title: Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729.
abstract: Apoptosis is a complex process essential for normal tissue development and cellular homeostasis. While biochemical events that occur late in the apoptotic process are better characterized, early physiological changes that initiate the progression of cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to growth factor withdrawal, experienced a rapid and transient rise in cytosolic pH. We found that the protein responsible was the pH-regulating, plasma membrane protein Na(+)/H(+) exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of the stress-activated kinase, p38 MAP kinase. In the current study, we examined how NHE1 is activated during apoptosis. We identified the phosphorylation sites on NHE1 that regulate its alkalinizing activity in response to a cell death stimulus. Performing targeted mutagenesis, we observed that substitution of Ser726 and Ser729 for alanines produced a mutant form of NHE1 that did not alkalinize in response to an apoptotic stimulus, and expression of which protected cells from serum withdrawal- induced death. In contrast, substitution of Ser726 and Ser729 for glutamic acids raised the basal pH and induced susceptibility to death. Analysis of serine phosphorylation showed that phosphorylation of NHE1 during apoptosis decreased upon mutation of Ser726 and Ser729. Our findings thus confirm a necessary function for NHE1 during apoptosis and reveal the critical regulatory sites that when phosphorylated mediate the alkalinizing activity of NHE1 in the early stages of a cell death response.
authors.affiliation: BioMolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 12722 Research Parkway, Orlando, FL 32826, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Grenier
authors.forename: Khaled
authors.initials: K
authors.lastname: Abu-ihweij
authors.forename: Ge
authors.initials: G
authors.lastname: Zhang
authors.forename: Shannon Moore
authors.initials: SM
authors.lastname: Ruppert
authors.forename: Rebecca
authors.initials: R
authors.lastname: Boohaker
authors.forename: Emily R
authors.initials: ER
authors.lastname: Slepkov
authors.forename: Kathryn
authors.initials: K
authors.lastname: Pridemore
authors.forename: Jian-Jian
authors.initials: JJ
authors.lastname: Ren
authors.forename: Larry
authors.initials: L
authors.lastname: Fliegel
authors.forename: Annette R
authors.initials: AR
authors.lastname: Khaled
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D016466:CHO Cells; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D051381:Rats; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers