DAP kinase activity is critical for C-2-ceramide-induced apoptosis in PC12 cells

Exposure of PC12 cells to C-2-ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-gamma-induced apoptosis of HeLa cells, in the C-2-ceramide-induced apoptosis of PC 12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC 12 cells to 30 muM C-2-ceramide, both the total (assayed in the presence of Ca2+/calmodulin) and Ca2+/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 mum C-2-ceramide, the Ca2+/calmodulin independent activity/total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C-2-ceramide concentration. C-8-ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C-2-dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C-2- and C-8-ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C-2-, and C-8-ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis.