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

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 Akt_i. 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. Thisdomain (amino acids 475-589) is predicted to be α-helical and contains a conserved cluster of three serines (Ser⁵¹⁵, Ser⁵²², and Ser⁵²⁶). Point mutations of two of these (S515A, S515D, or S526A) reduced basal NHE3 activity and surface expression and had no Akt_i inhibition. S526D had NHE3 activity equal to wild type with normal Akt _i inhibition. Ezrin binding to NHE3 was regulated by Akt, being eliminated by Akt_i. NHE3-S515A and -S526D did not bind ezrin;NHE3-S515D had reduced ezrin binding;NHE3-S526D bound ezrin normally. NHE3-Ser⁵²⁶ 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.