R. Efendiev et al., Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na+,K+-ATPase molecules to the plasma membrane, BIOCHEM, 39(32), 2000, pp. 9884-9892
Renal sodium homeostasis is a major determinant of blood pressure and is re
gulated by several natriuretic and antinatriuretic hormones. These hormones
, acting through intracellular second messengers, either activate or inhibi
t proximal tubule Na+,K+-ATPase. We have shown previously that phorbol este
r (PMA) stimulation of endogenous PKC leads to activation of Na+,K+-ATPase
in cultured proximal tubule cells (OK cells) expressing the rodent Na+,K+-A
TPase or-subunit. We have now demonstrated that the treatment with PMA lead
s to an increased amount of Na+,K+-ATPase molecules in the plasmalemma, whi
ch is proportional to the increased enzyme activity. Colchicine, dinitrophe
nol, and potassium cyanide prevented the PMA-dependent stimulation of activ
ity 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 trigg
ering mechanism for recruitment of Na+,K+-ATPase molecules to the plasma me
mbrane. 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 al
pha-subunit by stimulation of PKC-beta, and the extent of this phosphorylat
ion was greatly reduced in the S11A and S18A mutants. These results indicat
e that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stim
ulation of Na+,K+-ATPase activity, and that these amino acids are phosphory
lated during this process. The results presented here support the hypothesi
s that PMA regulation of Na+,K+-ATPase is the result of an increased number
of Na+,K+-ATPase molecules in the plasma membrane.