Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

Abstract

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…The striking point in all these studies is that the intracellular alkalinization seems to be an early event in the apoptotic cascade, occurring prior to caspase activation and DNA fragmentation. Furthermore, when tested, this phenomenon appears to be only transient, followed 56,60 or not 54,59 by an intracellular acidification (i.e. reaching pH i values below control levels).…”

“…Such a pH i change has been observed in cells undergoing situations as various as cytokine deprivation, 54 gamma radiation, 55 ceramide application 56 and exposure to xenobiotics, such as drugs targeting proteasome, 57 staurosporine, 58 potassium antimonyl tartrate (PAT) 59 or polycyclic aromatic hydrocarbons (PAHs). 60 Therefore, it appears that such a pH i change might represent a more generalized apoptotic feature and should be looked for in a more systematic way.…”

“…35 Besides endonucleases, the proapoptotic protein Bax appears to be another possible candidate for mediating the action of transient alkalinization. Indeed, Khaled et al 54 have shown that upon pH elevation this protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the apoptotic cascade. Such a relationship between changes in Bax conformation and pH i has also been reported by others, 56 this being reinforced by the fact that clamping pH to an acidic value was shown to inhibit such conformational Apoptosis-associated intracellular alkalinization Figure 7 Putative targets of early intracellular alkalinization associated with apoptosis.…”

“…Such a relationship between changes in Bax conformation and pH i has also been reported by others, 56 this being reinforced by the fact that clamping pH to an acidic value was shown to inhibit such conformational Apoptosis-associated intracellular alkalinization Figure 7 Putative targets of early intracellular alkalinization associated with apoptosis. Whereas endonucleases [63][64][65] and some of the Bcl-2 family proteins 54,56 have been identified to be sensitive to apoptosis-associated early intracellular alkalinization, the other illustrated targets remain to be tested. MAPKs: mitogen-activated protein kinases; ROS: reactive oxygen species changes in Bax.…”

Bax activation and mitochondrial insertion during apoptosis

“…These studies report physiologically relevant events, either a sub-lethal apoptotic stimulus or a change in the protein environment [10,12], namely a subtle pH modification, previously described to occur during the induction of apoptosis [26]. Both studies suggest that these conditions can result in the exposure of some epitopes without the release of mitochondrial cytochrome c and cell death.…”