Delineation of the Regions of Interleukin-2 (IL-2) Receptor beta  Chain Important for Association of Jak1 and Jak3
Jak1-INDEPENDENT FUNCTIONAL RECRUITMENT OF Jak3 TO IL-2Rbeta *

Ming-hua Zhu, Jennifer A. BerryDagger , Sarah M. Russell§, and Warren J. Leonard

From the Laboratory of Molecular Immunology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interleukin-2 (IL-2) induces heterodimerization of the IL-2 receptor beta  (IL-2Rbeta ) and gamma c chains of its receptor and activates the Janus family tyrosine kinases, Jak1 and Jak3. Whereas Jak1 associates with IL-2Rbeta , Jak3 associates primarily with gamma c but also with IL-2Rbeta . We analyzed four IL-2Rbeta mutations that diminish IL-2-induced proliferation and found that each also decreased IL-2-induced signal transducer and activator of transcription (STAT) activation. For this reason, and because the mutations were in the IL-2Rbeta membrane-proximal region, we investigated and found that each mutation diminished IL-2Rbeta association with both Jak1 and Jak3. This suggested that these Jaks might interact with the same region of IL-2Rbeta ; however, certain IL-2Rbeta internal deletions and C-terminal truncations differentially affected the association of Jak1 and Jak3. Interestingly, just as Jak1-IL-2Rbeta association is Jak3-independent and functionally important, we show that Jak3-IL-2Rbeta association is Jak1-independent and implicate this association as being important for IL-2-induced Stat5 activation. Moreover, Jak1 and Jak3 could associate only in the presence of IL-2Rbeta , suggesting that these kinases can simultaneously bind to IL-2Rbeta . Thus, our data not only demonstrate that somewhat more distal as well as membrane-proximal cytoplasmic regions of a type I cytokine receptor are important for Jak kinase association but also suggest that two IL-2Rbeta -Jak kinase interactions are important for IL-2 signaling.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interleukin-2 (IL-2)1 is the principal growth factor for T lymphocytes and is responsible for regulating the magnitude and duration of the T cell immune response following antigen encounter (1-4). Three classes of IL-2 receptors exist, binding IL-2 with low (Kd = 10-8 M), intermediate (Kd = 10-9 M), and high (Kd = 10-11 M) affinity. The low affinity receptors contain only the IL-2 receptor alpha  chain (IL-2Ralpha ); intermediate affinity receptors contain IL-2Rbeta and the common cytokine receptor gamma  chain, gamma c; and high affinity receptors contain all three chains (3, 4). The intermediate and high affinity receptors are the functional forms, and heterodimerization of the IL-2Rbeta and gamma c cytoplasmic domains is necessary and sufficient for IL-2 signaling (5-7). The highly inducible alpha  chain has a very short cytoplasmic domain (8, 9) and presumably mainly functions to increase the affinity for IL-2, allowing cellular responsiveness to the low levels of IL-2 that are physiologically present in vivo. In contrast, IL-2Rbeta and gamma c have longer cytoplasmic domains that can associate with a number of signaling molecules, allowing the activation of signaling pathways (2-4). Stimulation of lymphocytes with IL-2 results in the rapid activation of the Janus family tyrosine kinases, Jak1 and Jak3 (10-14). Activated Jaks are critical for inducing rapid tyrosine phosphorylation of downstream substrates, including STATs (signal transducers and activators of transcription), which then dimerize, translocate into the nucleus, and regulate the transcription of target genes (4, 13-15).

It has been reported that IL-2Rbeta and gamma c constitutively associate with two of the four Jak family kinases in a selective manner, IL-2Rbeta with Jak1 and gamma c with Jak3 (10, 16, 17). The S region (amino acids 267-322) of IL-2Rbeta has been shown to be important for Jak1 association (17). In addition to its ability to constitutively interact with Jak1, although it is not well appreciated, IL-2Rbeta can also associate with Jak3 following IL-2 stimulation of lymphoid cells (10), but the regions of interaction between IL-2Rbeta and Jak3 have not previously been investigated.

