Phosphorylation of Yeast Plasma Membrane H+-ATPase by Casein Kinase I

• Published in: The Journal of biological chemistry Vol. 271; no. 50; pp. 32064 - 32072
• Main Authors: Estrada, Enrique, Agostinis, Patrizia, Vandenheede, Jackie R., Goris, Jozef, Merlevede, Wilfried, François, Jean, Goffeau, André, Ghislain, Michel
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.12.1996; American Society for Biochemistry and Molecular Biology
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Binding Sites; Casein Kinase I; Casein Kinases; Cell Membrane; Cell Membrane - enzymology; Chromatography, High Pressure Liquid; enzyme activity; enzymology; ESTRUCTURA CELULAR; FOSFORILACION; GLUCOSA; GLUCOSE; Glucose - pharmacology; HIDROLASAS; Hydrogen-Ion Concentration; HYDROLASE; Kinetics; metabolism; MUTACION; MUTANT; MUTANTES; mutants; MUTATION; pharmacology; PHOSPHORYLATION; plasma membrane; Protein Kinases; Protein Kinases - metabolism; PROTEINA QUINASA; PROTEINE KINASE; Proton-Translocating ATPases; Proton-Translocating ATPases - metabolism; pyrophosphatases; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae Proteins; site-directed mutagenesis; STRUCTURE CELLULAIRE
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.50.32064

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a “down-regulating” casein kinase I activity and another, yet uncharacterized, “up-regulating” kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.