Simultaneous phosphorylation of Ser11 and Ser18 in the α-subunit promotes the recruitment of Na+,K+-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na+,K+-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na+,K+-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na+,K+-ATPase or-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na+,K+-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na+,K+-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na+,K+-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na+,K+-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na+,K+-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na+,K+-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na+,K+-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na+,K+-ATPase is the result of an increased number of Na+,K+-ATPase molecules in the plasma membrane.