©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Signal Transduction by the Human Interleukin-2 Receptor Requires Cytoplasmic Tyrosines of the Chain and Non-tyrosine Residues of the Chain (*)

(Received for publication, March 18, 1995; and in revised form, June 1, 1995)

Mark A. Goldsmith (1) (2)(§) Stephen Y. Lai (1)(¶) Weiduan Xu (1)(**) M. Catherine Amaral (1)(**) Elizabeth S. Kuczek (1)(**) Leslie J. Parent (4)(§§) Gordon B. Mills (5) Kathleen L. Tarr (6) Gregory D. Longmore (6) Warner C. Greene (1) (2)(**) (3)

From the  (1)Gladstone Institute of Virology and Immunology, Departments of (2)Medicine and (3)Microbiology and Immunology, School of Medicine, University of California, San Francisco, California 94141-9100, the (4)Department of Microbiology and Immunology, Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033, the (5)Toronto General Hospital, Toronto, Ontario M4X 1K9, Canada, and the (6)Departments of Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To evaluate the possible role for receptor-based tyrosine phosphorylation in growth signaling induced by interleukin-2 (IL-2), a series of substitution tyrosine mutants of the IL-2 receptor beta and (c) chains was prepared and analyzed. Concurrent mutation of all six of the cytoplasmic tyrosines present in the beta chain markedly inhibited IL-2-induced growth signaling in both pro-B and T cell lines. Growth signaling in a pro-B cell line was substantially reconstituted when either of the two distal tyrosines (Tyr-392, Tyr-510) was selectively restored in the tyrosine-negative beta mutant, whereas reconstitution of the proximal tyrosines (Tyr-338, Tyr-355, Tyr-358, Tyr-361) did not restore this signaling function. Furthermore, at least one of the two cytoplasmic tyrosines that is required for beta chain function was found to serve as a phosphate acceptor site upon induction with IL-2. Studies employing a chimeric receptor system revealed that tyrosine residues of the beta chain likewise were important for growth signaling in T cells. In contrast, although the (c) subunit is a target for tyrosine phosphorylation in vivo, concurrent substitution of all four cytoplasmic tyrosines of this chain produced no significant effect on growth signaling by chimeric IL-2 receptors. However, deletion of either the Box 1, Box 2, or intervening (V-Box) regions of (c) abrogated receptor function. Therefore, tyrosine residues of beta but not of (c) appear to play a pivotal role in regulating growth signal transduction through the IL-2 receptor, either by influencing cytoplasmic domain folding or by serving as sites for phosphorylation and subsequent association with signaling intermediates. These findings thus highlight a fundamental difference in the structural requirements for IL-2Rbeta and (c) in receptor-mediated signal transduction.


INTRODUCTION

Interleukin-2 (IL-2) (^1)is a helical cytokine that induces the proliferation of T and B lymphocytes as well as the expression of a number of immune effector functions by binding to the heterotrimeric IL-2 receptor complex (IL-2R). The 70-75-kDa beta (IL-2Rbeta) and 64-kDa ((c)) subunits of the IL-2R share structural homology with other members of a cytokine receptor superfamily (1) and together form a receptor complex that is competent to bind IL-2 with intermediate affinity and to transduce growth and differentiation signals (reviewed in (2) ). As in other receptor systems, evidence has accumulated indicating that signal transduction is initiated upon ligand-induced heterodimerization of the beta and (c) cytoplasmic tails(3, 4) . Interestingly, IL-2Rbeta is also employed in the receptor for IL-15(5, 6) , whereas (c) participates in the formation of the receptors for IL-4(7) , IL-7(8, 9) , IL-9(10) , and IL-15(6) .

Among the earliest biochemical changes induced by ligation of the IL-2 receptor is activation of cytoplasmic tyrosine kinases resulting in the phosphorylation of certain recognized and unrecognized cellular substrates. The biologic relevance of IL-2-induced tyrosine kinase activity is supported by the finding that selective tyrosine kinase inhibitors (herbimycin A and genistein) concomitantly block these intracellular phosphorylation events as well as growth signal transduction(11, 12) . Although none of the known IL-2R subunits contain recognizable kinase catalytic domains, tyrosine kinase activity has been coimmunoprecipitated with the IL-2R(13, 14, 15, 16, 17, 18) . Recent evidence indicates that the Janus kinases JAK1 and JAK3 (19, 20, 21) as well as various src family kinases (13, 15, 18, 22) are among the signaling molecules that are physically and functionally linked to the IL-2R. However, the specific role of each of these kinases and their substrates in IL-2R signal transduction remains to be defined.

Like many growth factor receptors containing intrinsic tyrosine kinase activity (for review, see (23) ), the cytoplasmic domains of the beta and subunits of the interleukin-2 receptor itself undergo inducible tyrosine phosphorylation upon engagement by IL-2(24, 25, 26) . The biological significance of such receptor phosphorylation is poorly defined for cytokine receptors lacking intrinsic tyrosine kinase activity. Since the IL-2 receptor itself is a major substrate of tyrosine phosphorylation following the binding of IL-2, the present investigation was undertaken to determine the potential regulatory role played by the cytoplasmic tyrosine residues of the IL-2Rbeta and (c) subunits. Our results demonstrate that tyrosines within the cytoplasmic tail of IL-2Rbeta are critical for full growth signaling in pro-B and T cells. In contrast, the tyrosine residues of the (c) chain are dispensable for this function, revealing an important distinction between the IL-2Rbeta and (c) subunits. These findings, along with a delineation of essential membrane-proximal domains of (c), may have general implications for the functional design of cytokine receptors, particularly those employing the common (c) subunit.


