Evidence that ceramide selectively inhibits protein kinase C-alpha translocation and modulates bradykinin activation of phospholipase D.
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.
Jones
M J
MJ
School of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, Australia.
Murray
A W
AW
D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology*; D002460:Cell Line; D002518:Ceramides / Q000502:physiology*; D004789:Enzyme Activation; D004817:Epidermis; D005347:Fibroblasts; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012867:Skin / Q000201:enzymology; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
D002518:Ceramides; D007295:Inositol Phosphates; D007527:Isoenzymes; C064769:N-acetylsphingosine; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase; D010739:Phospholipase D; D013110:Sphingosine; D001920:Bradykinin