PHOSPHORYLATION OF THE CATALYTIC ALPHA-SUBUNIT CONSTITUTES A TRIGGERING SIGNAL FOR NA-ATPASE ENDOCYTOSIS(,K+)

Inhibition of Na+,K+-ATPase activity by dopamine is an important mecha nism by which renal tubules modulate urine sodium excretion during a h igh salt diet. However, the molecular mechanisms of this regulation ar e not clearly understood. Inhibition of Na+,K+-ATPase activity in resp onse to dopamine is associated with endocytosis of its alpha- and beta -subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from o possum kidney and demonstrate that dopamine-induced endocytosis of Na,K+-ATPase and inhibition of its activity were accompanied by phosphor ylation of the alpha-subunit. Inhibition of both the enzyme activity a nd its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes sugg ests a causal link between phosphorylation and inhibition of enzyme ac tivity. However, after 10 min of dopamine incubation, the alpha-subuni t was no longer phosphorylated, whereas enzyme activity remained inhib ited due to its removal from the plasma membrane. Dephosphorylation oc curred in the late endosomal compartment. To further examine whether p hosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cD NA. Treatment of this cell line with dopamine resulted in phosphorylat ion and endocytosis of the alpha-subunit with a concomitant decrease i n Na+,K+-ATPase activity. In contrast, none of these effects were obse rved in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser(11) and Ser(18)) . Our results support the hypothesis that protein kinase C-dependent p hosphorylation of the alpha-subunit is essential for Na+,K+-ATPase end ocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.