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J. Biol. Chem., Vol. 282, Issue 9, 6292-6299, March 2, 2007

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Mitogen-activated Protein Kinase-dependent Activation of the Na⁺/H⁺ Exchanger Is Mediated through Phosphorylation of Amino Acids Ser⁷⁷⁰ and Ser^771*

Mackenzie E. Malo¹, Liang Li, and Larry Fliegel, Supported by a scientist award from the Alberta Heritage Foundation for Medical Research²

From the Departments of Biochemistry and Chemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

We investigated regulation of the type 1 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 NHE1. 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, Ser⁷⁷⁰, 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 NHE1. Our results suggest that amino acids Ser⁷⁷⁰ and Ser⁷⁷¹ mediate ERK-dependent activation of the Na⁺/H⁺ exchanger in vivo.

Received for publication, December 1, 2006 , and in revised form, January 3, 2007.

^* This work was supported by a grant from the Canadian Institutes of Health Research (to L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Canadian Institutes of Health Research strategic training initiative in membrane proteins and cardiovascular disease.

² To whom correspondence should be addressed. Tel.: 780-492-1848; Fax: 780-492-7751; E-mail: lfliegel{at}ualberta.ca.

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