Background: STAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain.
Results: Expression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3.
Conclusion: Our findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.