Regulation and identification of Na,K-ATPase α1 subunit phosphorylation in rat parotid acinar cells

Abstract

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser222, Ser407), three sites (Ser217, Tyr260, Ser 47) previously found from large scale proteomic screens, and two sites (Ser23, Ser16) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser23 and Ser16 and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca2+ ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser23 α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser 23 α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser23 α1 subunit was not modified byERKor PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser23 and Ser16, respectively, the latter because ouabain itself increased Ser16 phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser16 α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.