engleski

cAMP-induced phosphorylation and inhibition of Na(+)/H(+) exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor

The members of the regulatory factor (RF) gene family, Na+/H+ exchanger (NHE)-RF and NHE3 kinase A regulatory factor (E3KARP) are necessary for cAMP to inhibit the epithelial brush border NHE isoform 3 (NRE3). The mechanism of their action was studied using PS120 fibroblasts stably transfected with rabbit NHE3 and wild type rabbit NHE-RF or wild type human E3KARP, 8-Bromo-cAMP (8-Br-cAMP) had no effect on Na+/H+ exchange activity in cells expressing NHE3 alone. In contrast, in cells co-expressing NHE-RF, 8-Br-cAMP inhibited NHE3 by 39%. In vivo phosphorylation of NHE3 demonstrated that cAMP increased phosphorylation in two chymotrypsin-generated phosphopeptides of NHE3 in cells containing NHE-RF or E3KARP but not in cells lacking these proteins. The requirement for phosphorylation of NHE-RF in this cAMP-induced inhibition of NHE3 was examined by studying a mutant NHE-RF in which serines 287, 289, and 290 were mutated to alanines, Wild type NHE-RF was a phosphorylated protein under basal conditions, but treatment with 8-Br-cAMP did not alter its phosphorylation, Mutant NHE-RF was not phosphorylated either under basal conditions or after 8-Br-cAMP, 8-Br-cAMP inhibited NHE3 similarly in PS120/NHE3 cells containing wild type or mutant NHE-RF, NHE-RF and NHE3 co-precipitated and did so similarly with and without cAMP. Mutant NHE-RF also similarly immunoprecipitated NHE3 in the presence and absence of 8-Br-cAMP, This study shows that members of the regulatory factor gene family, NHE-RF and E3KARP, are necessary for cAMP inhibition of NHE3 by allowing NHE3 to be phosphorylated, This inhibition is not dependent on the phosphorylation of NHE-RF.

protein-kinase-a; H+ exchanger; mediated inhibition; identification; phosphoprotein; cofactor; isoform; domain; ebp50; ezrin

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