Stat5a serine phosphorylation - Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity

The activity of transcription factors of the Stat family is controlled by p hosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on s pecific serine residues has also been described. We have previously shown t hat in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) i n a prolactin-sensitive manner, whereas serine phosphorylation is constitut ive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighaus en, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser (779), located in a unique Stat5a SP motif, as the site of serine phosphory lation. By using phospho-Ser(779)-specific antiserum, we have determined th at Ser(779) is constitutively phosphorylated in mammary glands taken from d ifferent developmental stages. Stat5a isolated from spleen, heart, brain, a nd lung was also found to be phosphorylated on Ser(779) Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Che m. 273, 30218-30224). Here we show that mutagenesis of Ser(725) Ser(779), o r a combination of Ser(725/779) to an Ala had no effect on prolactin-induce d transcriptional activation of a beta-casein reporter construct, However, following prolactin induction the Ser(725) mutant displayed sustained DNA b inding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.