CONFORMATION-DEPENDENT PHOSPHORYLATION OF NA,K-ATPASE BY PROTEIN-KINASE-A AND PROTEIN-KINASE-C

Phosphorylation of sodium and potassium ion-activated adenosine tripho sphatase (Na,K-ATPase) by protein kinase A (PKA) and protein kinase C (PKC) was investigated in vitro, where substrate conformation, kinase activity, and consequent effects on Na,K-ATPase activity could be cont rolled. With Na, K-ATPase from rat kidney, optimal stoichiometries wer e close to 1 mol P-32/mol Na,K-ATPase for both kinases. Addition of Na +, K+, P-i, or ouabain is known to stabilize the Na,K-ATPase in differ ent states and was found to affect phosphorylation by the two kinases in a reciprocal way. This indicates that exposure of the phosphorylati on sites varies with conformation and suggests a structural basis for the variable responses to kinase activation in intact cells. Further e vidence for the importance of Na,K-ATPase conformation in its interact ion with kinase came from the autophosphorylation of PKC, which varied in proportion to both the concentration and conformation of rat Na,K- ATPase. With pig and dog Na,K-ATPase, little phosphorylation by PKC wa s detected, and yet the PKC phosphorylated itself when the Na,K-ATPase was in the optimal conformation. The location of the PKA phosphorylat ion site was con firmed to be Ser-938 by sequence analysis of a trypti c peptide. Effects of PRA on Na,K-ATPase activity could not be measure d because of inhibition by the Triton X-100 needed to obtain phosphory lation. Phosphorylation by PKC, even in optimal conditions, failed to result in inhibition of Na,K-ATPase activity. This suggests that any p hysiological role of phosphorylation either entails a subtle modulatio n of enzyme properties, or requires additional regulatory proteins.