Type 1 protein phosphatase participates in a large number physiological processes, ranging from glycogen metabolism to cell cycle control. Specificity is thought to be regulated by subunits that tether the catalytic subunit to the substrate or otherwise regulate its activity. However, recent evidence suggests that some PP1 subunits may also directly alter the catalytic activity of PP1. To fully understand the regulation of PP1 it is necessary to identify its physiological substrates and required targeting/regulatory subunits as well the precise mechanism through which the regulatory subunits exert control. Towards this end, we will attempt to answer the following questions: 1) What are the substrates and regulatory subunits of PP1 that are essential for its cell-cycle specific activities? In yeast and mammals, PP1 exhibits dynamic changes in its location during the cell cycle. The phenotypes of PP1 mutants in yeast, other fungi, and Drosophila indicates that PP1 has cell cycle specific roles. Recent evidence suggests that PP1 may be required for proper attachment of microtubules to kinetochores and may also dephosphorylate histone H3. However, the mechanism of regulation and relevant targeting subunits have not been identified. We will use genetic approaches to investigate the cell cycle-specific functions of yeast PP1 with the goal of identifying critical substrates and regulatory subunits. 2) What is the consequence of tethering PP1 to the bud neck? Yeast PP1 is tethered to the bud neck via Bni4p, a scaffold protein that also binds to septins and a regulatory subunit of chitin synthase Ill. We propose that PP1 negatively regulates the activity of chitin synthase. To test this hypothesis we will assay the sub-cellular location, phosphorylation state, and activity of chitin synthase in PP1 mutants that are not tethered to the bud neck and in BNI4 mutants that are specifically defective binding PP1. 3) Do regulatory/targeting subunits directly alter the activity of PP1, independent of any targeting role? Recent evidence suggests that the glycogen-specific targeting subunit of yeast PP1, Gac1 p, directly alters the activity of the phosphatase, independent of any target role. To test this hypothesis we will assay activity of wild type and mutant phosphatases using the bona fide substrate, glycogen synthase in the presence and absence of Gac1p.
Showing the most recent 10 out of 19 publications
