CIS Associates with the Interleukin-2 Receptor β Chain and Inhibits Interleukin-2-dependent Signaling

CIS is a cytokine-inducedSH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor β chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor β chain (IL-2Rβ). This interaction requires the A region of IL-2Rβ (residues 313–382), which also mediates the association of IL-2Rβ with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rβ and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rβ in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action. CIS is a cytokine-inducedSH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene. CIS is known to associate with the IL-3 receptor β chain and erythropoietin receptor and to inhibit signaling mediated by IL-3 and erythropoietin. We now demonstrate that CIS also interacts with the IL-2 receptor β chain (IL-2Rβ). This interaction requires the A region of IL-2Rβ (residues 313–382), which also mediates the association of IL-2Rβ with Lck and Jak3. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rβ and IL-2-mediated activation of Stat5. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Although a functional SH2 binding domain of CIS was not required for its interaction with IL-2Rβ in vitro, its phosphotyrosine binding capability was essential for the inhibitory action of CIS. On this basis, we have generated a mutant form of CIS protein with an altered SH2 domain that acts as a dominant negative and should prove useful in further understanding CIS action. interleukin interleukin-2 receptor peripheral blood lymphocytes monoclonal antibody phytohemagglutinin Src homology 2 signal transducers and activators of transcription Following antigen encounter, the magnitude and duration of the subsequent T-cell immune response is critically controlled by the interaction of IL-21 with specific high affinity receptors (1Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar, 2Lin J.-X. Leonard W.J. Cytokine Growth Factor Rev. 1997; 8: 313-332Crossref PubMed Scopus (143) Google Scholar). High affinity IL-2 receptors (IL-2Rs) are composed of three chains, denoted IL-2Rα, IL-2Rβ, and the common cytokine receptor γ chain, γc. IL-2 induces the heterodimerization of IL-2Rβ and γc, which together are necessary and sufficient for IL-2 signaling (3Nakamura Y. Russell S.M. Mess S.A. Friedmann M. Erdos M. Francois C. Jacques Y. Adelstein S. Leonard W.J. Nature. 1994; 369: 330-333Crossref PubMed Scopus (283) Google Scholar, 4Nelson B.H. Lord J.D. Greenberg P.D. Nature. 1994; 369: 333-336Crossref PubMed Scopus (280) Google Scholar). Although neither IL-2Rβ nor γc have intrinsic protein-tyrosine kinase catalytic activity, IL-2 rapidly induces tyrosine phosphorylation of these chains and of intracellular proteins (1Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar, 2Lin J.-X. Leonard W.J. Cytokine Growth Factor Rev. 1997; 8: 313-332Crossref PubMed Scopus (143) Google Scholar). This is accomplished through activation of receptor-associated tyrosine kinases, which in turn phosphorylate cellular substrates responsible for the transmission of IL-2-induced signals. Two principal groups of kinases have been reported to associate with the IL-2 receptor subunits: the Src family kinase Lck (1Taniguchi T. Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar) and the Janus kinases Jak1 and Jak3 (5Boussiotis V.A. Barber D.L. Nakarai T. Freeman G.J. Gribben J.G. Bernstein G.M. D'Andrea A.D. Ritz J. Nadler L.M. Science. 1994; 266: 1039-1042Crossref PubMed Scopus (307) Google Scholar, 6Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalstieg G.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (586) Google Scholar, 7Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z.J. Oishi I. Silvennoinen O. 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Science. 1995; 268: 251-255Crossref PubMed Scopus (674) Google Scholar) associate with IL-2Rβ, whereas Jak3 associates primarily with γc (5Boussiotis V.A. Barber D.L. Nakarai T. Freeman G.J. Gribben J.G. Bernstein G.M. D'Andrea A.D. Ritz J. Nadler L.M. Science. 1994; 266: 1039-1042Crossref PubMed Scopus (307) Google Scholar, 6Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalstieg G.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (586) Google Scholar, 7Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z.J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (500) Google Scholar) but also can interact with IL-2Rβ following stimulation with IL-2 (6Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. 