A number of membrane proximal cytoplasmic point mutants of IL-2Rbeta that diminish IL-2-induced proliferation have been identified (18-20). We found that these mutants also diminish IL-2-induced STAT protein activation and the association of both Jak1 and Jak3 with IL-2Rbeta . This led us to further characterize the regions of IL-2Rbeta required for the binding of Jak1 and Jak3, and we demonstrate that membrane distal as well as membrane proximal regions of IL-2Rbeta are vital for Jak kinase interaction. Moreover, the association between Jak3 and IL-2Rbeta is Jak1-independent and both Jak3 and Jak1 can be coprecipitated only in the presence of IL-2Rbeta . Finally, we provide evidence indicating that the association between IL-2Rbeta and Jak3 is important for potent Stat5 activation in response to IL-2 and, thus, that more than one IL-2Rbeta -Jak kinase interaction is involved in IL-2 signaling.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Constructs and in Vitro Mutagenesis-- The IL-2Rbeta constructs with point mutations in the cytoplasmic chain were prepared using an in vitro mutagenesis kit (5 Prime-3 Prime, Inc.) and wild-type IL-2Rbeta as the template. The oligonucleotides used for mutagenesis were as follows (mutant nucleotides are underlined): 5'-AAGTGTAACACCTCAGACCCCTCG-3' (for P257S); 5'-GTAACACCCCAGCGCCCTCGAAGTTC-3' (for D258A); 5'-GACGTCCAGAAGGGGCTCTCTTCG-3' (for W277G); and 5'-CTGGCACCTGAGATATCGCCAGCAGAAGTGCTGGAG-3' (for L299A). Successful mutagenesis was confirmed by DNA sequencing.

Wild-type IL-2Rbeta or IL-2Rbeta constructs containing internal deletion or truncation mutations in its cytoplasmic domain (see Fig. 3A), were cloned in the expression vector, pME18S, in which transcription is directed by the SRalpha promoter. pME18S also contains the SV40 origin of replication and is expressed at high copy number in either 293T+ or COS-7 cells, both of which express the SV40 large T antigen. Internal deletion mutants of IL-2Rbeta (beta Delta A, beta Delta S) were generated by loop-out mutagenesis using single-stranded M13 bacteriophage as a template. IL-2Rbeta truncation mutants (beta 379, beta 371, beta 362, beta 350, beta 330, beta 313, beta 300, beta 290, and beta 267) were prepared using the polymerase chain reaction to amplify BclI to XbaI fragments of IL-2Rbeta with premature termination codons, followed by subcloning into pME18S-IL-2Rbeta in which the BclI to XbaI fragment was excised. The IL-2Rbeta construct with four tyrosines changed to phenylalanines (beta FFFFYY) was described previously (21).

The murine Jak1 cDNA in pMLCMV was provided by Dr. J. Ihle; the human Jak3 cDNA was provided by Dr. J. O'Shea. Jak3 was subcloned in pME18S. Wild-type gamma c (gamma c-wt) and truncated mutant of gamma c (gamma c-Delta CT) were previously described (22, 23). Human Stat5a and Stat5b cDNAs (24) were cloned into pCi (Promega).

Cell Lines and Transfections-- COS-7 cells (ATCC), 293T+ cells (provided by Dr. N. Rice, National Cancer Institute), and E1C3 cells (Jak1-deficient HeLa cells, provided by Dr. R. Flavell, Yale University) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 100 units/ml each of penicillin and streptomycin. Transient transfections were performed using either DEAE-dextran (for COS-7 cells) or calcium phosphate (for 293T+ and E1C3 cells) methods. For immunoprecipitation experiments, cells were transfected in 150-mm dishes using 2-3 µg of each plasmid. Transfectants were harvested 36-48 h later. For experiments in which IL-2-induced STAT DNA binding activity was reconstituted, cells were transfected in 100-mm dishes using 1-2 µg of each plasmid, and nuclear extracts were made 36-48 h later.

32D cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 5% from WEHI-3B cells conditioned medium (WEHI-CM) as a source of IL-3, 2 mM glutamine, and 100 units/ml each of penicillin and streptomycin. Stable transfectants expressing wild-type or mutant forms of IL-2Rbeta were created by electroporation using a Gene Pulser (Bio-Rad) at 300 V and 960 microfarads. Cells (5 × 106/condition) were cotransfected with linearized IL-2Rbeta constructs and pcDNA3neo. 24 h after electroporation, cells were aliquoted into 24-well plates using medium containing 1 mg/ml G418 (Life Technologies, Inc.). Resistant clones were stained for IL-2Rbeta expression with fluorescein isothiocyanate-conjugated anti-p75 (IL-2Rbeta ) monoclonal antibody (Endogen) and analyzed on a FACSort (Becton-Dickinson).

Reagents and Antibodies-- Anti-IL-2Rbeta hMikbeta 1 (humanized Mikbeta 1; Ref. 25) or anti-Jak3 antibody (provided by J. O'Shea) and protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) were used for immunoprecipitation. Immunoblots were performed using antibodies to Jak1 (Transduction Labs and Santa Cruz Biotechnology), Jak3, or IL-2Rbeta (goat anti-human IL-2Rbeta , R&D Systems).

Immunoprecipitation and Western Blotting-- COS-7, 293T+, or E1C3 transient transfectants (one 150-mm culture dish) or 32D stable transfectants (5-10 × 106 cells) were harvested by washing with phosphate-buffered saline and lysed with lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 0.4 mM Na3VO4, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 mM leupeptin, 1 mM aprotinin). IL-2Rbeta -Jak3 or Jak3-IL-2Rbeta -Jak1 associations were assessed by immunoprecipitation with hMikbeta 1 or anti-Jak3 at 4 °C for 1-2 h. Samples were washed four times with lysis buffer, analyzed on 8 or 10% SDS-polyacrylamide gel electrophoresis (NOVEX), transferred to Immobilon-P membranes (Millipore Corp.), immunoblotted with different antibodies, and developed with ECL (Amersham Pharmacia biotech or Pierce).

Preparation of Nuclear Extracts and Electrophoresis Mobility Shift Assays (EMSAs)-- Extracts were prepared from 293T+ or COS-7 transfectants (cells from one 100-mm culture dish) or from 32D cells (1 × 107 cells) that were starved of growth factor for 4 h in RPMI 1640 medium and treated with 2 nM IL-2 for 30 min at 37 °C. Cells were washed with ice-cold phosphate-buffered saline, nuclear extracts were prepared as described previously (26), and 1 µg of protein from 293T+ or COS-7 transfectants or 5-10 µg of protein from 32D cells were used in EMSAs. For EMSAs, 1 µg of poly(dI-dC) was used as a nonspecific competitor and 15,000 cpm of 32P-labeled double-stranded oligonucleotide containing a trimer of the GAS sequence (5'-AGATTTCTAGGAATTC-3') from the beta -casein promoter (a motif capable of binding IL-2-activated STAT proteins) was used as the probe. The reactions were separated on 6% polyacrylamide gels in 0.5 × Tris borate-EDTA and autoradiographed.

Thymidine Incorporation Assays-- 32D cells were washed and starved of growth factor for 4 h in RPMI medium. Cells were aliquoted at 2-4 × 104 cells/well in a 96-well plate in triplicate in 200 µl of medium or medium containing 2 nM IL-2 or 5% WEHI-CM. After 20 h of incubation at 37 °C, 1 µCi of 3H-labeled thymidine (NEN Life Science Products) was added, and the cells were incubated at 37 °C for 4 h. Cells were harvested using a cell harvester (Tom Tec), and thymidine incorporation was assayed using a Betaplate 1205 counter (Wallac). For each transfectant, at least three clones with similar IL-2Rbeta expression were assayed.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IL-2Rbeta Point Mutants That Affect Proliferation Diminish IL-2-induced Stat5 DNA Binding Activity-- Four IL-2Rbeta mutants, including P257S (proline 257 replaced by serine), D258A, W277G, and L299A, have been reported to impair IL-2-induced proliferation in Ba/F3 or MOLT4 cells (18-20) even though they exhibit similar surface expression and IL-2 binding affinities (19). We sought to investigate the basis for the decreased proliferation of these mutants. We first made stable transfectants of each of these mutants in 32D cells and confirmed similar cell surface expression by flow cytometry (Fig. 1A). As expected, we confirmed that these mutants mediated greatly diminished proliferation, as compared with wild-type IL-2Rbeta , in 32D cells, which lack IL-2Rbeta but can proliferate in response to IL-2 after IL-2Rbeta is transfected and expressed (Refs. 21 and 27; Fig. 1B). Moreover, each of these mutations also diminished IL-2-induced STAT binding activity in transfected 32D cells (Fig. 1C) as well as in transiently transfected COS-7 cells (Fig. 1D). In 32D cells, previous studies indicate that the IL-2-induced STAT binding activity is due to Stat5 rather than Stat3 (28). For the COS-7 cell experiments, cells were transfected with gamma c, Jak3, Stat5a, Stat5b, and the different IL-2Rbeta constructs using a system previously shown to reconstitute IL-2-induced Stat5 DNA binding activity with wild-type IL-2Rbeta (24).


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Fig. 1.   IL-2Rbeta mutants containing individual substitutions at Pro-257, Asp-258, Trp-277, or Leu-299 each exhibit diminished IL-2-induced proliferation and STAT activation. A, flow cytometric analyses indicating IL-2Rbeta expression of representative 32D cell clones transfected with wild-type or mutant IL-2Rbeta constructs. The mean fluorescent intensity for each construct is indicated. B, diminished proliferation by IL-2Rbeta mutants. Untransfected 32D cells (32D), 32D cells transfected with mutant IL-2Rbeta constructs (P257S, D258A, W277G, and L299A), or wild-type IL-2Rbeta construct (beta wt) were treated with medium alone (open bars), 2 nM IL-2 (stippled bars), or 5% WEHI-CM as a source of IL-3 (hatched bars). 3H-labeled thymidine incorporation was determined. Three independent stable transfectants with similar expression levels of IL-2Rbeta (selected by flow cytometry with fluorescein isothiocyanate-conjugated anti-IL-2Rbeta monoclonal anitbody and similar IL-2Rbeta expression confirmed by Western blotting with a polyclonal anti-IL-2Rbeta antiserum) were assayed for each construct. C and D, defective STAT binding activity in the cells transfected with IL-2Rbeta mutants P257S, D258A, W277G, and L299A. C, untransfected 32D cells or stable 32D transfectants expressing either wild-type or mutant IL-2Rbeta constructs were washed, starved in medium without any growth factors, and then left untreated (lanes 1, 3, 5, 7, 9, and 11) or treated with 2 nM IL-2 for 15 min (lanes 2, 4, 6, 8, 10, and 12). D, COS-7 cells were transfected with Stat5a, Stat5b, Jak3, gamma c, and either wild-type or mutant forms (P257S, D258A, W277G, and L299A) of IL-2Rbeta . Two days after transfection, nuclear extracts were prepared from cells not treated (lanes 1, 3, 5, 7, 9, and 11) or treated with 2 nM IL-2 for 15 min (lane 2, 4, 6, 8, 10, and 12). C and D, EMSAs were performed using the beta -casein probe.

IL-2Rbeta Point Mutants Also Exhibit Diminished Association with Both Jak1 and Jak3-- Given the diminished STAT activation and that each of these mutations are contained in a region of IL-2Rbeta where Jak kinase interactions might be affected (Box B1/Box B2 region, see Refs. 29-32), we tested if these mutations diminished the association of Jak1 or Jak3 as a possible explanation for the decreased IL-2 signaling. Because the IL-2Rbeta -Jak3 interaction is only well seen in T cells following IL-2 stimulation, we used an overexpression system to map the regions of IL-2Rbeta that mediate association with Jak1 and Jak3. COS-7 cells were transfected with Jak1 or Jak3 and IL-2Rbeta mutants, cells were lysed, and lysates were immunoprecipitated with hMikbeta 1 antibody to IL-2Rbeta and then blotted with antibodies to IL-2Rbeta (Fig. 2A), Jak1 (Fig. 2B), or Jak3 (Fig. 2C). Jak1 and Jak3 each exhibited less binding to each of the IL-2Rbeta mutants than to wild-type IL-2Rbeta (Fig. 2, B and C).


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Fig. 2.   IL-2Rbeta mutants containing individual substitutions at Pro-257, Asp-258, Trp-277, or Leu-299 decreased interaction with Jak1 and Jak3. A, similar expression levels for the mutant and wild-type IL-2Rbeta . Cell lysates were immunoprecipitated with hMikbeta 1 and then Western blotted with anti-IL-2Rbeta . Cell surface expression of each IL-2Rbeta construct was confirmed by flow cytometry (data not shown). B, the P257S, D258A, W277G, and L299A IL-2Rbeta point mutants exhibit decreased association with Jak1. COS-7 cells were transfected with Jak1, Jak3, and either mutant or wild-type IL-2Rbeta , were immunoprecipitated with hMikbeta 1, and then were Western blotted with anti-Jak1 (top panel). Lysates were Western blotted with anti-Jak1 to confirm the expression of Jak1 in different transfectants (bottom panel). C, the P257S, D258A, W277G, and L299A point mutations in IL-2Rbeta also resulted in decreased association of Jak3. The blots in panel C were stripped and reblotted with anti-Jak3.

