Evidence That Ceramide Selectively Inhibits Protein Kinase C- Translocation and Modulates Bradykinin Activation of Phospholipase D

doi:10.1074/jbc.270.10.5007

Evidence That Ceramide Selectively Inhibits Protein Kinase C- Translocation and Modulates Bradykinin Activation of Phospholipase D (*)

Meril J. Jones(§) and
Andrew W. Murray

From the (1)School of Biological Sciences, Faculty of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia

^§To whom correspondence should be addressed. Tel.: 061-8-201-3836; Fax: 061-8-201-3015.

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

Footnotes

↵* This work was supported by grants from Anticancer Foundation of the Universities of South Australia and the National Health and Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SM

sphingomyelin

PKC

protein kinase C

PS

phosphatidylserine

SMase

sphingomyelinase

diC₈

dioctanoylglycerol

DAG

diacylglycerol

C₂-Cer

C₂-ceramide

C₈-cer

C₈-ceramide

PBut

phosphatidylbutanol

PEth

phosphatidylethanol

TLC

thin layer chromatography

TPA

12-O-tetradecanoylphorbol-13-acetate.
- Received September 14, 1994.
- Revision received November 11, 1994.