Phosphorylation of the catalyic α-subunit constitutes a triggering signal for Na⁺,K⁺-ATPase endocytosis

Inhibition of Na⁺,K⁺-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na⁺,K⁺-ATPase activity in response to dopamine is associated with endocytosis of its α- and β-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na⁺,K⁺-ATPase and inhibition of its activity were accompanied by phosphorylation of the α-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the α- subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na⁺,K⁺-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser¹¹ and Ser¹⁸). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na⁺,K⁺-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.