Apoptotic alkalinization induced by loss of a growth signal is mediated by phosphorylation of the Na+/H+ Exchanger isoform 1 at Ser726 and Ser729 (35.12)

Abstract

Abstract Apoptosis is a complex process essential for cellular homeostasis. Early physiological changes that initiate cell death remain poorly understood. Previously, we observed that lymphocytes, undergoing apoptosis in response to loss of a growth factor experienced a rapid rise in cytosolic pH. We found that the protein responsible was the Na+/H+ Exchanger isoform 1 (NHE1), and that its activity was impeded by inhibition of p38 MAP kinase (MAPK). 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 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 apoptosis, 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. Serine phosphorylation of NHE1 during apoptosis decreased upon mutation of S726 and S729. 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 cell death.