Type 1 serine-threonine protein phosphatase (PP1) is an abundant phosphatase found in all eukaryotes. This phosphatase has many physiological roles, ranging from the regulation of mitosis to the control of glycogen synthesis and has been implicated in the etiology of non- insulin dependent diabetes. The substrate specificity for PP1 is hypothesized to be determined by regulatory subunits. The ultimate goal of this proposal is to determine how these regulatory proteins alter the specificity, the activity, and the physiological roles of PP1. Yeast will be used as a model system for these investigations. The yeast and human enzymes are over 80% identical and are involved in some of the same physiological processes. This proposal addresses the following questions: 1) How do the putative regulatory proteins recognize PP1? The unusually high degree of sequence conservation in PP1 suggests that different regulatory proteins bind to different sites on PP1. This hypothesis will be tested by mutation residues on the surface of PP1 and using the yeast two-hybrid assay and co-association in immune complexes to analyze the effect that these mutation have on the PP1: regulatory protein interactions. 2) How does the interaction between the Gac1p regulatory protein and PP1 effect its activity and substrate specificity? The hypothesis that a yeast glycogen regulatory subunit (Gac1p) alters both activity and specificity of PP1 will be tested by assaying phosphatase activity using specific and non-specific substrates. These studies will allow us to identify the determinants of substrate specificity for PP1, which unlike those for protein kinases, may not reside in the primary sequence surrounding the phosphorylation site. 3) What additional pathways are regulated by PP1? A series of genetics experiments are proposed to identify the physiological process that requires Reg1p and Reg2p, two PP1 binding proteins. A dominant lethal PP1 mutant will be used to screen for dosage and extragenic suppressors that alleviate the growth defect.
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