Evidence that ceramide selectively inhibits protein kinase C-α translocation and modulates bradykinin activation of phospholipase D

Abstract

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-α and PKC-ε to particulate material. Treatment with SMase or C2-Cer specifically blocked the translocation of PKC-α but not that of PKC-ε. 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-α but not PKC-ε and further suggest that PKC-α may be involved in regulating phospholipase D activity.