Regulation of Stat1 transcription factor activity by phosphorylation of serine 727: Studies addressing the relevant kinase(s) and the implications of their activity in macrophages

The most prominent function of the T cell-derived cytokine IFN-gamma during an immune response is to activate macrophages for increased immunological performance, for example the killing of ingested microorganisms. The transcription factor Stat1 is instrumental in gene induction in response to Interferon-gamma (IFN- gamma). Consistent with its role in the IFN- gamma response of macrophages, targeted disruption of the Stat1 gene in mice results in severely compromised innate immunity against bacteria. Stat1 is a latent cytoplasmatic protein which becomes activated in response to signalling from the IFN- gamma receptor. Avtivation occurs in two distinct steps. Initially the protein is phosphorylated on a single tyrosine residue by Janus kinases (Jaks). This causes dimerization of Stat1, ist translocation to the cell nucleus and DNA binding. Activity as a transcription factor requires a second phosphorylation event which adds a phosphate to serine 727 (S727) in the carboxy terminus of the protein. Therefore, the protein serine kinase(s) phosphorylating S727 are thought to make an essential contribution to Stat1 function during IFN- gamma -mediated macrophage activation. The nature of these kinases is currently unknown. We will address the question which kinase(s) phosphorylate Stat1 S727. Our previous results strongly suggest that at least one of these kinases is among the stress-activated kinases and that the stress-activated Stat1 serine kinase most likely differs from that activated by IFN- gamma. Based on these results, we will start by testing candidate kinases among stress-activated kinases for their ability to phosphorylate Stat1. The ultimate goal will be to identify a Stat1 kinase activated by cellular stress signals and to clarify ist reationship to the serine kinase activated by IFN- gamma. A second important goal is to investigate the relevance of Stat1 S727 phosphorylation in macrophages. We would like to know whether an absence of S727 phosphorylation results in a defective IFN- gamma response, or whether this has less effect on macrophage activation than the complete absence of the protein. To this end, we will reconstitute Stat1 -deficient macrophages with mutants of Stat1 that allow to judge the importance of the phosphorylation event in the cell`s different biological functions. Finally, we will attempt to reconstitute Stat1 - deficient mice with the phosphorylation mutant of Stat1. Phenotypic analysis of such animals will complement the studies with isolated macrophages and reveal the importance of S727 phosphorylation in responses of the entire immune system. The overall aim of our studies is to identify a Stat1 kinase and assess its relative contribution to antimicrobial immune responses and to the process of inflammation.