Abstract
Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.
Highlights
From the $Laboratory of Molecular and Cellular Neuroscience, the Rockefeller University, New York, New York 10021, the IjDepartment of Pediatrics, St
We show that Ser943is the site phosphorylated by protein kinase A in therat kidney a1 isoform of Na+,K+-ATPase
A 13-amino acid peptide corresponding to amino acid residues 936-948of the rat a1 isoform was found to be a very good substrate for seryl phosphorylation by protein kinase A, lending further support to the notion that Ser943is the phosphorylation site for protein kinase A. In agreementwith these data,two-dimensional phosphopeptide mapping experiments demonstrated that anidentical phosphopeptide is generated by thermolytic cleavage either of Na+,K+-ATPase or of thesynthetic peptide after phosphorylation by protein kinase A
Summary
Thermolytic Phosphopeptide Mapping-Purified Na+,K+ATPase a subunit from shark rectal gland and from rat renal cortex and the synthetic peptide were subjected to phosphoeluate containinghighly acidic material (3-6 min eluate). Phosphoamino acid analysis revealed that radioactivity was subunit from shark rectal gland or from rat renal cortex when associated only with phosphorylation of seryl residues. This the enzyme had been phosphorylated in vitro using protein. Two-dimensionalphosphopeptidemapping of Na+,K+-ATPasea subunit and a subunit peptidee3"w8after phosphorylation by protein kinase k Purified Na',K+-ATPase from shark rectal gland and synthetic peptide were phosphorylated with protein kinasAe and digested with thermolysin (see "Experimental Procedures"). Ainhibitor, H89 [20],abolished the inhibitory effect of forskolin plus IBMX on Na+,K+-ATPaseactivity observed in membranes from wild-type cells (data not shown),supporting theidea that forskolin inhibits Na+,K+-ATPaseactivity in wild-type cells by virtue of stimulating thephosphorylation of Na+,K+-ATPaseon Ser"4"
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