Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C.
Phosphorylation of sodium and potassium ion-activated adenosine triphosphatase (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 controlled. With Na, K-ATPase from rat kidney, optimal stoichiometries were close to 1 mol 32P/mol Na,K-ATPase for both kinases. Addition of Na+, K+, P(i), or ouabain is known to stabilize the Na,K-ATPase in different states and was found to affect phosphorylation by the two kinases in a reciprocal way. This indicates that exposure of the phosphorylation sites varies with conformation and suggests a structural basis for the variable responses to kinase activation in intact cells. Further evidence for the importance of Na,K-ATPase conformation in its interaction 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 was detected, and yet the PKC phosphorylated itself when the Na,K-ATPase was in the optimal conformation. The location of the PKA phosphorylation site was confirmed to be Ser-938 by sequence analysis of a tryptic peptide. Effects of PKA on Na,K-ATPase activity could not be measured because of inhibition by the Triton X-100 needed to obtain phosphorylation. Phosphorylation by PKC, even in optimal conditions, failed to result in inhibition of Na,K-ATPase activity. This suggests that any physiological role of phosphorylation either entails a subtle modulation of enzyme properties, or requires additional regulatory proteins.
Feschenko
M S
MS
Laboratory of Membrane Biology, Massachusetts General Hospital, Charlestown 02129.
Sweadner
K J
KJ
D000818:Animals; D001345:Autoradiography; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D010761:Phosphorus Radioisotopes; D010042:Ouabain; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase