KINETICS OF PHOSPHORYLATION OF NA+/K+-ATPASE BY PROTEIN KINASE-C

The kinetics of phosphorylation of an integral membrane enzyme, Na+ K+-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic α-subunit of Na+ K+-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka ≈ 1.0 μM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 μM). PKC phosphorylation of Na+ K+-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+ K+-ATPase as a substrate was 0.5 μM for dog kidney enzyme and 0.3 μM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+ K+-ATPase greater than 1.0 μM. Phosphorylation of purified kidney and salt-gland Na+ K+-ATPase occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated α subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+ K+-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+ K+-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins. © 1990.