Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis.
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.
Fisone
G
G
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Cheng
S X
SX
Nairn
A C
AC
Czernik
A J
AJ
Hemmings
H C
HC
Höög
J O
JO
Bertorello
A M
AM
Kaiser
R
R
Bergman
T
T
Jörnvall
H
H
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010768:Phosphoserine / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017931:DNA Primers; D010455:Peptides; D011994:Recombinant Proteins; D010768:Phosphoserine; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D015056:1-Methyl-3-isobutylxanthine