Abstract

Two isoforms of Stat3 (signal transducer and activator of transcription 3) are expressed in cells, alpha (p92) and beta (p83), both derived from a single gene by alternative mRNA splicing. The 55-residue C-terminal transactivation domain of Stat3alpha is deleted in Stat3beta and replaced by seven unique C-terminal residues (CT7) whose function remains uncertain. We subcloned the open reading frames of Stat3alpha and Stat3beta into the C terminus of green fluorescent protein (GFP). Fluorescent microscopic analysis of HEK293T cells transiently transfected with GFP-Stat3alpha or GFP-Stat3beta revealed similar kinetics and cytokine concentration dependence of nuclear accumulation; these findings were confirmed by high throughput microscope analysis of murine embryonic fibroblasts that lacked endogenous Stat3 but stably expressed either GFP-Stat3alpha or GFP-Stat3beta. However, although time to half-maximal cytoplasmic reaccumulation after cytokine withdrawal was 15 min for GFP-Stat3alpha, it was >180 min for GFP-Stat3beta. Furthermore, although the intranuclear mobility of GFP-Stat3alpha was rapid and increased with cytokine stimulation, the intranuclear mobility of GFP-Stat3beta in unstimulated cells was slower than that of GFP-Stat3alpha in unstimulated cells and was slowed further following cytokine stimulation. Deletion of the unique CT7 domain from Stat3beta eliminated prolonged nuclear retention but did not alter its intranuclear mobility. Thus, Stat3alpha and Stat3beta have distinct intracellular dynamics, with Stat3beta exhibiting prolonged nuclear retention and reduced intranuclear mobility especially following ligand stimulation. Prolonged nuclear retention, but not reduced intranuclear mobility, mapped to the CT7 domain of Stat3beta.

Highlights

  • Nuclear export of Stat[1] appears to be linked to dephosphorylation (44), results with Stat[3] are less clear (36, 38)

  • Cellular biology of Stat[3] in the past 15 years, more remains to be discovered regarding its intracellular trafficking and intranuclear dynamics, especially whether or not these features differ between isoforms and the domains within each responsible for any differences observed

  • Basal co-localization has been attributed to constitutive shuttling of Stat3␣ between the cytoplasm and nucleus

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Summary

Introduction

Nuclear export of Stat[1] appears to be linked to dephosphorylation (44), results with Stat[3] are less clear (36, 38). Stat3␣ out of the nucleus mapped to two nuclear export sequences, one contained within residues 404 – 414 within the DNA-binding domain and the other within residues 524 – 36 –38, 41, 42), including those reported have demonstrated accumulation of Stat3␣ in the nucleus with ligand stimulation.

Results
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