MATERIALS AND METHODS

Cell Lines

The cell line BA/F3(27) , an IL-3-dependent murine pro-B cell line, was maintained as described previously(28) . Supernatant from WEHI-3 cells (ATCC) was used as a source of IL-3. HT-2, an IL-2-dependent murine helper T cell line (ATCC), was maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 55 µM beta-mercaptoethanol, 2 mML-glutamine, and 200 units/ml recombinant human IL-2 (a gift of the Chiron Corp.). Transfection of either BA/F3 or HT-2 cells was performed by electroporation as described previously(28) ; stable transfectants were obtained by selection in G418 (Geneticin, 1 mg/ml, Life Technologies, Inc.) and clones isolated by limiting dilution were screened by radioligand binding analysis with I-IL-2 or I-EPO (see below) or by Northern blot analysis to identify clones expressing the transfected receptor (see text and figure legends). HT-2EPObeta was established by transfecting HT-2EPObeta cells with pEPOneo and culturing in recombinant human EPO (10 units/ml, Amgen, Inc.) without IL-2. The COS-7 cell line (ATCC) was maintained as described(29) .

Proliferation Assays

Conventional 24-h thymidine incorporation assays and transfection proliferation assays were performed essentially as described previously (28) . In transfection studies using the chimeric receptors, HT-2 cells and their derivatives (see text) were transfected with expression plasmids encoding chimeric receptors and were then selected for approximately 10 days in EPO (50 units/ml) in the absence of IL-2; cell growth was assessed by [^3H]thymidine incorporation on the indicated days.

Plasmid Constructs

All receptor cDNAs were subcloned into the expression vectors pCMV4(30) , pCMV4Neo(28) , or pCMV4Delta (a pCMV4 derivative containing a deletion of a vestigial second polylinker downstream of the cytomegalovirus expression cassette). For all constructs requiring synthetic oligonucleotides or PCR reactions, sequences were confirmed by DNA sequencing. The murine EPOR cDNA from pXM-nEPOR (31) was inserted into the KpnI/XbaI sites of pCMV4Neo to yield pEPORneo, and the human IL-2Rbeta cDNA from pIL2R30 (provided by T. Taniguchi) was inserted into the HindIII/BamHI sites of pCMV4Neo to yield pbetaneo.

The tyrosine substitution mutants of IL-2Rbeta and (c) (tyrosine (TAC) to phenylalanine (TTC)) were prepared by a combination of oligonucleotide-directed mutagenesis in M13 bacteriophage and PCR-based methods. For constructs involving the (c) cytoplasmic tail, a full-length cDNA was obtained by reverse transcription PCR based on the IL-2R sequence reported by Takeshita et al.(32) . Deletion and substitution mutants described under ``Results'' (see figure legends) were prepared by PCR using IL-2Rbeta or (c) cDNAs as templates.

pEPObetaneo, constructed by PCR using an NheI site at the fusion junction, encodes a chimeric receptor (see Fig. 6A) containing the extracellular domain of the EPOR fused just above the transmembrane segment to the human IL-2Rbeta transmembrane and cytoplasmic segments (resulting sequence: . . . (EPOR-T-A-S)-(G-K-D-IL-2Rbeta) . . . ). pEPOneo, also constructed by PCR using the NheI site, encodes a receptor (see Fig. 6A) containing the extracellular domain of the EPOR fused to the human (c) transmembrane and cytoplasmic segments (resulting sequence: . . . (EPOR-T-A-S)-(S-K-E-(c)) . . . ). Expression plasmids encoding the mutants described in the text were prepared by subcloning appropriate DNA fragments spanning the indicated mutations into the parental pEPObetaneo and pEPOneo plasmids.


Figure 6: Chimeric EPOR/IL-2R receptor subunits. A, schematic representation of native (IL-2Rbeta, (c), and EPOR) and chimeric (EPObeta and EPO) subunits fused at a unique NheI site located immediately N-terminal to the transmembrane segment. Some conserved features of the cytokine receptor superfamily shown are indicated (the extracellular WSXWS sequence and the intracellular signaling domain, including Box 1, Box 2, and the intervening V-(variable) Box). B, immunoblot analysis of native and chimeric receptor subunits. The EPOR and EPObeta variants are shown on the left, and the EPO variants are shown on the right. The betaDeltaA, betaDeltaAB, and betaD258A mutations have been described(28) ; in the betaYF and YF cytoplasmic tails all tyrosines (TAC) have been replaced with phenylalanines (TTC). See Fig. 9legend for description of additional mutant (c) cytoplasmic tails. C, parental HT-2 cells(-) or stable transfectants expressing EPOR, EPObeta, or EPO were stimulated with either EPO (closed bars, 50 units/ml) or IL-2 (open bars, 10 nM) for 24 h, and [^3H]thymidine incorporation was assessed as in Fig. 2. Results are expressed relative to the level of incorporation occurring with IL-2 stimulation (100%); error bars represent standard errors of the mean (n = 3).




Figure 9: Functional analyses of EPOR/(c) chimeras in transfection assays of proliferation. Transfection growth assays using the HT-2EPObeta line as a host to assess the responses of the the indicated EPO mutants. The 336, 294, and TM mutants are truncated immediately after amino acids 336, 294, and 286, respectively, in the mature (c) protein. DeltaBox 1 is deleted of residues 281-294, DeltaV-Box is deleted of residues 295-320, and DeltaBox 2 is deleted of residues 321-334. Results are expressed as the incorporation of [^3H]thymidine for each line relative to that of the wild type (wt) cytoplasmic tail, with standard errors of the mean (n geq 3).




Figure 2: Growth signaling and expression characteristics of stable transfectants expressing tyrosine-negative IL-2Rbeta chains. BA/F3 cells expressing wild type IL-2Rbeta (BafbetaWT) or tyrosine-negative mutant IL-2Rbeta chains (BafbetaYF) were analyzed. Cells stimulated for 24 h with IL-2 at the indicated concentration (in the absence of IL-3) were pulsed with [^3H]thymidine for the final 4 h of the culture and harvested. Results are expressed relative to the level of incorporation occurring with IL-3 stimulation (100%); error bars represent standard errors of the mean (n = 3).



