Stat3 is activated by phosphorylation on Tyr-705, which leads to dimer formation, nuclear translocation, and regulation of gene expression. Serine phosphorylation of Stat3 by mitogen-activated protein kinase has also been observed in cells responding to epidermal growth factor and shown to affect its tyrosine phosphorylation and transcriptional activity. Serine phosphorylation of Stat3 is also induced by interleukin-6 (IL-6) stimulation, which is shown to be independent of mitogen-activated protein kinase and sensitive to the Ser/ Thr kinase inhibitor H7. In this study, we investigated whether protein kinase C (PKC) is the kinase that is induced and responsible for Stat3 serine phosphorylation by IL-6 stimulation and which isoform of PKCs is likely to be involved. Here, we report that Stat3 was specifically associated with PKC ␦ in vivo in an IL-6-dependent manner in several cell types. Furthermore, Stat3 was phosphorylated by PKC ␦ in vivo on Ser-727, which could be inhibited either by a specific PKC ␦ inhibitor or by a dominant-negative mutant of PKC ␦. Finally, we showed that the phosphorylation of Stat3 by PKC ␦ led to a negative regulation of Stat3 DNA binding and transcriptional activity. These results indicate that PKC ␦ is likely to be the kinase that phosphorylates Stat3 in response to IL-6 stimulation and suggest a possible regulatory role of PKC ␦ on Stat3 function.

STAT proteins are a family of latent transcription factors that mediate the response to various cytokines and growth factors. Upon stimulation by cytokines, STAT proteins are recruited to the receptors via their SH2 domains, phosphorylated on a specific tyrosine, dimerized, and translocated into the nucleus, where they bind specific DNA sequences and activate the target gene transcription. STATs share highly conserved structures, including an N-domain, a coiled-coil domain, a DNA-binding domain, a linker domain, and an SH2 domain. To investigate the role of the coiled-coil domain, we performed a systematic deletion analysis of the N-domain and each of the ␣-helices and mutagenesis of conserved residues in the coiled-coil region of Stat3. Our results indicate that the coiled-coil domain is essential for Stat3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding, stimulated by epidermal growth factor (EGF) or interleukin-6 (IL-6). Single mutation of Asp170 or, to a lesser extent, Lys177 in ␣-helix 1 diminishes both receptor binding and tyrosine phosphorylation. Furthermore, the Asp170 mutant retains its ability to bind to DNA when phosphorylated on Tyr705 by Src kinase in vitro, implying a functional SH2 domain. Finally, we demonstrate a direct binding of Stat3 to the receptor. Taken together, our data reveal a novel role for the coiled-coil domain that regulates the early events in Stat3 activation and function.Growth factors and cytokines regulate cell growth and differentiation by triggering various intracellular signaling pathways that lead to gene expression. JAK-STAT is a key pathway that mediates cellular responses to a variety of cytokines (reviewed in references 11, 20, and 30). As the name suggests, STATs (signal transducers and activators of transcription) represent a family of proteins with dual functions that transduce the signal in the cytoplasm and activate gene expression in the nucleus. Seven STATs with different functions have been identified so far in mammalian cells, and over 40 different polypeptides are known to activate one or more STATs (reviewed in reference 10). Among them, Stat3 was identified both as an acute-phase response factor activated by interleukin-6 (IL-6) in mouse liver and by homology to Stat1 (2, 46). Stat3 is also activated by other members of the IL-6 family, such as IL-11, ciliary neurotrophic factor, oncostatin M, and leukemia-inhibitory factor, which share the common transducing gp130 receptor subunit (2, 25, 33). IL-6, as a pleiotropic cytokine, exhibits various functions in immune response, hematopoiesis, and neuronal differentiation (37).The model of activation of Stat3 by IL-6 has been established. Binding of IL-6 to its receptor gp80 (subunit ␣) induces homodimerization of gp130 (subunit ␤) and phosphorylation of the gp130-associated Janus kinases (JAKs). JAKs phosphorylate the tyrosine residues on gp130 that serve as docking sites fo...