Mitogen-activated protein kinases (MAPKs) are a conserve family of protein kinases. These enzymes mediate intracellular phosphorylation events that link receptor activation to the control of cell proliferation and differentiation. Defining the architecture and regulation of these signal pathways is pertinent to understanding events that cause various cancers. This premise is reinforced by the finding that oncogenes such as raf and ras activate these pathways. While an understanding of vertebrate MAPK activation is just beginning to emerge, we know more about analogous pathways in yeast. Separate but structurally related MAPK activation pathways in S. cerevisiae control three distinct physiological responses. The best understood of these is the pheromone induced pathway that simulates cells to differentiate into a mating competent state. The pheromone induced signal is coupled though a G protein to the intracellular components that involves five protein kinases, STE20, STE11, STE7 and a redundant pair of MAPK homologs, FUS3 and KSS1. My objective are to: [1] Reconstitute the STE11-STE7-FUS3 phosphorylation cascade using purified components. [2] Define the molecular basis for pheromone induced stimulation of STE7 and STE11. After physical mapping of phosphorylation sites will be analyzed for effects on signal transduction and enzyme activity. Because the N-terminal negative regulatory domain of STE11 has an inhibitory role,kinase assays with isolated recombinant polypeptides will be used to test a pseudosubstrate inhibition model. Finally, we will use a dosage suppression approach to identity novel components involved in promoting signal transduction. [3] Investigate mechanisms causing desensitization to the pheromone induced signal. We will evaluate whether feed back phosphorylation and a predicted protein tyrosine phosphatase have specific roles in the desenitization response. A genetic screen will be used to identity novel components that promote desensitization. [4] Evaluate parameters that prevent cross interactions of structurally related kinases in different signal pathways. We will examine enzyme- substrate selectivity using phosphorylation assay with STE7 and different yeast MAP-kinase family members. We will generate STE7 mutations in vitro and use genetic selection to identity changes that promote interactions with inappropriate MAPK activation pathways.
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