Protein Expression and Phosphorylation Studies

COS-7 cells (ATCC) were transfected with the indicated plasmids (see text) using Lipofectamine (Life Technologies, Inc.) as per the manufacturer's instructions. For expression analysis of chimeric receptors, immunoblotting analyses were performed on cell lysates using an anti-EPOR N-terminal peptide antiserum and I-protein A as described previously(31) . For phosphorylation analyses, the indicated cell lines were stripped of bound ligands by a 1-min acidic wash (10 mM sodium citrate, 0.14 M NaCl, pH 4) and then were rested in medium without serum or cytokines for 4 h. Cells were then stimulated with either IL-2 (10 nM) or EPO (50 units/ml) for 10 min at 37 °C, lysed (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 8.0, 50 mM NaF, 100 µM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 µg/ml pepstatin A) and immunoprecipitated with either the anti-IL-2Rbeta monoclonal antibody 561 (kindly provided by Dr. R. Robb) or an anti-JAK1 antiserum (Upstate Biotechnology, Inc.) and protein A-Sepharose. Immunoblotting studies were performed with anti-phosphotyrosine antibody (4G10, Upstate Biotechnology, Inc.) per the manufacturer's instructions followed by ECL (Amersham Corp.) signal development.

Radioligand Binding and Cross-linking

Equilibrium binding analyses were performed as described (28, 33) with I-IL-2 (DuPont NEN) and either BA/F3 cell lines or COS-7 cells transfected by the DEAE-dextran method (34) with human IL-2R and the indicated IL-2Rbeta expression plasmids. Cross-linking analyses were performed by cotransfecting COS-7 cells with IL-2Ralpha and IL-2Rbeta expression plasmids followed by incubation with I-IL-2, cross-linking with disuccinimidyl suberate (Pierce), immunoprecipitation with the anti-beta monoclonal antibody DU-2(14) , and analysis by SDS-polyacrylamide gel electrophoresis and autoradiography, as described previously(35, 36) .


RESULTS

Substitution Mutation of all Six Cytoplasmic Tyrosine Residues in IL-2Rbeta Impairs Growth Signal Transduction in a Transient Assay System

The cytoplasmic tail of the human interleukin-2 receptor (IL-2R) beta chain contains six tyrosine residues (37) (Fig. 1), including four in the ``acidic'' region (A) (38) and one in each of two distal segments (B, C)(28) . To investigate the possibility that growth signaling through the IL-2R is regulated by tyrosine phosphorylation, a mutant IL-2Rbeta chain (betaYF) containing concurrent substitutions of phenylalanine at all six cytoplasmic tyrosine positions was prepared and analyzed in a transient assay of lymphocyte growth signal transduction. In this method(28) , IL-3-dependent murine pro-B cells (BA/F3) (27) containing endogenous IL-2R chains are transfected with expression plasmids encoding wild type or mutant IL-2Rbeta and selected in medium containing IL-2 in the absence of IL-3. Cells transfected with wild type IL-2Rbeta (betaWT) chains proliferated vigorously as indicated by substantial incorporation of [^3H]thymidine within 7 to 9 days, whereas cells receiving the vector control died in culture (Fig. 1A). Using this assay system, lymphocytes transfected with the all tyrosine-negative IL-2Rbeta mutant (betaYF) demonstrated a dramatically impaired proliferative response to IL-2 (Fig. 1B). Thus, one or more of these cytoplasmic tyrosines of IL-2Rbeta appeared to be critically required for full growth signal transduction through the IL-2R.


Figure 1: Growth signal transduction properties and expression of tyrosine-negative mutant IL-2beta chains in transient assays. A, proliferation of BA/F3 cells transfected with expression vector encoding wild type IL-2Rbeta (betaWT, closed circles) or empty vector (Vector, open circles), as measured by incorporation of [^3H]thymidine on the indicated days following initiation of IL-2 selection (10 nM) in the absence of IL-3. Each data point is the mean of triplicates, and each experiment shown is representative of several independent experiments. The relative positions of the six cytoplasmic tyrosine residues of IL-2Rbeta are indicated by closed symbols in the schematic: 1, Tyr-338; 2, Tyr-355; 3, Tyr-358; 4, Tyr-361; 5, Tyr-392; 6, Tyr-510. B, proliferation of cells transfected with vector encoding tyrosine-negative mutant of IL-2Rbeta (betaYF, closed squares). C, surface expression of IL-2Rbeta and betaYF mutant receptors. Autoradiograph of immunoprecipitates of COS cells cotransfected with vectors encoding wild type human IL-2Ralpha and either IL-2Rbeta (betaWT) or the tyrosine-negative mutant (betaYF) prepared following disuccinimidyl suberate-mediated cross-linking with I-IL-2. Molecular mass markers are shown on left (kDa). D and E, equilibrium I-IL-2 binding analysis of COS cells cotransfected with vectors encoding wild type human IL-2R and either IL-2Rbeta (D) or betaYF (E).



Two independent types of experiments were performed to ensure that the impaired function of betaYF was not simply the result of ineffective surface expression or faulty binding of ligand. First, to monitor surface expression COS cells were transiently transfected with expression vectors encoding the IL-2Ralpha chain and either native IL-2Rbeta or betaYF, followed by incubation with I-IL-2, chemical cross-linking with disuccinimidyl suberate, and immunoprecipitation with the anti-beta monoclonal antibody DU-2(14) . Following SDS-polyacrylamide gel electrophoresis, bands of comparable intensity and migration were observed for cells transfected with the wild type beta and betaYF, indicating the unimpaired surface expression of the mutant betaYF receptor (Fig. 1C). To investigate potential changes in receptor affinity, radioligand binding analyses were performed with I-IL-2 in COS cells transfected with IL-2R and betaWT or betaYF. These studies revealed the expected single class of intermediate affinity IL-2 binding sites for both betaWT and betaYF (K 300-400 pM) (Fig. 1, D and E). Thus, surface expression and ligand binding by betaYF appeared indistinguishable from wild type beta and therefore do not account for its impaired signaling function in the transfection assay system.

The Tyrosine-negative Mutant of IL-2Rbeta Demonstrates Impaired Responsiveness to IL-2 in a Stable Transfectant

To confirm the phenotype of betaYF, stable sublines of BA/F3 were prepared by transfection with the plasmid pbetaYFNeo. Radioligand analysis demonstrated that the BafbetaWT and BafbetaYF cell lines expressed receptors that bound IL-2 with comparable intermediate affinities (data not shown), although for unknown reasons the BafbetaYF lines consistently expressed the receptor at somewhat lower levels than did BafbetaWT (BafbetaWT, 3000 receptors/cell; BafbetaYF, 700 receptors/cell). Nevertheless, in analyses of numerous sublines we have seen no correlation between expression levels in this range and proliferative signaling capacity.

Analysis of [^3H]thymidine incorporation in response to IL-2 revealed marked unresponsiveness of the stable BafbetaYF cell line to IL-2 compared with BafbetaWT (Fig. 2). As expected, the BafbetaWT cell line demonstrated detectable proliferation even at very low doses of IL-2 (10 pM) well below the K of IL-2 binding to IL-2Rbeta complexes, whereas the BafbetaYF line demonstrated no response even at very high doses of IL-2 (100 nM) vastly exceeding the measured K. These findings confirmed the impaired proliferation signaling exhibited by the betaYF mutant initially detected in the transient system.

Selective Mutation of Individual Tyrosine Residues Does Not Alter IL-2Rbeta Growth Signaling in a Pro-B Cell Line

The results in both transient and stable assay systems indicated that at least one tyrosine residue contributes importantly to IL-2R growth signaling competence in pro-B cells. To identify the relevant functional tyrosine residue(s), IL-2Rbeta mutants containing selective phenylalanine for tyrosine substitutions were constructed and characterized using the BA/F3 transient assay system. Surprisingly, substitution of phenylalanine at Tyr-338 (betaY1F), Tyr-355/Tyr-358/Tyr-361 (betaY234F), Tyr-392 (betaY5F), or Tyr-510 (betaY6F) had little or no effect on growth signal transduction in response to IL-2 (Fig. 3). In contrast to betaYF, each of these selective tyrosine mutants mediated substantial proliferation; only a subtle compromise in receptor function was intermittently observed with betaY5F and betaY6F. These results revealed that no single cytoplasmic tyrosine is essential to growth signaling function, implying that a functional redundancy may exist involving two or more of these residues.


Figure 3: Peak proliferative responses of various tyrosine and deletion mutants of IL-2Rbeta. Transient proliferation assays were performed as in Fig. 1. Peak [^3H]thymidine incorporation is shown as a percentage of the peak response by betaWT transfectants in each assay. Each value is the mean of triplicate determinations with standard errors of the mean, and results shown are representative of multiple independent experiments. betaWT, wild type IL-2Rbeta; betaYF, tyrosine-negative IL-2Rbeta; betaY1F, beta with Tyr-338 (1) mutated to Phe; betaY234F, beta with Tyr-355(2) , Tyr-358(3) , and Tyr-361 (4) mutated to Phe; betaY5F, beta with Tyr-392 (5) mutated to Phe; betaY6F, beta with Tyr-510 (6) mutated to Phe; betaYF:56Y, beta with Tyr-338(1) , Tyr-355(2) , Tyr-358(3) , and Tyr-361 (4) mutated to Phe; betaY56F, beta with Tyr-392 (5) and Tyr-510 (6) mutated to Phe; betaDeltaAB, IL-2Rbeta deleted from amino acid 313-431; betaDeltaABC, IL-2Rbeta truncated after amino acid 312.



Either Tyr-392 or Tyr-510 Alone Is Sufficient to Permit IL-2Rbeta Growth Signaling Function in Pro-B Cells

Previous reports with stable transfectants expressing IL-2Rbeta mutants had demonstrated that the ``A'' segment spanning the first four cytoplasmic tyrosine residues is dispensable for growth signaling function(38) , an observation confirmed in our previous studies employing the transient assay system in BA/F3 cells(28) . This finding implied that the C-terminal tyrosines (Tyr-392 and Tyr-510) may be sufficient for full growth signaling. To evaluate this possibility, a mutant was prepared (betaYF:56Y) containing substitutions of phenylalanine for the proximal four tyrosines, leaving the distal tyrosines intact; this mutant mediated a full proliferative response to IL-2 in BA/F3 cells (Fig. 3). In contrast, a mutant with phenylalanines replacing exclusively these two distal tyrosines (betaY56F) was substantially impaired in its growth signal transduction capacity in the BA/F3 cells, further demonstrating the importance of Tyr-392 and Tyr-510 to growth signaling by IL-2Rbeta (Fig. 3).

We further observed that internal deletion of a 119-amino acid cytoplasmic region of IL-2Rbeta spanning the A region as well as the contiguous ``B'' segment exhibited fully preserved growth signaling (Fig. 3, betaDeltaAB), suggesting that the first five tyrosines are dispensable. In contrast, extension of this deletion to include the C-terminal region containing the sixth tyrosine (betaDeltaABC) abrogated receptor function (Fig. 3). These results suggested that the sixth tyrosine (Tyr-510) is sufficient to permit growth signal transduction. Indeed, an IL-2Rbeta mutant in which only this single tyrosine was restored in the betaYF background (betaYF:6Y) exhibited substantial IL-2 growth signaling (Fig. 4A).


