were deprived of IL-2 for 16 h and then either left untreated as a control (C) or treated with IL-4 for the indicated periods of time or with IL-2 for 10 min. B, murine LPS-activated splenic B cells were either left untreated as a control (C) or treated for 10 min with IL-4 (4), 10 min with IL-13 (13), or 2 min with insulin (I). Samples were separated through 7.5% acrylamide gels by SDS-PAGE and immunoblotting performed with 4G10. The molecular mass standards are indicated in kDa. The position of p170 is also indicated.



p170, Tyrosine Phosphorylated in Response to IL-13, Co-immunoprecipitates with p85 PI3`-kinase.

We were interested in determining if the p170 protein, tyrosine phosphorylated in response to IL-13, had similar properties to the tyrosine-phosphorylated 170/4PS, which we³(³) and others have shown to coimmunoprecipitate with the p85 subunit of PI3`-kinase (20, 25, 27) . TF-1 cells were stimulated with IL-13, IL-4, insulin, and IL-3 or left untreated as a control. Immunoprecipitations were performed using antibodies directed against the p85 subunit of PI3`-kinase, and resulting precipitates were immunoblotted with antiphosphotyrosine antibodies. The results are shown in Fig. 3A (upper panel, also see Fig. 8B). In the IL-13-, IL-4-, and insulin-treated samples, a tyrosine-phosphorylated 170-kDa protein coimmunoprecipitated with the p85 subunit of PI3`-kinase. Similar results were observed in B9 cells (see Fig. 3B, upper panel) and also in FD-5, LPS-activated splenic B cells, and ConA-activated splenic T cells in response to IL-4 or insulin (data not shown). In each case equal amounts of p85 were immunoprecipitated (Fig. 3, A and B, lower panels). Thus, the IL-13 p170 substrate behaved in a similar manner to 4PS and IRS-1 in associating with p85 (20, 50) .


Figure 3: IL-13 induces coimmunoprecipitation of p170 with PI3`-kinase. A, factor-deprived TF-1 cells were either left untreated as a control (C) or treated for 10 min with hIL-4 (4), 10 min with mIL-13 (13), 2 min with insulin (I), or 10 min with gIL-3 (3). Cell extracts, from the equivalent of 2 10 cells/sample, were immunoprecipitated with 2 µl of anti-p85 PI3`-kinase antibody. B, B9 cells were either left untreated as a control (C) or stimulated for 10 min with mIL-4 (4), 10 min with mIL-13 (13), or 2 min with insulin (I). The equivalent cell extract from 2 10 cells were used for immunoprecipitation with 2 µl of anti-PI3`-kinase antibody. C, factor-deprived TF-1 cells were treated as follows: control, untreated (C); 10 min IL-3 (3); 10 min hIL-4 (4); 10 min mIL-13 (13), or 2 min insulin (I). Cell extracts were divided equally into two aliquots, each containing the equivalent of 2.8 10 cells/sample and precipitates performed using 2 µg of either purified N-SH2-GST or C-SH2-GST. Immunoblotting was performed first with 4G10, antiphosphotyrosine antibody (A and B, upper panels, and C). The same immunoblots as in A and B were stripped and reprobed with antibodies directed against p85 to monitor for efficiency of immunoprecipitation in each case (A and B, lower panels). The positions of molecular mass standards are shown and expressed in kDa. The positions of immunoglobulin heavy chain (IgG, contributed by the immunoprecipitating antibody), p170, and p85 are indicated.




Figure 8: Activation of Jak family kinases by IL-13 and IL-4 in TF-1 cells. A, factor-deprived TF-1 cells were either left untreated (C) or treated for 10 min with either IL-3 (3), hIL-4 (4), or mIL-13 (13). Extracts from the equivalent of 5 10 cells were then immunoprecipitated with antibodies specifically recognizing either Jak-1, Jak-3, or Tyk-2. After extensive washing, immunoprecipitates were separated by SDS-PAGE through 7.5% acrylamide gels and immunoblotting carried out first with 4G10 (upper panels in each case). Blots were stripped and reprobed with the appropriate immunoprecipitating antibody (indicated underneath the lower panel in each case). Positions of Jak-1, Jak-3, and Tyk-2 are indicated, as are the molecular mass standards (in kDa). The small arrowhead denotes the position of tyrosine phosphorylated Jak-1. B, TF-1 cells were treated with IL-4 (4) or IL-13 (13) for 10 min or left untreated as a control (C). Samples containing the same number of cell equivalents were removed both prior to (Pre IP) and following immunoprecipitation with either Jak-3 or p85 PI3`-kinase antibodies (Post IP). Immunoprecipitates are also shown. Immunoblotting was performed with 4G10. Blots were then stripped and sequentially reprobed with anti-Jak-3 and anti-p85 antibodies. The positions of p170, Jak-3, and p85 are indicated. The upper small arrowhead denotes p170 and the lower small arrowhead p115/Jak-3. C, B9 cells, and D, LPS-activated B cell blasts were treated for 10 min with either mIL-4 (4) or mIL-13 (13), for 2 min with insulin (I) or left untreated as a control (C). Immunoprecipitations were performed with anti-Jak-3 antibodies (B9 and LPS-activated B cells) or anti-Jak-1 antibodies (LPS-activated B cells) and immunoblotted with 4G10. The same blots were stripped and reprobed with anti-Jak-3 or anti Jak-1 antibodies as appropriate.



The SH2 Domains of p85 PI3`-kinase Direct Interaction with p170

We investigated the mechanism responsible for the interaction between p170 and PI3`-kinase. The p85 subunit of PI3`-kinase contains two SH2 domains, which mediate interaction with phosphotyrosine residues having a consensus sequence of Y*XXM (51) . We investigated the ability of fusion proteins, containing either the amino-terminal (N-SH2) or carboxyl-terminal (C-SH2) SH2 domain of p85 fused to GST (48) to precipitate p170 from IL-13-, IL-4-, or insulin-treated TF-1 cells. The results are shown in Fig. 3C. Both the amino-terminal and the carboxyl-terminal SH2 domains of p85 immunoprecipitated tyrosine phosphorylated p170 from IL-13, IL-4, and insulin-treated cells. This complex was stable to 0.1% SDS and 0.5 M NaCl, indicating that it was a stable, high affinity interaction (data not shown). An additional protein of 115 kDa also coimmunoprecipitated with the N-SH2 and C-SH2 constructs from IL-4-treated cells. Similar results were obtained using the myeloid cell line FD-5 (data not shown).

