title: Cyclic GMP kinase II (cGKII) inhibits NHE3 by altering its trafficking and phosphorylating NHE3 at three required sites: identification of a multifunctional phosphorylation site.
abstract: 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 Ser(554), Ser(607), and Ser(663), equivalent to mouse Ser(552), Ser(605), and Ser(659)), all of which had to be present at the same time for cGMP to inhibit NHE3. NHE3-Ser(663) 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 Ser(554), Ser(607), and Ser(663), all of which are necessary for cGMP/cGKII to inhibit NHE3. 2) Dexamethasone stimulates NHE3 by phosphorylation of a single site, Ser(663). 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.
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Tiane
authors.initials: T
authors.lastname: Chen
authors.affiliation: the Departments of Pediatrics and.
authors.forename: Hetal S
authors.initials: HS
authors.lastname: Kocinsky
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Rakhilya
authors.initials: R
authors.lastname: Murtazina
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: C Ming
authors.initials: CM
authors.lastname: Tse
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Varsha
authors.initials: V
authors.lastname: Singh
authors.affiliation: the Biological Chemistry Department, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Robert
authors.initials: R
authors.lastname: Cole
authors.affiliation: Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, and.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Aronson
authors.affiliation: the GI Division, Erasmus Medical Center, 3015CN Rotterdam, Netherlands.
authors.forename: Hugo
authors.initials: H
authors.lastname: de Jonge
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and.
authors.forename: Rafiquel
authors.initials: R
authors.lastname: Sarker
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, and mdonowit@jhmi.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000378:metabolism; D062967:Cyclic GMP-Dependent Protein Kinase Type II / Q000378:metabolism*; D003907:Dexamethasone / Q000737:chemistry; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008871:Microvilli / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012694:Serine / Q000737:chemistry; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013499:Surface Properties; D014162:Transfection
chemical_list: C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D012694:Serine; D003907:Dexamethasone; D062967:Cyclic GMP-Dependent Protein Kinase Type II
keywords: Cyclic GMP (cGMP); Epithelial Cell; Intestine; Phosphorylation; Sodium-proton Exchange; Trafficking