Kinetics of phosphorylation of [formula omitted] by protein kinase C

Abstract

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 ( K a ≈ 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 K m 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.