Cytokine‐Induced Slowing of STAT3 Nuclear Import; Faster Basal Trafficking of the STAT3β Isoform

The STAT3 signal transducer and activator of transcription is a key mediator of gene transcription in response to cytokines such as oncostatin M (OSM). We performed direct live cell imaging of GFP‐tagged STAT3 proteins for the first time, showing transient relocalization of STAT3α to the nucleus following OSM exposure, in contrast to sustained nuclear relocalization of the shorter STAT3β spliceform. To explore this further, we applied fluorescence recovery after photobleaching (FRAP) to determine the nuclear import kinetics of STAT3α and β, as well as of a C‐terminal truncation derivative STAT3ΔC comprising only the sequence shared by the spliceforms, in the absence or presence of OSM. The rates of basal nuclear import for STAT3β and STAT3ΔC were significantly faster than those for STAT3α. Strikingly, OSM slowed the import rates of all the three STAT3 proteins, whereas the import rates of GFP alone or a classical importin‐mediated cargo were unaffected, with analysis of Y705F mutant derivatives for all the three STAT3 constructs, or of a S727A mutant within the unique C‐terminus of STAT3α, reinforcing the contribution of specific phosphorylation to the cytokine‐stimulated changes. The results introduce a new paradigm where cytokine treatment prolongs nuclear retention simultaneous with decreasing rather than increasing the rate of nuclear import. The α and β spliceforms of the inducible transcription factor STAT3 play key roles in various important biological processes. Here we show that STAT3α and β show distinct differential cytokine‐stimulated nuclear kinetics, with STAT3β exhibiting faster basal shuttling, and prolonged nuclear retention upon cytokine treatment. Importantly, cytokine stimulation does not enhance but slows the STAT3 nuclear import rate, suggesting a new paradigm for STAT3 signalling where cytokine treatment increases nuclear retention rather than nuclear import per se.