Yamamoto Mutsuya, Hioki Takeshi, Ishii Takehisa, Nakajima-Iijima Sadayo, Uchino Shigeo
Pharmaceuticals Discovery Laboratory, Yokohama Research Center, Mitsubishi-Tokyo Pharmaceuticals Inc., Aoba-ku, Yokohama, Japan.
Eur J Biochem. 2002 Jan;269(1):139-47. doi: 10.1046/j.0014-2956.2002.00029.x.
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.
将PC12细胞暴露于C(2)-神经酰胺会导致剂量依赖性凋亡。在此，我们研究死亡相关蛋白（DAP）激酶（最初被鉴定为干扰素-γ诱导的HeLa细胞凋亡的正向调节因子）在C(2)-神经酰胺诱导的PC12细胞凋亡中的作用。DAP激酶在这些细胞中内源性表达。将PC12细胞暴露于30微摩尔的C(2)-神经酰胺后，在10分钟时，总DAP激酶活性（在Ca(2+)/钙调蛋白存在下测定）和Ca(2+)/钙调蛋白非依赖性DAP激酶活性（在EGTA存在下测定）分别瞬时增加了5.0倍和12.2倍，然后在90分钟时降至1.7倍和3.4倍。暴露于30微摩尔的C(2)-神经酰胺10分钟后，Ca(2+)/钙调蛋白非依赖性活性与总活性的比值从0.22增加到0.60。这些效应取决于C(2)-神经酰胺的浓度。另一种活性神经酰胺类似物C(8)-神经酰胺也诱导凋亡并激活DAP激酶，而无活性的神经酰胺类似物C(2)-二氢神经酰胺则未能诱导凋亡和增加DAP激酶活性。此外，转染研究表明，野生型DAP激酶的过表达增强了对C(2)-和C(8)-神经酰胺的敏感性，而催化无活性的DAP激酶突变体以及含有DAP激酶死亡结构域和C末端尾巴的构建体（以显性负性方式起作用）可使细胞免受C(2)-和C(8)-神经酰胺诱导的凋亡。这些发现表明，DAP激酶是参与神经酰胺诱导凋亡的凋亡机制的重要组成部分，并且内在的DAP激酶活性对于神经酰胺诱导的凋亡至关重要。
