Multiple Stat complexes interact at the interferon regulatory factor-1 interferon-gamma activation sequence in prolactin-stimulated Nb2 T cells

Interferon regulatory factor-1 (IRF-1) is a major immediate early gene induced by prolactin (PRL) in a biphasic, cell cycle-dependent manner in Nb2 T cells. This biphasic expression (30 min and 10 h) is mediated in part by an interferon-gamma activation sequence (GAS) in the IRF-1 promoter which binds factors belonging to the Signal Transducers and Activators of Transcription (Stat) family. By electrophoretic mobility shift assays (EMSA), Stat1 alpha was found to be the major and Stat5a a minor component of the 30 min complex. At 10 h, Stat-like factors were again found at the IRF-1 GAS. Western blot analyses show that Stat5a was rapidly induced by PRL to enter the nucleus, but unexpectedly, Stat1 alpha and the alternatively-spliced Stat1 beta were already present in the uninduced nucleus. Further, Stat1 alpha but not Stat1 beta is preferentially tyrosine phosphorylated in response to PRL stimulation. Our studies suggest that multiple Stat complexes may contribute to the biphasic transcription of the IRF-1 gene in PRL-stimulated T cells.