Subcellular Localization of p170

Initial reports suggested that 4PS was a membrane-associated molecule (20) . We investigated the localization of p170 in FD-5, TF-1, and B9. Cells were fractionated into cytosol (C) and membrane components (M), either by sonication in buffer containing 150 mM NaCl (isotonic) or in homogenization buffer containing no NaCl (hypotonic). Cell fractions were separated by SDS-PAGE and immunoblotting performed with 4G10-antiphosphotyrosine antibodies. The results are shown in Fig. 4 . The subcellular location of tyrosine-phosphorylated p170 appeared to be dependent on the salt concentration of the buffer used during the fractionation procedure. In the presence of 150 mM NaCl (isotonic), the majority of tyrosine phosphorylated p170/4PS was found in the cytosolic fractions of all the cells, with only small amounts present at the membrane (Fig. 4, A-C, NaCl 150 mM). In the absence of NaCl (hypotonic conditions), a larger percentage of tyrosine-phosphorylated p170 was located in the crude membrane fraction (Fig. 4, A-C, NaCl 0 mM). Comparable results were obtained for FD-5 (Fig. 4A) and TF-1 (Fig. 4B) and suggest that tyrosine-phosphorylated p170 interacts with membrane proteins in a salt-sensitive manner. In B9 cells the overall tyrosine phosphorylation of p170 appears to increase in the homogenized samples, the reason for which is unclear, but may simply reflect variability between stimulations. In addition in B9, the association of tyrosine-phosphorylated p170 with the membrane following IL-4 and IL-13 treatment in the absence of NaCl is less pronounced than the results we observed with insulin in B9 cells. Further studies are underway to investigate this in more detail. In previous studies we have shown that fractionation in isotonic buffer does result in the expected localization of other proteins in the crude membrane fraction. Thus, integral membrane proteins, e.g. c-kit and IL-3 receptor c, were found only in crude membrane fractions, and we could detect the characteristic PMA-induced translocation of PKC to the plasma membrane in mast cells (17) . Reprobing the immunoblots shown here with anti-c-src antibodies demonstrated that greater than 90% of c-src was associated with the crude membrane fraction, irrespective of the buffer system used (data not shown).


Figure 4: Subcellular localization of p170. FD-5 (A), TF-1 (B), and B9 (C) cells were factor deprived and then either left untreated (CON), or treated for 10 min with either IL-4, or IL-13, or for 2 min with insulin (INS). Cells were fractionated using either sonication (150 mM NaCl) or homogenization buffer (0 mM NaCl) as described under ``Materials and Methods.'' All samples were disrupted by sonication except those in A (0 mM NaCl), where the samples disrupted in a Dounce homogenizer. Similar results were obtained in three independent experiments for each cell line with a representative experiment shown in each case. Results were independent of the mechanical procedure used. Total cytosol (C) and crude membrane fractions (M), containing the same cell equivalents were separated by SDS-PAGE and immunoblotted with 4G10. Note the order of the fractions in panel C. The molecular mass standards are indicated (in kDa) and the position of p170 is indicated.



p170 Is a Distinct Species from IRS-1

It has been reported, based on weak antibody cross-reactivity, that 4PS/p170 is distinct, but related, to the insulin receptor substrate-1 molecule, IRS-1 (27) . However, these data can be interpreted in two ways; either the myeloid cells being studied could have expressed extremely low levels of authentic IRS-1 or the polyclonal anti-IRS-1 serum contained a minor subset of antibodies which cross-reacted with 4PS. One-dimensional phosphopeptide mapping of P-labeled material demonstrated some differences between 4PS and IRS-1 (27) , but these could simply reflect different patterns of phosphorylation of IRS-1 in different cells. We used two approaches to attempt to clarify this issue using antibodies with different specificities.

We prepared antibodies against the conserved pleckstrin homology domain of IRS-1 (-IRS-1PH) and compared these antibodies with a commercially available polyclonal antibody raised against the entire rat IRS-1 protein (-IRS-1) for the ability to immunoprecipitate tyrosine-phosphorylated p170/4PS from myeloid cells and IRS-1 from 3T3 fibroblasts. The commercial antiserum immunoprecipitated tyrosine-phosphorylated IRS-1 efficiently from insulin-treated Swiss 3T3 cells, but precipitated only low amounts of tyrosine-phosphorylated p170 from FD-5 cells or TF-1 cells (Fig. 5A). Interestingly, tyrosine-phosphorylated p170 from FD-5 and TF-1 consistently migrated more slowly on SDS-PAGE than did IRS-1 from Swiss 3T3 cells, suggesting that it may be a distinct protein. The anti-IRS-1PH antibody was equally efficient at immunoprecipitating a tyrosine-phosphorylated p170 species from both FD-5 and Swiss 3T3 following insulin treatment (Fig. 5B) and also precipitated tyrosine-phosphorylated p170 from IL-4-treated FD-5 (Fig. 5B) and TF-1 cells (data not shown). The immunoprecipitates from insulin-treated Swiss 3T3 cells appeared to contain a doublet of tyrosine-phosphorylated proteins in the 170 kDa range. In sequential immunoprecipitation analyses, extracts of 3T3 or FD-5 were first immunoprecipitated with the anti-IRS-1PH antibody. After extensive washing, samples were eluted, denatured by boiling in SDS-PAGE sample buffer (without bromphenol blue), and half of the sample subjected to a secondary immunoprecipitation using antibodies raised against the entire IRS-1 protein. The primary immunoprecipitates are those shown in Fig. 5B. After extensive washing, reprecipitated proteins were eluted and immunoblotted with 4G10. The results of the secondary immunoprecipitations are shown in Fig. 5C. While the IRS-1PH antibody precipitated similar amounts of tyrosine-phosphorylated IRS-1 and p170 from 3T3 and FD-5, respectively (Fig. 5B), the commercial anti-IRS-1 antibody reimmunoprecipitated only the lower of the tyrosine-phosphorylated proteins, presumably IRS-1, from 3T3 cells (Fig. 5C). This provides clear evidence that IRS-1 and p170 are distinct species, and it is likely that p170/4PS plays a major role in transducing IL-4, IL-13, and insulin signals in lymphohemopoietic cells.


Figure 5: p170 is distinct from IRS-1. Factor-deprived FD-5 were either left untreated as a control (C) or stimulated for 10 min with IL-4 (4) or 2 min with insulin (I). Factor-deprived TF-1 cells were either left untreated as a control (C), stimulated for 10 min with either IL-3 (3), IL-4 (4), or IL-13 (13) or for 2 min with insulin (I). Swiss 3T3 cells (3T3) were deprived of serum for 24 h prior to stimulation for 2 min with insulin (I) or left untreated as a control (C). A, cell extracts were immunoprecipitated using a commercially available polyclonal antibody raised against the entire coding sequence of rat IRS-1, termed -IRS-1. B, samples were removed prior to immunoprecipitation (Pre IP) to demonstrate that the relative levels of the tyrosine-phosphorylated 170-kDa protein (p170 or IRS-1) in the different cells was similar. Cell extracts were then immunoprecipitated with a polyclonal antibody raised against the conserved pleckstrin homology domain of IRS-1, termed -IRS-1PH. C, aliquots of the same primary immunoprecipitations shown in B were used for secondary immunoprecipitation with the aIRS-1 antibody. Primary -IRS-1PH precipitates were eluted, denatured by boiling in SDS sample buffer (without bromphenol blue) and reimmunoprecipitated using the commercial -IRS-1 antibody. Immunoprecipitates were separated by SDS-PAGE and immunoblotting performed with 4G10. The position of tyrosine phosphorylated p170 and IRS-1 are indicated, as are the molecular mass standards (in kDa).



Effects of IL-13 on Shc Tyrosine Phosphorylation and Association of Shc and p170 with grb2

Given the similarities in molecular weight, subcellular localization and the ability to coimmunoprecipitate with p85 PI3`-kinase, it was important to examine whether 4PS/p170 interacted with the same proteins as IRS-1 and was involved in similar functions. We examined the ability of IL-13, IL-4, and insulin to induce tyrosine phosphorylation of the adaptor molecule Shc, which has been implicated as an upstream regulator of ras(52, 53) . TF-1 cells were treated with either IL-3 (which is known to induce Shc tyrosine phosphorylation, 17), IL-13, IL-4, or insulin or left untreated as a control. Precipitations were performed with either anti-Shc polyclonal antiserum, or with a grb2-GST fusion protein which only binds Shc that has been tyrosine phosphorylated at Tyr (the grb2 recognition site, 54). Precipitates were subjected to SDS-PAGE, and immunoblotting was performed with antiphosphotyrosine antibodies. The results are shown in Fig. 6, A and B. As we had observed in other cell types (17) , IL-3 induced an increase in tyrosine phosphorylation of p50 and p55 in TF-1 cells (Fig. 6A), although there was a detectable basal level of Shc tyrosine phosphorylation in these cells. This tyrosine-phosphorylated Shc was also recognized by the grb2-GST fusion protein, along with a number of other phosphotyrosine-containing proteins (Fig. 6B). IL-13, IL-4, and insulin all failed to induce increased tyrosine phosphorylation of Shc proteins as judged by precipitation with anti-Shc antibodies, or with the grb2-GST fusion protein. Reprobing the precipitates with anti-Shc antibodies (Fig. 6, A and B, lower panels), demonstrated equal amounts of Shc in the anti-Shc immunoprecipitates (Fig. 6A) and an increase in Shc present in the grb2-GST fusion protein precipitates from IL-3, but not IL-4-, IL-13-, or insulin-treated TF-1 cells (Fig. 6B). However, the grb2-GST fusion protein was able to coprecipitate p170 from cells treated with IL-13, IL-4, or insulin, indicating that these factors induce phosphorylation of p170/4PS on a tyrosine that is recognized by grb2.


Figure 6: IL-13, IL-4, and insulin fail to induce tyrosine phosphorylation of Shc or its association with grb2 in TF-1 cells. TF-1 cells were factor deprived for 16 h, and cells were then either left untreated as a control (C) or treated for 10 min with either hIL-4 (4), mIL-13 (13), gIL-3 (3), or for 2 min with insulin (I). A, cell extracts equivalent to 2 10 cells were subjected to immunoprecipitation with 2 µl/sample anti-human Shc antibodies. IPs were separated by SDS-PAGE and immunoblotting performed first with 4G10 (upper panel). The blot was stripped and reprobed with anti-Shc antibodies (lower panel). B, cell extracts equivalent to 3 10 cells were used per sample, and following lysis 5 µg of full-length human grb2-GST fusion protein was added per sample. Coprecipitating proteins were analyzed by immunoblotting with 4G10 (upper panel). A duplicate immunoblot was probed with antibodies to Shc (lower panel). The positions of the molecular mass standards are shown (in kDa), as are the positions of p170 and Shc. C, factor-deprived TF-1 cells were treated for 10 min with either gIL-3 (IL-3), hIL-4 (IL-4), or mIL-13 (13) or treated with insulin for 2 min (INS). Cells were fractionated into cytosol and membrane components using sonication buffer and aliquots containing the equivalent of 2.6 10 cells/sample separated through 7.5% acrylamide gels by SDS-PAGE. Immunoblotting was performed using anti Sos1 antibodies (17, 54).



Effects of IL-13 on mSos1

Shc and grb2 are believed to be key adaptor molecules in mediating the relocation of the ras nucleotide-exchange factor, Sos1, to the plasma membrane, where ras is located (55) . The observation that tyrosine-phosphorylated p170 can be detected at the membrane (see Fig. 4 ) and that grb2 can bind to p170/4PS raised the possibility that this interaction could mediate the relocation of Sos1 to the plasma membrane. In addition, Sos1 is modified in its electrophoretic mobility following stimulation of cells with epidermal growth factor (54) , IL-3, and SLF, but not IL-4 (17) . To determine whether IL-13 induced either a translocation of Sos1 to the membrane or a change in its electrophoretic mobility, TF-1 cells were treated with either IL-3, IL-4, IL-13, or insulin, or left untreated as controls and were fractionated into cytosol and crude membrane components, in the presence of 150 mM NaCl. Aliquots, containing equal cell equivalents, were subjected to SDS-PAGE and immunoblotted with anti Sos1 antibodies (54) . The results shown in Fig. 6C indicate that IL-13, IL-4, and insulin all failed to induce detectable translocation of Sos1 to the membrane fraction. As expected from our previous experiments (17) , IL-3, but not IL-4, induced a retardation in electrophoretic mobility of Sos1. Insulin and IL-13 both resembled IL-4 in failing to induce this change in Sos1.

