Is phosphorylation of the alpha1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?
The alpha1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo alpha1 subunits. In cells incubated at 37 degrees C, phorbol 12, 13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally ( approximately 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18 degrees C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing alpha1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase alpha1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.
Féraille
E
E
Division de Néphrologie, Hôpital Cantonal Universitaire, CH-1211 Geneva 14, Switzerland. feraille@cmu.unige.ch
Béguin
P
P
Carranza
M L
ML
Gonin
S
S
Rousselot
M
M
Martin
P Y
PY
Favre
H
H
Geering
K
K
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016296:Mutagenesis; D010042:Ouabain / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014162:Transfection
D004791:Enzyme Inhibitors; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; D012694:Serine; D010042:Ouabain; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase