The experiments described in this proposal deal with the action on animal cells of extracellular signalling polypeptides through a newly discovered signalling pathway, the JAKSTAT pathway. First uncovered by studying the molecular mechanism of action of interferon alpha and gamma, this pathway is now known to be the direct route by which many different cytokine and growth factors bound to their cognate receptors at the cell surface activate transcription of specific genes in the cell nucleus. This gene activation is usually transient with long lasting effects on cells due to a cascade of events dependent on the first round of gene activation. The specific experiments proposed will advance knowledge of the physical and biochemical properties of the STAT proteins, so-called because they carry out the dual function of signal transduction and activators of transcription. The first two of these proteins to be recognized, Stat1 and Stat2, will be studied intensively to determine specific amino acid contact sites for homo- and heterodimerization, a necessary step in activation, for precise amino acid DNA contact sites and for contact sites with proteins in the transcriptional machinery. Crystallographic analysis of a portion (amino acids 133-712) of Stat1 will be carried out. The mechanism of inactivation of the activated STATs, which occurs within a few minutes to a few hours will be investigated to determine whether nuclear dephosphorylation or protein turnover occurs. Among the most important biologic events concerned with signalling from the cell surface are growth control and differentiation. STAT activation functions in both events and will be studied in both contexts. IFN-alpha and gamma block entry of cells into the S phase and the IFN-alpha and gamma sensitive genes contributing to cell growth restraint will be identified. Finally, genetic and biochemical studies of development is most advanced using Drosophila and we have discovered the first Drosophila Stat molecule that will be studied for its role in developmental pathways. Because many cytokines and growth factors that are crucial in inflammation, immunity, growth regulation and normal development act through this pathway these studies have obvious implication in human disease.
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