Activation of NHE3 by dexamethasone requires phosphorylation of NHE3 at Ser663 by SGK1

Abstract

Glucocorticoids stimulate Na+ absorption by activation of the epithelial Na+/H+ exchanger NHE3 in the kidney and intestine. It has been thought that glucocorticoid-induced activation of NHE3 is solely dependent on transcriptional induction of the NHE3 gene. While the transcriptional regulation remains an essential part of the chronic effect of glucocorticoids, a previous study by us identified the serum- and glucocorticoid-inducible kinase 1 (SGK1) as an important component of the activation of NHE3 by glucocorticoids. In this work, we have demonstrated phosphorylation of NHE3 by SGK1 as the key mechanism for the stimulation of the transport activity by glucocorticoids. By using in vitro SGK1 kinase assay and site-directed mutagenesis, we have identified Ser663 of NHE3 to be the major site of phosphorylation by SGK1. Ser663 is invariantly conserved in all NHE3 proteins from several species, and the mutation of Ser663 to Ala blocks the effect of dexamethasone, demonstrating the importance of phosphorylation at Ser663. We also show that phosphorylation of NHE3 precedes the changes in NHE3 activity, and the increased activity is associated with an increased amount of NHE3 proteins in the surface membrane. These data reveal that dexamethasone activates NHE3 activity by phosphorylating the NHE3 protein, which initiates trafficking of the protein into the plasma membrane.

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

“…To this end, we used Caco2BBE cells stably transfected with NHE3V to boost the overall NHE3 activity. NHE3 activity in Caco2BBE/NHE3V cells were measured in the presence of 50 M HOE694, which blocked all Na ϩ /H ϩ exchange activity of NHE1 and NHE2 (20). As shown in Fig.…”

“…Surface Biotinylation-Surface biotinylation of NHE3 was performed as previously described (20). Briefly, cells grown in 10-cm Petri dishes were treated with 100 nM thapsigargin, 500 nM ionomycin, or ethanol for 10 min, followed by rinsing twice in PBS and 10 min incubation in borate buffer composed of 154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl 2 , and 10 mM H 3 BO 3 (pH 9.0).…”

“…Densitometric analysis was performed using Scion Image software (National Institutes of Health). Na ϩ -dependent Intracellular pH Recovery-The Na ϩ -dependent changes in intracellular pH (pH i ) by NHE3 was determined with the use of the ratio fluorometric, pH-sensitive dye 2Ј,7Ј-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester (BCECF-AM) as previously described (20,21). Briefly, cells were seeded on coverslips, grown to 70 -80% confluence for PS120 or 5 days post-confluence for Caco2 cells, and then serum starved overnight.…”

“…Photometric data were acquired using the Metafluor software (Molecular Devices). Na ϩ /H ϩ exchange rate was described by the rate of pH i recovery, which was calculated by determining slopes along the pH i recovery by linear least-squares analysis over a minimum of 9 s (20).…”

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

“…Glucocorticoid hormones regulate approximately 70% of basal and 50%-70% of acidosisstimulated ammonia excretion (69,70). Their role appears to involve regulation of SNAT3, PEPCK, and NHE3 (71)(72)(73)(74). In addition, glucocorticoids contribute to acidosis-induced skeletal muscle protein degradation (65), which by contributing to extrarenal glutamine production, enables maintenance of normal plasma glutamine levels (70).…”