Naturally Occuring N-Terminal Truncated STAT5 Isoforms Are Widely Expressed, Functional, and Have Enhanced Nuclear Localization.

Signal transducer and activator of transcription 5(STAT5) plays major roles in hematopoietic stem cell and progenitor cell development. Our previous studies have shown that mast cells derived from STAT5ab^−/− mice in which the first two exons were deleted, had severe defects in interleukin (IL)-3 and stem cell factor (SCF) induced proliferation and survival signaling. However, expression of an endogenous hypomorphic N-terminal truncated form of STAT5 through cryptic start codons found at position 102 and 136 was uncovered, suggesting the possibility of compensation for STAT5-mediated functions. ΔN isoforms of STAT5 were also identified in different wild-type and STAT5ab^−/− mouse tissues including significant expression in brain, liver, spleen, lung, kidney, but not in heart. Here we describe that endogenous mast cell STAT5ΔN had surprisingly increased nuclear localization in STAT5ab^−/− mast cells even without cytokine stimulation but which increased in response to IL-3 and SCF. In general, the N-terminus is responsible for tetramerization and regulates receptor recognition and phosphatase recruitment. Others have reported that N-terminal truncated mutants of STAT1 and STAT4 failed to migrate into nuclei upon cytokine stimulation. Therefore, the N-terminus of STAT5 functions differently. To further characterize this function of STAT5aΔN¹³⁶, we co-transfected STAT5aΔN¹³⁶ with mouse erythropoietin receptor (EPOR) into 293T cells, and subsequently, stimulated the cells with EPO (20 μg/ml). Consistent with the mast cell studies, immunostaining revealed that STAT5aΔN¹³⁶ had enhanced nuclear retention relative to full-length STAT5a. Both STAT5a and STAT5aΔN¹³⁶ migrated to the nucleus upon EPO stimulation for 30 minutes. EPO stimulation also showed a similar pattern of phosphorylation, however, STAT5aΔN¹³⁶ phosphorylation was weaker and more transient, compared to full-length STAT5a. A known constitutively active STAT5a^S711F mutant used as a control, showed extremely high and persistent phosphorylation levels. To test for functional activity in mast cells, we used MSCV-based retroviral vectors expressing either STAT5a or STAT5aΔN¹³⁶ upstream of GFP and transduced STAT5ab^−/− mast cells. Initial transduction efficiencies of 20–40% were obtained with all vectors. After 10–25 days of culture, %GFP⁺ cells rose to 85–100% for both STAT5a and STAT5aΔN¹³⁶, while the mast cells transduced with an MSCV control vector remained about 20% GFP^+. Notably, in mast cells and GP+E86 ecotropic producer cells, overexpession of retroviral full-length STAT5a resulted in detectable STAT5aΔN¹³⁶ co-expression from the same vector, indicating that effects due to STAT5 expression are potentially modulated by ΔN isoforms generated through alternative internal start codons. Overall, this work indicates that ΔN isoforms of STAT5 are capable of enhanced nuclear localization and can promote growth in STAT5ab^−/− mast cells, suggesting that they likely participate in normal signaling functions. Studies to assess transcriptional activity of these isoforms using STAT promoter luciferase constructs is ongoing. A better understanding of the regulation of these natural isoforms of STAT5a and STAT5b and their physiological significance may lead to novel insights into JAK/STAT signaling.