©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of JAK3, but Not JAK1, Is Critical to Interleukin-4 (IL4) Stimulated Proliferation and Requires a Membrane-proximal Region of IL4 Receptor (*)

M. Grazia Malabarba (1)(§), Robert A. Kirken (2), Hallgeir Rui (2)(¶), Karl Koettnitz (3), Masaru Kawamura (4), John J. O'Shea (4), Frank S. Kalthoff (3), , William L. Farrar (2)

From the (1) Biological Carcinogenesis and Development Program, Program Resources Inc./DynCorp., National Cancer Institute, Frederick Cancer Research and Development Center, the (2) Cytokine Molecular Mechanisms Section, Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, (3) Sandoz Research Institute, Brunnerstr. 59, A-1235 Wien, Austria, and the (4) Leukocyte Cell Biology Section, Laboratory of Experimental Immunology, Biological Response Modifiers Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The tyrosine kinases JAK1 and JAK3 have been shown to undergo tyrosine phosphorylation in response to interleukin-2 (IL), IL4, IL7, and IL9, cytokines which share the common IL2 receptor -chain (IL2R), and evidence has been found for a preferential coupling of JAK3 to IL2R and JAK1 to IL2R. Here we show, using human premyeloid TF-1 cells, that IL4 stimulates JAK3 to a larger extent than JAK1, based upon three different evaluation criteria. These include a more vigorous tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays and a more manifest presence of JAK3 in activated IL4- receptor complexes. These observations suggest that IL4 receptor signal transduction does not depend on equimolar heterodimerization of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R and IL2R. Indeed, when human IL4R was stably expressed in mouse BA/F3 cells, robust IL4-induced proliferation and JAK3 activation occurred without detectable involvement of JAK1, JAK2, or TYK2. The present study suggests that JAK1 plays a subordinate role in IL4 receptor signaling, and that in certain cells exclusive JAK3 activation may mediate IL4-induced cell growth. Moreover, mutational analysis of human IL4R showed that a membrane-proximal cytoplasmic region was critical for JAK3 activation, while the I4R motif was not, which is compatible with a role of JAK3 upstream of the recruitment of the insulin receptor substrate-1/4PS signaling proteins by IL4 receptors.


INTRODUCTION

Interleukin-4 (IL4)() is a T-cell derived growth factor and differentiation agent that acts on a variety of cells, including T cells, B cells, thymocytes, mast cells, and granulocytes (1) . IL4 signals across the cell membrane via two receptor proteins which belong to the hematopoietin receptor superfamily, the unique IL4R which is exclusive to IL4, and the shared IL2R, which is also used by IL2, IL7, IL9, and IL15 (2, 3, 4, 5, 6) . All of these cytokines are lymphoid growth factors, but each factor may also induce distinct and even opposing effects in certain cells. For example, IL4 has been shown to inhibit IL2-induced proliferation in peripheral blood mononuclear cells, monoclonal B-cells, natural killer cells, and TALL-103/2 cells (7, 8, 9, 10) . Dissimilar cellular impact of these individual cytokines can most likely be attributed to recruitment of separate intracellular effector proteins by their unique receptor chains, while shared functions may be mediated by the common receptor subunit.

Interestingly, the tyrosine kinase JAK3 has been shown to undergo marked tyrosine phosphorylation in response to all cytokines tested that use the common IL2R, including IL2, IL4, IL7, and IL9 (4, 11, 12, 13) .() In addition, JAK1 but not JAK2 or TYK2 has also been found to undergo tyrosine phosphorylation in response to these cytokines in T cells and NK cells (4) .Similar paired activation of different JAK enzymes has been shown for interferons and IL6-related ligands (15, 16) . A series of initial studies have indicated that cytokines which cause homodimerization of their respective receptors are able to signal via a single form of JAK, i.e. JAK2 by erythropoietin, growth hormone, and prolactin (17, 18, 19) , while cytokines which induce heterooligomerization of at least two distinct receptor subunits may rely on the activation of more than one JAK kinase (20) . Genetic complementation studies of interferon receptor signaling have shown that simultaneous involvement of two separate JAKs is indeed required for signal to occur (21, 22) . It has been proposed that ligand-induced hetero- or homooligomerization of receptor subunits cause preassociated JAK molecules to hetero- or homooligomerize, resulting in intermolecular transphosphorylation and activation in a fashion similar to that reported for receptor tyrosine kinases (20) .

Indirect support for this model has been provided by evidence for a selective preference of JAK3 for IL2R (5) and JAK1 for IL2R (5, 23) . On the other hand, comparison of the relative involvement of JAK1 and JAK3 in IL2 receptor signaling in human T cells and NK cells showed a disproportionately higher recruitment of JAK3 than JAK1, and did not favor the concept of equimolar stoichiometry of the two enzymes in the IL2 receptor complex.Indeed, semiquantitative analysis indicated at least a 10-fold higher recruitment of JAK3 over JAK1 by IL2 receptors. It is therefore currently unclear whether both JAK enzymes are needed for signal transduction via IL2R-containing receptor complexes.

A key to understanding the mechanism of JAK activation by cytokine receptors will be to define the nature of receptor-JAK interactions. Moderately conserved membrane-proximal regions of cytoplasmic receptor domains have emerged as probable binding sites that are critical for JAK activation and signal transduction (13, 17, 24, 25) . More specifically, a membrane-proximal region of the shared IL2R has been shown to be essential for IL2-induced JAK3 activation and growth signal (26) . Similarly, mutational mapping of the IL4R has indicated that a cytoplasmic region of 130-140 membrane-proximal amino acids is indispensible for IL4-induced growth signaling (27, 28, 29) . This part of IL4R contains the proline-rich homology box 1 and two conserved acid-rich elements. Intriguingly, a second separate motif has been shown to be important for the growth-stimulatory effect of IL4 (27, 30) . This motif contains a positionally conserved tyrosine residue that is also found in the receptors for insulin and insulin-like growth factor-I and has been designated I4R, because it couples IL4R to the insulin receptor substrate-1 (IRS-1) or its homologue, 4PS (30) . However, the relative contribution of the I4R-motif to growth induction by IL4 in different cell lines may largely depend on the cellular expression levels of IRS-1 or 4PS (30, 31) .

The current investigation was undertaken to characterize the involvement of JAK tyrosine kinases in IL4 receptor signal transduction, addressing not only the relative IL4-induced tyrosine phosphorylation of various JAK enzymes, but also evaluating their catalytic activation and receptor association. For these initial studies of IL4 receptor function we primarily used the human IL4-responsive premyeloid cell line TF-1 (32) . Moreover, we sought to determine the importance of cytoplasmic regions of IL4R for IL4-induced JAK tyrosine phosphorylation and activation, and to what extent JAK activation correlated with mitogenesis. Functional testing of a series of human IL4R variants with internal deletions was carried out by stable expression in murine lymphoblastoid BA/F3 cells, which consequently proliferate in response to human IL4 (27) .


EXPERIMENTAL PROCEDURES

Materials

Polyclonal rabbit antisera to synthetic peptides derived from human/mouse JAK1, mouse JAK2, mouse JAK3, and human TYK2 sequences were purchased from UBI (catalog no. 06-272, 06-255, 06-342, and 06-275, respectively). These antibodies recognize both human and mouse forms with the exception of the anti-mouse JAK3 serum, and each could be used for immunoprecipitation and immunoblotting independent of the phosphorylation state of the JAK enzymes. Antiserum to human JAK3, previously named L-JAK, was raised against a peptide corresponding to the 20 COOH-terminal amino acids (amino acids 1104-1124) which are unique to human JAK3 (33) . We also generated polyclonal rabbit antibodies against an eight-residue peptide corresponding to the COOH terminus of human IL2R (NH-CYTLKPET-COOH) and affinity purified the antibodies as described by others (4) . Monoclonal mouse anti-phosphotyrosine antibodies were purchased from UBI (4G10; catalog no. 05-321) and anti-human IL2R (561) was a generous gift from Dr. Richard Robb (34) . Biotinylation of IL4 was carried out by incubating 1 mg of human IL4 (PeproTech, Rock Hill, NJ) in 1 ml of carbonate buffer (pH 8.5) with 1 m M NHS-LC-biotin (Pierce, catalog no. 21335) for 4 h at room temperature. The reaction was stopped by addition of 20 µl of 1 M NHCl and uncoupled biotin was removed by dialysis against phosphate-buffered saline.

Cell Culture and Treatment

Human T lymphocytes from normal donors were grown in RPMI 1640 medium containing 10% fetal calf serum (Sigma, catalog no. F2442), 2 m M L-glutamine, 5 m M HEPES buffer (pH 7.3), and penicillin-streptomycin (50 IU/ml and 50 mg/ml, respectively), while the human erythroleukemic cell line, TF-1, was grown in the same medium supplemented with 5 ng/ml granulocyte macrophage-colony stimulating factor. T lymphocytes were activated for 72 h with phytohemagglutinin (1 µg/ml) and were subsequently made quiescent by washing and incubating for 24 h in RPMI 1640 medium containing only 1% fetal calf serum before exposure to cytokines, while TF-1 cells were made quiescent in 5% gelded horse serum (Sigma, catalog no. H1885). The IL3-dependent murine BA/F3 cell line containing the IL4R mutants were grown in RPMI 1640 medium with 10% fetal calf serum supplemented with 0.8 mg/ml geneticin sulfate (G-418 sulfate, Life Technologies, Inc.), 10 m M HEPES and 2% WEHI-3B supernatant as a source of IL3. Cells were normally stimulated with 100 n M recombinant human IL4 (PeproTech, Rock Hill, NJ) at 37 °C as indicated in the corresponding figure legends. Cell pellets were frozen at -70 °C.

Solubilization of Membrane Proteins and Immunoprecipitation

Frozen cells were thawed on ice and solubilized in lysis buffer (10cells/ml) containing 10 m M Tris-HCl (pH 7.6), 5 m M EDTA, 50 m M NaCl, 30 m M sodium pyrophosphate, 50 m M sodium fluoride, 200 µ M sodium orthovanadate, 1% Triton X-100, 1 m M phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, and 2 µg/ml leupeptin. Cell lysates were rotated end over end at 4 °C for 60 min, and insoluble material was pelleted at 12,000 g for 20 min. Depending on the experiment, supernatants were incubated rotating end over end for 2 h at 4 °C with anti-phosphotyrosine monoclonal antibody (4G10; 4 µg/ml), anti-IL2R monoclonal antibody (561; 5 µg/ml), normal rabbit serum or polyclonal sera to individual JAK enzymes (5 µl/ml). Antibodies were captured by incubation for 30 min with protein A-Sepharose beads. Cells incubated with biotinylated IL4 were lysed and treated as described above and biotin-IL4 was subsequently captured with streptavidin-agarose beads (Life Technologies, Inc., BRL). For immunoblotting, anti-phosphotyrosine and anti-IL2R antibodies were used at 1 µg/ml, while all anti-JAK sera were used at a dilution of 1:1000. Precipitated material was eluted by boiling in SDS sample buffer for 4 min, subjected to 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions, and transferred to polyvinylidene difluoride membrane (Immobilion, Millipore, catalog no. 1PVH 00010). Immunoblot analysis was performed as described previously (35) .

Tyrosine Kinase Assays

JAK1 and JAK3 immune-complex tyrosine kinase assays were carried out by incubating the individually immunoprecipitated tyrosine kinases from lysates of unstimulated and IL4-stimulated cells in the presence and absence of ATP, and visualizing incorporated phosphate on tyrosines by immunoblotting. Receptor complex tyrosine kinase assays were carried out in a similar manner, but were based on capture of activated receptor complexes from cell lysates by means of either anti-IL2R antibodies or biotinylated IL4. Immobilized proteins were washed three times with lysis buffer followed by a single wash with kinase buffer containing 25 m M HEPES (pH 7.3), 0.1% Triton X-100, 100 m M NaCl, 10 m M MgCl, 3 m M MnCl, and 200 µ M sodium orthovanadate. Isotope-free tyrosine kinase reactions were initiated by the addition of 15 µ M unlabeled ATP and allowed to incubate at 37 °C for 15 min. The reactions were quenched by washing the streptavidin-agarose or protein A-Sepharose beads with lysis buffer and eluting bound material by boiling in SDS sample buffer for 4 min. The material in each lane represents immunoprecipitates from approximately 2 10cells unless specified otherwise.

Proliferation Assay

Quiescent BA/F3 cells (2.5 10/well) were plated in flat-bottom 96-well microtiter plates in starvation medium (100 µl final) in the presence of various concentrations of human IL4 or IL3. Cells were pulsed for 4 h with [H]thymidine (0.5 µCi/100 µl) after 24 h of stimulation, harvested onto glass fiber filters, and analyzed for [H]thymidine uptake by liquid scintillation counting.


RESULTS

Immunoblotting of anti-phosphotyrosine immunoprecipitated proteins revealed that JAK3 was the predominant protein inducibly tyrosine phosphorylated in TF-1 cells after 5 min of IL4 treatment (Fig. 1). Specifically, IL4 stimulated the tyrosine phosphorylation of a protein migrating with an apparent molecular mass of 116 kDa by SDS-polyacrylamide gel electrophoresis (Fig. 1, lane b), and this protein was recognized by anti-JAK3 serum (Fig. 1, lane j). Moreover, the 116-kDa protein comigrated with inducibly phosphorylated JAK3 which had been immunoprecipitated with anti-JAK3 serum (Fig. 1, lane d), as well as with positive controls of immunoprecipitated and immunoblotted JAK3 (Fig. 1, lanes k and l). These results corresponded well with our previous demonstration of IL2-induced tyrosine phosphorylation of JAK3 (11) . Phosphorylated JAK3 was not readily detectable in total cell lysates (Fig. 1, lanes g and h), demonstrating the necessity of amplifying the signal by prior immunoprecipitation. The IL4-induced tyrosine phosphorylation of JAK3 was time- and dose-dependent as judged from parallel anti-phosphotyrosine immunoblots of either anti-JAK3 or anti-phosphotyrosine immunoprecipitates from lysates of stimulated TF-1 cells (Fig. 2). Peak phosphorylation levels were reached after 5-10 min of IL4 stimulation (Fig. 2 A) and with an ECvalue of 1-10 n M (Fig. 2 B).


Figure 1: IL4-induced tyrosine phosphorylation of JAK3 in human TF-1 cells. Anti-phosphotyrosine ( PY; lanes a-h) or anti-JAK3 ( JAK3; lanes i-p) immunoblots of two parallel sets of protein samples from lysates of human TF-1 cells which had been stimulated without (-) or with (+) 100 n M IL4 for 10 min at 37 °C. Individual lanes represent immunoprecipitates ( IP) with anti-phosphotyrosine ( PY; lanes a, b, i, and j), anti-JAK3 ( JAK3; lanes c, d, k, and l), control antibodies ( CTRL; lanes e, f, m, and n), or total cellular lysates ( TCL; lanes g, h, o, and p). Arrow denotes JAK3 and bracket indicates immunoglobulin heavy chains ( IgG). Molecular size markers are indicated on the left (kDa).




Figure 2: Time kinetics and concentration dependence of JAK3 tyrosine phosphorylation induced by IL4 in human TF-1 cells. Anti-phosphotyrosine immunoblots of proteins that had been immunoprecipitated with either anti-phosphotyrosine ( PY) or anti-JAK3 antibodies ( JAK3) from lysates of TF-1 cells (1 10cells/lane) which had been stimulated with 100 n M hIL4 from 0 to 20 min ( Panel A) or with varying concentrations of hIL4 (0-100 n M) for 10 min ( Panel B). Arrows denote JAK3 and brackets indicate immunoglobulin heavy chains ( IgG). Molecular size markers are indicated on the left (kDa).



Anti-phosphotyrosine immunoprecipitates in these first two experiments did not indicate the presence of significant quantities of a second IL4-modulated JAK of higher molecular weight than JAK3. However, specific testing with antibodies to other known JAK kinases showed that JAK1 was also phosphorylated to a certain extent in TF-1 cells in response to IL4 (Fig. 3, lane d). In contrast, the phosphorylation state of JAK2 and TYK2 was not modulated by IL4. Since the anti-JAK2 serum shows partial cross-reactivity with the faster migrating JAK3 (5) , phosphorylated JAK3 was detected to some extent in the JAK2 immunoprecipitates from IL4-stimulated cells (Fig. 3, lane f). Moreover JAK2, but not TYK2, for unknown reasons showed high basal tyrosine phosphorylation levels in quiescent TF-1 cells (Fig. 3, lanes e and f).


Figure 3: IL4-induced tyrosine phosphorylation of JAK1 and JAK3 but not JAK2 or TYK2 in human TF-1 cells. Anti-phosphotyrosine ( PY) immunoblot of TF-1 cells (1 10cells/lane) which had been incubated in the absence (-) or presence (+) of 100 n M hIL4 for 10 min at 37 °C. Lysates were immunoprecipitated with anti-JAK3 ( JAK3; lanes a and b), anti-JAK1 ( JAK1; lanes c and d), anti-JAK2 ( JAK2; lanes e and f), or anti-TYK2 ( TYK2; lanes g and h) antibodies. Arrows denote JAK1, JAK2, or JAK3, and bracket indicates immunoglobulin heavy chains ( IgG). Molecular size markers are indicated on the left (kDa).



We next sought to establish the relative presence of JAK kinases in activated IL4-receptor complexes. Since no antibody was available that recognized IL4-complexed human IL4R, we used biotinylated IL4 (biotin-IL4) to assess indirectly the binding of JAK3 and JAK1 to IL4 receptors. Initial testing of the biotin-IL4 showed that it retained its bioactivity and induced tyrosine phosphorylation of JAK3 and JAK1 to the same extent as unconjugated IL4 (data not shown). While JAK3 coprecipitated with biotin-IL4 from lysates of cells after immobilization on streptavidin-agarose beads and subsequent visualization by anti-JAK3 immunoblotting (Fig. 4 A, lanes b and d), biotin-IL4 receptor complexes did not contain detectable amounts of JAK1 (data not shown). This observation combined with the observed lower degree of IL4-induced tyrosine phosphorylation of JAK1 indicated a quantitatively lower recruitment of JAK1 than JAK3 by IL4. Biotin-IL4 complexes also contained IL2R as demonstrated by immunoblotting of parallel samples with anti-IL2R antibodies (Fig. 4 B, lane b) in agreement with previous reports (2, 4) .


Figure 4: Association of JAK3 with the IL4-receptor complex. Panel A, anti-JAK3 immunoblot of proteins captured with streptavidin-agarose beads from lysates of TF-1 cells (2 10cells/lane) which had been incubated with (+) or without (-) 100 n M biotinylated hIL4 (bIL4) for 10 min ( lanes a and b) or 30 min ( lanes c and d) at 37 °C. Panel B, anti-IL2R immunoblot of proteins captured with streptavidin-agarose beads from lysates of TF-1 cells treated with bIL4 for 10 min ( lane b). Anti-IL2R immunoprecipitated material from human YT cells treated with IL2 provided a positive control ( lane d). Lanes a and c represent untreated control cells ( C). IL2R chain is denoted by arrow, bracket indicates immunoglobulin heavy chains ( IgG). Molecular size markers are indicated on the left (kDa).



The effect of IL4 on the catalytic activities of JAK1 and JAK3 in immune-complex autokinase assays was also evaluated. Several studies have revealed a close correlation between cytokine-induced tyrosine phosphorylation and catalytic activation of JAK kinases (12, 13, 16, 17, 18, 19) .We here extend this correlation to include IL4-modulated JAK3 and JAK1 in TF-1 cells by demonstrating that both JAK kinases were activated by IL4 (Fig. 5, lanes a-d). Although a certain degree of basal JAK3 activity was present in unstimulated cells (Fig. 5, lane b), a significant increase in phosphate incorporation on tyrosine residues was seen when JAK3 from IL4-stimulated cells was incubated with ATP in vitro (Fig. 5, lanes c and d). Control immunoprecipitates were negative (Fig. 5, lanes e-h), and reprobing of the blots with JAK3 antibody verified equal loading (data not shown). In order to detect IL4-induced JAK1 autokinase activity in vitro, three times as many cells had to be used, so that immunoprecipitates from as many as 6 10cells per lane had to be loaded (Fig. 5 A, lanes i-l). A similar need for higher cell numbers had also been required for the detection of IL2-induced JAK1 activity in YT cells.These results provided a third line of evidence for a quantitatively lower involvement of JAK1 in IL4 receptor signaling. Moreover, we observed comparably disproportionate activity levels of JAK3 and JAK1 in IL4-stimulated human T-lymphocytes and several lymphoid cell lines, including HuT-101, YT, A-301, rat Nb2-11C, and mouse CTLL-2,() suggesting that a skewed recruitment of JAK3 over JAK1 is a widespread phenomenon in IL4 target cells.


Figure 5: IL4-induced activation of JAK3 analyzed by autophosphorylation assay using unlabeled ATP and anti-phosphotyrosine immunoblotting. Anti-phosphotyrosine immunoblot of immunoprecipitated lysates of TF-1 cells, which had been incubated with (+) or without (-) 100 n M IL4 for 5 min at 37 °C. Anti-JAK3 ( JAK3; lanes a-d) or control antibody ( CTRL; lanes e-h) immunoprecipitates were washed and subsequently incubated for 20 min at 37 °C in the absence (-) or presence (+) of 15 µ M unlabeled ATP. For detection of JAK1 activity, immunoprecipitates from 6 10cells were loaded per lane, in contrast to 2 10cells per lane for JAK3 and CTRL. Arrows denote JAK1 and JAK3. Molecular size markers are indicated on the left (kDa).



The presence of catalytically active JAK enzymes in activated IL4 receptor complexes was also assessed, using either anti-IL2R antibodies or biotin-IL4 to purify receptor complexes for in vitro tyrosine kinase assays. IL4-stimulated the in vitro activity of JAK3 which could be coprecipitated with anti-IL2R antibodies, whereas no detectable JAK1 activity was observed (Fig. 6 A, lane d). Parallel analysis of biotin-IL4 receptor complexes revealed similar selective detection of JAK3 activity (Fig. 6 B, lane d). The identity of the 116-kDa protein as JAK3 was verified by reprobing of the stripped blot with anti-JAK3 serum (data not shown). The absence of detectable amounts of catalytically active JAK1 from IL4-receptor complexes represents another indication of a subordinate role for JAK1 in IL4 signaling.


Figure 6: Detection of JAK3 autokinase activity in IL4 receptor complexes. Panel A, anti-phosphotyrosine immunoblot of proteins coprecipitating with IL2R captured with protein A-Sepharose beads from lysates of TF-1 cells which had been treated treated with (+) or without (-) 100 n M IL4 and for 5 min at 37 °C, subsequently incubated in the absence (-) or presence (+) of 15 µ M unlabeled ATP. Arrow denotes JAK3 and molecular size markers are indicated on the left (kDa). Panel B, anti-phosphotyrosine immunoblot of proteins captured with streptavidin-agarose beads from lysates of TF-1 cells which had been treated treated with (+) or without (-) 100 n M biotinylated IL4 ( bIL4) for 5 min at 37 °C, and subsequently incubated for 20 min at 37 °C in the absence (-) or presence (+) of 15 µ M unlabeled ATP. Arrow denotes JAK3 and molecular size markers are indicated on the left (kDa).



To analyze the role of the cytoplasmic domain of human IL4R in the IL4-induced activation of JAK enzymes, we tested a set of previously described IL4R variants with systematic internal deletions which had been individually introduced in the murine IL3-dependent lymphoblastoma line BA/F3 (27) . The human IL4R can form functional complexes with the murine IL2R and is able to mediate proliferative signals in these cells (27, 28, 29) . The structures of wild type IL4R and the mutant variants Cyt, R1, R2, R3, and R4, are reviewed in Fig. 7 A. Previous Scatchard analysis of the various cloned cell populations revealed between 2,000 and 14,000 binding sites for IL4 per cell (27) .

A comparison of the ability of the engineered IL4R variants to mediate IL4-stimulated thymidine incorporation in BA/F3 cells is shown in Fig. 7B. In parental BA/F3 cells and each of the sublines expressing IL4R, IL3 induced comparable increases in thymidine incorporation after 24 h of stimulation. However, only cells expressing wild type, R4, R3, and R2 forms of IL4R responded with IL4-induced thymidine incorporation. The data shown in Fig. 7 B represent maximal incorporation levels induced by 1 n M IL4, and repeated dose-response studies ( n = 3) established that the four active receptor variants had similar ECvalues of 5-50 p M IL4 (data not shown). Of the four functional forms of IL4R, wild type, R4, and R2 mediated average IL4 responses that were 52, 67, and 39% of the corresponding IL3-induced effects, respectively. In contrast, IL4-induced proliferation mediated by R3, which lacks the I4R motif, was as low as 13% of the corresponding IL3-induced response. These results are compatible with the suboptimal IL4-induced thymidine incorporation observed in several BA/F3 clones which were stably expressing another I4R-deficient form of human IL4R, the P-mutant reported by Harada and colleagues (29) . This P-mutant only included the first 176 cytoplasmic amino acids of the IL4R. However, BA/F3 cells expressing R3 did not survive prolonged culture in IL4 in the absence of IL3 as reported previously (27) .


Figure 7: Ability of IL4R mutants to mediate IL4-induced thymidine incorporation. Panel A, structural review of the various human IL4R mutants that have been stably introduced into the murine IL3-dependent BA/F3 cell line. The wild type ( wt) receptor was not modified, while Cyt lacks the entire cytoplasmic domain. The internal deletion mutants R1, R2, R3, and R4 are defined by the following excluded segments which are indicated in the sketch: Ile-Ser(R1), Ser-Thr(R2), Thr-Aal(R3), and Ala-Thr(R4), respectively. The diagram also depicts the relative position of homology box 1 ( Box 1), acidic domains 1 ( Acid1) and 2 ( Acid2), as well as conserved tyrosine residues. Panel B, thymidine incorporation assay comparing the ability of the various IL4R variants to mediate IL4-induced proliferation. Quiescent cells (2 10cells/well) were incubated with or without 1 n M human IL4 or murine IL3 for 24 h, followed by addition of [H]thymidine (0.5 µCi/well) for 4 h, before harvesting and quantification of the incorporated thymidine. Results are expressed as counts/min, reflecting incorporation into 2 10cells. BA/F3 indicates the parental cell line, whereas the denotation of the IL4R constructs are used to indicate the various stably transfected BA/F3 subclones.



Subsequent analyses ( n = 4) of the ability of the IL4R variants to mediate IL4-induced tyrosine phosphorylation of JAK3 demonstrated that wild type, R4, R3, and R2 were equally efficient, while the growth-defective mutants R1 and Cyt mediated no JAK3 phosphorylation. The results from one representative experiment is shown in Fig. 8. Intriguingly, repeated analyses failed to detect any IL4-induced tyrosine phosphorylation of a second JAK enzyme, including JAK1, JAK2, or TYK2, in these BA/F3 cells (data not shown), raising the possibility that IL4 receptors under certain circumstances may signal via only one JAK kinase. Moreover, the ability of wild type and mutant forms of IL4R to mediate IL4-induced JAK3 tyrosine phosphorylation coincided with their ability to mediate catalytic activation of JAK3, as assessed by in vitro JAK3 immune-complex tyrosine kinase assay (Fig. 9).


