Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A

Phosphorylation of Bcl2 at serine 70 is required for its potent anti- apoptotic function. We have recently shown that Bcl2 phosphorylation is a dynamic process that involves the protein kinase C α and protein phosphatase 2A (PP2A) (Ruvolo, P. P., Deng, X., Carr, B. K., and May, W. S. (1998) J. Biol. Chem. 273, 25436-25442; and Deng, X., Ito, T., Carr, B. K., Mumby, M. C., and May, W. S. (1998) J. Biol. Chem. 273, 34157-34163). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2- dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain- of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC₅₀ > 50 μM). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC₅₀ = 14 μM). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.