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The signal transducer and activator of transcription-3 (Stat3) protein is activated by the interleukin 6 (IL-6) family of cytokines, epidermal growth factor, and leptin. A protein named PIAS3 (protein inhibitor of activated STAT) that binds to Stat3 was isolated and characterized. The association of PIAS3 with Stat3 in vivo was only observed in cells stimulated with ligands that cause the activation of Stat3. PIAS3 blocked the DNA-binding activity of Stat3 and inhibited Stat3-mediated gene activation. Although Stat1 is also phosphorylated in response to IL-6, PIAS3 did not interact with Stat1 or affect its DNA-binding or transcriptional activity. The results indicate that PIAS3 is a specific inhibitor of Stat3.

The binding of cytokines to their cell surface receptors activates, by tyrosine phosphorylation, a family of latent cytoplasmic transcription factors termed STATs (signal transducer and activator of transcription). After cytokine receptor activation, STATs dimerize and translocate into the nucleus to activate genes. Seven members of the STAT family, activated by a variety of cytokines, have been cloned (1-5). Genetic knockout studies indicate that STATs have highly specific functions. Stat1, the founding member of the STAT family, is essential for innate response to either viral or bacterial infection (6, 7). Stat1 is phosphorylated on a single residue, Tyr-701, in response to stimulation by a number of ligands including interferons (IFNs), interleukin 6 (IL-6), and epidermal growth factor (8-11). The phosphorylation on the Tyr-701 residue of Stat1 is required for its nuclear translocation, dimerization, DNA binding, and gene activation (8,12).Although great progress has been made toward the understanding of STAT activation, little is known about how STAT signals are down-regulated. Several mechanisms to downregulate STAT signaling have been proposed. (i) Because the activities of STATs depend on tyrosine phosphorylation, the precise recognition and dephosphorylation of STATs by their protein tyrosine phosphatases (PTPases) is expected to be crucial for gene regulation (13-16). However, it is not known what and how PTPases can dephosphorylate STATs. (ii) Inhibitors of proteosome activity are shown to prolong the activation of Stat1, implying the involvement of ubiquitination in the degradation of Stat1 (17). However, because these inhibitors also affect the half-life of IFN receptor, the importance of the degradation of Stat1 through ubiquitination pathway remains unclear (4, 16). (iii) Recently, a family of cytokine-inducible inhibitors of signaling have been isolated (18)(19)(20). This family of proteins, named SOCS͞JAB͞SSI, are relatively small protein molecules that contain mainly SH2 domains. SOCS͞JAB͞SSI proteins can directly bind to JAKs and can inhibit the tyrosine kinase activity of JAKs.We have identified recently a protein named PIAS3 (protein inhibitor of activated Stat3) that functions as a specific inhibitor of Stat3 signaling (21). We report here the identification of four additional members of the PIAS family. We found that PIAS1 was associated with Stat1, but not with Stat2 or Stat3 in vivo in cells treated with IFN or IL-6. The PIAS1-Stat1 interaction requires the phosphorylation of Stat1 on Tyr-701. Furthermore, PIAS1 but not other PIAS proteins blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation. Our results suggest that PIAS1 is a specific inhibitor of Stat1-mediated gene activation. The mode of the PIAS-mediated inhibition on STAT activity is distinct from other known inhibitory mechanisms involved in STAT signaling. MATERIALS AND METHODSCells. U3A and U3A-derived cell lines were maintained in DMEM containing 10% fetal bovine serum at 10% CO 2 . Human Daud...

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