Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A

Phosphorylation of Bcl2 at serine 70 is required for its potent anti-apopto tic function. We have recently shown that Bcl2 phosphorylation is a dynamic process that involves the protein kinase C alpha and protein phosphatase 2 A (PP2A) (Ruvolo, P. P., Deng, X., Carr, B. K., and May, W. S. (1998) J. Bi ol. 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 apopto tic agent ceramide can activate a PP2A, suggesting that one potential compo nent of the ceramide-induced death signal may involve the inactivation of B cl2. Results indicate that CB-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 cera mide-induced cell death. Using a genetic approach, the gain-of-function S70 E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis e ven with the addition of high doses of ceramide (IC50 > 50 mu M). In contra st, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramid e at dosages where PP2A is fully active and Bcl2 would be expected to be de phosphorylated (IC50 = 14 mu M). These findings indicate that in cells expr essing functional Bcl2, the mechanism of death action for ceramide may invo lve, at least in part, a mitochondrial PP2A that dephosphorylates and inact ivates Bcl2.