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

Department of Anatomy and Cell Biology, University of Florida, Gainesville, Florida, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2006; 281(27):18859-67. DOI: 10.1074/jbc.M512543200
Source: PubMed

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.

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    • "Activation by ceramides of serine/threonine protein phosphatase PP2A is involved in regulation of the apoptotic/anti-apoptotic activity of Bcl-2 family proteins by changing their phosphorylation status. Ceramide-activated PP2A increases the pro-apoptotic potential of Bcl-2 family proteins by dephosphorylation of Bax (activation) (Xin and Deng, 2006), or Bcl-2 (inactivation) (Ruvolo et al., 1999). An additional substrate for PP2A is serine/threonine kinase Akt/PKB (Pettus et al., 2002). "
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    • "Activation by ceramides of serine/threonine protein phosphatase PP2A is involved in regulation of the apoptotic/anti-apoptotic activity of Bcl-2 family proteins by changing their phosphorylation status. Ceramide-activated PP2A increases the pro-apoptotic potential of Bcl-2 family proteins by dephosphorylation of Bax (activation) (Xin and Deng, 2006), or Bcl-2 (inactivation) (Ruvolo et al., 1999). An additional substrate for PP2A is serine/threonine kinase Akt/PKB (Pettus et al., 2002). "
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    • "The Ku protein mainly localizes and functions in nucleus [30]. In contrast, PP2A can be localized in both cytoplasm and nucleus [11] [31]. A previous report has demonstrated that CPT-induced DSB signal promotes PP2A accumulation in the nucleus [11], suggesting a potential mechanism by which PP2A regulates DSB repair. "
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    ABSTRACT: Protein phosphatase 2A (PP2A) functions as a potent tumor suppressor, but its mechanism(s) remains enigmatic. Specific disruption of PP2A by either expression of SV40 small tumor antigen or depletion of endogenous PP2A/C by RNA interference inhibits Ku DNA binding and DNA-PK activities, which results in suppression of DNA double-strand break (DSB) repair and DNA end-joining in association with increased genetic instability (i.e., chromosomal and chromatid breaks). Overexpression of the PP2A catalytic subunit (PP2A/C) enhances Ku and DNA-PK activities with accelerated DSB repair. Camptothecin-induced DSBs promote PP2A to associate with Ku 70 and Ku 86. PP2A directly dephosphorylates Ku as well as the DNA-PK catalytic subunit (DNA-PKcs) in vitro and in vivo, which enhances the formation of a functional Ku/DNA-PKcs complex. Intriguingly, PP2A promotes DSB repair in wild type mouse embryonic fibroblast (MEF) cells but has no such effect in Ku-deficient MEF cells, suggesting that the Ku 70/86 heterodimer is required for PP2A promotion of DSB repair. Thus, PP2A promotion of DSB repair may occur in a novel mechanism by activating the nonhomologous end-joining pathway through direct dephosphorylation of Ku and DNA-PKcs, which may contribute to maintenance of genetic stability.
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