DISCUSSION

The present study verified that IL4 may induce parallel tyrosine phosphorylation and activation of JAK1 and JAK3, but found that the recruitment of individual JAK enzymes by IL4 in human TF-1 cells was highly skewed in favor of JAK3. These results therefore did not favor the concept of equimolar transphosphorylation and activation of JAK1 and JAK3 following IL4-induced heterodimerization of IL4R and IL2R, which has been proposed as a generic activation model for cytokine receptor complexes (20) . Our conclusion of a predominant involvement of JAK3 in IL4 receptor signaling was based upon several independent evaluation criteria. These included a preferential tyrosine phosphorylation of JAK3 as measured by anti-phosphotyrosine immunoblotting, a more marked activation of JAK3 as determined by in vitro tyrosine kinase assays, and a more manifest presence of JAK3 in activated IL4 receptor complexes.

The overall quality and efficiency of the anti-JAK1 and anti-JAK3 sera used for these experiments were comparable, justifying semiquantitative assessments to be made. In addition, this evaluation was in part based on methods that did not rely directly on anti-JAK sera, including the dominant degree of JAK3 tyrosine phosphorylation observed in anti-phosphotyrosine immunoprecipitated material from IL4-stimulated cells (Figs. 1 and 2), and the detectable catalytic activation of JAK3, but not JAK1, in biotin-IL4 receptor complexes (Fig. 6 B). Neither of these two approaches is biased toward the detection of one specific JAK enzyme. The possibility that the antiserum to the cytoplasmatic domain of IL2R might selectively displace JAK1 and not JAK3 from the activated IL4 receptor complex (Fig. 6 A) is unlikely in light of the recent demonstration that in the IL2 receptor complexes JAK3 associates with IL2R and JAK1 with IL2R (5) . We have also observed similar predominant JAK3 recruitment by IL4 in activated human T cells and several lymphoid cell lines (data not shown), indicating that this phenomenon is not specific to TF-1 cells. Moreover, a more extreme imbalance of JAK activation was observed in murine BA/F3 cells expressing human IL4R. In these cells IL4 induced detectable tyrosine phosphorylation and activation exclusively of JAK3, and not of JAK1, JAK2, or TYK2. Nonetheless, IL4 induced robust proliferation in IL4R-expressing BA/F3 cells, suggesting that activation of JAK3 alone is sufficient to mediate growth signal by IL4 receptors. However, it is possible that the assay is not sensitive enough to detect minor, but essential phosphorylation of JAK1 in these cells. Specific immunoblotting did indeed reveal the presence of low amounts JAK1 in BA/F3 cells (data not shown), but it cannot at present be excluded that the human IL4R does not recognize mouse JAK1. Parallel anti-phosphotyrosine immunoblotting of anti-phosphotyrosine immunoprecipitated proteins from IL4-stimulated BA/F3 cells did not indicate any other modulated protein than JAK3 in the size range of known JAK kinases (110-150 kDa; data not shown), arguing against the possibility that a novel JAK kinase might be activated in these cells. Reconstitution of IL4 receptors in JAK1-deficient cells will help to determine the importance of JAK1 in IL4 receptor signaling.

Another question of significance for understanding the mechanism of JAK activation by IL4 is to determine the basis for the interdependence of IL4R and IL2R. As mentioned previously, a preferential coupling of JAK3 to IL2R has been proposed (5, 11) and a membrane-proximal region of IL2R is critical for JAK3 activation by IL2 (26) . In the present study we demonstrate that a membrane-proximal region of 132 amino acids of IL4R is also critical for the mediation IL4-induced JAK3 activation. In agreement with previous reports, this region is essential for IL4-induced growth signal. Interestingly, a corresponding membrane-proximal region of the IL2R has recently been shown to be of similar importance for JAK3 activation by IL2 (13) . These observations therefore suggest that although the common IL2R may serve as the principal interaction partner for JAK3, the cytoplasmic domains of the unique components of IL4 and IL2 receptor complexes, i.e. IL4R and IL2R, are also indispensible for JAK3 activation. Studies directed toward identifying the exact localization and nature of the interaction sites between receptors and JAKs will be needed to understand how JAK kinases become activated by cytokines.

Mutational analyses of IL4R have previously shown that a second more distally located motif of the cytoplasmic domain of IL4R is important for IL4-induced growth signal transduction (27, 30) . This motif has been designated I4R, and couples IL4R to the IRS-1 or 4PS signaling proteins (30) . The present study found that selective deletion from IL4R of a region containing the I4R motif did not affect JAK3 activation, but caused a profound reduction in IL4-induced proliferation as measured by thymidine incorporation assay. However, a residual growth promotional activity remained that was approximately 22% of the response mediated by wild type IL4R, suggesting that a JAK-activated IRS-1/4PS-independent proliferative pathway exists. In support of this view, two previous studies have shown moderate IL4-induced thymidine incorporation in BA/F3 cells mediated by human IL4R mutants that had been truncated above the I4R-motif (28, 29) . On the other hand, since R3 expressing cells do not survive long-term culture (>1 week) in medium containing only IL4 (27) , it appears that this residual IL4-stimulated growth signal mediated by the R3 mutant is incapable of overcoming the apoptotic drive induced by IL3 depletion.

