Our long-term objectives are to understand the conserved cell cycle regulations that control mitotic exit, and regulate centrosome duplication. Both processes are poorly understood, yet are critical for ensuring the viability and genomic stability of dividing cells. Defects in genomic stability are common among cancerous cells. The goals of this proposal are to gain insight to these processes by elucidating the cell cycle functions of MOB1, an essential and conserved the Saccharomyces cerevisiae gene required for mitotic exit and maintenance of ploidy. MOB1 encodes a protein (Mob1p) that interacts with several protein kinases, including Mps1p, a protein kinase required for SPB duplication and mitotic checkpoint regulation, and Dbf2p/Dbf20p, a pair of redundant protein kinases required for mitotic exit. Mob1p is differentially phosphorylated throughout the cell cycle and transiently localizes to spindle pole bodies (SPBs, the functional equivalent of centrosomes) and the cytokinesis ring in late mitosis. Three objectives are proposed to elucidate MOB1's cell cycle function(s). The first objective is to determine the functional relationship between Mob1p and Mps1p, Dbf2p and Dbf20p protein kinases. Experiments are proposed to determine 1) whether Mob1p interacts with each kinase in a cell cycle dependent fashion 2) whether Mob1p functions as substrate or effector of each protein kinase and 3) whether Mob1p regulates the subcellular distribution of each protein kinase. The second objective is to identify the functional domains of Mob1p by assaying the binding properties, subcellular distribution and function of mutant Mob1p. These approaches should reveal the functional significance of Mob1p's interactions and subcellular localizations. The third objective is to determine MOB1 function by identifying additional components in its regulatory pathway(s) via a genetic approach designed to investigate MOB1's role for the maintenance of ploidy and a biochemical approach designed to identify Mob1p binding proteins. Both approaches are unbiased with respect to any model of Mob1p function. Collectively, the proposed approaches will reveal insight to the molecular mechanisms of Mob1p function. Given the conservation of MOB1 among eukaryotes, it is likely that data from these approaches will be relevant for understanding the conserved regulations that are essential for maintaining the genomic stability and viability of dividing cells.
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