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 activa te 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 phosp horylation 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 transfecte d into COS cells. In COS cells expressing wild-type Na+-K+-ATPase the beta-adrenergic agonist isoproterenol (1 mu M) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation wa s accompanied by a significant inhibition of the enzyme activity, as r eflected by a decrease in ATP hydrolysis and Rb-86(+) transport. The e ffect of isoproterenol was reproduced by the PKA activator forskolin u sed in combination with the phosphodiesterase inhibitor 3-isobutyl-1-m ethylxanthine and was abolished by the specific PKA inhibitor H-89. Ok adaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced ph osphorylation and inhibition of Na+-K+-ATPase induced by isoproterenol . The changes in activity of Na+-K+-ATPase linearly correlated with th e extent of the alpha-subunit of Na+-K+-ATPase being phosphorylated. W hen Ser-943 was replaced by alanine, stimulation of the phosphorylatio n and inhibition of the activity of Na+-K+-ATPase induced by isoproter enol, alone or in combination with okadaic acid, were not observed. Th ese results indicate that, in intact cells, modulation of the activity of Na+-K+-ATPase can be achieved by regulation of the state of phosph orylation of Ser-943. Moreover, they provide a biochemical mechanism b y which beta-adrenergic agonists can regulate Na+-K+-ATPase activity.