title: AKT and GSK-3 are necessary for direct ezrin binding to NHE3 as part of a C-terminal stimulatory complex: role of a novel Ser-rich NHE3 C-terminal motif in NHE3 activity and trafficking.
abstract: Basal activity of the BB Na(+)/H(+) exchanger NHE3 requires multiprotein complexes that form on its C terminus. One complex stimulates basal NHE3 activity and contains ezrin and phosphoinositides as major components; how it stimulates NHE3 activity is not known. This study tested the hypothesis that ezrin dynamically associates with this complex, which sets ezrin binding. NHE3 activity was reduced by an Akti. This effect was eliminated if ezrin binding to NHE3 was inhibited by a point mutant. Recombinant AKT phosphorylated NHE3 C terminus in the domain ezrin directly binds. This domain (amino acids 475-589) is predicted to be α-helical and contains a conserved cluster of three serines (Ser(515), Ser(522), and Ser(526)). Point mutations of two of these (S515A, S515D, or S526A) reduced basal NHE3 activity and surface expression and had no Akti inhibition. S526D had NHE3 activity equal to wild type with normal Akti inhibition. Ezrin binding to NHE3 was regulated by Akt, being eliminated by Akti. NHE3-S515A and -S526D did not bind ezrin; NHE3-S515D had reduced ezrin binding; NHE3-S526D bound ezrin normally. NHE3-Ser(526) is predicted to be a GSK-3 kinase phosphorylation site. A GSK-3 inhibitor reduced basal NHE3 activity as well as ezrin-NHE3 binding, and this effect was eliminated in NHE3-S526A and -S526D mutants. The conclusions were: 1) NHE3 basal activity is regulated by a signaling complex that is controlled by sequential effects of two kinases, Akt and GSK-3, which act on a Ser cluster in the same NHE3 C-terminal domain that binds ezrin; and 2) these kinases regulate the dynamic association of ezrin with NHE3 to affect basal NHE3 activity.
authors.affiliation: From the Departments of Physiology and Medicine, Gastroenterology Division, Johns, Hopkins University School of Medicine, Baltimore, Maryland 21205 and.
authors.forename: Varsha
authors.initials: V
authors.lastname: Singh
authors.forename: Rong
authors.initials: R
authors.lastname: Lin
authors.forename: Jianbo
authors.initials: J
authors.lastname: Yang
authors.forename: Boyoung
authors.initials: B
authors.lastname: Cha
authors.forename: Rafiquel
authors.initials: R
authors.lastname: Sarker
authors.forename: Chung Ming
authors.initials: CM
authors.lastname: Tse
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D000099149:Ezrin
chemical_list: D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D051057:Proto-Oncogene Proteins c-akt; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C000613385:SLC9A3 protein, human
keywords: Absorption; Akt; Alpha Helix; Ezrin; Membrane; Na+/H+ Exchanger; Phosphorylation; Trafficking