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Commun. 1997; 237: 79-83Crossref PubMed Scopus (140) Google Scholar) proteins (reviewed in Ref. 22Aman M.J. Leonard W.J. Curr. Biol. 1997; 12: R784-R788Abstract Full Text Full Text PDF Google Scholar). CIS (now also denoted as CIS-1) is the prototype member of the CIS/JAB/SOCS/SSI family of proteins (reviewed in Ref. 22Aman M.J. Leonard W.J. Curr. Biol. 1997; 12: R784-R788Abstract Full Text Full Text PDF Google Scholar). It is induced in hematopoietic cells within 30 min of stimulation by IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin, but not by stem cell factor, granulocyte colony-stimulating factor, or IL-6 (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). STAT response elements have been identified in the promoter region of CIS, allowing its induction by a variety of cytokines including IL-2 (23Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar). Once expressed, CIS/JAB/SOCS/SSI proteins interfere with signaling events and suppress cytokine-specific cellular responses. JAB/SOCS-1/SSI-1 has been shown to associate with Jak kinases and to inhibit their catalytic activities. Previously, it was demonstrated that CIS could associate with the IL-3 receptor β chain and erythropoietin receptor upon appropriate stimulation. Furthermore, CIS was shown to reduce the proliferative responsiveness of cells to IL-3 (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar) and to partially inhibit erythropoietin-induced Stat5 phosphorylation and transactivation in HEK293 cells reconstituted with the erythropoietin receptor and Stat5 (23Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar). We now demonstrate that CIS can associate with IL-2Rβ and that it can inhibit more than one IL-2-related signaling pathway. Peripheral blood lymphocytes (PBL) were prepared from normal donors using standard methods. To generate "preactivated PBL," freshly isolated PBL were cultured for 72 h in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml each of penicillin and streptomycin ("complete medium"), and 2 μg/ml PHA-L (Roche Molecular Biochemicals), and then washed and rested for 24 h in complete medium. NK-like YT cells were cultured and maintained in complete medium. 32D cells were maintained in complete medium supplemented with 10−5m2-mercaptoethanol and 5% WEHI-3B conditioned medium as a source of IL-3. Transfectants expressing IL-2Rβ were generated by electroporating cells (5 × 106 cells/400 μl) with linearized pCDNA3zeo (InVitrogen) containing IL-2Rβ using a Gene Pulser (Bio-Rad; 300 V, 960 microfarads; average time constant = 30 ms). After 24 h, cells were aliquoted into a 24-well plate and selected in 0.8 mg/ml ZeocinTM (InVitrogen). Resistant clones were tested for IL-2Rβ expression by Western blotting using goat anti-human IL-2Rβ antiserum (R & D Systems, Minneapolis, MN). 293T+ cells were cultured in Dulbecco's modified Eagle's medium (Biofluids) supplemented with 10% fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. 293T+ cells at 50% confluency were transfected using calcium phosphate precipitation reagents (5 Prime → 3 Prime, Inc., Boulder, CO), as described previously (24Aman M.J. Tayebi N. Obiri N.I. Puri R.K. Modi W.S. Leonard W.J. J. Biol. Chem. 1996; 271: 29265-29270Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Transient transfections of YT and 32D-IL-2Rβ cells for luciferase reporter assays were performed using the DEAE-dextran method. Briefly, 1–2 × 106 cells were incubated with up to 10 μg of plasmid DNA and 200 μg of DEAE-dextran in 1 ml of STBS buffer (25 mm Tris, pH 7.4, 137 mm NaCl, 0.5 mm MgCl2, 0.7 mm CaCl2, 5 mm KCl) at 37 °C for 30 min, followed by washing once with medium. For YT cells, 18 h after transfection, cells were stimulated with 2 nm IL-2 for an additional 12–24 h and then harvested. 32D-IL-2Rβ cells were incubated with either 0.05% WEHI-3B conditioned medium (which is sufficient to maintain cell viability but not growth) or with 2 nm IL-2 or 5% WEHI-3B conditioned medium for 24–36 h. For luciferase assays, lysates were prepared using the Luciferase Assay System kit (Promega). Protein concentrations were measured with a protein assay kit (Bio-Rad), and 5–20 μg of protein were used for luciferase assays according to the manufacturer's instructions (Promega). Luciferase activity was measured using a Monolight 2010 luminometer (Analytical Luminescence Laboratory). Ba/F3 cells were stably transfected with IL-2Rβ to create Ba/F3-IL-2Rβ cells. Ba/F3-IL-2Rβ-CIS cells additionally express CIS in pMAMneo (so that its expression can be induced by steroids; see Ref. 15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). Poly(A)+ RNA was extracted from PBL using the FastTrack 2.0 Kit (InVitrogen). Northern blotting was performed using 2 μg/lane of poly(A)+ RNA on 0.8% formaldehyde-agarose gels as described previously (25Ascherman D.P. Migone T.