Figure 4: Proliferative responses of IL-2Rbeta mutants with selective reconstitution of cytoplasmic tyrosines. Transient proliferation assays were performed as in Fig. 1. A, betaYF:6Y, betaYF with Phe-510 (6) back-mutated to Tyr; B, betaYF:5Y, betaYF with Phe-392 (5) back-mutated to Tyr; C, betaYF:234Y, betaYF with Phe-355(2) , Tyr-358(3) , and Tyr-361 (4) back-mutated to Tyr; D, betaYF:1Y, betaYF with Phe-338 (1) back-mutated to Tyr. Constructs were prepared by recombination of cytoplasmic restriction fragments derived from mutants shown in Fig. 3and were verified by DNA sequence analysis.



Although Tyr-510 alone is sufficient for receptor competence, selective substitution of phenylalanine at this position had little effect on the signaling function (Fig. 3). These results strongly implied that at least one other tyrosine site also could support growth signal transduction, a hypothesis that was tested by evaluating additional tyrosine add-back mutants. Interestingly, reconstitution of Tyr-392 (betaYF:5Y) substantially restored the IL-2Rbeta signaling function (Fig. 4B). In contrast, restoration of tyrosines in the first four positions in two additional add-back mutants (betaYF:234Y and betaYF:1Y) failed to reconstitute receptor function (Fig. 4, C and D, respectively). Importantly, the betaYF:56Y, betaY56F, betaYF:1Y, betaYF234Y, betaYF:5Y, and betaYF:6Y proteins were all expressed abundantly as detected by immunoblotting analysis (data not shown). Thus, either the fifth tyrosine (Tyr-392) or sixth tyrosine (Tyr-510) is necessary and sufficient for IL-2 growth signaling in BA/F3 cells.

Tyrosine 392 of IL-2Rbeta Is Phosphorylated upon Engagement of the IL-2R

The present findings indicating a functional role for certain cytoplasmic tyrosine residues of IL-2Rbeta raised the important question of whether or not these tyrosine residues serve as phosphate acceptor sites, a possibility suggested by the recognition that this chain undergoes rapid tyrosine phosphorylation during receptor activation(24, 25) . To address this question, stable transfectants of the BA/F3 line were prepared using expression plasmids encoding tyrosine add-back mutants (pbetaYF:5YNeo and pbetaYF:6YNeo). Both of the resulting cell lines (BafbetaYF:5Y and BafbetaYF:6Y) proliferated vigorously in IL-2 despite the unresponsiveness of the BafbetaYF line (Fig. 5). These results confirmed in permanent BA/F3 cell lines the reconstitution of growth signaling function upon restoration of either Tyr-392 or Tyr-510.


Figure 5: Signaling function and receptor phosphorylation in stable transfectants expressing IL-2Rbeta tyrosine add-back mutants. A, BA/F3 cells expressing tyrosine-negative IL-2Rbeta (BafbetaYF) or IL-2Rbeta tyrosine add-back mutants with restoration of Tyr-392 (BafbetaYF:5Y) or Tyr-510 (BafbetaYF:6Y) IL-2Rbeta chains (BafbetaYF) were analyzed. A, [^3H]Thymidine incorporation assay with the indicated doses of IL-2, as described in the legend to Fig. 2. B, tyrosine phosphorylation analysis of BafbetaWT (betawt), BafbetaYF (betaYF), and BafbetaYF:5Y (betaYF:5Y). Anti-IL-2Rbeta immunoprecipitates from cells stimulated with IL-2 for 10 min were subjected to immunoblotting with anti-phosphotyrosine antibody.



Phosphorylation studies were next performed using these stable transfectants. In these experiments, cell lines were rested without growth factors and then exposed to IL-2. Stimulated cells were lysed, immunoprecipitated with anti-IL-2Rbeta monoclonal antibody, and then subjected to immunoblot analysis with anti-phosphotyrosine antibody. Upon induction with IL-2 the BafbetaWT line yielded a strong phosphotyrosine signal at the appropriate molecular weight for IL-2Rbeta chains, whereas the BafbetaYF line yielded no discernible signal (Fig. 5). Like BafbetaWT, BafbetaYF:5Y cells also yielded a phosphotyrosine-containing protein band (Fig. 5B). Since this add-back cell line expresses IL-2Rbeta chains containing only a single cytoplasmic tyrosine residue (Tyr-392) with all others replaced by phenylalanine, a phosphotyrosine signal generated in the immunoblot experiment is clearly attributable to this tyrosine. These results thus indicated that Tyr-392 of IL-2Rbeta serves as a phosphate acceptor site during receptor activation.

Similar experiments were performed with the BafbetaYF:6Y line to assess the role of Tyr-510 in receptor phosphorylation. Surprisingly, no IL-2Rbeta chain tyrosine phosphorylation was detectable in experiments with cells expressing the Tyr-510 add-back mutant (data not shown). Such experiments were performed with multiple, independently derived lines, and stimulations were performed for various lengths of time ranging from 3 to 30 min. It remains possible that this functional tyrosine residue of IL-2Rbeta does indeed undergo phosphorylation and that this site is perhaps particularly sensitive to phosphatase attack after detergent solubilization of the cells. Nonetheless, phosphorylation of this tyrosine has not yet been detected (see ``Discussion'').

Establishment of EPOR/IL-2R Chimeric Receptors to Study the Cytoplasmic Domains of the IL-2Rbeta and (c)Receptor Subunits in T Cells

To permit study of the functional interactions of the IL-2Rbeta and (c) cytoplasmic domains in T lymphocytes already expressing endogenous IL-2 receptors, we developed a chimeric receptor system in which the intracellular domains of interest (derived from IL-2Rbeta and (c)) were fused to an extracellular ligand binding domain not present in the host cell lines (Fig. 6A). Extracellular domains of the homodimeric EPOR extracellular domain were employed for this purpose, since the EPOR, IL-2Rbeta, and (c) subunits are all members of the cytokine receptor superfamily. Because the EPOR homodimerizes in the presence of EPO, these chimeric receptors were expected to promote dimerization of the IL-2Rbeta and/or (c) cytoplasmic domains following ligand binding. Plasmids encoding the chimeric EPObeta and EPO receptors expressed proteins of the predicted masses as detected by immunoblot analysis of lysates from transfected COS-7 cells (Fig. 6B): the native EPOR and wild type EPObeta and EPO constructs yielded bands of approximately 70, 75, and 40 kDa, respectively. Frequently protein doublets were observed with all of these constructs, which result from variable glycosylation.

