The objective of this proposal is to study Ras and Cdc42-dependent signal transduction in the fission yeast, Schizosaccharomyces pombe. The fission yeast Ras protein homolog, Ras1, is required for two distinct cellular functions that closely parallel known Ras functions in higher organisms: (1) regulation of mating pheromone-induced MAP kinase cascade and (2) control of cytoskeletal-dependent cellular morphology. Ras1 is linked to the MAP kinase cascade via a direct interaction with the MAP kinase kinase kinase, Byr2. A homolog of the mammalian Cdc42 GTP-binding protein acts downstream of Ras1 in regulating cell morphology, and the two proteins are part of a multiprotein signal transduction complex. In our PRELIMINARY STUDIES, we have demonstrated that Shk1, a homolog of the mammalian p65PAK and Saccharomyces cerevisiae Ste20 protein kinases, is a downstream target of Cdc42 in fission yeast. Our results suggest that Shk1 links the Ras1/Cdc42 signaling complex to an as yet uncharacterized morphology control pathway and participates in the regulation of the Ras1-dependent MAP kinase cascade. Our results, combined with data from others, suggest that signaling modules homologous to the Ras1/Cdc42/Shk1 module are conserved in evolution. We will use the genetically tractable fission yeast as a powerful model system for characterizing the signal transduction pathways regulated by these newly discovered signaling modules. Our primary efforts will focus on characterizing the biological functions and regulation of the Shk1 protein kinase. We will use yeast genetic screens to identify potential regulators and downstream targets of Shk1. Clones isolated from these screens will be characterized by examining their effects on Ras- and/or Cdc42-dependent signaling pathways using well-established yeast, amphibian (Xenopus laevis), and mammalian in vivo and in vitro assay systems. These experiments should provide important information on the functional properties and biological roles of the products encoded by our cloned sequences. We will also use a combination of genetic and biochemical approaches to characterize the functional properties of Shk1 and mechanisms involved in its regulation. Our proposed studies should increase our fundamental understanding of Ras and Cdc42-dependent signal transduction. Mutationally activated Ras oncogenes are among the most prevalent of known human oncogenes. Cdc42-related G proteins have recently been implicated as playing essential roles in Ras-induced transformation of mammalian cells. Our long-term objective is to apply the knowledge obtained from our studies on Ras1/Cdc42/shk1-dependent signal transduction in fission yeast toward gaining an understanding of related signaling pathways in higher organisms.
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