Effects of IL-13 on erk-1 and erk-2 MAP Kinase Activity

The family of extracellular signal regulated (erk) or mitogen-activated protein (MAP) kinases is a group of serine/threonine kinases which are thought to play a central role in mitogenesis. We had previously failed to detect any increases in either erk-1 or erk-2 activity following treatment of different lymphohemopoietic cells with IL-4 (16, 18) . To examine the effects of IL-13 on regulation of activity of the erk-1 and erk-2 members of the MAP kinase family, TF-1 were stimulated either with IL-3, PMA, IL-4, IL-13, or insulin or left untreated as controls. MAP kinases were immunoprecipitated from cell extracts and in vitro kinase assays performed on the immunoprecipitates. Results of a typical experiment are shown in Fig. 7 , and summarizes the results of multiple analyses. In no case did we detect significant increases in erk-1 or erk-2 activity following treatment of TF-1 cells with IL-13, IL-4, or insulin. erk-1 and erk-2 MAP kinases were activated by IL-3 or PMA in TF-1 cells. Thus, IL-13 like IL-4 (16, 18) , failed to activate erk-1 or erk-2. In B9 cells, IL-13 and IL-4 also failed to induce activation of either erk-1 or erk-2 (data not shown). Interestingly, although insulin failed to activate erk-1 in TF-1 cells, in Swiss 3T3 cells insulin induced activation of both erk-1 and erk-2 (see ). In addition, insulin did induce activation of erk-1 in another hemopoietic cell line, FD-5 (see ), although insulin is not mitogenic for these cells.()


Figure 7: IL-13 does not induce erk-1 activity in TF-1 cells. 4.6 10 factor-deprived TF-1 cells were either left untreated as controls (C) or treated for 10 min with either IL-3 (3), hIL-4 (4), IL-13 (13), or PMA (P) or for 2 min with insulin (INS). Immunoprecipitations were carried out with anti erk-1 beads, and in vitro immune complex kinase assays were carried out in the presence of MBP. A, the incorporation of P into MBP (indicated). B, immunoblotting with an anti-erk-1 antibody was performed to confirm immunoprecipitation of erk-1. The positions of the heavy chain of IgG (from the immunoprecipitating antibody) and p44 are indicated. C, MBP bands were excised and counted in a scintillation counter. The counts obtained were used to calculate the fold activation in erk-1 activity following factor treatment. The levels in the controls were taken as 1, and the results are depicted graphically.



Effects of IL-13, IL-4, and Insulin on Jak Family Kinases

It has recently been reported that IL-4 induces activation of Jak-1 and Jak-3 kinases in human and murine T cells (31, 32) and that Jak-3 associates with the c chain shared by the IL-2, 4, 7, and 9 receptors (34, 35) . It has been suggested that the c chain may also be part of the IL-13 receptor (8, 9) . Therefore, we examined the repertoire of Jak kinases activated by these individual growth factors in TF-1. The results are shown in Fig. 8A and summarized in . In all cases immunoblotting was carried out with 4G10 first (upper panels), and blots were then stripped and reprobed with the appropriate anti-Jak antibody to control for efficiency of immunoprecipitation (lower panels). Both IL-13 and IL-4 stimulated increased tyrosine phosphorylation of Tyk-2 and Jak-1 kinases, although the levels of phosphorylated Jak-1 were extremely low in TF-1 cells. Tyk-2 appeared to be constitutively tyrosine phosphorylated, even in factor-deprived TF-1 cells, although both IL-13 and IL-4 consistently induced increased tyrosine phosphorylation of Tyk-2. As predicted from studies of IL-4 on T cells, we observed increased tyrosine phosphorylation of Jak-3 in response to IL-4 treatment in TF-1 cells. This tyrosine-phosphorylated Jak-3 appears to correspond to the prominent p115 protein substrate we had observed in total cell extracts (Fig. 1A and 8B). In immunodepletion studies (Fig. 8B), the intensity of the p115 band (denoted by the lower small arrowhead in IL-4 Post IP samples, Fig. 8B) was decreased in extracts following Jak-3 immunoprecipitation, whereas p170 was not depleted (denoted by the upper small arrowhead). In extracts which had been immunoprecipitated with anti-p85 PI3`-kinase antibodies, p170 was depleted but p115 was not (see Fig. 8B). Reprobing the immunoblots with either anti-Jak-3 (Fig. 8B, centerpanel), or anti-p85 PI3`-kinase (Fig. 8B, lower panel) antibodies demonstrated the expected partial immunodepletion of both Jak-3 and p85 in the Post IP samples. Therefore, in TF-1 cells Jak-3 appears to be one of the major proteins phosphorylated on tyrosine in response to IL-4. The p115 tyrosine-phosphorylated protein observed in CT.4S cells following treatment with IL-4 or IL-2 (Fig. 1F) also appears to correspond to Jak-3. Interestingly, in repeated experiments IL-13 failed to cause an increase in Jak-3 tyrosine phosphorylation.

We also examined Jak kinase tyrosine phosphorylation in FD-5-, B9-, and LPS-activated splenic B cells. The results are summarized in . In B9 cells we detected low levels of tyrosine-phosphorylated Jak-1 and Tyk-2 in response to IL-4 and IL-13, but not insulin. Jak-3 appeared to have a high constitutive level of tyrosine phosphorylation, which did not alter after factor deprivation or treatment with cytokines (see Fig. 8C). In the LPS-activated splenic B cells, IL-4 induced Jak-1 and Jak-3 tyrosine phosphorylation, in contrast to insulin which failed to induce tyrosine phosphorylation of either of these kinases. IL-13 treatment of this population of murine B cells induced no detectable effects on Jak kinases (Fig. 8D).

IL-13 Induces Tyrosine Phosphorylation of IL-4R 140-kDa Subunit

The failure of IL-13 to induce tyrosine phosphorylation of Jak-3 argued against the c chain having a role in the functional IL-13 receptor complex. Jak-1 is reported to associate with the IL-4 140-kDa receptor subunit (33) and the activation of Jak-1 by IL-13, albeit at low levels, prompted us to examine the possible functional role of the 140-kDa IL-4 R chain in the IL-13 receptor complex. Therefore, we examined the ability of IL-13 to induce tyrosine phosphorylation of the IL-4 140-kDa subunit in the murine cell line B9. Cells were either left untreated as a control or stimulated for 10 min with either IL-4 or IL-13 and immunoprecipitates prepared using monoclonal antibodies specific for the 140-kDa subunit of the IL-4 receptor. Immunoblotting was performed with 4G10. As is apparent in Fig. 9A, both IL-4 and IL-13 induced tyrosine phosphorylation of the 140-kDa IL-4 R in B9 cells. In LPS-activated splenic B cells, only IL-4 was able to induce tyrosine phosphorylation of the 140-kDa IL-4 receptor, consistent with the lack of effect of IL-13 on these cells (Fig. 9B).


