Is phosphorylation of the alpha 1 subunit at Ser-16 involved in the control of Na,K-ATPase activity by phorbol ester-activated protein kinase C?

The alpha 1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol e ster-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylatio n was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S 16A and S16D-E) ouabain-resistant Bufo alpha 1 subunits. In cells incubated at 37 degrees C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport a ctivity and decreased the cell surface expression of wild-type and mutant N a,K-pumps equally (similar to 20-30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A(2), and cytochro me P450-dependent monooxygenase. In contrast, incubation of cells at 18 deg rees C suppressed the down-regulation of Na,K-pumps and revealed a phosphor ylation-dependent stimulation of the transport activity of Na,K-ATPase. Na, K-ATPase from cells expressing (alpha 1-mutants mimicking Ser-16 phosphoryl ation (S16D or S16E) exhibited an increase in the apparent Na affinity. Thi s finding was confirmed by the PDBu-induced increase in Na sensitivity of t he activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 c ells. These results illustrate the complexity of the regulation of Na,K-ATP ase alpha 1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosph orylation-independent decrease in cell surface expression and 2) a phosphor ylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity.