Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-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 alpha-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.
College of Pharmacy, University of Houston, Houston, Texas 77204, Department of Molecular Medicine, Karolinska Institutet, S-171 76 Stockholm, Sweden.
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Efendiev
A M
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Bertorello
T A
TA
Pressley
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M
Rousselot
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Féraille
C H
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Pedemonte
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology*; D003078:Colchicine / Q000494:pharmacology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011190:Potassium Cyanide / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D012377:Rodentia; D012413:Rubidium / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D007527:Isoenzymes; D008565:Membrane Proteins; D012694:Serine; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; D011190:Potassium Cyanide; D013755:Tetradecanoylphorbol Acetate; D019297:2,4-Dinitrophenol; D003078:Colchicine