The Regions of IL-2Rbeta Required for Jak1 and Jak3 Binding Partially Overlap-- Because each of the IL-2Rbeta point mutations interfered with the association of both Jak1 and Jak3, we hypothesized that the regions of IL-2Rbeta that were important for Jak kinase interaction might be similar. To investigate this possibility and to map the regions of IL-2Rbeta involved in the binding of both Jak kinases, COS-7 cells were transiently transfected with Jak1, Jak3, and wild-type IL-2Rbeta or a series of IL-2Rbeta truncation mutants (Fig. 3A). Cells were lysed, and lysates were immunoprecipitated with anti-IL-2Rbeta antibody, followed by Western blotting with antibodies to IL-2Rbeta (to control for expression and the efficiency of immunoprecipitation, Fig. 3B), Jak1 (Fig. 3C), or Jak3 (Fig. 3D). IL-2Rbeta truncation mutants retaining 350 (beta 350 construct) or more residues (beta 362, beta 371, and beta 379 constructs) could bind efficiently to Jak1 (Fig. 3C); beta 330 and beta 313 bound to Jak1 weakly; whereas beta 300, beta 290, and beta 267 could not bind to Jak1. Therefore, the region between residues 300 and 350 of IL-2Rbeta is important for its interaction with Jak1. In contrast, the region between residues 330 and 362 was important for the Jak3-IL-2Rbeta interaction, given that there was efficient coprecipitation of Jak3 with beta 362 but no detectable coprecipitation of Jak3 with beta 330 even at longer exposure times (Fig. 3D and data not shown). Wild-type IL-2Rbeta and IL-2Rbeta mutants retaining the first 362, 371, or 379 amino acids could associate with both Jak1 and Jak3. These results in COS-7 cells were confirmed using 293T+ cells (data not shown). Thus, the 300-350 and 330-362 regions of IL-2Rbeta are important for Jak1 and Jak3 binding, respectively (summarized below in Fig. 8).


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Fig. 3.   Interaction of Jak1 and Jak3 with non-identical regions of IL-2Rbeta . A, schematic of IL-2Rbeta and its truncated mutants showing the extracellular domain, transmembrane (TM) domain, and cytoplasmic domains. In the cytoplasmic domain, the S, A, Box B1, and Box B2 regions are shown. B, expression of wild-type and truncated forms of IL-2Rbeta . COS-7 cells were transfected with Jak1, Jak3, and either wild-type IL-2Rbeta (beta wt) or truncated mutants of IL-2Rbeta , were immunoprecipitated with hMikbeta 1, and then were Western blotted with anti-IL-2Rbeta . Cell surface expression of each IL-2Rbeta construct was confirmed by flow cytometry (data not shown). C, importance of the amino acids 300 to 350 region of IL-2Rbeta for Jak1 binding. Top panel, lysates from COS-7 cells transfected with Jak1, Jak3, and either wild-type or mutant forms of IL-2Rbeta were immunoprecipitated with hMikbeta 1 and then Western blotted with anti-Jak1. Bottom panel, the lysates were Western blotted with anti-Jak1 to confirm the expression of Jak1 in different transfectants. D, importance of the amino acids 330 to 362 region of IL-2Rbeta for Jak3 binding. The blots described in panel C were stripped and reblotted with anti-Jak3.

We next tested the effect of internal deletions of the S region (residues 267 to 322) or the A region (residues 313 to 382) on the binding of Jak1 (Fig. 4A) and Jak3 (Fig. 4B). Deletion of the S region (beta Delta S) resulted in a dramatic decrease in IL-2Rbeta association with Jak1, consistent with previously reported results (17), whereas deletion of the A region only modestly decreased Jak1 association (Fig. 4A). In contrast to the findings for Jak1, deletion of the A region had a much greater effect on the association of Jak3 than did deletion of the S region (Fig. 4B). Thus, the A region of IL-2Rbeta is more important for Jak3 association, whereas the S region is more important for Jak1 association. Consistent with the data in Fig. 3, these data indicate that Jak3 binding extends to a more distal region of the IL-2Rbeta cytoplasmic domain than does Jak1. Therefore, the data contained in Figs. 3 and 4 demonstrate that Jak1 and Jak3 interact with different, albeit overlapping regions of IL-2Rbeta .


