Stat5a Serine Phosphorylation

The activity of transcription factors of the Stat family is controlled by phosphorylation 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 specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr694 in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863–31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser779, located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser779-specific antiserum, we have determined that Ser779 is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser779. Ser725 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. Chem. 273, 30218–30224). Here we show that mutagenesis of Ser725, Ser779, or a combination of Ser725/779 to an Ala had no effect on prolactin-induced transcriptional activation of a β-casein reporter construct. However, following prolactin induction the Ser725 mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser725 phosphorylation has an impact on signal duration.