Signal transducers and activators of transcription (Stat) proteins play an important role in signaling through a variety of cytokine and growth factor receptors. Each of the Stat proteins is activated in a ligandspecific manner. Only the Src homology 2 (SH2) domains of Stat1 and Stat2 are critical for the ligand-specific activation of interferon signaling. In this study we determined the domains in Stat3 protein that contribute to interleukin 6 (IL-6)-specific phosphorylation. Based on evidence

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... that Stat3, but not Stat1, is activated in the presence of low levels of IL-6 and soluble IL-6 receptor, we constructed various swap mutants between Stat3 and Stat1 and examined their response to IL-6 after their transient expression into COS7 cells. The region upstream of the SH2 domain was exchangeable between Stat1 and Stat3, whereas the region carboxyl-terminal to the SH2 domain of Stat3 was critical to phosphorylation by IL-6. However, unlike Stat1 and Stat2 in interferon signaling, the swap mutant in which 5 amino acid residues just carboxyl-terminal to the tyrosine phosphorylation site (Tyr 705 ) in Stat3 was replaced by the corresponding region derived from Stat1 was not phosphorylated in response to IL-6. Substituting 1 amino acid (Lys 709 ) at position ؉4 relative to Tyr 705 abolished the tyrosine phosporylation of Stat3 in response to IL-6. Co-immunoprecipitation experiments demonstrated that these mutants were associated with gp130 at an extent similar to wild-type Stat3. Taken together, these results show that the amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site are involved in IL-6-specific activation of Stat3. Cytokines and growth factors mediate their biological effects through interaction with their receptors. Investigations into the transcriptional response to interferons have identified the Janus kinases (Jak)-signal transducers and activators of transcription (Stat) 1 signaling pathway, which is in fact used by a large number of cytokines and growth factors (1). The Jak family of known nonreceptor protein kinases consists of Jak1, Jak2, Jak3, and Tyk2. The family of Stat proteins constitutes a new class of transcription factors that contains SH2, SH3-like domains, and a carboxyl-terminal tyrosine phosphorylation site (2). Six Stat family members (Stat1-Stat6) have been cloned. Jak kinases associate with the cytoplasmic membrane proximal region of receptors and are catalytically activated after ligand binding. The activated Jak kinases then phosphorylate Stat proteins at their tyrosine residues. Thereafter, the Stat proteins become homodimerized or heterodimerized and translocate to the nucleus to activate transcription by interaction with specific DNA sequences. The Stat proteins are activated in a ligand-specific manner. In in vitro studies, Stat1 and Stat2 are phosphorylated in response to IFN-␣, whereas Stat1, but not Stat2, is phosphorylated in response to IFN-␥. Besides the IFN signaling systems, Stat1 is activated by signaling through various cytokine receptors (3-6), growth factor receptors (6 -8), and the G protein-coupled receptor for angiotensin II (9, 10). Stat3 is phosphorylated by stimulation with cytokines using gp130 and gp130-related receptors such as IL-6, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor, and granulocyte colony-stimulating factor, as well as EGF (11-13). Stat4 and Stat6 are phosphorylated in response to respectively (14, 15) . Stat5 is phosphorylated by a variety of cytokines, including IL-2, IL-3, IL-5, granulocyte-macrophage colony-stimulating factor, prolactin, and thrombopoietin (16 -21). The targeted disruption of the Stat genes in mice has revealed the involvement of each Stat protein in the cytokine signal pathways in vivo (22) (23) (24) (25) (26) (27) . Although Stat1 is activated by many cytokines and growth factors, it appears specific for IFN pathways in vivo. Stat1Ϫ/Ϫembryonic stem cells are unresponsive to IFN but retain responsiveness to LIF and remain LIFdependent for undifferentiated growth (22), indicating that Stat1 does not play a distinctive role in LIF signaling. Stat6 plays an essential and specific role in 25) , and Stat4 does in 27) . To understand how the specificity of the signal is achieved after the interaction of a ligand with the corresponding receptor and subsequent activation of Jak kinases and Stat proteins, the molecular mechanism of their interaction has been investigated. In response to IFN-␥, Stat1 is activated by Jak1 and Jak2. A functionally critical membrane-distal tyrosine residue (Tyr 440 ) in the IFN-␥␣ receptor is a target of activated Jak kinases, and the sequence YpDKPH, containing the phosphotyrosine of the IFN-␥ receptor, provides the specific association site for Stat1 (28). In IFN-␣ signaling, Leung et al. showed that Stat1 and Stat2 proteins might be sequentially phosphorylated in response to IFN-␣, and that phosphorylated Stat2 might be required, so that unphosphorylated Stat1 can bind to the activated IFN-␣ receptor (29). For IL-6 signaling, gp130 becomes homodimerized when IL-6 binds the IL-6 receptor (30, 31).