Selective STAT3-α or -β expression reveals spliceform-specific phosphorylation kinetics, nuclear retention and distinct gene expression outcomes

Phosphorylation of STAT3 (signal transducer and activator of transcription 3) is critical for its nuclear import and transcriptional activity. Although a shorter STAT3 beta spliceform was initially described as a negative regulator of STAT3a, gene knockout studies have revealed that both forms play critical roles. We have expressed STAT3 alpha and STAT3 beta at comparable levels to facilitate a direct comparison of their functional effects, and have shown their different cytokine-stimulated kinetics of phosphorylation and nuclear translocation. Notably, the sustained nuclear translocation and phosphorylation of STAT3 beta following cytokine exposure contrasted with a transient nuclear translocation and phosphorylation of STAT3 alpha. Importantly, coexpression of the spliceforms revealed that STAT3 beta enhanced and prolonged the phosphorylation and nuclear retention of STAT3 alpha, but a STAT3 beta R609L mutant, with a disrupted SH2 (Src homology 2) domain, was not tyrosine phosphorylated following cytokine stimulation and could not cross-regulate STAT3a. The physiological importance of prolonged phosphorylation and nuclear retention was indicated by transcriptome profiling of STAT3(-/-) cells expressing either STAT3 alpha or STAT3 beta, revealing the complexity of genes that are up- and down-regulated by the STAT3 spliceforms, including a distinct set of STAT3 beta-specific genes regulated under basal conditions and after cytokine stimulation. These results highlight STAT3 beta as a significant transcriptional regulator in its own right, with additional actions to cross-regulate STAT3 alpha phosphorylation and nuclear retention after cytokine stimulation.