Asymmetric post-translational modifications regulate the intracellular distribution of unstimulated STAT3 dimers

Signal Transducer and Activator of Transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus-STAT3 bimolecular fluorescence complementation (BiFC) assay that allows the visualization and study of STAT3 dimerization and protein-protein interactions in living cells. Inactivating mutations on residues susceptible to post-translational modifications (K49R, K140R, K685R, Y705F and S727A) did not alter the basal dimerization of unstimulated STAT3, but changed significantly the intracellular distribution of STAT3 dimers. Surprisingly, the distribution of specific asymmetric STAT3 dimers (i.e. the STAT3 molecules carry different mutations) was different from symmetric dimers. Our results indicate that asymmetric post-translational modifications on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. This set of Venus-STAT3 BiFC constructs provides yet unexplored means to advance our understanding of STAT3 behavior and function in cancer and beyond.