The mating pheromone response pathway of budding yeast, Saccharomyces cerevisiae, is arguably the best understood multi-tiered, mitogen- activated protein kinase (MAPK)-stimulating signaling cascade yet eludicated in any eukaryotic cell. It is now clear, however, that to coordinate the changes in gene expression and cell morphology necessary for mating, response to pheromone requires an elaborate network of interlocking events rather than a simple linear pathway. In addition, feedback mechanisms exist that modulate the efficiency and duration of the events required for signaling at essentially every step. Moreover, this signaling pathway must evoke an appropriate response upon the correct stimulus, yet avoid adventitious activation under inappropriate circumstances. In this regard, it has been shown recently that many of the same components required for pheromone response are also utilized for a different developmental outcome, termed filamentous or invasive growth, that occurs in response to nutrient limitation. How different extracellular signals impinge on the same MAPK cascade, yet are deciphered differently, is not fully understood in any organism. Thus, yeast continues to provide an opportunity to examine fundamental aspects of the organization, specificity, fidelity, and regulation of signal transmission, including how the same signaling components can be coupled to different upstream inputs and downstream responses in the same cell type.
Specific aims i nclude: further eludication of the specificity of MEK-MAPK recognition; genetic and biochemical analysis of the regulatory function of the RING-H2 domains of the scaffold proteins, Ste5 and Far1, and their interaction with Gbetagamma (Ste4-Ste18), including development of a new, Gbetagamma-based method for identification of coiled-coil interactions applicable to functional genomics; determination of the crystal structure of the various domains of Ste5 and the mechanism of its regulated nuclear import and export; elucidation of the function and solution structure of Ste50, whose role is as yet ill-defined; biochemical characterization of the novel transcriptional regulators, Dig1 and Dig2, and their function in down- modulation and promoter-specific discrimination in Ste12-dependent gene expression; and, implementation of genetic screens to pinpoint as yet unidentified components unique to the pheromone-response and filamentous-growth signaling pathways. Knowledge gained by studying MAPK signaling in yeast may provide insights ultimately of clinical value for developing therapies against cancers because inappropriate MAPK activation, such as that evoked by well-known oncoproteins (including Ras and Raf), leads to tumor formation.
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