The suppressor of cytokine signaling (SOCS) proteins are a family of cytokine-inducible negative regulators of cytokine signaling. Interferon (IFN)-␥ treatment induces the expression of SOCS1, SOCS2, and SOCS3 mRNAs. To examine the effect of SOCS proteins on IFNmediated Janus-activated kinase/signal transducers and activators of transcription (STAT) signaling, HeLaand MCF-7-derived stable cell lines expressing SOCS1, SOCS2, and SOCS3 proteins were established. SOCS1 and SOCS3 but not SOCS2 inhibited the tyrosine phosphorylation and nuclear translocation of STAT1 in response to IFN stimulation. The IFN-mediated antiviral and antiproliferative activities were consistently blocked by the constitutive expression of SOCS1 and SOCS3 but not SOCS2 proteins. The maximum inhibitory activities of SOCS1 and SOCS3 proteins toward the activation of STAT1 were observed at very low levels of SOCS protein expression. In addition, SOCS1 exhibited a much stronger inhibitory activity toward the activation of STAT1 than did SOCS3. These results suggest that SOCS1 and SOCS3 but not SOCS2 are inhibitors of IFN-mediated Janus-activated kinase/STAT signaling pathways. Interferons (IFNs)1 have antiviral, immunomodulatory, and antiproliferative activities (1). The binding of IFNs to their cell surface receptors activates receptor-associated Jak tyrosine kinases that then phosphorylate a family of latent cytoplasmic transcription factors named signal transducers and activators of transcription (STATs; Ref. 2). Phosphorylated STAT proteins dimerize and translocate into the nucleus to activate genes. Jak1 and Tyk2, which are associated with IFN-␣ receptor subunits, phosphorylate STAT1 and STAT2 upon IFN-␣ stimulation. In cells treated with IFN-␥, which binds to a distinct receptor complex, Jak1 and Jak2 are activated and then phosphorylate STAT1 (3-7). Tyrosine phosphorylation of STAT1 is required for its dimerization, nuclear translocation, DNA binding, and gene activation (8, 9).Biochemical and genetic studies suggest that STAT1 plays an important role in IFN-mediated biological activities. Cells lacking STAT1 are defective in IFN-mediated antiviral activity and are resistant to IFN-␥-induced growth arrest (10 -13). Overexpression of a constitutively activated STAT1 mutant protein can enhance IFN-induced antiproliferative activity (14). Studies with STAT1 knockout mice demonstrated that STAT1 is essential for the innate response to either viral or bacterial infection (15, 16).Although great progress has been made toward the understanding of the molecular mechanism responsible for the activation of Jak/STAT signaling pathways, little is know about how Jak/STAT signals are down-regulated. Recently, a family of cytokine-inducible inhibitors of signaling has been isolated (17-21). The proteins in this family, including cytokine-inducible SH2-containing protein and SOCS/Jak-binding protein/ STAT-induced STAT inhibitor proteins, are relatively small protein molecules containing SH2 domains. It has been shown that SOCS1 protein can directly bind...

Gamma interferon (IFN-␥) signals to the nucleus through the activation, by tyrosine phosphorylation, of the latent cytoplasmic transcription factor Stat1 (signal transducer and activator of transcription). It has been demonstrated that the activity of Stat1 is dependent on tyrosine phosphorylation which is regulated by Jak tyrosine kinases as well as by the as-yet-unidentified protein tyrosine phosphatase. We report that the N-terminal domain of Stat1, which is highly conserved among all STAT family members, is required for its tyrosine dephosphorylation. A single amino acid substitution (Arg-31 to Ala) in the Stat1 N-terminal domain inhibited Stat1 tyrosine dephosphorylation. The deletion of the Stat1 N-terminal domain resulted in a mutant Stat1 protein which was constitutively phosphorylated on Tyr-701. Upon IFN-␥ stimulation, the tyrosine phosphorylation of this mutant protein was further enhanced but was not down-regulated by protein tyrosine phosphatase in vivo. When expressed in NIH 3T3 cells, this mutant protein greatly enhanced the antiproliferative activity of IFN-␥. We suggest that the N-terminal domains of STATs are crucial for modulating STAT activities through regulating the tyrosine dephosphorylation of STATs.Interferons (IFNs) have antiviral, immunomodulatory, and antiproliferative properties (8). A direct signal transduction pathway from the IFN receptor to the nucleus has been discovered (4,22,36,45). The binding of IFN to its receptor leads to receptor dimerization and the activation of Janus kinase (Jak) tyrosine kinases (30,41,42,46,48). Specific tyrosine residues on the receptor are then phosphorylated by activated Jaks and serve as docking sites for recruiting a family of latent cytoplasmic transcription factors termed signal transducers and activators of transcription (STATs) (12, 13). The STATs are then phosphorylated on tyrosine, most likely by Jaks, and form homo-or heterodimers which subsequently translocate into the nucleus and direct immediate gene activation. It has been shown that gamma IFN (IFN-␥) activates the Stat1 homodimer, which binds to the IFN-␥ response element and activates transcription (6,38,39).STATs belong to a growing family of proteins involved in signal transduction by many cytokines and growth factors. Sequence analysis indicates that members of the STAT family of proteins are highly homologous in several regions. The SH2 domains of STATs have been shown to be crucial for both the activation and dimerization of STAT proteins (10,38). It has been shown that the specificity of STAT activation is largely determined by the SH2 domains of STATs (16) as well as the specific STAT binding motifs present on ligand receptors (13,43).

Human γδ T cells have the ability to rapidly expand and produce IFN-γ in response to nonpeptide Ags of microbial pathogens, in particular a class of compounds known as the prenyl phosphates. We investigated the ability of IL-15, a T cell growth factor, to modulate prenyl phosphate-induced γδ T cell proliferation and cytokine production. IL-15 significantly enhanced the expansion of γδ T cells in the peripheral blood after stimulation in vitro with isopentenyl pyrophosphate. Moreover, using γδ T cell clones, we determined that IL-15-induced T cell proliferation was dependent on the IL-2Rβ chain but not the IL-2Rα chain. We therefore studied the IL-15Rα chain expression in human γδ T cells in the presence or absence of nonpeptide Ags. We found IL-15Rα mRNA expression in IL-15-stimulated and Ag-stimulated human γδ T cells but not in resting γδ T cells. Although IL-15 itself had little effect on the production of IFN-γ, IL-15 plus IL-12 acted synergistically to augment IFN-γ production by γδ T cells. Moreover, we showed that this increase in IFN-γ could be explained by the dual activation of STAT1 and STAT4 by IL-15 and IL-12, respectively. Taken together, these results suggest that IL-15 may contribute to activation of human γδ T cells in the immune response to microbial pathogens.