Inhibition of PKB/Akt1 by C2-ceramide involves activation of ceramide-activated protein phosphatase in PC12 cells

Accumulation of ceramide has been reported in stress- and receptor-induced apoptosis in the nervous system. However, its role in apoptosis signaling remains elusive. We describe here the inhibition of the NGF-activated phosphoinositide 3-kinase (PI3K)-PKB/Akt1 survival pathway by the cell permeable analog CS-ceramide. C2-ceramide did not inhibit ERK, PI3K, or PDK1 activities and did not alter the translocation of PDK1 and Akt1 to the plasma membrane, but blocked nuclear translocation of Akt1. Down-regulation of the Akt pathway was due to enhanced dephosphorylation of Akt1 at residues T308 and S473. Moreover, Akt1 was dephosphorylated in vitro by a cation-independent phosphatase involving ceramide-activated protein phosphatase (CAPP). Membrane-anchored Akt1 was more resistant to dephosphorylation/inactivation by CP-ceramide than wild-type Akt1. Consistently, N-myristylated-Akt1 conferred resistance to the apoptosis induced by Ca-ceramide in PC12 cells. These results provide a novel mechanism for induction of apoptosis by ceramide in nerve-derived cells.