Conversion of threonine 757 to valine enhances stat5a transactivation potential

The growth hormone family of cytokines transduces intracellular signals thr ough the Jak2-Stat5 pathway to activate the transcription of target genes. Amino acids within the C termini of Stats constitute the transactivation do main but also regulate the time course of tyrosine phosphorylation and exte nt of DNA binding. We mutated Thr(757) in the C-terminal of Stat5a (Thr-Sta t5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the effect on nucle ar translocation, DNA binding. and prolactin-induced transcriptional activa tion of a Stat5-responsive luciferase reporter gene. Val-Stat5 produced a 5 -fold higher increase in transcriptional activity relative to Thr-Stat5; As p-Stat5 produced a similar response to Thr-Stat5. The increased transactiva tion was ligand induced and was not due to differences in basal expression of Val-Stat5 or to a constitutively activated Stat5 protein. Similar rates of loss of DNA binding ability and phosphorylation of Val- and Thr-Stat5 we re observed following a single pulse of prolactin, indicating that the deph osphorylation pathways were unaltered. The serine-threonine kinase inhibito r H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 t o a similar extent, eliminating phosphorylation of Thr757 as a regulatory m echanism. The results suggest that Thr757 modulates the transactivation pot ential of Stat5 by a mechanism(s) that is dependent on the formation of Sta t5 dimers and/or their nuclear translocation.