Ceramide and Reactive Oxygen Species Generated by H2O2 Induce Caspase-3-independent Degradation of Akt/Protein Kinase B

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H 2 O 2 -induced cell death. H 2 O 2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H 2 O 2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H 2 O 2 . Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H 2 O 2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H 2 O 2 -induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H 2 O 2 -treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.