DAP kinase activity is critical for C2‐ceramide‐induced apoptosis in PC12 cells

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Abstract
Exposure of PC12 cells to C2‐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‐γ‐induced apoptosis of HeLa cells, in the C2‐ceramide‐induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 µm C2‐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 µm C2‐ceramide, the Ca2+/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C2‐ceramide concentration. C8‐ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C2‐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 C2‐ and C8‐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 C2‐, and C8‐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.