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293T + cells were cultured in Dulbecco's modified Eagle's medium (Biofluids) supplemented with 10% fetal bovine serum, 2 m m glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Resistant clones were tested for IL-2Rβ expression by Western blotting using goat anti-human IL-2Rβ antiserum (R & D Systems, Minneapolis, MN). After 24 h, cells were aliquoted into a 24-well plate and selected in 0.8 mg/ml Zeocin TM (InVitrogen). Transfectants expressing IL-2Rβ were generated by electroporating cells (5 × 10 6 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). 32D cells were maintained in complete medium supplemented with 10 −5 m2-mercaptoethanol and 5% WEHI-3B conditioned medium as a source of IL-3. NK-like YT cells were cultured and maintained in complete medium. To generate “preactivated PBL,” freshly isolated PBL were cultured for 72 h in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 m m 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.

Peripheral blood lymphocytes (PBL) were prepared from normal donors using standard methods. 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. 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. Thus, we demonstrate that CIS can negatively control at least two independent IL-2 signaling pathways. Correspondingly, CIS inhibits functions associated with both of these kinases: Lck-mediated phosphorylation of IL-2Rβ and IL-2-mediated activation of Stat5. This interaction requires the A region of IL-2Rβ (residues 313–382), which also mediates the association of IL-2Rβ with Lck and Jak3. We now demonstrate that CIS also interacts with the IL-2 receptor β chain (IL-2Rβ). 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. Glycobiology and Extracellular MatricesĬIS is a cytokine- induced SH2-containing protein that was originally cloned as an interleukin (IL)-3-inducible gene.

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