The IL-2-dependent murine helper T cell line, HT-2, was employed for analysis of EPObeta and EPO signaling. Initially, stable HT-2 transfectants expressing the EPOR, EPObeta, or EPO subunits were established. In 24-h [^3H]thymidine incorporation assays, the EPOR was found to mediate a modest response to EPO, whereas neither of the chimeric receptor subunits alone produced a detectable response in multiple transfected clones (Fig. 6C). The failure of EPObeta and EPO to mediate a response was not due to lack of expression, since Northern blotting, Western blotting, and radioligand binding analyses with I-EPO confirmed the expression and ligand binding competence of these chimeras in the HT-2EPObeta and HT-2EPO cell lines (data not shown).

Since neither chimera alone (EPObeta or EPO) demonstrated detectable growth signal transduction, combinations of these chimeras in HT-2 cells were tested for growth signaling in response to EPO as a means of promoting heterodimerization of the IL-2Rbeta and (c) cytoplasmic tails. For these studies the transfection assay originally described for BA/F3 cells (28) was adapted to HT-2 cells. When the EPO expression plasmid was introduced by electroporation into multiple HT-2 clones stably expressing EPObeta (HT-2EPObeta), addition of EPO without IL-2 produced marked proliferation and vigorous incorporation of [^3H]thymidine during the 12-day assay (Fig. 7A). Similarly, multiple HT-2 clones stably expressing EPO (HT-2EPO) displayed marked proliferative responses to EPO following introduction of the EPObeta expression plasmid (Fig. 7B). Additionally, double transfectants arising from such experiments were easily maintained in long term culture by addition of EPO alone, allowing the isolation of a stable transfected cell line (HT-2EPObeta) for further studies of early signal transduction events. Thus, concurrent engagement of both the beta and (c) chimeras is required for effective growth signaling, as has been reported in studies with other chimeric receptors(3, 4) .


Figure 7: Functional analyses of EPOR/IL-2R chimeras. A, parental HT-2 cells or HT-2EPO cells described in the legend to Fig. 6were transfected with the EPObeta expression plasmid, selected in EPO (50 units/ml) without other cytokines, and assayed for growth by measuring [^3H]thymidine incorporation on the indicated days. B, parental HT-2 cells or HT-2EPObeta cells described in Fig. 6were transfected with the EPO expression plasmid, selected in EPO, and assayed for growth. C, HT-2EPO cells were transfected with the EPObetaYF expression plasmid, selected in EPO, and assayed for growth. Each experiment shown was performed multiple times with similar results. D, to assess phosphorylation of JAK1 during receptor activation, the stable transfectants HT-2EPObeta and HT-2EPObetaYF/ were stimulated with no cytokine(-), EPO (E), or IL-2 (2) followed by immunoprecipitation with the anti-JAK1 antiserum and immunoblotting with the anti-phosphotyrosine antibody.



Tyrosine Residues of IL-2Rbeta Are Required for Full Growth Signaling in Mature T Cells

The functional contribution of IL-2Rbeta cytoplasmic tyrosines in T cells was assessed using the chimeric receptor system and the HT-2 cell line. HT-2EPO cells transfected with expression plasmids encoding either wild type EPObeta or a mutant, tyrosine-negative EPOR/IL-2Rbeta chimera (EPObetaYF) were selected in EPO and assessed for proliferation. Unlike the parental EPObeta (Fig. 7A), the tyrosine-negative EPObetaYF exhibited no detectable growth response to EPO (Fig. 7C). Similarly, stable double transfectants of HT-2 expressing both EPObetaYF and EPO demonstrated no proliferation response to EPO (data not shown). These findings demonstrated that the cytoplasmic tyrosines of the IL-2Rbeta chain strongly influence receptor growth signaling independently of ligand specificity in both pro-B and mature T cells.

To analyze further the disruption in signal transduction by the betaYF mutant, Janus kinase induction in response to receptor engagement was assessed. Lysates prepared from HT-2 cells stimulated with no cytokine, IL-2, or EPO were subjected to immunoprecipitation with an anti-JAK1 antiserum followed by immunoblot analysis with an anti-phosphotyrosine antibody. Cells expressing chimeric (c) chains and either wild type chimeric beta chains (HT-2EPObeta) or tyrosine-negative beta chains (HT-2EPObetaYF/) both exhibited strong induction of JAK1 phosphorylation in response to either ligand (Fig. 7D). Likewise, preserved induction of JAK3 phosphorylation by receptor complexes containing EPObetaYF was observed in parallel experiments employing an anti-JAK3 antiserum (data not shown). Therefore, at least one early phase of receptor-mediated signaling by the betaYF mutant is intact despite the failure to achieve full growth signaling.

Characterization of Cytoplasmic (c)Mutant Function in T Lymphocytes

Development of the chimeric receptor system also permitted an examination in T cells of the functional contributions of tyrosine residues and other elements within the (c) cytoplasmic tail. We therefore introduced EPO mutants into the HT-2EPObeta stable cell line for functional analysis in the transfection assay. Protein expression from the various mutant EPO chimeric constructs was first verified by immunoblot analysis of lysates from transiently transfected COS-7 cells (Fig. 6B). As predicted, the substitution mutant construct (EPOYF, see below) produced protein comparable with that of the wild type EPO construct, and the deletion mutants (EPO336, EPO294, EPODeltaBox1, and EPODeltaV-Box) produced slightly faster migrating species.

