Cyclic GMP Kinase II (cGKII) Inhibits NHE3 by Altering Its Trafficking and Phosphorylating NHE3 at Three Required Sites

The epithelial brush-border Na+/H+ exchanger NHE3 is acutely inhibited by cGKII/cGMP, but how cGKII inhibits NHE3 is unknown. This study tested the hypothesis that cGMP inhibits NHE3 by phosphorylating it and altering its membrane trafficking. Studies were carried out in PS120/NHERF2 and in Caco-2/Bbe cells overexpressing HA-NHE3 and cGKII, and in mouse ileum. NHE3 activity was measured with 2′,7′-bis(carboxyethyl)-S-(and 6)carboxyfluorescein acetoxy methylester/fluorometry. Surface NHE3 was determined by cell surface biotinylation. Identification of NHE3 phosphorylation sites was by iTRAQ/LC-MS/MS with TiO2 enrichment and immunoblotting with specific anti-phospho-NHE3 antibodies. cGMP/cGKII rapidly inhibited NHE3, which was associated with reduced surface NHE3. cGMP/cGKII increased NHE3 phosphorylation at three sites (rabbit Ser554, Ser607, and Ser663, equivalent to mouse Ser552, Ser605, and Ser659), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser663 phosphorylation was not necessary for cAMP inhibition of NHE3. Dexamethasone (4 h) stimulated wild type NHE3 activity and increased surface expression but failed to stimulate NHE3 activity or increase surface expression when NHE3 was mutated to either S663A or S663D. We conclude that 1) cGMP inhibition of NHE3 is associated with phosphorylation of NHE3 at Ser554, Ser607, and Ser663, all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser663. The requirement for three phosphorylation sites in NHE3 for cGKII inhibition, and for phosphorylation of one of these sites for dexamethasone stimulation of NHE3, is a unique example of regulation by phosphorylation.cGKII acutely inhibits brush-border NHE3, but the mechanism is unknown. cGMP/cGKII phosphorylates NHE3 at three sites. All are necessary for NHE3 inhibition. One of these is also phosphorylated by SGK1 to stimulate NHE3. cGKII inhibits NHE3 by phosphorylating and reducing NHE3 surface amount. Phosphorylation of the same site in a protein can alter function differently based on phosphorylation of additional sites.