Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules

We have previously shown that, in oxygenated rat kidney proximal convoluted tubules (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) directly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. PKC modulation of Na(+)-K(+)-ATPase activity by phosphorylation of its alpha-subunit was the postulated mechanism. The present study was therefore designed to investigate the relationship between PKC-mediated phosphorylation of the catalytic alpha-subunit and the cation transport activity of the Na(+)-K(+)-ATPase. In a suspension of rat kidney cortical tubules, activation of PKC by 10(-7) M PDBu increased the level of phosphorylation of the Na(+)-K(+)-ATPase alpha-subunit and stimulated the ouabain-sensitive 86Rb uptake by 47 and 42%, respectively. Time and dose dependence of the PDBu-induced increase in Na(+)-K(+)-ATPase activity and phosphorylation was strongly linearly correlated. The effects of PDBu on phosphorylation and activity of Na(+)-K(+)-ATPase were prevented by GF-109203X, a specific PKC inhibitor, whereas H-89, a specific PKA inhibitor, was ineffective. These results demonstrate that PKC activation induces phosphorylation of the catalytic alpha-subunit of Na(+)-K(+)-ATPase, which may participate in the stimulation of its cation transport activity in the rat PCT.