9400696 Matthews Protein phosphorylation is a pervasive protein modification in both prokaryotic and eucaryotic cells. A common function of protein phosphorylation is in signal transduction. In prokaryotes, phosphohistidine formation is a key step in a two-component regulatory system. Although signal transduction roles for phosphoserine, phosphothreonine and phosphotyrosine are known in eucaryotes, the phosphohistidine system is poorly characterized. Recently with improved methods for studying phosphohistidine in proteins, it is now clear that histidine kinase and histidine phosphatases both exist in eucaryotic cells. To begin to understand the role of histidine phosphorylation in these cells, we will identify protein substrates for the enzymes. Studies will first determine how much histidine phosphate/protein occurs in cells and how many proteins are involved in this change. Studies will be performed with Saccharomyces cerevisiae cells. Proteins shown to contain phosphohistidine will be purified to homogeneity and their amino acid sequence will be determined. This information will be used to clone the gene(s) for each phosphorylated protein involved. Cloned genes will then be used to determine the copy number and the presence of near homologues in parent yeast cells. Cloned genes may also be used as tools in gene disruption and over- expression experiments to get a better understanding of the substrate requirements and metabolic role of protein/histidine phosphorylation eucaryotic cells. %%% Most cells respond to external signals. For example, bacteria can sense the concentration of nutrients and swim in their direction. Animal cell growth and development is regulated by hormonal signals also from outside the cell. The hormonal signal is passed from the sensor (the molecule in the animal cell that detects the signal) to the receptor (a molecule that responds.) In bacteria, many different signal systems have been described, and one common feature of them all is a reversible change in chemical structure of a regulatory protein that links the sensor to the effector. For example, the change may be the presence, or absence, of a phosphate on a specific histidine in a protein. Similar protein changes also occur in animal/yeast cells but their role is not as well understood. The idea in this study is that a specific change, such as a protein histidine phosphorylation, is involved in animal cell signal transduction, as in bacteria. To test this, the first aim of our research is to determine how much histidine phosphate/protein there is in animal/yeast cells, and how many different proteins are histidine phosphorylated. The histidine phosphorylated proteins will be isolated and cloned. Finally, the cloned genes will provide the basis for molecular genetic studies to determine the function(s) of protein histidine phosphorylation in animal cells. ***