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Fig. 4.   The A and S regions of IL-2Rbeta are differentially important for binding Jak3 and Jak1, respectively. A, deletion of either the A or S regions of IL-2Rbeta diminished the association of Jak1. COS-7 cells were transfected with Jak1, Jak3, and either mutant or wild-type IL-2Rbeta , were immunoprecipitated with hMikbeta 1, and then were Western blotted with anti-Jak1. Lysates were Western blotted with anti-Jak1 to confirm the expression of Jak1 in different transfectants. B, deletion of the A region of IL-2Rbeta more greatly diminished the association with Jak3 than did the S region. The blots described in panel A were stripped and reblotted with anti-Jak3. C, similar expression levels for beta wt, beta Delta A, and beta Delta S. The cell lysates were immunoprecipitated with hMikbeta 1 and then Western blotted with anti-IL-2Rbeta . Cell surface expression of each IL-2Rbeta construct was confirmed by flow cytometry (data not shown). D, association of IL-2Rbeta with Jak1 and Jak3 does not depend on the phosphorylation of the tyrosine residues located on the A region. Cells were transfected with Jak1 + Jak3 + either beta wt, beta FFFFYY, or pME18S. Cell lysates were immunoprecipitated with hMikbeta 1 and then blotted with either anti-Jak3 (top panel), anti-Jak1 (middle panel), or anti-IL-2Rbeta (bottom panel). The bands corresponding to Jak3, Jak1, and IL-2Rbeta are indicated.

As the A region contains four tyrosines (Tyr-338, Tyr-355, Tyr-358, and Tyr-361), we evaluated the ability of Jak3 to associate with IL-2Rbeta containing mutations in these tyrosines (IL-2Rbeta FFFFYY). As shown in Fig. 4D, Jak3 efficiently associated with this mutant, indicating that the interaction does not depend on phosphorylated tyrosine residues.

Jak3 Can Bind to IL-2Rbeta in Jak1-deficient HeLa Cells-- Given that Jak1 is ubiquitously expressed, it was possible that the interaction of Jak3 with IL-2Rbeta required Jak1. To investigate this possibility, we transfected Jak1-deficient HeLa cells (E1C3 cells) with Jak3 + wild-type IL-2Rbeta  ± Jak1. Transfected cells were lysed and immunoprecipitated with hMikbeta 1, followed by blotting with an antiserum to Jak3. We found that IL-2Rbeta and Jak3 could interact even in the absence of Jak1, and the presence of Jak1 did not enhance this interaction (Fig. 5A, first two lanes). The uniformity of expression of Jak3, Jak1, and IL-2Rbeta was verified by immunoblotting with appropriate antibodies (Fig. 5B). We also used E1C3 cells to map the region of IL-2Rbeta required for its interaction with Jak3, and confirmed the findings reported above in Figs. 3 and 4 (data not shown).


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Fig. 5.   Jak3 can interact with IL-2Rbeta in the absence of Jak1. A, HeLa cells lacking Jak1 (E1C3 cells) were transfected with beta wt or pME18S and Jak3, Jak1, or Jak3 + Jak1. Cell lysates were immunoprecipitated with hMikbeta 1 and then blotted with either anti-Jak3. B, lysates of the E1C3 transfectants were Western blotted with anti-Jak3, anti-Jak1, or anti-IL-2Rbeta to confirm the expression levels of transfected cDNAs.

Jak1 and Jak3 Can Only Be Coprecipitated in the Presence of IL-2Rbeta -- Because the association between Jak3 and IL-2Rbeta was Jak1-independent, and Jak1 could be coprecipitated with IL-2Rbeta in the absence of Jak3, we next investigated whether Jak1 and Jak3 could be coprecipitated through IL-2Rbeta . COS-7 cells were transfected with Jak1, Jak3, and either pME18S, wild-type IL-2Rbeta , or IL-2Rbeta deletion constructs (beta Delta A, beta Delta S, and beta 350) that were missing regions important for the interaction of either Jak1 and/or Jak3 (see Figs. 3 and 4). Coprecipitation of Jak3 and Jak1 required IL-2Rbeta (Fig. 6A, lane 2 versus lane 1); this association was markedly decreased when the beta Delta A, beta Delta S, or beta 350 mutants were used instead of wild type IL-2Rbeta (lanes 3-5), further confirming that the association between Jak1 and Jak3 is dependent on the presence of IL-2Rbeta .