Figure 9: IL-13 induces tyrosine phosphorylation of the IL-4 receptor 140-kDa subunit. B9 cells (A) and LPS-activated B cell blasts (B) were treated for 10 min with either mIL-4 (4), or mIL-13 (13), or for 2 min with insulin (I) or left untreated as controls (C). Immunoprecipitations were performed with 5 µg of anti IL-4 receptor antibody M2. Immunoblotting was performed with 4G10. The positions of the molecular mass standards are indicated, as is the position of the 140-kDa IL-4 receptor.



DISCUSSION

In this report we have demonstrated that IL-13 shares major signal transduction pathways with the structurally and functionally related cytokine, IL-4. We have defined the major substrate of IL-13-activated tyrosine kinases as the molecule termed p170, which is likely to be equivalent to the IL-4 substrate termed 4PS (20) . IL-13, IL-4, and insulin induced tyrosine phosphorylation of a 170-kDa protein in both human myeloid cells (TF-1) and murine lymphoid cells (B9). IL-4 also induced the tyrosine phosphorylation of p170 in primary cultures of murine splenic T cells and B cells and FD-5, whereas IL-13 was not active on these populations. The tyrosine-phosphorylated p170 molecule bound with high affinity to the p85 subunit of PI3`-kinase, an interaction mediated by the SH2 domains of p85. Although p170 was also bound by the adaptor protein grb2, and under certain conditions could be found associated with the cell membrane, IL-13, like IL-4 (16, 17, 18) failed to activate components of the ras/MAP kinase pathway, including Shc, Sos1, and erk-1 and erk-2 MAP kinases. IL-13 also resembled IL-4 in that it activated the Janus family kinases Jak-1 and Tyk-2. The one difference was that IL-13, in contrast with IL-4, failed to activate the Jak-3 kinase. This is consistent with the notion that the IL-13 receptor does not use the IL-2 receptor chain and dissociates activation of Jak-3 alone from tyrosine phosphorylation of p170. Importantly, we observed that IL-13-induced tyrosine phosphorylation of the 140-kDa subunit of the IL-4 receptor in B9 cells, indicating that this receptor subunit may be responsible for the shared effects mediated by IL-13 and IL-4.

The similarities in molecular mass, in tyrosine phosphorylation in response to insulin and in association with the p85 subunit of PI3`-kinase via high affinity interactions directed by the SH2 domains of p85, suggested that p170/4PS was identical, or related to IRS-1. We found that antibodies raised against the conserved pleckstrin homology domain of IRS-1 were able to immunoprecipitate tyrosine-phosphorylated p170/4PS from myeloid cells and a doublet in the 170 kDa range from insulin-treated 3T3 cells with similar efficiencies (Fig. 5, A and B). However, additional observations by ourselves and others (27) suggest that p170 and IRS-1, while related, are distinct molecules. A commercial anti-IRS-1 antibody was extremely inefficient at immunoprecipitating tyrosine-phosphorylated p170 from FD-5 and TF-1 cells, but efficiently precipitated IRS-1 from insulin-treated Swiss 3T3 cells. Moreover, when immunoprecipitates from IL-4 or insulin-treated FD-5 cells, made with the anti-IRS-1PH antibodies, were subjected to a secondary immunoprecipitation with the commercial anti-IRS-1 antibody, no tyrosine-phosphorylated p170 species could be reprecipitated. In contrast, when the same procedure was performed on extracts of insulin-treated 3T3 cells, clearly tyrosine-phosphorylated IRS-1 (the lower species of the doublet) was reprecipitated. In addition, in sensitive reverse transcription-polymerase chain reaction analyses, it has proven extremely difficult to detect amplified IRS-1-specific sequences from FD-5, B9, and TF-1 cells, whereas it was consistently easy to detect amplified IRS-1 from Swiss 3T3 mRNA.² It has recently been reported that mice lacking IRS-1 exhibit a second molecule of 170 kDa which is tyrosine phosphorylated in response to insulin and which binds to PI3`-kinase p85 subunit (56, 57) . The molecular mass of this protein was 10 kDa greater than IRS-1, a difference in size similar to that we have observed when comparing p170 in FD-5 and TF-1 cells to IRS-1 in Swiss 3T3 fibroblasts (see Fig. 5). This molecule has been termed IRS-2 and may indeed be the same molecule as p170/4PS. Taken together these results suggest that although p170 contains key structural similarities, especially within the pleckstrin homology region, it is a distinct molecule from IRS-1.

In further characterization we found that p170 was located in crude membrane preparations in a salt-sensitive manner (Fig. 4). In the absence of NaCl, a greater proportion of tyrosine-phosphorylated p170 was located at the membrane following treatment of cells (TF-1 and FD-5), with either IL-4, IL-13, or insulin than when fractionation of the same cells was performed in the presence of NaCl, at 150 mM (isotonic conditions), when the majority of tyrosine-phosphorylated p170 was located in the cytosol. This was the case, irrespective of the mechanical disruption procedure used. Similar, although less dramatic, alterations in p170 location were also observed in B9 cells, where treatment with insulin had the most noticeable effects. The reason for these differences is unclear and is under further investigation. These findings contrast with a previous report in which 4PS was reported to be membrane-associated (20) . Other reports have demonstrated, albeit at low stoichiometry, association of IRS-1 with the insulin receptor (58) and the IL-4 receptor (29) . Our findings suggest that these and other potential interactions of p170 with membrane proteins are dependent on salt-sensitive interactions. Molecular characterization of p170/4PS/IRS-2 and the development of specific probes will enable these interactions to be characterized further.

We propose that despite certain structural and functional similarities, including association with p85 PI3`-kinase, p170/4PS and IRS-1 mediate some distinct downstream signaling events. In myeloid cells, which in our hands express barely detectable levels of IRS-1, IL-13, IL-4, and insulin, all failed to induce tyrosine phosphorylation of Shc or its association with grb2. Despite the fact that we observed binding of grb2 to p170 in TF-1 cells treated with IL-13, IL-4, or insulin none of these factors induced modification of mSos1 or activation of erk-1 or erk-2 MAP kinases. This indicates that association of p170 with grb2 was not sufficient for activation of the ras/MAP kinase signaling pathway. We had previously observed similar results with IL-4 in FD-5 cells (16, 17) . Our findings with insulin were somewhat surprising given that insulin has been shown to induce tyrosine phosphorylation of Shc (59, 60) , association of Shc and IRS-1 with grb-2 (60, 61, 62) , and activation of ras and MAP kinases (63, 64) . Moreover, we were able to detect activation of erk-1 and erk-2 MAP kinases by insulin in Swiss 3T3 cells. In many of the previous reports, cells over-expressing the insulin receptor (up to 1.4 10 IR/cell; 59, 60) had been used, so the differences in our findings could merely reflect differences in receptor numbers. However, it is also formally possible that insulin may induce qualitatively different signals in hemopoietic cells because its downstream effects are mediated by p170/4PS and not IRS-1. With respect to this, it would be interesting to determine if IL-4 treatment of the 32D cells cotransfected with IRS-1 and the IL-4 receptor (28, 29) transduce signals similar to those we have described here and previously (16, 17, 18, 21) , or whether in such a cell IL-4 could activate the ras/MAP kinase pathway.

