Protein Phosphatase 2A Enhances the Proapoptotic Function of Bax through Dephosphorylation

Xingming Deng,Meiguo Xin

doi:10.1074/jbc.m512543200

Abstract

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.

Highlights

BH3 domain of Bax or Bak to block their proapoptotic function
Because Bax is extensively co-expressed with serine/threonine protein Phosphatase 2A (PP2A) in both small cell lung cancer and non-small cell lung cancer cells (Fig. 1A), PP2A may act as a physiological phosphatase to dephosphorylate Bax
Because okadaic acid (OA) is an inhibitor for both PP2A and PP1 [32], these findings suggest that an OA-sensitive phosphatase (PP2A or PP1) may be involved in regulating Bax dephosphorylation

Summary

EXPERIMENTAL PROCEDURES

Materials—Nicotine and cisplatin were purchased from Sigma. Okadaic acid, C2-ceramide, and purified PP2A were obtained from Calbiochem. Cells were incubated with mouse anti-human Bax and rabbit anti-human PP2A/C primary antibodies for 90 min. Subcellular Fractionation—Cells (2 ϫ 107) were washed with cold 1ϫ PBS and resuspended in isotonic mitochondrial buffer (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM Hepes, pH 7.5) containing protease inhibitor mixture set I (Calbiochem), homogenized with a Polytron homogenizer operating for four bursts of 10 s each at a setting of 5, and centrifuged at 2000 ϫ g for 3 min to pellet the nuclei and unbroken cells. The alkali-resistant Bax (i.e. nonextractable or integral) was determined by Western blot using a Bax antibody as described previously [34, 35]. The heavy membrane fraction was lysed, and lysate protein (40 ␮g) was subjected to SDS-PAGE followed by Western blotting with an anti-Bax antibody. The percentage of Annexin-Vlow cells (percentage of viable cells) or Annexin-Vhigh cells (percentage of apoptotic cells) was determined by fluorescence-activated cell sorter analysis as described previously [36]

RESULTS

DISCUSSION

Meiguo Xin and Xingming Deng