tailieunhanh - Báo cáo Y học: DAP kinase activity is critical for C2-ceramide-induced apoptosis in PC12 cells

Exposure of PC12 cells to C2 -ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identi®ed as a positive mediator of the interferon-c-induced apoptosis ofHeLa cells, in theC2-ceramide-inducedapoptosis of PC12 isendogenouslyexpressedinthesecells. | Eur. J. Biochem. 269 139-147 2002 FEBS 2002 DAP kinase activity is critical for C2-ceramide-induced apoptosis in PC12 cells Mutsuya Yamamoto Takeshi Hioki Takehisa Ishii Sadayo Nakajima-Iijima and Shigeo Uchino Pharmaceuticals Discovery Laboratory Yokohama Research Center Mitsubishi-Tokyo Pharmaceuticals Inc. Aoba-ku Yokohama Japan Exposure of PC12 cells to C2-ceramide results in dosedependent apoptosis. Here we investigate the involvement of death-associated protein DAP kinase initially identified as a positive mediator of the interferon-y-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 M 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 and respectively at 10 min and then decreased to and at 90 min. After 10 min exposure to 30 M C2-ceramide the Ca2 calmodulin independent activity total activity ratio increased from to . These effects were dependent on the C2-ceramide concentration. C8-cera-mide 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. Keywords apoptosis DAP .