Stat3 Isoforms, α and β, Demonstrate Distinct Intracellular Dynamics: Prolonged Nuclear Retention of Stat3β Maps to Its Unique C-Terminal End.

Abstract Two isoforms of signal transducer and activator of transcription (STAT) 3 are expressed in all cells—alpha] (p92) and β (p83)—both derived from a single gene by alternative mRNA splicing. Structurally, Stat3α contains a 55-residue C-terminal transactivation domain (TAD), which is deleted in Stat3β and replaced by 7 unique C-terminal residues (CT7) whose function remains uncertain. Functionally, Stat3α is transcriptionally active while Stat3β is not despite binding DNA more tightly than Stat3α. Stat3α is an oncogene activated in many cancers including multiple myeloma, acute myelogenous leukemia and lymphoma, while Stat3β appears to antagonize the oncogenic functions of Stat3α. To gain additional insight into the distinct biological and oncogenic functions of Stat3α and β at the single-cell level and, in particular, to determine the function of the unique CT7 domain of Stat3β, we subcloned the open reading frames of Stat3α, Stat3β and Stat3β missing the CT7 domain (ΔStat3β) into the C-terminus of GFP. Immunoblot analysis and DNA binding studies demonstrated that each GFP-tagged construct encoded proteins of the expected size that bound DNA. Similar to their non-tagged counterparts, GFP-Stat3α, but neither GFP-Stat3β nor GFP-ΔStat3β, activated a reporter construct containing acute phase response elements. Each GFP-Stat3 construct was stably expressed in murine embryonic fibroblasts (MEF) in which Stat3 was deleted previously using Cre/lox technology. Following selection, clones were isolated and sorted to ensure levels of expression of each GFP-Stat3 construct similar to levels found in wild type MEF cells. Fluorescence microscopic analysis of unstimulated MEF/α, MEF/β and MEF/Δβ cells revealed predominantly diffuse cytoplasmic localization of each GFP-tagged protein. Stimulation of cells with IL-6 (200 ng/ml) and soluble IL-6 receptor (sIL-6R, 250 ng/ml) revealed similar kinetics of cytoplasmic-to-nuclear translocation for each GFP-Stat3 construct with translocation starting at 10 min and becoming maximal at 30 min. High throughput microscopy analysis of 1,000 or more cells following removal of IL-6/sIL-6R revealed a nuclear-to-cytoplasmic translocation half life of 15 min for GFP-Stat3α and >3hr for GFP-Stat3β. In contrast to GFP-Stat3β, GFP-ΔStat3β had a nuclear-to-cytoplasmic translocation half life of <30 min similar to GFP-Stat3α indicating that the CT7 domain was responsible for prolonged nuclear retention of GFP-Stat3β. The intranuclear mobility of the GFP-Stat3 constructs was determined by fluorescence recovery after photobleaching (FRAP). The mobility of GFP-Stat3α assessed by fluorescence recovery half-time (t1/2) increased 33% with IL-6/sIL-6R stimulation (t1/2=1.07 ± 0.58 s before stimulation; t1/2=0.72 ± 0.45 s after stimulation; p<0.05). The mobility of GFP-Stat3β in unstimulated cells (1.89 ± 0.51 s) was 77% slower than GFP-Stat3α without stimulation (p<0.05) and was slowed 52% further following IL-6/sIL-6R simulation (2.88 ± 0.60 s; p<0.05). While deletion of the unique CT7 domain from Stat3β eliminated prolonged nuclear retention, it did not substantially reduce its intranuclear mobility (1.48 ± 0.47 s without stimulation; 2.63 ± 0.65 s following stimulation). Thus, Stat3α and Stat3β have distinct intracellular dynamics with Stat3β exhibiting prolonged nuclear retention and reduced intranuclear mobility both without and with stimulation; prolonged nuclear retention, but not reduced intranuclear mobility, map to the CT7 domain of Stat3β.