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Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729 - PubMed

Apoptosis-induced alkalinization by the Na+/H+ exchanger isoform 1 is mediated through phosphorylation of amino acids Ser726 and Ser729

Amy L Grenier et al. Am J Physiol Cell Physiol. 2008 Oct.

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.

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Figures

Fig. 1.
Fig. 1.

Alkalinization is a general response to apoptotic stimuli that activates death factors such as BAX and is prevented by inhibition of Na+/H+ exchanger isoform 1 (NHE1) and p38 MAPK. A: cytosolic pH changes were examined in four cell lines deprived of their respective growth factors. IL-3-dependent FL5.12A pro-B cells, IL-7-dependent D1 pro-T cells, IL-2-dependent CTLL-2 T cells, and NGF-dependent PC-12 cells were cultured in the presence or absence of their respective growth factors for 2–8 h. Inhibition of the cytokine-withdrawal-induced rise in pH was achieved with an NHE1 inhibitor [dimethyl ameloride (DMA); 200 μM] or a p38 MAPK inhibitor (PD-169316, 20 μM). Hours shown at top indicate peak time of transient alkalinization for each cell line. Cytosolic pH was measured with the pH-sensitive probe BCECF-AM. A pH calibration curve was established using the K+ ionophore nigericin and high-K+ buffers at pH 6.8, 7.2, 7.8, and 8.0 (data not shown). BCECF fluorescence was acquired on a FACSAria flow cytometer (BD Biosciences). Actual pH values were determined by correlating the ratio between 525 nm and 610 nm of experimental samples to a pH value on the preestablished calibration curve. Results shown are means ± SD of at least three experiments. B: Western blot analysis of the mitochondrial translocation of BAX in D1 cells in response to IL-7 deprivation. D1 cells were cultured with or without IL-7 for 6 h in the presence or absence of inhibitors of pH-regulatory proteins DMA (NHE1 inhibitor) (200 μM), SITS, and DIDS (inhibitors of anion exchanger) (200 μM) or a caspase inhibitor, zVAD-FMK (1–100 μM). Mitochondrial protein lysates were isolated, and the presence of BAX was determined by immunoblotting. Prohibitin was blotted as a loading control for mitochondrial content. Shown is a representative experiment of three that were performed.

Fig. 2.
Fig. 2.

Four potential p38 MAPK phosphorylation sites on NHE1 are targets for mutagenesis. A: model of NHE1 displaying the cytosolic domain and sites for interaction of known regulatory binding proteins and kinases. Highlighted are the four residues, T718, S723, S726, and S729 (human NHE1 sequence), previously identified as potential sites for phosphorylation (P) by p38 MAPK. Solid arrows indicate target residues that may regulate the alkalinizing activity of NHE1 during apoptosis. B: list of the NHE1 mutants generated for study. Membrane surface expression of NHE1 and NHE1 mutants in AP1 cells stably cotransfected with cDNA for yellow fluorescent protein (YFP) was demonstrated by confocal microscopy. Cells were fixed and stained for hemagglutinin (HA)-tagged NHE1 proteins using an anti-HA antibody followed by an anti-Cy5 secondary antibody. Images shown were acquired at ×100 magnification. Multiple cells were visualized, and representative cells are shown. ERM, ezrin/radixin/moesin; PIP2, phosphatidylinositol 4,5-bisphosphate; CHP, calcineurin homologous protein; CAII, carbonic anhydrase II.

Fig. 3.
Fig. 3.

Mutation of T718, S723, S726, and S729 does not impair NHE1-mediated recovery from acid load. NHE1 activity in response to an acid load (described in

materials and methods

) was assessed in AP1 cells stably transfected with cDNA for YFP or YFP and NHE1 or NHE1-F4 (A), T718A, NHE1-3S, S723A (B), and NHE1-2S, S726A, S729A (C). Recovery of intracellular pH was examined for 0–45 min by detecting pH-sensitive changes in YFP fluorescence by flow cytometry. Representative time points of 0, 15, and 45 min are plotted and are typical of four experiments that were performed.

Fig. 4.
Fig. 4.

NHE1 mediates apoptotic alkalinization. A: calibration curves of pH-sensitive changes in YFP fluorescence for AP1 cells and AP1 cells expressing NHE1 or NHE1-F4 mutant (MUT). To establish a pH calibration curve for YFP fluorescence, cells were resuspended in a high-potassium HEPES buffer (as described in

materials and methods

) at defined pH standards (6.8, 7.2, and 7.8) and supplemented with nigericin. pH-sensitive changes in YFP fluorescence were detected by flow cytometry. Similar calibration curves were established for each of the NHE1 mutant proteins listed in Fig. 2B. B: AP1 cells stably expressing YFP and NHE1 were induced to undergo apoptosis with STS (1 μM), and changes in intracellular pH were detected by flow cytometry. Briefly, cells were treated with STS for 30 min to 1 h, and pH-sensitive changes in YFP fluorescence were detected by flow cytometry as described in

materials and methods

. To confirm that NHE1 was the mediator of alkalinization, cells were pretreated for 2 h with DMA (200 μM) followed by STS treatment. To confirm that p38 MAPK was required for apoptotic alkalinization, cells were pretreated for 2 h with a p38 MAPK inhibitor [PD-169316 (PD), 20 μM] followed by STS treatment. Experimental pH values were determined from the previously established calibration curve as described in A. ΔpH values were derived from difference between the pH of STS-treated cells (pHEXP) and the pH of mock (DMSO) control cells (pHCTRL). The median ΔpH values were determined as described in

materials and methods

. The empirical distribution functions of the median ΔpH values are shown in the figure. P values were calculated by performing permutation tests. For determining significance, the threshold P value was 0.05. These results, along with sample sizes, are summarized in the table. C: AP1 cells stably expressing YFP alone or YFP and NHE1 or NHE1-F4 were induced to undergo apoptosis with STS (1 μM), and changes in intracellular pH were detected by flow cytometry as described in B. Median ΔpH values were determined as described in B. The empirical distribution functions of the median ΔpH values are shown in the figure. Sample sizes, the median ΔpH values, and P values (threshold P value was 0.05), calculated by performing permutation tests, can be found in the table.

