Cyclic GMP Kinase II (cGKII) Inhibits NHE3 by Altering Its Trafficking and Phosphorylating NHE3 at Three Required Sites IDENTIFICATION OF A MULTIFUNCTIONAL PHOSPHORYLATION SITE

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