PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone.
The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.
Cheng
X J
XJ
Department of Woman and Child Health, St. Göran's Children's Hospital, Karolinska Institute, Stockholm, Sweden.
Fisone
G
G
Aizman
O
O
Aizman
R
R
Levenson
R
R
Greengard
P
P
Aperia
A
A
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000409:Alanine; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012694:Serine; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018411:Spodoptera; D013449:Sulfonamides; D014162:Transfection
D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013449:Sulfonamides; D005576:Colforsin; D019319:Okadaic Acid; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase; D007545:Isoproterenol; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D012413:Rubidium; D000409:Alanine; D015056:1-Methyl-3-isobutylxanthine