Since tyrosine phosphorylation of the (c) subunit upon ligand binding has been well described(26) , we investigated the putative role of the tyrosine residues present in the (c) subunit by phenylalanine substitution of all four tyrosine residues (EPOYF). Surprisingly, growth signal transduction by EPOYF was nearly indistinguishable from that by EPO both in transfection assays (Fig. 8, A and B) and in 24-h [^3H]thymidine incorporation assays of stable transfectants arising from transfection of HT-2EPObeta cells with the EPOYF expression plasmid (Fig. 8, C and D). Thus, the cytoplasmic tyrosine residues of (c) appeared to be dispensable for growth signaling, which stands in sharp contrast to their importance in the IL-2Rbeta subunit.


Figure 8: Preserved growth signal transduction function of tyrosine-negative (c) chains. A and B, transfection growth assays were performed by transfecting HT-2EPObeta cells with expression vectors encoding either EPO (A) or EPOYF (B) and selecting in EPO, as in Fig. 7. C and D, stable HT-2 transfectants expressing EPObeta and either EPO (C) or EPOYF (D) were analyzed by 24-h [^3H]thymidine incorporation assays, as in Fig. 6C. Shown is the dose response to EPO for each cell line relative to the response to IL-2, with closed bars representing the means and error bars indicating the standard errors of the mean (n = 3).



Although the tyrosine residues are non-essential, other regions of the (c) cytoplasmic tail proved important for growth signaling. EPO mutants truncated at the cell membrane (EPOTM) or at the end of the Box 1 (39) homology region (EPO294) mediated no detectable proliferation signaling (Fig. 9). Similarly, internal deletion of Box 1 (EPODeltaBox1), of a segment with distant relationship to the Box 2 motif (EPODeltaBox2), or of the segment connecting Box 1 to Box 2 (EPODeltaV-Box), also abolished proliferation signaling. However, truncation of the (c) subunit at the C-terminal end of the Box 2 region (EPO336) resulted in levels of growth signaling similar to that obtained with the wild type subunit. Thus, unlike the IL-2Rbeta subunit, the distal portion of the (c) subunit is dispensable for proliferation signal transduction, and full growth-signaling function resides in the proximal 53 amino acids containing the Box 1, Box 2, and intervening (V-Box) segments.


DISCUSSION

Like many other cytokine receptor systems, the binding of IL-2 to the IL-2R induces the tyrosine phosphorylation of a variety of intracellular substrates, including the IL-2Rbeta and (c) chains(24, 25, 26) . Although no tyrosine kinase domain is identifiable within the recognized ligand-binding subunits of the IL-2R, the Janus kinases JAK1 and JAK3 as well as the src family kinase p56 and p59 are now recognized to associate noncovalently with the cytoplasmic tails of IL-2R subunits(10, 15, 19, 40) . The activation of such receptor-associated kinases may represent a mechanism for signal transduction that is fundamentally the same as that for receptors containing intrinsic kinase activity. Indeed, as in such kinase-containing receptors, some evidence has accumulated from mutagenesis and in vitro analyses that certain tyrosine residues of the IL-4 and interferon receptors are crucial for signal transduction competence(41, 42, 43, 44) .

The present studies were undertaken to evaluate the potential regulatory role of cytoplasmic tyrosines of the IL-2Rbeta and (c) chains. In these studies employing both native and chimeric receptors, substitution of phenylalanine for all six cytoplasmic tyrosine residues of IL-2Rbeta substantially impaired growth signaling in both a pro-B and a mature T cell line ( Fig. 1and Fig. 7). A panel of add-back mutants revealed that both Tyr-392 and Tyr-510 individually exhibit signaling potential in the BA/F3 pro-B cell line while the four more proximal tyrosines demonstrate no functional capacity in this specific cellular environment (Fig. 4). We conclude from these experiments that, in BA/F3 cells, the two C-terminal cytoplasmic tyrosines serve important but redundant functions in determining the signal transduction competence of the IL-2Rbeta chain.

The finding that C-terminal tyrosines of IL-2Rbeta influence growth signaling in this system appears to contrast with an earlier report that the IL-2Rbeta segment encompassing these tyrosines is dispensable for proliferative signaling(38) . However, point substitutions and deletions of identical regions may have different phenotypic consequences, particularly if the protein region in question exerts regulatory effects via conformational changes. For example, the C terminus of IL-2Rbeta may negatively regulate proximal domains through steric hindrance, which might be relieved by receptor activation. Such a model would also explain the negative regulatory domain identified within the EPOR C terminus(45) . A deletion mutant thus may obscure a role of tyrosine residues within this region. Therefore, we conclude that tyrosines within the IL-2Rbeta cytoplasmic tail are indeed important for the growth signaling competence of IL-2Rbeta.

The mechanism(s) underlying the importance of Tyr-392 and Tyr-510 to IL-2R function remain uncertain. In the platelet-derived growth factor receptor system, several distinct signaling pathways are activated selectively by individual phosphotyrosine residues through interactions with proteins via SH2 domains(46, 47) . Recent reports have described the inducible binding of p52 to the IL-2Rbeta chain upon the binding of IL-2(48, 49) , although the molecular basis of this interaction is unknown. Similarly, phosphatidylinositol 3-kinase has also been found to associate with the IL-2Rbeta chain in the presence of IL-2(50, 51) , an event which may be facilitated by phosphorylation of IL-2Rbeta Tyr-392 as revealed in studies with phosphopeptides(51) . Finally, following completion of the present work, we (52) and others (53) have demonstrated that phosphopeptides encompassing either Tyr-392 or Tyr-510 are potent and specific inhibitors of the in vitro DNA binding activity of STAT-5, a STAT factor that is regulated by the IL-2R(52, 53, 54) . Interestingly, tyrosine residues of IL-2Rbeta are dispensable for Janus kinase activation by the IL-2R (Fig. 7D) but are essential for the effective induction of STAT-5 (52) . Together, these findings are consistent with the popular model of cytokine receptor function (55) in which ligand-induced phosphorylation of certain tyrosine residues of the receptor is a critical step in the generation of downstream intracellular signals.

