136: Cytokine addition slows STAT3 transport to the nucleus as revealed by live cell imaging and fluorescence recovery after photobleaching

Abstract

STAT3 (Signal Transducer and Activator of Transcription 3) is a latent transcription factor mediating cellular proliferation and differentiation in response to cytokines such as oncostatin M (OSM). Two STAT3 spliceforms, STAT3α and STAT3β, that differ only in their C-terminal transactivation domain sequences are encoded by the Stat3 gene, however these splice forms differ in both their OSM-stimulated duration of Y750 phosphorylation and nuclear residency. To address the mechanisms underlying these spliceform differences, we performed live cell-imaging using GFP-tagged STAT3 proteins to investigate their nuclear trafficking in response to OSM. These studies showed that STAT3 β relocalisation to the nucleus was sustained following OSM exposure while STAT3 α relocalisation to the nucleus was transient. These observations paralleled the sustained STAT3 β Y750 phosphorylation but transient STAT3 α Y750 phosphorylation in response to OSM. We subsequently employed the use of fluorescence recovery after photobleaching (FRAP) protocols to investigate the kinetics of STAT3 splice form movement into the nucleus both under control conditions and following OSM treatment. Quantitative analyses of the rates of basal nuclear import showed that nuclear entry of STAT3β was significantly faster than the nuclear entry rates for STAT3α. Strikingly, the nuclear import rates of STAT3α and STAT3β were slowed by OSM stimulation, whereas parallel control studies indicated that OSM did not impact on protein diffusion across the nuclear envelope or classical import in-mediated nuclear transport mechanisms. Further analyses of STAT3α/β Y705F mutants or a STAT3α S727A highlighted the contributions of STAT3 phosphorylation to these cytokine-stimulated changes in STAT3 nuclear import kinetics. Taken together, these results support a new paradigm where cytokines prolong STAT3 nuclear retention simultaneous but decrease rather than increase their rates of nuclear import.