Like human cells, budding yeast (Saccharomyces cerevisiae) cells contain protein kinases that control virtually all aspects of their physiology, morphology and development, especially multi-tiered protein kinase cascades, such as mitogen- / messenger-activated protein kinase (MAPK) pathways and the Target of Rapamycin (TOR) complexes, TORC1 and TORC2, and the protein kinases regulated by them. Moreover, all of the classes of protein kinases that evolved in yeast have been conserved in humans. The yeast mating pheromone response pathway (Fus3 MAPK), initiated by a G-protein-coupled receptor (GPCR), is arguably the best understood MAPK pathway in any eukaryote. Likewise, the existence and discrete functions of TORC1 and TORC2 were first delineated in yeast. However, many basic questions remain about how such protein kinase-based signaling pathways are arranged to maintain specificity, how such pathways are integrated, and how they modulate the processes and behaviors under their control, especially coordination of changes in cell growth, plasma membrane expansion and polarity. The overall goals of this project are to develop novel methodological tools for globally interrogating the logic circuitry of protein kinase action and to use them, and the experimental advantages of yeast, to continue to examine fundamental properties of the organization, fidelity, regulation and function of protein kinase signaling pathways, as a means of undercovering additional new principles and processes generally applicable to the highly homologous pathways in human cells. Signaling mediated by a protein kinase often elicits a complex network of interlocking events, rather than a simple linear output; and, it is not well understood how changes in metabolism, gene expression, and biosynthesis (especially membrane lipid synthesis) are properly coordinated in space and time to achieve appropriate, and sometimes dramatic, changes in cell morphology. Moreover, temporal and spatial aspects of protein kinase-evoked signaling are often imposed by negative feedback mechanisms, or must be integrated with the cell cycle machinery, but our understanding of the mechanisms that modulate the efficiency and duration of signaling events, and avoid adventitious activation of the wrong response, is not fully understood in any organism. To address many of these issues experimentally, our specific aims include: (1) mutational and structural analysis of a-arrestin-GPCR recognition and its control by phosphorylation and genome-wide analysis of the targets of the 14 recognized a-arrestins and their phospho-regulation; (2) global screening, and subsequent genetic and biochemical studies of new substrates of the TORC2-regulated protein kinase Ypk1; (3) assembly, organization and control of TORC2 by stress, especially perturbation of plasma membrane lipid composition, and the role of lipid-anchored Ras2 in TORC2 function; and, (4) genetic and biochemical studies of protein kinase control of flippase function in remodeling of plasma membrane lipids during the cell division cycle and in pheromone- and nutrient limitation-induced polarized growth.
This proposed project has substantial public health relevance because mutations that alter protein kinase signaling pathways are known to be causal in human cancers, inflammatory syndromes and other maladies. Thus, further elucidation of the fundamental aspects of protein kinase signaling may provide new insights for the development of novel and more effective therapies to ameliorate many human diseases.
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