Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771

We investigated regulation of the type I isoform of the Na+/H+ exchanger by phosphorylation. Four specific groups of serine and threonine residues in the regulatory carboxyl-terminal tail were mutated to alanine residues: group 1, S693A; group 2, T718A and S723A/S726A/S729A; group 3, S766A/S770A/ S771A; and group 4, T779A and S785A. The proteins were expressed in Na+/H+ exchanger-deficient cells, and the activity was characterized. All of the mutants had proper expression, localization, and normal basal activity relative to wild type NHEI. Sustained intracellular acidosis was used to activate NHE1 via an ERK-dependent pathway that could be blocked with the MEK inhibitor U0126. Immunoprecipitation of 32p-labeled Na+/H+ exchanger from intact cells showed that sustained intracellular acidosis increased Na+/H+ exchanger phosphorylation in vivo. This was blocked by U0126. The Na+/H+ exchanger activity of mutants 1 and 2 was stimulated similar to wild type Na+/H+ exchanger. Mutant 4 showed a partially reduced level of activation. However, mutant 3 was not stimulated by sustained intracellular acidosis, and loss of stimulation of activity correlated to a loss of sustained acidosis-mediated phosphorylation in vivo. Mutation of the individual amino acids within mutant 3, Ser766, Ser171, and Ser771, showed that Ser770 and Ser771 are responsible for mediating increases in NHE1 activity through sustained acidosis. Both intact Ser770 and Ser771 were required for sustained acidosis-mediated activation of NHEL Our results suggest that amino acids Ser770 and Ser771 mediate ERK-dependent activation of the Na+/H+ exchanger in vivo.