title: DAP kinase activity is critical for C(2)-ceramide-induced apoptosis in PC12 cells.
abstract: 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 PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 microm C(2)-ceramide, both the total (assayed in the presence of Ca(2+)/calmodulin) and Ca(2+)/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 microm C(2)-ceramide, the Ca(2+)/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.
authors.affiliation: Pharmaceuticals Discovery Laboratory, Yokohama Research Center, Mitsubishi-Tokyo Pharmaceuticals Inc., Aoba-ku, Yokohama, Japan. mutu@phy.med.kyoto-u.ac.jp
authors.forename: Mutsuya
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hioki
authors.forename: Takehisa
authors.initials: T
authors.lastname: Ishii
authors.forename: Sadayo
authors.initials: S
authors.lastname: Nakajima-Iijima
authors.forename: Shigeo
authors.initials: S
authors.lastname: Uchino
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002518:Ceramides / Q000494:pharmacology*; D064550:Death-Associated Protein Kinases; D004789:Enzyme Activation; D016716:PC12 Cells; D051381:Rats
chemical_list: D051017:Apoptosis Regulatory Proteins; D002518:Ceramides; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium