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.

Role of the Anion Exchanger 2 in the Pathogenesis and Treatment of Primary Biliary Cirrhosis

“…Defining the factor(s) that may trigger apoptosis, oxidative stress and autophagy in those biliary epithelial cells remains a major challenge. Previous reports on the association of oxidative stress, apoptosis-like cell death, and changes in the intracellular pH (pH i ) [67,68,69] may provide some clues.…”

Regulation of the Na+/H+ Exchanger (NHE1) in Breast Cancer Metastasis

“…In vascular smooth muscle cells, angiotensin II stimulates NHE1 by ERK-dependent p38MAPK phosphorylation and, in pro-B cells, NHE1-mediated apoptosis is regulated by phosphorylation at amino acids T 718 , S 723 , S 726 , and S 729 (21). Interestingly, the mutation of S 726/729 to nonphosphorylatable alanine is protective against serum deprivation-induced cell death (22). In myocardial cells, Erk 1/2-dependent pathways are critical in the regulation of NHE1; Erk 1/2 phosphorylates NHE1 at S 770 and S 771 , while p90 rsk , a kinase downstream of Erk 1/2, phosphorylates NHE1 at S 703 and, by doing so, forms a 14-3-3 ligand binding site (18).…”

The major isoforms of Bim contribute to distinct biological activities that govern the processes of autophagy and apoptosis in interleukin-7 dependent lymphocytes

“…3B). While we previously showed that D1 cells transiently alkalinize 6–8 hours after IL-7 withdrawal due to the activity of the sodium hydrogen exchanger 1 (NHE1) [16], this is an active mechanism induced by apoptotic stimulus [45]. In contrast, the increased intracellular pH of SMoR cells was detectable in the presence of IL-7 and did not change even during IL-7 withdrawal.…”

“…Intracellular pH-dependent changes in fluorescence were measured by flow cytometry, using methods previously described [45,46]. To establish a pH calibration curve, a separate group of cells (D1, SMoR, primary C57BL/6 and BimKO) were re-suspended in high-potassium HEPES buffer (25 mM HEPES, 145 mM KCl, 0.8 mM MgCl2, 5.5 mM glucose, and DDH2O) at pH standards (5.9, 6.5, 7.0, 7.2, and 7.8) and nigericin (10 μM) added followed by BCECF-AM.…”