Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I.
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.
Estrada
E
E
Unité de Biochimie Physiologique, Université Catholique de Louvain, Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium. goffeau@fysa.ucl.ac.be
Agostinis
P
P
Vandenheede
J R
JR
Goris
J
J
Merlevede
W
W
François
J
J
Goffeau
A
A
Ghislain
M
M
D001665:Binding Sites; D047389:Casein Kinase I; D047388:Casein Kinases; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; D047389:Casein Kinase I; D047388:Casein Kinases; C479664:YCK1 protein, S cerevisiae; C479666:YCK2 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D005947:Glucose