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Fig. 6.   Jak3 can associate with Jak1 in the presence of wild-type IL-2Rbeta . COS-7 cells were transfected with Jak1, Jak3, and either wild-type IL-2Rbeta , mutant IL-2Rbeta , or pME18S. Cell lysates were immunoprecipitated with anti-Jak3 and then blotted with anti-Jak1 (A). The blot was stripped and reblotted with anti-Jak3 to control the immunoprecipitation of Jak3 (B). The expression of Jak1 is shown in (C).

Association between IL-2Rbeta and Jak3 Is Required for IL-2-induced Stat5 DNA Binding Activity-- It has previously been shown that disruption of the Jak1-IL-2Rbeta interaction diminished IL-2 signaling (2). To investigate the functional significance of the association between IL-2Rbeta and Jak3, we used Jak3-deficient 293T+ cells in which IL-2-induced Stat5 DNA binding activity could be reconstituted following transfection with IL-2Rbeta , gamma c, Jak3, Stat5a, and Stat5b (Fig. 7A, lanes 5 and 6; Fig. 7B, lanes 1 and 2). Previous studies indicate the vital role of Jak3 for IL-2-induced STAT activation (24). Both IL-2Rbeta and gamma c were required since little, if any, IL-2-induced Stat5 DNA binding activity was seen in the absence of either gamma c (Fig. 7A, lanes 1 and 2) or IL-2Rbeta (Fig. 7A, lanes 3 and 4). However, a truncated form of gamma c (gamma c-Delta CT) that is missing 80 of 86 amino acids of the gamma c cytoplasmic domain and contributes to IL-2 binding (22, 23) but does not interact with Jak3 (10) still allowed partial IL-2-induced DNA binding activity (Fig. 7B, lanes 3 and 4). This activity was diminished when Jak3 (Fig. 7B, lanes 5 and 6) was deleted, implicating the IL-2Rbeta -Jak3 interaction as being important for STAT activation.


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Fig. 7.   Reconstitution of Stat5 DNA binding activation in 293T+ cells. Jak3 association with IL-2Rbeta is required for IL-2-induced Stat5 activation. A, 293T+ cells were transfected with IL-2Rbeta , Jak3, Stat5a, and Stat5b (lanes 1 and 2); gamma c, Jak3, Stat5a, and Stat5b (lanes 3 and 4); or IL-2Rbeta , gamma c, Stat5a and Stat5b (lanes 5 and 6). 36-48 h after transfection, cells were either not treated (lanes 1, 3, and 5) or treated with 2 nM IL-2 for 30 min (lanes 2, 4, and 6), and nuclear extracts were made. EMSAs were performed using the beta -casein probe. B, 293T+ cells were transfected with IL-2Rbeta , gamma c, Jak3, Stat5a and Stat5b (lanes 1 and 2); IL-2Rbeta , gamma c-Delta CT, Jak3, Stat5a, and Stat5b (lanes 3 and 4); or IL-2Rbeta , gamma c-Delta CT, Stat5a, and Stat5b (lanes 5 and 6). IL-2 treatment, nuclear extracts, and EMSAs were performed as described in panel A.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IL-2 signaling requires the dimerization of both IL-2Rbeta and gamma c. As Jak1 has been shown to associate with IL-2Rbeta and Jak3 with gamma c, an attractive model has been that each receptor chain associates with a different Jak family kinase in a selective manner and that IL-2-mediated activation of Jak1 and Jak3 initiates a signaling cascade(s). It is well established that the gamma c-Jak3 interaction (10) and Jak3 activation (33, 34) are vital for signaling. We now provide evidence that Jak3 and IL-2Rbeta can associate with each other in a Jak1-independent fashion. The fact that IL-2Rbeta provides interaction sites for Jak3 as well as Jak1 (see Figs. 8 and 9) suggests that a function of gamma c might be not only to recruit Jak3 but also to facilitate the "delivery" of Jak3 to IL-2Rbeta . Moreover, the ability of Jak3 to associate with both IL-2Rbeta and gamma c suggests that Jak3 might stabilize the receptor complex and promote downstream signaling. Our studies on the reconstitution of IL-2-induced Stat5 activation in 293T+ cells provide evidence that the full activation of Stat5 requires IL-2Rbeta association with both Jak1 and Jak3, and that the heretofore poorly appreciated IL-2Rbeta -Jak3 association has physiological significance.


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Fig. 8.   Regions of IL-2Rbeta important for the association of Jak1 and Jak3. The extracellular, transmembrane (TM), and cytoplasmic domains are shown. In the cytoplasmic domain, the Box B1, Box B2, S region, and A region are shown on the left. On the right are shown the regions and residues important for the association of Jak1 and Jak3.


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Fig. 9.   Schematic model showing an important role for Jak3 association with IL-2Rbeta for IL-2-induced Stat5 activation in both transfected 293T+ cells (A) and IL-2-dependent cells (B). A, in 293T+ cells, Jak3 associates with both IL-2Rbeta and gamma c in order to achieve full Stat5 activation upon IL-2 stimulation (shown as ++++ on the left). When the truncated gamma c, which cannot bind Jak3, was present instead of wild-type gamma c, Jak3 associates with IL-2Rbeta ; in this setting, there is still Stat5 activation, albeit decreased (shown as ++ in the middle). Finally, in the absence of Jak3, there is very little Stat5 activation (shown as +/- on the right). B, in IL-2-dependent cells, IL-2Rbeta and gamma c form a heterodimer after IL-2 stimulation. Jak3 binds both IL-2Rbeta and gamma c, perhaps stabilizing the receptor complex, allowing for potent downstream signaling.

We have now delineated regions on IL-2Rbeta that are important for the interaction of Jak1 and Jak3. We show that four point mutations in the Box B1/Box B2 region of IL-2Rbeta that diminished proliferation also decreased the binding of both Jak1 and Jak3. This is consistent with the important role of this region of a number of type I cytokine receptors for Jak interaction (14, 29-32). Interestingly, however, analysis of a series of deletion and truncation mutants not only demonstrated differences in the regions of IL-2Rbeta that mediate recruitment of Jak1 versus Jak3, but unexpectedly also provided evidence that regions more distal than previously suspected play major roles in the recruitment of the Jak kinases (see Fig. 8). To our knowledge, these data represent the most detailed mapping on a cytokine receptor of the region/residues involved in Jak kinase association. Previously, for all cytokine receptors studied, including IL-2Rbeta , only the membrane proximal and Box1/Box2 regions have been shown to be important for the association of Jak kinases; thus, our findings have implications regarding the interaction sites of Jak kinases for other type I cytokine receptors as well. Although some receptor chains, such as gamma c, appear to be uniquely associated with a single Jak, the gp130 signal transducing receptor component that is shared by the receptors for IL-6, IL-11, leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, and cardiotrophin-1, can associate with more than one Jak. gp130 has been reported to associate with Jak1, Jak2, and Tyk2 (35, 36), but it remains unknown whether these three Jak family kinases serve completely distinctive roles and how they associate with gp130. Our data therefore provide the first example wherein more than one Jak (Jak1 and Jak3) can independently interact with a single receptor molecule (IL-2Rbeta ) via overlapping but non-identical regions.

    ACKNOWLEDGEMENTS

We thank J. Hakimi for anti-IL-2Rbeta hMikbeta 1; J. Ihle for the Jak1 cDNA; J. O'Shea for the Jak3 cDNA and antisera; R. Flavell for E1C3 cell line; M. Friedmann for assistance in preparing the IL-2Rbeta truncation mutants; and M. J. Aman, S. John, J.-X Lin, H. Nakajima, D. Finbloom, and A. M. Weissman for critical comments.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Howard Hughes Medical Institute Research Scholar award and present address: Johns Hopkins University, School of Medicine, Baltimore, MD 21287.

§ Present address: Austin Research Institute, Austin Hospital, Studley Road, Heidelberg, Victoria 3084, Australia.

To whom correspondence should be addressed: NHLBI, National Institutes of Health, 10 Center Dr., Bethesda, MD 20892. Tel.: 301-496-0098; Fax: 301-402-0971.

1 The abbreviations used are: IL-2, interleukin-2; IL-2R, interleukin-2 receptor; STAT, signal transducer and activator of transcription; EMSA, electrophoretic mobility shift assay.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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