Jones M J, Murray A W
School of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, Australia.
J Biol Chem. 1995 Mar 10;270(10):5007-13. doi: 10.1074/jbc.270.10.5007.
Sphingomyelinase (SMase) treatment (0.1 unit/ml for up to 30 min) of mouse epidermal (HEL-37) or human skin fibroblast (SF 3155) cells preincubated with [3H]serine to label the sphingomyelin pool caused the accumulation of labeled ceramide but not sphingosine or ceramide 1-phosphate. Incubation of HEL-37 cells with dioctanoylglycerol (diC8) or SF 3155 cells with bradykinin caused translocation of calcium/phosphatidylserine-dependent protein kinase C (PKC) activity to particulate material. In both cell lines the translocation was blocked by SMase treatment of the cells or by incubation with the cell-permeable ceramide analogue N-acetylsphingosine (C2-Cer). Western blot analysis indicated that treatment of HEL-37 cells with diC8 or SF 3155 cells with bradykinin resulted in the translocation of both PKC-alpha and PKC-espilon to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-alpha but not that of PKC-epsilon. Pretreatment of cells with SMase or C2-Cer also inhibited the activation of phospholipase D activity induced by either diC8 (HEL-37 cells) or bradykinin (SF 3155 cells). The data provide strong evidence that ceramide can negatively regulate the translocation of PKC-alpha but not PKC-epsilon and further suggest that PKC-alpha may be involved in regulating phospholipase D activity.
用[3H]丝氨酸预孵育小鼠表皮（HEL-37）或人皮肤成纤维细胞（SF 3155）细胞以标记鞘磷脂池，然后用鞘磷脂酶（SMase）处理（0.1单位/毫升，处理30分钟），结果导致标记的神经酰胺积累，但鞘氨醇或神经酰胺1-磷酸未积累。用二辛酰甘油（diC8）孵育HEL-37细胞或用缓激肽孵育SF 3155细胞会导致钙/磷脂酰丝氨酸依赖性蛋白激酶C（PKC）活性转位至颗粒物质。在这两种细胞系中，细胞经SMase处理或与细胞可渗透的神经酰胺类似物N-乙酰鞘氨醇（C2-Cer）孵育后，转位均被阻断。蛋白质免疫印迹分析表明，用diC8处理HEL-37细胞或用缓激肽处理SF 3155细胞会导致PKC-α和PKC-ε均转位至颗粒物质。用SMase或C2-Cer处理可特异性阻断PKC-α的转位，但不能阻断PKC-ε的转位。用SMase或C2-Cer对细胞进行预处理也可抑制由diC8（HEL-37细胞）或缓激肽（SF 3155细胞）诱导的磷脂酶D活性的激活。这些数据提供了强有力的证据，证明神经酰胺可负向调节PKC-α的转位，但不能调节PKC-ε的转位，并且进一步表明PKC-α可能参与调节磷脂酶D活性。
