This proposal is to study the activity of protein phosphatase 1 (PP1) in budding yeast. Protein phosphatases in antagonism to protein kinases are engaged in regulation of cellular functions on multiple levels. PP1, encoded by Glc7p, is an essential kinase in yeast and deficiencies in its function are accompanied by slowed growth, inability to synthesize glycogen and glucose derepression. Interestingly, there are apparently fewer species of phosphatases and those characterized display lower levels of substrate specificity than typical of kinases. This lack of specificity is apparently compensated by association of kinases like PP1 with regulatory subunits. In the case of PP1, there are several such candidate or known subunits. These include Gac1p and Gac2p, Egp1p, Reg1p and 2p, Glc8p, Shp1p, and Scd5p. Only in the case of Gac1p is the substrate for dephosphorylation known. The Tatchell laboratory showed in the past funding period that Gac1p together with PP1 dephosphorylate glycogen synthetase resulting in its activation. Thus it performs a function similar to its mammalian skeletal muscle homologue RG1. Because the yeast PP1 has a very high degree of amino acid identity with a representative mammalian PP1 for which both the sequence and X-ray structure are available, it represents a useful model for studies focused on the modulation of its activities by a multitude of regulatory subunits. The proposed work will focus on three issues: (I) identification of the domains of interaction between PP1 and its various regulatory subunits and determination of the extent to which they may be overlapping; (II) determination of the mode of action of Gac1p; and (III) performing genetic screens for genes which suppress mutations in REG1 and REG2, which affect the growth rate of cells, in order to identify interacting functions, such as antagonistic kinases or substrates, of the Reg1p/Reg2p PP1 regulatory cascade. The first objective will be accomplished exploiting a set of 23 GLC7 genes containing Ala scanning mutations of known position in the tertiary structure based on comparison with the rabbit protein. A subset of appropriate mutants will be subcloned into two hybrid vectors and tested against various regulatory subunit genes expressed from the other two hybrid vector. Combinations in which interaction is disrupted will be pursued by co-immunoprecipitation of the native proteins from yeast extract. Gaps in Ala mutants on the surface of PP1 will be filled in by construction of additional mutants. Mutations in PP1 which are specific for a particular regulatory subunit will be examined for phenotype and compared to mutations which are null for the regulatory subunit in order to assess the likelyhood that only one subunit interacts at the mutant site. The second goal, investigation of the mode of action of Gac1p has two parts. RG1, the homologue of Gac1p, is itself regulated by phosphorylation and phosphorylation of Gac1p was documented in the previous funding period. The significance of this modification of Gac1p will be examined. Whether Gac1p modifies the activity of PP1 by changing its affinity for glycogen synthase or by changing its Vmax will be determined by immunoprecipitating various mutant forms of Gac1p together with PP1 from cells and measuring activity. The third objective, that of identifying interacting proteins in the PP1-Reg1,2p cascade will be accomplished using yeast genetics to search for mutations which reverse reg slow growth phenotypes and to identify high copy suppressors of a putative dominant negative mutant, glc7-135p.
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