One principal mechanism of signal transduction subsequent to JAK kinase activation and phosphorylation of tyrosine residues of cytoplasmic receptor domains and JAK molecules, is the secondary recruitment of a variety of phosphotyrosyl-binding effector proteins with SRC-homology 2 (SH2) domains (36) . However, it has been argued that tyrosine residues of the IL4R are not essential to IL4-induced signal transduction, based upon the ability of a tyrosine-deficient truncated form of IL4R to mediate IL4-induced thymidine incorporation (28) . The present paper supports the view that at least a portion of the IL4-induced growth signal may not depend on phosphorylated tyrosine residues of IL4R or the I4R motif, suggesting that recruitment of some growth-inducing SH2 domain proteins may occur directly via phosphorylated tyrosine residues of IL2R or JAK3. However, it has been shown that IL4R becomes tyrosine phosphorylated after IL4 stimulation (37) , and it is evident that the I4R motif contributes significantly to an enhanced IL4-induced growth signal, both in BA/F3 cells as shown in the present work (Fig. 7 B) and in 32D cells (31) . Indeed, overexpression of IRS-1 in 32D cells dramatically increased the growth induction by IL4 in these cells (30) . It is possible that Tyr-472 of the I4R motif is phosphorylated by JAK3 and serves as a docking site for IRS-1 via the SH2 domain of an unidentified adaptor protein, since IRS-1 does not itself contain an SH2 domain. It will therefore be interesting to determine whether a Y472F substitution will abolish the recruitment of IRS1 and 4PS by IL4R. Other conserved tyrosine residues in the cytoplasmic domain of IL4R may interact selectively with different SH2 domain proteins upon phosphorylation, and contribute to cell-specific effects of IL4 that do not overlap with those induced by IL2 and IL7.

It also interesting that tyrosine kinase activity from IL4-stimulated cells spontaneously associates with I4R-containing fusion proteins (30) , indicating that tyrosine kinases other than JAKs may also be activated by IL4. Since an involvement of SRC family tyrosine kinases have been implicated in signaling downstream of JAK enzymes by several hematopoietin receptors, it is possible that SRC kinases or other SH2 domain kinases such as FES (14) associate with IRS-1 or 4PS, and may indeed phosphorylate these docking proteins.

The present paper has demonstrated that a membrane-proximal region of human IL4R is essential for IL4-induced activation of the tyrosine kinase JAK3 in murine myeloid BA/F3 cells. In these cells, no detectable tyrosine phosphorylation of other JAK kinases than JAK3 was induced by IL4, although robust IL4-stimulated cell proliferation was observed. In human T lymphocytes and human TF-1 cells minor, but consistent, IL4-induced tyrosine phosphorylation of JAK1 was found in addition to the more dominant JAK3 phosphorylation. We have recently observed similar disproportionate tyrosine phosphorylation and activation of JAK1 and JAK3 by IL2 in human T cells and YT cells.Whereas interferon receptor signal transduction may depend on the simultaneous presence of two distinct JAK kinases (21, 22) , the present work suggests that IL4 receptors can mediate growth signal solely through JAK3 activation. This study therefore does not support a symmetric model of interdependent and equimolar transactivation of JAK3 and JAK1 associated with each of the heterodimerizing IL4R and IL2R. Ongoing studies are addressing the qualitative and quantitative roles of individual JAK enzymes in IL4 receptor signal transduction.


FOOTNOTES

*
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.

§
Supported in part by the University of Milan and the Italian Association for Cancer Research.

Recipient of Fogarty Fellowship 5FO5 TWO4300-02. To whom correspondence should be addressed.

The abbreviations used are: IL, interleukin; IL2R/, IL2 receptor /; IL4R, IL4 receptor ; JAK, Janus kinase.

R. A. Kirken, H. Rui, M. G. Malabarba, M. Kawamura, J. J. O'Shea, and W. L. Farrar, unpublished data.

M. G. Malabarba, R. A. Kirken, H. Rui, and W. L. Farrar, unpublished information.


ACKNOWLEDGEMENTS

We thank Dr. Richard Robb for providing generous amounts of monoclonal anti-human IL2R and Terry Williams for expert technical help with the preparation of the figures. We also acknowledge the support and critical review of the manuscript by Dr. Joost Oppenheim and Dr. Dan Longo.


REFERENCES
  1. Paul, W. E. (1991) Blood 77, 1859-1870 [Medline] [Order article via Infotrieve]
  2. Kondo, M., Takeshita, T., Ishi, N., Nakamura, M., Watanabe, S., Arai, K.-I., and Sugamura, K. (1993) Science 262, 1874-1877 [Medline] [Order article via Infotrieve]
  3. Noguchi, M., Nakamura, Y., Russell, S. M., Ziegler, S. F., Tsang, M., Cao, X., and Leonard, W. (1993) Science 262, 1877-1880 [Medline] [Order article via Infotrieve]
  4. 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. L. (1993) Science 262, 1880-1883 [Medline] [Order article via Infotrieve]
  5. Russell, S. M., Johnston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedman, M., Berg, M., Witthuhn, B. A., 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]
  6. Giri, J. G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D. (1994) EMBO J. 13, 2822-2830 [Abstract]
  7. Han, X., Itoh, K., Balch, C. M., and Pellis, N. R. (1988) Lymphokine Res. 141, 227-235
  8. Karray, S., DeFrance, T., Merle-Béral, H., Banchereau, J., Debré, P., and Galanaud, P. (1988) J. Exp. Med. 168, 85-94 [Abstract]
  9. Nagler, A., Lanier, L. L., and Phillips, P. H. (1988) J. Immunol. 141, 2349-2351 [Abstract/Free Full Text]
  10. Torigoe, T., O'Connor, R., Fagard, R., Fischer, S., Santoli, D., and Reed, C. J. (1992) Cytokine 4, 369-376 [Medline] [Order article via Infotrieve]
  11. Kirken, R. A., Rui, H., Malabarba, M. G., and Farrar, W. L. (1994) J. Biol. Chem. 269, 19136-19141 [Abstract/Free Full Text]
  12. Johnston, J. A., Kawamura, M., Kirken, R. A., Chen, Y-I., Blake, T. B., Ortaldo, J. R., McVicar, D. W., and O'Shea, J. J. (1994) Nature 370, 151-153 [Medline] [Order article via Infotrieve]
  13. Witthuhn, B. A., Silvennoinen, O., Miura, O., Lai, K. S., Cwik, C., Liu, E. T., and Ihle, J. N. (1994) Nature 370, 153-157 [Medline] [Order article via Infotrieve]
  14. Izuhara, K., Feldman, R. A., Greer, P., and Harada, N. (1994) J. Biol. Chem. 269, 18623-18629 [Abstract/Free Full Text]
  15. Igarashi, K., Garotta, G., Ozmen, L., Ziemiecki, A., Wilks, A., Harpur, A. G., Larner, A. C., and Finbloom, D. S. (1994) J. Biol. Chem. 269, 14333-14336 [Abstract/Free Full Text]
  16. Stahl, N., Boulton, T. G., Farruggella, T., Ip, N. Y., Davis, S., Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Barbieri, G., Pellegrini, S., Ihle, J. N., and Yancopoulos, G. D. (1994) Science 263, 92-95 [Medline] [Order article via Infotrieve]
  17. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N. (1993) Cell 74, 227-236 [Medline] [Order article via Infotrieve]
  18. Argetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., and Carter-Su, C. (1993) Cell 74, 237-244 [Medline] [Order article via Infotrieve]
  19. Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 5364-5368 [Abstract/Free Full Text]
  20. Stahl, N., and Yancopoulos, G. D. (1993) Cell 74, 587-590 [Medline] [Order article via Infotrieve]
  21. Müller, M., Briscoe, J., Laxton, C., Guschin, D., Ziemiecki, A., Silvennoinen, O., Harpur, A. G., Barbieri, G., Witthuhn, B. A., Schindler, C., Pellegrini, S., Wilks, A. W., Ihle, J. N., Stark, G. R., and Kerr, I. M. (1993) Nature 366, 129-135 [Medline] [Order article via Infotrieve]
  22. Watling, D., Guschin, D., Müller, M., Silvennoinen, O., Witthuhn, B. A., Quelle, F. W., Rogers, N. C., Schindler, C., Stark, G. R., Ihle, J. N., and Kerr, I. M. (1993) Nature 366, 166-170 [Medline] [Order article via Infotrieve]
  23. Tanaka, N., Asao, H., Ohbo, K., Ishii, I., Takeshita, T., Nakamura, M., Sasaki, H., and Sugamura, K. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7271-7275 [Abstract]
  24. DaSilva, L., Howard, O. M. Z., Rui, H., Kirken, R. A., and Farrar, W. L. (1994) J. Biol. Chem. 269, 18267-18270 [Abstract/Free Full Text]
  25. Quelle, F. W., Sata, N., Witthuhn, B. A., Inhorn, R. C., Eder, M., Miyajima, A., Griffin, J. D., and Ihle, J. N. (1994) Mol. Cell. Biol. 14, 4335-4341 [Abstract]
  26. Asao, H., Tanaka, N., Ishii, N., Higuchi, M., Takeshita, T., Nakamura, M., Shirasawa, T., and Sugamura, K. (1994) FEBS Lett. 351, 201-206 [CrossRef][Medline] [Order article via Infotrieve]
  27. Koettnitz, K., and Kalthoff, F. S. (1993) Eur. J. Immunol. 23, 988-991 [Medline] [Order article via Infotrieve]
  28. Seldin, D. C., and Leder, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2140-2144 [Abstract]
  29. Harada, N., Yang, G., Miyajima, A., and Howard, M. (1992) J. Biol. Chem. 267, 22752-22758 [Abstract/Free Full Text]
  30. Keegan, A. D., Nelms, K., White, M., Wang, L., Pierce, J. H., and Paul, W. E. (1994) Cell 76, 811-820 [Medline] [Order article via Infotrieve]
  31. Wang, L., Myers, M. G., Sun, X., Aaronson, S. A., White, M., and Pierce, J. H. (1993) Science 261, 1591-1594 [Medline] [Order article via Infotrieve]
  32. Kitamura, T., Takaku, F., and Miyajima, A. (1991) Int. Immunol. 3, 571-577 [Abstract]
  33. Kawamura, M., McVicar, D. W., Johnston, J. A., Blake, T. B., Chen, Y. C., Lal, B. K., Lloyd, A. R., Kelvin, D. J., Staples, J. E., Ortaldo, J. R., and O'Shea, J. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6374-6378 [Abstract]
  34. Voss, S. D., Sondel, P. M., and Robb, R. J. (1992) J. Exp. Med. 176, 531-541 [Abstract]
  35. Kirken, R. A., Rui, H., Evans, G. A., and Farrar, W. L. (1993) J. Biol. Chem. 268, 22765-22770 [Abstract/Free Full Text]
  36. Schlessinger, J. (1994) Curr. Opin. Gen. Dev. 4, 25-30 [Medline] [Order article via Infotrieve]
  37. Wang, L., Keegan, A. D., Paul, W. E., Heidaran, M. A., Gutkind, J. S., and Pierce, J. H. (1992) EMBO J. 11, 4899-4908 [Abstract]

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