ABSTRACT
The budding yeast Glc7 serine/threonine protein phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian phosphatase inhibitor-2. In this work, we demonstrated that similarly to inhibitor-2, Glc8 function is regulated by phosphorylation. The cyclin-dependent protein kinase, Pho85, in conjunction with the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase in vivo and in vitro. Several glc7 mutations are dependent on the presence of Glc8 for viability. For example, glc7 alleles R121K, R142H, and R198D are lethal in combination with a glc8 deletion. We found that glc7-R121K is lethal in combination with a pho85 deletion. This finding indicates that Pho85 is the sole Glc8 kinase in vivo. Furthermore, glc7-R121K is also lethal when combined with deletions of pcl6, plc7, pcl8, and pcl10, indicating that these related cyclins redundantly activate Pho85 for Glc8 phosphorylation in vivo. In vitro kinase assays and genetic results indicate that Pho85 cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Enzyme Inhibitors , Fungal Proteins/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinases/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genes, Fungal , Mutation , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Phosphatase 1 , Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.