Our observations on the activation of different Jak kinases by IL-13 and IL-4 may be useful in clarifying which components of the IL-4 receptor are shared by the IL-13 receptor. Both IL-4 and IL-13 were able to activate Jak-1 and Tyk-2 in TF-1 cells (Fig. 8A) and B9 cells (). IL-4 has been previously shown to activate Jak-1 in T cells (31, 32, 33) , but this is the first demonstration that IL-4 and IL-13 can induce tyrosine phosphorylation of Jak-1 in myeloid cells. It is also the first report that these cytokines can activate Tyk-2. In other systems, there is some precedent for what appears to be cell type-dependent activations of specific Jak family kinases by particular growth factors (65) .

IL-4 was the only cytokine examined which was capable of activating Jak-3 in TF-1, FD-5, and populations of normal murine splenic B cells. In TF-1 cells we demonstrated Jak-3 as being the major 115-kDa substrate tyrosine phosphorylated in response to IL-4. Importantly, IL-13 failed to induce tyrosine phosphorylation of Jak-3 and insulin failed to induce significant tyrosine phosphorylation of any of the Jak kinases examined.

These studies address two key issues. First, it appears that Jak-3 alone is not responsible for tyrosine phosphorylation of p170/4PS because it is not tyrosine phosphorylated in response to IL-13, which induces p170 tyrosine phosphorylation in B9 and TF-1 cells. Moreover, while IL-2, IL-7, IL-9, and presumably IL-15 induce tyrosine phosphorylation and activation of Jak-3 kinase, IL-2 fails to induce consistently detectable tyrosine phosphorylation of p170/4PS (Fig. 1, E and F).³ The levels of tyrosine phosphorylation of Jak-1 and Tyk-2 were very low in the cells examined, and we feel it unlikely that activation of either of these kinases is related to tyrosine phosphorylation of p170. The case of FD-5 is particularly instructive as in these cells IL-4 induces very marked p170 tyrosine phosphorylation, but there is no detectable tyrosine phosphorylation of Jak-1 or Tyk-2. Secondly, it has previously been suggested that the cross-competition observed between IL-4 and IL-13 for binding to TF-1 cells was due to both factors using the IL-2 c chain in their receptors (8, 9) . It has recently been shown that Jak-3 binds the IL-2 c chain (34, 35) , and all growth factors which use this receptor subunit activate Jak-3 (34, 35) . The fact that IL-13 failed to activate Jak-3 suggests that it does not use the c subunit in its receptor structure, and the similarities in signaling observed between IL-13 and IL-4 must then be due to an alternate shared receptor chain. Our evidence that in B9 cells both IL-4 and IL-13 induced tyrosine phosphorylation of the IL-4 receptor 140-kDa subunit (Fig. 9) is more consistent with this being the IL-4/IL-13 shared receptor subunit. We feel it is unlikely, but formally possible, that IL-13 induces activation of a kinase, which cross-phosphorylates the IL-4 receptor. Moreover, we propose that the shared features of IL-13 and IL-4 signaling are due to certain key signals being mediated by the IL-4 140-kDa receptor subunit. Jak-1 is one candidate, since it is found associated with the IL-4 receptor (33) . Also, tyrosine 472 of the IL-4 receptor has been shown to be important for IL-4-induced tyrosine phosphorylation of IRS-1 in 32D cell transfectants (29) . Differences in IL-4 and IL-13 signals, e.g. involvement of Jak-3, may reflect the participation of c in the IL-4 receptor and the probable involvement of a molecule specific to the IL-13 receptor. Insights to how the early events in signaling which we have characterized here relate to the specific functions of IL-4 and IL-13, such as immunoglobulin class switching, generation of Th2 helper cells, and anti-inflammatory effects, await the molecular characterization of p170/4PS/IRS-2 and comparison of its cell specific expression with that of IRS-1 and elucidation of the structure of the IL-13 receptor.

  
Table: Effects of IL-13, IL-4, and insulin on erk1 MAP kinase activation

The average of four independent experiments are shown for the TF-1 and 3T3 samples, with the exception of the erk-2 activity in TF-1 cells, which is the average of two independent experiments. The average of at least three independent experiments are shown for the FD-5 samples.


  
Table: Effects of IL-13, IL-4, and insulin on Jak kinase tyrosine phosphorylation

The symbols used are: , no detectable tyrosine phosphorylation; +, detectable levels of tyrosine phosphorylation; ++, good levels of tyrosine phosphorylation; +++, high levels of tyrosine phosphorylation.



FOOTNOTES

*
This work was supported by grants from CIBA GEIGY, Canada, The Medical Research Council of Canada and The Arthritis Society of Canada (to J. W. S.). 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.

§
To whom correspondence should be addressed: School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom. Tel.: 01225-826-428; Fax: 01225-826-114; E mail: prsmjw@bath.ac.uk.

¹
The abbreviations used are: IL, interleukin; erk, extracellular-regulated kinase; GST, glutathione S-transferase; IRS-1, insulin receptor substrate-1; MAP, mitogen-activated protein; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; PY, phosphotyrosine; SH2, src homology 2; LPS, lipopolysaccharide.

²
K. B. Leslie, unpublished data.

³
M. J. Welham, unpublished data.

L. Learmonth and M. J. Welham, unpublished data.


ACKNOWLEDGEMENTS

We thank Kevin Leslie for communication of unpublished results and helpful discussions, Ian Clark-Lewis for synthetic cytokine preparations, John O'Shea for anti-human Jak-3 antibodies, David Bowtell for anti-Sos1 antibodies, and Immunex Corporation for anti-IL-4 receptor antibodies.


