Abstract
The regulation of Na-K-ATPase in various tissues is under the control of a number of hormones and peptides that exert both short- and long-term control over its activity. The present study was performed to investigate the effect of chronic insulin treatment on Na-K-ATPase in renal proximal tubular cells. Incubation of opossum kidney (OK) cells, transfected with the rat Na-K-ATPase α(1)-subunit, with 1 nmol/l insulin for 48 h decreased Na-K-ATPase activity. Insulin decreased α(1)-protein content and increased α(1)-serine phosphorylation and α(1)-adaptor protein 2 (AP2) interaction. Removal of the 26 NH(2)-terminal (-NT) amino acid from the α(1)-subunit containing serine/threonine sites abolished the insulin-mediated serine phosphorylation and inhibition of Na-K-ATPase. Substitution of serine 16 and 23 with alanine showed a comparable effect on -NT. Insulin increased the activity of protein kinase C (PKC), which was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. Both PI3K and PKC inhibitors abolished the insulin-mediated inhibition of Na-K-ATPase. Insulin increased the expression of PKC-β(1), -δ, -ξ, and-λ; however, only PKC-ξ/λ-specific inhibitors blocked insulin-induced phosphorylation and inhibition of Na-K-ATPase. Our data demonstrate that insulin activates the atypical PKC isoforms-ξ/λ via the PI3K pathway. PKC-ξ/λ-induced phosphorylation of the α(1)-subunit at serine 16 and 23 leads to AP2 recruitment, degradation, and a decrease in Na-K-ATPase activity.