The signal transducer and activator of transcription 3 (Stat3) gene mediates vital processes such as involution, maintenance of pluripotent stem cells, and inflammation. Stat3 is activated by phosphorylation at Tyr705(Y705) and Ser727(S727) residues, that has independent and overlapping functions when studied in overexpressing cells in vitro. In this study, we sought to characterize the roles of Y705 and S727 residues in vivo, using transgenic mice which harbor the inactivating mutants Stat3-Y705F (tyrosine to phenylalanine), Stat3-S727A (serine to alanine) and activation mutant Stat3-S727E (serine to glutamic acid). The Stat3-S727E mimics constitutively phosphorylated Stat3 at S727, while the other two mutants cannot be phosphorylated. We, for the first time, have generated transgenic mice with the Knock-In (KI) mutants of Stat3 at Y705 and S727, alone or in combination. Because previous studies have shown that Stat3 knockout mice are embryonic lethal, we determined the independent roles of the two phosphorylation sites, and their interactions during embryonic development of KI-mice. We have further analyzed if stability of the Stat3 protein is affected by these mutations to support our key observations. Our studies show that Y705F homozygous mutation is embryonic lethal at weaning, only wildtype and heterozygous Stat3Y705F-KI mice survived. Similar observation was made with Stat3Y705F/S727E –KI mice (Stat3Y-SE), where homozygous mutation was also embryonic lethal. However, the KI-mice with Stat3S727E homozygous mutation were viable suggesting that constitutive phosphorylation of 727 had no detrimental effect on embryonic development, but could not rescue the loss of Stat3 function due to Y705F mutation in the double mutant. We analyzed stability of Stat3 protein in mouse embryonic fibroblasts (MEFs) isolated from KI-mice. Stat3Y-SE/- had a significantly decreased level of Stat3 mRNA. Stat3 protein was undetectable in Stat3Y705F/- and Stat3Y-SE/- MEFs, suggesting that phosphorylation at Y705 is critical for stability of Stat3 protein and that phosphomimetic at S727 could not rescue Stat3 from degradation. It is also apparent that Stat3Y705F/- and Stat3Y-SE/- KI-mice phenocopies Stat3-/- mice as both are embryonic lethal. Studies are underway to characterize the Stat3S727A and Stat3Y705F/S727A (Stat3Y-SA) KI-mice and the mutant proteins. Our characterization of Y705 and S727 will result in better understanding of their key roles in activation of Stat3 in a physiological context.Ohio State University Second Year Transformative Experience ProgramMayers Undergraduate Research FellowshipArts and Sciences Undergraduate Research ScholarshipA five-year embargo was granted for this item.Academic Major: Molecular Genetic
