TRANSCRIPTIONAL INHIBITION BY STAT5 - DIFFERENTIAL ACTIVITIES AT GROWTH-RELATED VERSUS DIFFERENTIATION-SPECIFIC PROMOTERS

Prolactin (PRL) induces transcriptional activation of not only growth- related genes such as interferon regulatory factor-1 (LRF-1) but also differentiation-specific genes such as beta-casein through a signaling cascade consisting of Janus kinases and Stat (signal transducer and a ctivator of transcription) factors. To understand better the role of S tats in PRL signaling, we cloned rat Stat5b from a PRL-responsive T ce ll line Nb2. A Stat5b-specific peptide antibody was generated. In PRL receptor reconstituted COS cells cotransfected with Stat5b or Stat5a, both Stat5 proteins become tyrosine phosphorylated and bind to the IRF -1 GAS (interferon-gamma activation sequence) element in a PRL-inducib le manner. Unexpectedly, both Stat5b and Stat5a inhibit PRL induction of the IRF-1 promoter, but they mediate PRL stimulation of the beta-ca sein promoter. Stat5-mediated inhibition was observed only at the nati ve IRF-1 promoter and not at the isolated IRF-1 GAS element linked to a heterologous thymidine kinase promoter. Mutational analyses showed t hat the DNA binding activity of Stat5b is not required, but the carbox yl terminal transactivation domain is essential for Stat5b to inhibit PRL induction of the IRF-1 promoter. These results suggest that Stat5b mediates inhibition via protein protein interactions. In contrast, bo th DNA binding and transactivation domains of Stat5b are required to m ediate PRL induction of the beta-casein promoter. Furthermore, at carb oxyl-terminal truncated dominant negative Stat5b can reverse Stat5b in hibition at the IRF-1 promoter, These studies suggest that Stat protei ns can act as not only positive but also negative regulators of gene t ranscription. Further, Stat5 can modulate gene expression without bind ing to DNA but via protein-protein interactions.