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

Enrique Estrada,Patrizia Agostinis,Jackie R Vandenheede,Jozef Goris,Wilfried Merlevede,Jean François,André Goffeau,Michel Ghislain

doi:10.1074/jbc.271.50.32064

Abstract

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.

Highlights

When glucose is added to starved yeast cells, there is a transient rise in cAMP which induces a protein phosphorylation cascade [1]
Effect of Growth Conditions on the Level of Plasma Membrane Phosphorylation—We examined the phosphorylation pattern of plasma membranes isolated from LRB341 cells that had been incubated in glucose instead of sorbitol before membrane homogenization
We first show that the phosphorylation of PMA1 is severely affected by mutations of both YCK1 and YCK2, two similar and plasma membrane-associated isoforms of CK1 [13, 25]

Summary

Introduction

When glucose is added to starved yeast cells, there is a transient rise in cAMP which induces a protein phosphorylation cascade [1]. Among the many metabolic changes observed within minutes after glucose addition is an increased plasma membrane Hϩ-ATPase activity [2], which is encoded by the essential PMA1 gene [3] This observation initially led to the hypothesis that the cAMP-dependent protein kinase might be involved in regulation of the PMA1 protein during growth in glucose, an hypothesis consistent with the finding that HϩATPase activity increases after cAMP is added to starved Schizosaccharomyces pombe cells [4] or to Saccharomyces cer-. Evisiae mutants defective in cAMP synthesis [5] It is clear, that the regulation of Hϩ-ATPase activity by glucose is still observed in strains that are deficient in cAMP-dependent kinase activity [6], arguing against a direct role of the cAMP-dependent protein kinase in the phosphorylation of PMA1. We show that PMA1 is phosphorylated in vitro at a consensus site for CK1, which is located in the putative MgATP-binding domain of the enzyme

Methods

Results

Conclusion