We plan to investigate two signal transduction systems that regulate initiation of the cell cycle in the yeast Saccharomyces, both of which are homologous to signal transduction pathways in mammalian cells. The first system is mediated by Ras proteins, which are both structurally and functionally homologous to human ras proteins, and provides positive activation of cell cycle initiation. The second system, mediated by a novel lipid second messenger, ceramide, shows striking parallels to the pathway by which a variety of mammalian growth modulators - including TNFalpha and gamma-interferon - regulate cell proliferation. We have shown that activation of this pathway inhibits initiation of cell cycle progression in yeast, similar to the response of mammalian cells to activation of this pathway. Thus, the two pathways appear to work in opposition in both yeast and mammalian cells to provide balanced cell growth. For both signal transduction systems we plan to investigate the nature of the interactions of the components of the pathways, the mechanism by which signals impinge on each pathway, and the downstream targets that are responsible for the physiological consequences of pathway activation. For the Ras pathway, we will focus on the function and activation of Cdc25p, the Ras guanine nucleotide exchange factor, which appears to mediate input into the pathway. In addition, we will explore the role of the Ras pathway in protein secretion and in possible interactions between the Ras pathway and cytoskeletal organization of the cell. For ceramide signal transduction, we have shown that ceramide-induced inhibition of yeast cell proliferation is mediated by a ceramide activated protein phosphatase, which we have characterized. Yeast also contain a ceramide activated protein kinase quite similar to that found in mammalian cells. We propose a genetic analysis to determine (1) the role of the kinase in growth control, (2) the nature of the signal into the pathway and (3) the critical targets of the ceramide activated phosphatase and kinase. The proposed dissection of these pathways should provide valuable insight into the mechanism by which mammalian cells limit their proliferation, the lessons from which should have profound implications in understanding and treating cancer.
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