Fig. 5.
Fig. 5.

Mutation of S726 and S729 inhibits NHE1-mediated apoptotic alkalinization. AP1 cells stably coexpressing YFP and NHE1 or the NHE1 mutants were induced to undergo apoptosis with STS (1 μM) for 30 min to 1 h, and pH-sensitive changes in YFP fluorescence were detected by flow cytometry. pH values determined from previously established calibration curves and ΔpH values were determined from the difference in the pH of STS-treated cells and mock (DMSO) control cells. A: summary of the median ΔpH and sample sizes of each sample set, displaying the median ΔpH in ascending order. B: comparison of the empirical distribution functions of the median ΔpH for AP1 cells expressing YFP alone and AP1 cells coexpressing YFP and NHE1 or T718A mutant. P values were calculated by performing two permutation tests as described in

materials and methods

and are shown in the table. For determining significance, the threshold P value was 0.05. C: comparison of the empirical distribution functions of the median ΔpH for AP1 cells expressing YFP alone and AP1 cells coexpressing YFP and NHE1 or S723A mutant. P values were calculated by performing two permutation tests as described in

materials and methods

and are shown in the table. For determining significance, the threshold P value was 0.05. D: empirical distribution functions of the median ΔpH for AP1 cells expressing YFP alone in comparison with AP1 cells coexpressing YFP and NHE1-2S, S726A, or S729A mutants. P values were calculated by performing three permutation tests as described in

materials and methods

and are shown in the table. For determining significance, the threshold P value was 0.05. E: empirical distribution functions of the median ΔpH for AP1 cells coexpressing YFP and NHE1 in comparison with AP1 cells coexpressing YFP and NHE1-2S, S726A, or S729A mutants. P values were calculated by performing three permutation tests as described in

materials and methods

and are shown in the table. For determining significance, the threshold P value was 0.05.

Fig. 6.
Fig. 6.

During apoptosis, mutation of S726 and S729 impairs serine phosphorylation of NHE1. A: Western blot shows the serine phosphorylation of NHE1. Analysis of serine phosphorylation was performed by preparing whole cell lysates from AP1 cells stably expressing YFP and NHE1 or NHE1-2S mutant, which were treated with STS (1 μM) for 30 min. Lysates were immunoblotted for serine-phosphorylated proteins and reblotted for HA expression on HA-tagged NHE1 proteins. A molecular weight marker (98-kDa) is indicated. Arrows point to the specific protein bands of the different glycosylated forms of NHE1. Shown are representative results of three experiments performed. WT, wild type. B: Western blot shows results from immunoprecipitation of whole cell lysates from AP1 cells expressing NHE1, S726A, and NHE1-2S muteins for serine-phosphorylated proteins. Phosphoserine-immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotted for NHE1. Densitometry measurements were acquired relative to the signal from AP1 cells. A representative result from three experiments performed is shown. In-blot molecular weight (MW) markers are indicated.

Fig. 7.
Fig. 7.

S726 and S729 on NHE1 regulate cell viability during serum withdrawal-induced death. A: change in pH (ΔpH) for AP1 cells expressing YFP and NHE1, S726A, NHE1-2S, and the constitutively active NHE1-2E was measured by YFP fluorescence detected by flow cytometry. Bar chart summarizes the median ΔpH, baseline pH, and sample sizes of each sample set. B: ratio of dead (fluorescence at 400 nm) versus live cells (fluorescence at 485 nm) was determined using the MultiTox-Fluor cytotoxicity assay (Promega). AP1 cells expressing NHE1, S726A, NHE1-2S, or NHE1-2E were cultured for 24 h with or without serum. Dead and live cells were determined as described in

materials and methods

. Fluorescence was measured with a Synergy 2 plate reader (Biotek). A representative experiment of three performed is shown. C: viability of AP1 cells expressing YFP and NHE1, NHE1-2S, or NHE1-2E was visualized by fluorescence and brightfield microscopy. Images were acquired with a ×40 objective lens.

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References

    1. Ammar YB, Takeda S, Hisamitsu T, Mori H, Wakabayashi S. Crystal structure of CHP2 complexed with NHE1-cytosolic region and an implication for pH regulation. EMBO J 25: 2315–2325, 2006. - PMC - PubMed
    1. Baldursson O, Berger HA, Welsh MJ. Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia. Am J Physiol Lung Cell Mol Physiol 279: L835–L841, 2000. - PubMed
    1. Baumgartner M, Patel H, Barber DL. Na+/H+ exchanger NHE1 as plasma membrane scaffold in the assembly of signaling complexes. Am J Physiol Cell Physiol 287: C844–C850, 2004. - PubMed
    1. Belaud-Rotureau MA, Leducq N, Macouillard PdG, Diolez P, Lacoste L, Lacombe F, Bernard P, Belloc F. Early transitory rise in intracellular pH leads to Bax conformation change during ceramide-induced apoptosis. Apoptosis 5: 551–560, 2000. - PubMed
    1. Bertrand B, Wakabayashi S, Ikeda T, Pouyssegur J, Shigekawa M. The Na+/H+ exchanger isoform 1 (NHE1) is a novel member of the calmodulin-binding proteins. Identification and characterization of calmodulin-binding sites. J Biol Chem 269: 13703–13709, 1994. - PubMed

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