REFERENCES
  1. Zurawski, G., and de Vries, J. E.(1994) Immunol. Today 15, 19-26 [CrossRef][Medline] [Order article via Infotrieve]
  2. Brown, K. D., Zurawski, S. M., Mosmann, T. R., and Zurawski, G. (1989) J. Immunol. 142, 679-687 [Abstract/Free Full Text]
  3. de Vries J. E., Gauchat, J-F., Aversa, G. G., Punnonen, J., Gascan, H., and Yssel, H.(1991) Curr. Opin. Immunol. 3, 851-858 [Medline] [Order article via Infotrieve]
  4. Punnonen, J., Aversa, G., Cocks, B. G., Mckenzie, A. N. J., Menon, S., Zurawski, G., and de Waal Malefyt, R.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3730-3734 [Abstract]
  5. Mosely, B., Beckmann, M. P., March, C. J., Idzerda, R. L., Gimpel, S. D., Vanden Bos, T., Friend, D., Alpert, A., Anderson, D., Jackson, J., Wignall, J. M., Smith, C., Gallis, B., Sims, J. E., Urdal, D., Widmer, M., Cosman, D., and Park, L. S.(1989) Cell 59, 335-348 [Medline] [Order article via Infotrieve]
  6. Idzerda, R. L., March, C. J., Mosely, B., Lyman, S. D., Vanden Bos, T., Gimpel, S. D., Din, W. S., Grabstein, K. H., Widmer, M. B., Park, L. S., Cos man, D., and Beckmann, M. P.(1990) J. Exp. Med. 171, 861-873 [Abstract]
  7. Galizzi, J-P., Zuber, C. E., Harada, N., Gorman, D. M., Djossou, O., Kastelein, R., Bachereau, J. Howard, M., and Miyajima, A.(1990) Int. Immunol. 2, 669-679 [Medline] [Order article via Infotrieve]
  8. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Wanatabe, S., Arai, K-I., and Sugamura, K.(1993) Science 262, 1874-1877 [Medline] [Order article via Infotrieve]
  9. Russell., S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedmann, M. C., Miyajima, A., Puri, R. K., Paul, W. E., and Leonard, W. J.(1993) Science 262, 1880-1883 [Medline] [Order article via Infotrieve]
  10. Harada, N., Yang, G., Miyajima, A., and Howard, M.(1992) J. Biol. Chem. 267, 22752-22758 [Abstract/Free Full Text]
  11. Koettnitz, K., and Kalthoff, F. S.(1993) Eur. J. Immunol. 23, 989-991
  12. Seldin, D. C., and Leder, P.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2140-2144 [Abstract]
  13. Zurawski, S. M., Vega, F., Jr., Huyghe, B., and Zurawski, G.(1993) EMBO J. 12, 2663-2670 [Abstract]
  14. Satoh T., Nakafuku, M., Miyajima, A., and Kaziro, Y.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3314-3318 [Abstract]
  15. Duronio, V., Welham, M., Abraham, S., Dryen, P., and Schrader, J. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1587-1591 [Abstract]
  16. Welham, M. J., Duronio, V., and Schrader, J. W.(1994) J. Biol. Chem. 269, 5865-5873 [Abstract/Free Full Text]
  17. Welham, M. J., Duronio, V., Leslie, K. B., Bowtell, D., and Schrader, J. W.(1994) J. Biol. Chem. 269, 21165-21176 [Abstract/Free Full Text]
  18. Welham, M. J., Duronio, V., Sanghera, J. S., Pelech, S., and Schrader, J. W.(1992) J. Immunol. 149, 1683-1693 [Abstract/Free Full Text]
  19. Turner, B., Rapp., U., App, H., Greene, M., Dobahi, K., and Reed, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 89, 1227-1231
  20. Wang, L. M., Keegan, A. D., Paul, W. E., Heidaran, M. A., Gutkind, J. S., and Pierce, J. H.(1992) EMBO J. 11, 4899-4908 [Abstract]
  21. Welham, M. J., Dechert, U., Leslie, K. B., Jirik, F., and Schrader, J. W.(1994) J. Biol. Chem. 269, 23764-23768 [Abstract/Free Full Text]
  22. Justement, L., Chen, Z., Harris, L., Ransom, J., Sandoval, V., Smith, C., Rennick, D., Roehm, N., and Cambier, J.(1986) J. Immunol. 137, 3664-3670 [Abstract/Free Full Text]
  23. Mizuguchi, J., Beaven, M. A., Ohara, J., and Paul, W. E.(1986) J. Immunol. 137, 2215-2219 [Abstract/Free Full Text]
  24. Beckmann, M. P., Cosman, D., Fanslow, W., Maliszewski, C. R., and Lyman, S. D.(1992) Chem. Immunol. 51, 107-134 [Medline] [Order article via Infotrieve]
  25. Gold, M. R., Duronio, V., Saxena, S. P., Schrader, J. W., and Aebersold, R.(1994) J. Biol. Chem. 269, 5403-5412 [Abstract/Free Full Text]
  26. Izuhara, K., and Harada, N.(1993) J. Biol. Chem. 268, 13097-13102 [Abstract/Free Full Text]
  27. Wang, L-W., Keegan, A. D., Li, W., Lienhard, G. E., Pacini, S., Gutkind, J. S., Myers, M. G., Sun, X-J., White, M. F., Aaronson, S. A., Paul, W. E., and Pierce, J. H.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4032-4036 [Abstract]
  28. Wang, L-W., Myers, M. G., Sun, X-J., Aaronson, S. A., White, M., and Pierce, J. H.(1993) Science 261, 1591-1594 [Medline] [Order article via Infotrieve]
  29. Keegan, A. D., Nelms, K., White, M., Wang, L-W., Pierce, J. H., and Paul, W. E.(1994) Cell 76, 811-820 [Medline] [Order article via Infotrieve]
  30. Izuhara, K., Feldman, R. A., Greer, P., and Harada, N.(1994) J. Biol. Chem. 269, 18623-18629 [Abstract/Free Full Text]
  31. Johnston, J. A., Kawamura, M., Kirken, R. A., Chen, Y-Q., Blake, T. B., Shibuya, K., Ortaldo, J. R., McVicar, D. W., and O'Shea, J. J.(1994) Nature 370, 151-153 [CrossRef][Medline] [Order article via Infotrieve]
  32. Witthuhn, B. A., Silvennoinen, O., Miura, O., Lai, K. S., Cwik, C., Liu, E. T., and Ihle, J. N.(1994) Nature 370, 153-157 [CrossRef][Medline] [Order article via Infotrieve]
  33. Yin, T., Tsang, M. L-S., and Yang, Y-C.(1994) J. Biol. Chem. 269, 26614-26617 [Abstract/Free Full Text]
  34. Russell, S. M., Johnston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedman, M., Berg, M., McVicar, D. W., Wittuhn, B. A., Silvennoinen, O., Goldman, A. S., Schmalstieg, F. C., Ihle, J. N., O'Shea, J. J., and Leonard, W. J.(1994) Science 266, 1042-1045 [Medline] [Order article via Infotrieve]
  35. Miyazaki, T., Kawahara, A., Fujii, H., Nakagawa, Y., Minami, Y., Liu, Z-J., Oishi, I., Silvennoinen, O., Witthuhn, B. A., Ihle, J. N., and Taniguchi, T.(1994) Science 266, 1045-1047 [Medline] [Order article via Infotrieve]
  36. Schindler C., Kashleva, H., Pernis, A., Pine, R., and Rothman, P. (1994) EMBO J. 13, 1350-1356 [Abstract]
  37. Kotanides, H., and Reich, N. C.(1993) Science 262, 1265-1267 [Medline] [Order article via Infotrieve]
  38. Hou, J., Schindler, U., Henzel, W. J., Chun Ho, T., Brasseur, M., and McKnight, S. L.(1994) Science 265, 1701-1706 [Medline] [Order article via Infotrieve]
  39. Kohler, I., Alliger, P., Minty, A., Caput, D., Ferrara, P., Holl-Neugebauer, B., Rank, G., and Rieber, E. P.(1994) FEBS Lett. 345, 187-192 [CrossRef][Medline] [Order article via Infotrieve]
  40. Karasuyama, H., and Melchers, F.(1988) Eur. J. Immunol. 18, 97-104 [Medline] [Order article via Infotrieve]
  41. Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y. F., Miyazono, K., Arabe, A., and Takaku, F.(1989) J. Cell. Physiol. 140, 323-334 [Medline] [Order article via Infotrieve]
  42. Lansdorp, P. M., Aarden, L. A., Calafat, J., and Zeiljemaker, W. P. (1986) Curr. Top. Microbiol. Immunol. 132, 105-113 [Medline] [Order article via Infotrieve]
  43. Mosmann, T.(1983) J. Immunol. Methods 65, 55-63 [CrossRef][Medline] [Order article via Infotrieve]
  44. Cherewinski, H., Schumacher, J., Brown, K., and Mosmann, T.(1987) J. Exp. Med. 166, 1229-1244 [Abstract]
  45. Kaye, J., Porcelli, S., Tite, J., Jones, B., and Janeway, C. A.(1983) J. Exp. Med. 158, 836-843 [Abstract]
  46. Watts, J. Welham, M. J., Kalt, L., Schrader, J. W., and Aebersold, R. A.(1993) J. Immunol. 151, 6862-6871 [Abstract/Free Full Text]
  47. Andersonn, J., Sjoberg, O., and Moller, G.(1972) Eur. J. Immunol. 2, 349-353 [Medline] [Order article via Infotrieve]
  48. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayoutou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 65, 91-104 [Medline] [Order article via Infotrieve]
  49. Laemlli, U. K.(1970) Nature 227, 680-684 [Medline] [Order article via Infotrieve]
  50. White, M. F.(1994) Curr. Opin. Genet. Dev. 4, 47-54 [Medline] [Order article via Infotrieve]
  51. Songyang Z., Shoelson, S. E, Caudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R. B., Fajardo, J. E., Chou, M. M. N., Hanafusa, H., Schaffhausen, B., and Cantley, L. C.(1993) Cell 72, 767-778 [Medline] [Order article via Infotrieve]
  52. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Pawson, T., and Pelicci, P. G.(1992) Cell 70, 94-104
  53. Rozakis-Adcock, M., McGlade, J., Mbamalu, G., Pelicci, G., Daly, R., Li, W., Batzer, A., Thomas, S., Brugge, J., Pelicci, P. G., Schlessinger, J., and Pawson, T.(1992) Nature 360, 689-692 [CrossRef][Medline] [Order article via Infotrieve]
  54. Rozakis-Adcock, M., Fernley, R., Wade, J., Pawson, T., and Bowtell, D. (1993) Nature 363, 83-85 [CrossRef][Medline] [Order article via Infotrieve]
  55. Buday, L., and Downward, J.(1993) Cell 73, 611-620 [Medline] [Order article via Infotrieve]
  56. Araki, E., Lipes, M. A., Patti, M-E., Bruening, J. C., Haag, B., III, Johnson, R. S., and Kahn, C. R.(1994) Nature 372, 186-190 [CrossRef][Medline] [Order article via Infotrieve]
  57. Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Teruchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y., and Aizawa, S.(1994) Nature 372, 182-186 [CrossRef][Medline] [Order article via Infotrieve]
  58. Backer, J. M., Myers, M. G., Jr., Sun, X. J., Chin, D. J., Shoelson, S. E., Miralpeix, M., and White, M. F.(1993) J. Biol. Chem. 268, 8204-8212 [Abstract/Free Full Text]
  59. Pronk, G. J., McGlade, J., Pelicci, G., Pawson, T., and Bos, J. L. (1993) J. Biol. Chem. 268, 5748-7553 [Abstract/Free Full Text]
  60. Skolnik, E. Y., Lee, C-H., Batzer, A., Vincentini, L. M., Zhou, M., Daly, R., Myers, M. J., Backer, J. M., Ullrich, A., White, M. F., and Schlessinger, J.(1993) EMBO J. 12, 1929-1936 [Abstract]
  61. Skolnik, E. Y., Batzer, A., Li, N., Lee, C-H., Lowenstein, E., Mohammadi, M., Margolis, B., and Schlessinger, J.(1993) Science 260, 1953-1955 [Medline] [Order article via Infotrieve]
  62. Baltensperger, K., Kozma, L.M., Cherniack, A.D., Klarlund, J. K., Chawla, A., Banerjee, U., and Czech, M. P.(1993) Science 260, 1950-1952 [Medline] [Order article via Infotrieve]
  63. Ray, L. B., and Sturgill, T. W.(1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1502-1506 [Abstract]
  64. Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. H.(1990) Science 249, 64-67 [Medline] [Order article via Infotrieve]
  65. Stahl, N., Boulton, T. G., Farruggella, T., Ip, N. Y., Davis, S., Witthuhn, B. A., Quelle, F. W., Silvennoienen, O., Barbieri, G., Pellegrini, S., Ihle, J. N., and Yanacopoulos, G. D.(1994) Science 263, 92-95 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.