Convincing demonstration of the significance of this model for IL-2R function requires identification of the sites of IL-2-induced tyrosine phosphorylation of IL-2Rbeta in vivo. The present studies demonstrated that Tyr-392 serves as a phosphate acceptor site upon exposure of BA/F3 transfectants to IL-2 (Fig. 5). Unexpectedly we failed to detect phosphorylation of Tyr-510 in parallel experiments. It is possible that this lack of detection results from technical problems, such as insensitivity of the assay method or contaminating phosphatase activity released during cell lysis. Alternatively, this observation may indicate that Tyr-510 function is entirely independent of its phosphorylation status. Indeed, the published evidence supporting a critical role for receptor phosphotyrosines in the JAK-STAT pathway is largely circumstantial. For example, experimental demonstration of direct interactions between STAT factors and phosphotyrosine-containing receptor segments has proven difficult in most circumstances, and heavy emphasis has been placed instead on in vitro peptide approaches(44) . Therefore, the lack of detectable phosphorylation of Tyr-510 in the present studies raises the possibility that this and perhaps other tyrosine residues of IL-2Rbeta exert crucial influences on the tertiary conformation of IL-2Rbeta independently of their phosphorylation status. Although we tend to favor the tyrosine phosphorylation model, rigorous consideration of the published data demands further studies to distinguish effectively between these interpretations.

Other cytokine receptor superfamily members (1) may similarly be influenced by tyrosines. Functionally important tyrosine residues within the cytoplasmic domains of the IL-4 and interferon- receptors have been described recently(41, 42, 43) , although the significance of IL-4R phosphorylation has been disputed(56) . The functional redundancy described here for the distal IL-2Rbeta tyrosines may also be a feature of the human IL-4 receptor that could explain the incomplete impairment of function reported upon substitution of phenylalanine for Tyr-497 in the IL-4 receptor(41) . Further investigation is needed to clarify these events within the IL-2R.

The EPOR/IL-2R chimeric system also permitted an assessment of the role of tyrosine and other residues within the (c) cytoplasmic tail for growth signaling in T cells. In contrast to the IL-2Rbeta chain, the (c) subunit functioned fully in the absence of all four of its cytoplasmic tyrosine residues (Fig. 8). This finding indicates that growth signaling intermediates interacting with the (c) tail do so independently of phosphotyrosine docking sites, even though one or more of these tyrosine sites is phosphorylated after IL-2 stimulation in vivo. In view of the fact that both the IL-4 and IL-2 receptors employ the (c) subunit, these observations raise the intriguing possibility that the longer, unique chain in each receptor provides the docking sites for the specific signaling intermediates engaged by each receptor complex. In this arrangement, the shared (c) subunit would participate in general initiation of the signaling process, whereas the specialized subunits would contain unique sites for the inducible binding of specific components, such as STAT factors. Other cytokine receptors might employ a similar functional configuration. Of course, it remains possible that components involved in other pathways not measured here (such as differentiation) do indeed depend upon these (c) tyrosine sites.

Although the tyrosines of (c) proved to be dispensable for growth signaling by the IL-2R, a panel of truncation and internal deletion mutants revealed other elements within (c) that are critical for growth signaling in the T cell line. Remarkably, the C-terminal 33 amino acids of (c) are fully dispensable for growth signaling (Fig. 9), indicating that the proximal 53 amino acids are sufficient for full growth signal transduction. Mutations within this membrane-proximal region abrogated signaling function. For example, extension of the truncation N-terminal to a vestigial ``Box 2'' motif (39) abolished the signaling function, as did internal deletion of the 14 amino acids constituting a ``Box 1'' motif, the 14 amino acids constituting this vestigial Box 2 motif, or the 26 amino acids connecting Box 1 to Box 2 (V-Box) (Fig. 5). These observations in T cells extend the studies by others which employed certain truncated (c) subunits expressed in heterologous cell types(57, 58, 59) and demonstrate clearly that the (c) tail is needed for growth signal transduction by IL-2R heterodimers in T cells. Importantly, the impairment of these (c) domains undoubtedly contributes to the pathologic effects manifested in the X-linked severe combined immunodeficiency syndrome(60) .

The recognition that the growth signaling function of (c) resides in a relatively small portion of the cytoplasmic tail and that this segment functions independently of tyrosine residues is consistent with the receptor model described above. The essential, membrane-proximal region of (c) has been shown to be crucial for the assembly of the Janus kinase JAK3 with (c)(10, 40) . Perhaps the primary function of (c) in the IL-2, IL-4, and other receptors is to convey JAK3 into the receptor complex upon engagement of the appropriate ligand, which would thus allow trans-activation of JAK1 and JAK3 bound to their respective receptor subunits. Subsequent signaling activities may focus primarily upon the extended cytoplasmic tail of the unique IL-2Rbeta chain, including the inducible binding and activation of specific factors. Further studies are needed to determine whether or not the (c) chain has additional functions in addition to its conveyance role. One or both of the Janus kinases may be involved in phosphorylation substrates within the receptor complex. The present findings provide a rationale for further investigation of these intracellular events.


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 by the J. David Gladstone Institutes and by the National Institutes of Health. To whom correspondence should be addressed: Gladstone Institute of Virology and Immunology, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3775; Fax: 415-826-1514.

In the National Institutes of Health Medical Scientist Training Program.

**
Supported by the J. David Gladstone Institutes.

§§
Supported by the National Institutes of Health.

(^1)
The abbreviations used are: IL-2, interleukin-2; IL-2R, interleukin-2 receptor; EPO, erythropoietin; EPOR, erythropoietin receptor; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

Expression vectors and cDNA's were kindly provided by Drs. M. Stinksi, M. Feinberg, and T. Taniguchi. Dr. R. Robb kindly provided antibodies. IL-2 was a generous gift of Chiron Corp. We acknowledge the excellent assistance of Michelle Baskes in the preparation of this manuscript.


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