Differential control of the phosphorylation state of proline-juxtaposed serine residues Ser725 of Stat5a and Ser730 of Stat5b in prolactin-sensitive cells.

Transcription factors of the Stat family are controlled by protein kinases. Phosphorylation of a positionally conserved tyrosine residue is obligatory for Stat dimerization, nuclear translocation, and specific DNA binding. Studies of Stat1 and Stat3 have suggested that serine phosphorylation may also regulate function. We now identify serine residues located in a conserved PSP motif of Stat5a (Ser725) and Stat5b (Ser730) as major phosphorylation sites, using mutagenesis, phosphoamino acid analysis, and site-specific anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation control of this PSP motif differed between the highly homologous Stat5a and Stat5b proteins. Whereas Ser725 of Stat5a was constitutively phosphorylated both in COS-7 cells and Nb2 lymphocytes, phosphorylation of Ser730 of Stat5b was markedly stimulated by prolactin. The data also suggested the existence of a second major serine phosphorylation site in Stat5a. Interestingly, constitutive phosphorylation of the PSP motif was suppressed by PD98059 but not by staurosporine under conditions in which both agents inhibited mitogen-activated protein kinases. Furthermore, pretreatment of cells with staurosporine, PD98059, H7, or wortmannin did not prevent either Stat5a or Stat5b from becoming maximally serine-phosphorylated after prolactin exposure. We propose that two pathways regulate Stat5 serine phosphorylation, one that is prolactin-activated and PD98059-resistant and one that is constitutively active and PD98059-sensitive and preferentially targets Stat5a. Finally, phosphorylation of the PSP motif of Stat5a or Stat5b was not essential for DNA binding or transcriptional activation of a beta-casein reporter gene in COS-7 cells, suggesting that serine kinase control of Stat5 activity differs from that of Stat1 and Stat3.