Two hallmarks of malignant tumors are their potential for unlimited growth and metastasis. Among the genes that participate in these complex disease processes are the ras genes, which are mutated in about 25% of all human tumors. The ras proteins are members of a large superfamily of proteins, known as GTPases, which act as molecular switches by cycling between a GTP- bound, active form and a GDP-bound, inactive form. We have chosen the yeast Schizosaccharomyces pombe as our model system since ras signaling in this eukaryote is similar to ras signaling in humans and since yeast affords many experimental advantages. As part of the pheromone response pathway in S. pombe, ras binds the byr2 kinase. ras binding, in combination with an unknown signal, activates the byr2 kinase. The byr2 kinase is the first in a series of kinases, known as a MAP kinase cascade, that sequentially phosphorylate and activate each other. In mammals, ras activates a similar MAP kinase cascade by binding to the raf-1 kinase. Our long-term goal is to understand how ras activates intracellular signaling and how this signaling contributes to disease formation. To achieve this goal, the following specific aims are proposed: 1) To identify the missing molecules in the ras1-dependent activation of a MAP kinase cascade and to understand how they function. Two novel, allele- specific suppressors of ras1 effector mutants were identified and a molecular analysis of these genes will be conducted. The function of these genes in S. pombe and mammals will be explored using genetics and biochemistry. 2) To understand how the byr2 kinase is regulated and how the ras1 protein affects this regulation. Preliminary data suggest there are similarities between ras1 activation of byr2 and cyclin activation of cyclin-dependent kinases. To address this hypothesis, byr2 mutants will be tested using biological and biochemical assays for bry2 activity. 3) To identify additional components of the ras1 signaling pathway. Additional components in the ras1 signaling pathway will be found by screening novel sterile mutants and other suppressors of ras1 effector mutants using epistasis tests. New genes in the ras pathway will characterized as above.