-S. Friedmann M.C. Leonard W.J. J. Biol. Chem. 1997; 272: 8704-8709Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), using CIS or control pHe7 32P-labeled probes. pHe7(26Kaczmarek L. Calabretta B. Kao H.-T. Heinz N. Nevins J. Baserga R. J. Cell Biol. 1987; 104: 183-187Crossref PubMed Scopus (27) Google Scholar) is a "housekeeping" gene whose expression is similar before and after IL-2 stimulation (27Otani H. Erdos M. Leonard W.J. J. Biol. Chem. 1993; 268: 22733-22736Abstract Full Text PDF PubMed Google Scholar). The full-length murine CIS cDNA was subcloned into the mammalian expression vector pME18S. FLAG-tagged wild type CIS and truncation mutants of CIS were generated by polymerase chain reaction, subcloned into pME18S, and sequenced. A point mutation converting arginine 107 to lysine (R107K) in the SH2 domain was introduced into the CIS cDNA using standard polymerase chain reaction-based techniques, and the sequence was confirmed by DNA sequencing. The wild type human IL-2Rβ and IL-2Rβ mutant constructs used in this study have been described (8Zhu M. Berry J.A. Russell S.M. Leonard W.J. J. Biol. Chem. 1998; 273: 10719-10725Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10Friedmann M.C. Migone T.-S. Russell S.M. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2077-2082Crossref PubMed Scopus (168) Google Scholar). Myc-tagged wild type Lck (Lckwt) and LckY505F were provided by Dr. J. Ashwell (NCI, National Institutes of Health). The NF-κB-responsive chloramphenicol acetyltransferase reporter construct, pTKκB3, containing three copies of the human immunodeficiency virus κB site, has been described previously (28Lin J.-X. Bhat N. John S. Queale W.S. Leonard W.J. Mol. Cell. Biol. 1993; 13: 6201-6210Crossref PubMed Google Scholar). The Stat5a, Stat5b, IL-2Rβ, γc, and Jak3 cDNAs were all human cDNAs. The β-casein reporter construct was generated by cloning three repeats of the GAS motif from the β-casein promoter and the cytomegalovirus minimal promoter into pGL-2basic (Promega). Polyclonal rabbit anti-CIS antiserum was prepared as described previously (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). Anti-Myc mAb 9E10 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-FLAG M2 mAb was from Eastman Kodak Co.; anti-phosphotyrosine mAbs 4G10 and PY20 were from Upstate Biotechnology, Inc. (Lake Placid, NY) and Transduction Laboratories. Polyclonal rabbit anti-Lck antiserum was a gift of Dr. L. Samelson (NICHD, National Institutes of Health). Mikβ1 mAb to IL-2Rβ was provided by Drs. M. Tsudo (Tokyo Metropolitan Institute) and J. Hakimi (Hoffmann-La Roche), and Mikβ3 mAb to IL-2Rβ was provided by M. Tsudo. Cells were lysed in Nonidet P-40 lysis buffer (50 mmTris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, 1 mm Na3VO4, 5 mm NaF, 10 μg/ml each leupeptin and aprotonin, and 1 mm4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) and centrifuged at 14,000 × g at 4 °C for 15 min. Lysates were boiled in reducing SDS sample buffer and immunoblotted or were immunoprecipitated for 1–2 h at 4 °C using specific antibodies and protein A-Sepharose beads. To investigate the potential role of CIS in IL-2 signaling, we first examined the expression of CIS mRNA in response to IL-2 and phytohemagglutinin (PHA) in normal human PBL. In unstimulated freshly isolated PBL, CIS mRNA was not detected (Fig.1 A, lane 1); however, IL-2 induced CIS mRNA within 30 min (lane 2), and the levels of CIS mRNA increased with time, with high level expression being sustained for at least 24 h (lanes 4, 6, and8). Stimulation of PBL with PHA also induced CIS, but with a slower time course so that it was 4 h before even very low levels of CIS mRNA were detected (compare lanes 3,5, and 7 with lane 1). However, by 24 h, the level of CIS expression was comparable with that seen with IL-2 (lane 9). These data suggest that PHA may not induce CIS expression directly but rather indirectly through induction of IL-2 production, given that at least 4 h is typically needed before appreciable levels of IL-2 protein can be detected after PHA activation. When PBL were preactivated with PHA for 3 days and then rested overnight in IL-2-free medium, a treatment that induces maximal expression of high affinity IL-2 receptors and primes cells for potent cellular responsiveness to IL-2, CIS mRNA expression was rapidly induced by IL-2 and sustained at a high level for at least 24 h (Fig. 1 B, lanes 1–4). We also investigated the expression pattern of CIS protein in these cells. CIS protein was potently expressed within 24 h, and high levels were maintained for at least 72 h (Fig.1 C, lanes 1–4). As noted above, CIS is induced by both erythropoietin and IL-3 and can associate with both the erythropoietin receptor and the murine IL-3 receptor β chain (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). We therefore tested the ability of CIS to associate with the IL-2Rβ chain. Lysates from IL-2-stimulated, preactivated PBL were immunoprecipitated with Mikβ3 mAb to IL-2Rβ and then Western blotted with anti-CIS antibodies. As shown in Fig. 1 D, CIS coprecipitated with IL-2Rβ, indicating a physical interaction between these two proteins. Although the degree of apparent interaction was inducible (Fig. 1 D), it was not as marked as was the inducibility of CIS (Fig. 1 C). To further investigate this situation, we also evaluated coprecipitation in Ba/F3 cells stably transfected with IL-2Rβ (Fig. 2). In these cells, IL-2 stimulates CIS expression (Fig. 2 A). However, as in PBL, it was difficult to detect a CIS-IL-2Rβ interaction with anti-IL-2Rβ antibodies (data not shown), but this interaction was revealed with more sensitive anti-phosphotyrosine antibodies (Fig. 2 B). The relatively weak coprecipitation of CIS and IL-2Rβ could reflect low stoichiometry; alternatively, a higher stoichiometry might exist but might not be readily detected given that neither the anti-CIS nor anti-IL-2Rβ antibodies are particularly robust. It is also possible that the weak coprecipitation could also reflect a comparatively low affinity or transient interactions. To map the region of IL-2Rβ required for its interaction with CIS, we performed coimmunoprecipitation experiments in 293 T+ cells transfected with murine CIS plus wild type IL-2Rβ or IL-2Rβ mutants containing various internal deletions or C-terminal truncations (see Fig. 3 A for a schematic of IL-2Rβ, including the locations of the A (residues 313–382) and S (residues 267–323). For unclear reasons, we consistently observed lower CIS expression when CIS was co-expressed with mutants of IL-2Rβ lacking either the A or S regions. Therefore, we increased the amount of the CIS plasmid cotransfected with IL-2RβΔA or IL-2RβΔS in order to achieve more similar levels of CIS expression (Fig.3 B, lanes 4 and 5 versus lane 1; middle panel; note that murine CIS is routinely detected as a doublet). When CIS and wild type IL-2Rβ were cotransfected (lane 1) and then immunoprecipitated with Mikβ1 mAb to IL-2Rβ, CIS was efficiently coprecipitated (lane 1, top panel). Deletion of the S region partially decreased the degree of association of IL-2Rβ with CIS (lanes 3 and 5), whereas deletion of the A region of IL-2Rβ abrogated CIS interaction (lanes 2 and 4; note that no CIS was coprecipitated in lane 4 (upper panel) although expression of CIS in this lane was higher than in lane 3(middle panel), where CIS weakly coprecipitated with IL-2RβΔS). We next analyzed several C-terminal IL-2Rβ truncation mutants for their abilities to bind CIS. These experiments revealed that the sequences beyond amino acid 350 were dispensable for this interaction, while truncation at amino acid 330 abrogated the association (Fig.3 C, upper panel). Therefore, these results identify amino acids 330–350 as critical for CIS binding, complementing the deletion analysis. The fact that a mutant lacking the S region (residues 267–323) showed reduced association with CIS (Fig.3 B) suggests the presence of an additional direct or indirect contact point within the S region or suggests that deletion of the S region has conformational effects resulting in reduced association. To clarify the region of CIS that mediates its interaction with IL-2Rβ, we generated wild type CIS and C-terminal truncation mutants of CIS that were FLAG-tagged at their C termini. The truncation mutants contained either the first 82 amino acids (the residues N-terminal to the SH2 domain, denoted CISNT) or the first 177 amino acids (retaining the N-terminal region as well as the SH2 domain, denoted CISΔCT)(Fig.4 A). Following coexpression of these constructs with wild type IL-2Rβ in 293 T+ cells, immunoprecipitation experiments were performed (Fig. 4 B,top panel, lanes 1–3). Whereas CISΔCT could associate with IL-2Rβ as well as wild type CIS (Fig. 4 B, middle panel,lane 1 versus lane 3), CISNT (which lacks the SH2 domain) could not (lane 2), suggesting that the interaction of CIS with IL-2Rβ might involve a classical SH2/phosphotyrosine interaction. This hypothesis is consistent with the suggestion that tyrosine-phosphorylated forms of IL-3Rβ and the erythropoietin receptor could associate with CIS (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). Surprisingly, however, the association of CIS with IL-2Rβ did not appear to require the tyrosine phosphorylation of IL-2Rβ as demonstrated by the ability of CIS to associate with an IL-2Rβ mutant in which all six cytoplasmic tyrosines were mutated to phenylalanines (IL-2RβFFFFFF) (Fig. 4 C, middle panel,lane 3 versus lane 1). Even wild type IL-2Rβ does not appear to be phosphorylated in these 293 transfections (data not shown and shown below in Fig. 6 B, top panel,lane 1). However, we used the IL-2RβFFFFFF construct to exclude the possibility that a low but undetectable level of tyrosine phosphorylation of IL-2Rβ played a role in the interaction seen in Fig. 4 C. To more directly assess the role of the CIS SH2 domain in the CIS-IL-2Rβ interaction, we prepared a CIS mutant in which arginine 107 in the phosphotyrosine binding FLVR motif was changed to lysine (CISR107K). While this type of mutation is known to disrupt the ability of SH2 domains to bind phosphotyrosine (29Mayer B.J. Jackson P.K. Van Etten R.A. Baltimore D. Mol. Cell. Biol. 1992; 12: 609-618Crossref PubMed Scopus (236) Google Scholar, 30Waksman G. Kominos D. Robertson S.C. Pant N. Baltimore D. Birge R.B. Cowburn D. Hanafusa H. Mayer B.J. Overduin M. Resh M.D. Rios C.B. Silverman L. Kuriyan J. Nature. 1992; 358: 646-653Crossref PubMed Scopus (573) Google Scholar, 31Eck M.J. Shoelson S.E. Harrison S.C. Nature. 1993; 362: 87-91Crossref PubMed Scopus (443) Google Scholar), it had little effect on the ability of CIS to associate with IL-2Rβ (Fig.4 C, middle panel, lane 2). Together, the above results indicate that at leastin vitro the CIS-IL-2Rβ interaction does not require a classical SH2-phosphotyrosine interaction between CIS and IL-2Rβ. Therefore, the ability of IL-2Rβ to interact with CISΔCT but not CISNT suggests either that other residues distinct from the FLVR motif in the SH2 region are important for binding or that the CISNT construct has a severely altered structure resulting from deletion of the SH2 domain. The above results do not exclude a partial contribution of an SH2-mediated interaction of CIS with IL-2Rβ; they instead demonstrate that non-SH2-mediated interactions also exist. Interestingly, in 293 cells, the common β chain (βc) of the IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors associated with CIS and augmenting its phosphorylation by cotransfecting Jak1 did not significantly increase its association with CIS (Fig.5). Previously, CIS was shown to associate with IL-3Rβ and the EPO receptor after ligand stimulation (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (619) Google Scholar). However, the role of ligand requirement for this induction may have been in part related to the stronger induction of CIS in the presence of ligand. Thus, like IL-2Rβ, βc may at least in part associate with CIS independently of a phosphotyrosine-SH2 interaction.Figure 6CIS inhibits Lck-mediated phosphorylation of IL-2R β. A, 293T+ cells were transfected with the indicated plasmids (wild type or constitutively activated forms of Lck, both tagged with Myc epitopes, IL-2Rβ, Jak1, and/or wild type CIS or CISR107K), and lysed. Lysates were immunoprecipitated using Mikβ1 or 9E10 (anti-Myc) mAbs. Samples were run on gels and Western blotted with the indicated antibodies (4G10 or anti-IL-2Rβ). Note that analogous to the lack of effect of CIS on Jak1 phosphorylation of IL-2Rβ, there is no effect of CIS on Jak1 autophosphorylation (lanes 9 and10, upper panel). B andC, IL-2Rβ and LckY505F were cotransfected with CIS or LckY505F. Lysates were either directly blotted as indicated (lower panels) or were first immunoprecipitated with anti-IL-2Rβ and then blotted as indicated (upper panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Association of βc and CIS is not substantially affected by Jak1-mediated tyrosine phosphorylation of βc in 293T+ cells. 293T+ cells were transfected with CIS plus either IL-2Rβ, βc, or βc plus Jak1. Lysates were then immunoprec