Ceramide Inactivates Cellular Protein Kinase Cα*
- From the Departments of Medicine and Cell Biology, Duke University Medical Center, and the Veterans Administration Geriatric Research Education and Clinical Center, Durham, North Carolina 27710
- ‡ Recipient of a Paul Beeson Physician Faculty Scholars Award in Aging Research. To whom correspondence should be addressed: Division of Geriatrics, Dept. of Medicine, Box 3345, DUMC, Durham, NC 27710. Tel.: 919-684-2541; Fax: 919-681-8253.
Abstract
Ceramide mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. In this study, we explored the mechanisms by which ceramide induces its cellular effects. In Molt-4 cells, phorbol 12-myristate 13-acetate (PMA) induced retinoblastoma gene product (Rb) phosphorylation, and ceramide inhibited this effect, suggesting an inhibitory effect of ceramide on the protein kinase C (PKC) pathway, the primary target of PMA. Molt-4 cells contained primarily PKCα and βII isoforms of PKC. To determine the effects of ceramide on PKC, we developed an immunoprecipitation assay for PKCα activity. Exposure of Molt-4 cells to C6-ceramide resulted in a concentration and time-dependent inhibition of immunoprecipitated protein kinase Cα (PKCα). Initial inhibition was observed as early as 4.5 h after treatment of cells with C6-ceramide, and the activity was completely lost by 13 h. Inhibition of PKCα activity was seen at concentrations of ceramide as low as 5 μM with maximal effects occurring at a concentration of 15 μM. Both C2 and C6-ceramide were inhibitory, but C2 and C6 dihydroceramides were not. Ceramide did not directly inhibit PKCα in vitro or modulate the levels of PKCα protein, suggesting that ceramide acted indirectly. Moreover, ceramide did not inhibit PMA-induced translocation of PKCα. Taken together, these results suggested that ceramide caused inactivation of PKCα. Since PKC requires phosphorylation for activity, we determined the effects of ceramide on phosphorylation of PKCα. C6-ceramide inhibited basal and PMA-induced phosphorylation of PKCα. In addition, okadaic acid, a potent phosphatase inhibitor, slightly stimulated PKC activity and blocked the effects of ceramide on PKCα inhibition. These results demonstrate that ceramide causes inhibition/inactivation of PKCα and suggest these effects of ceramide may be mediated by a protein phosphatase.
Footnotes
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↵* This work was supported in part by Grants 5T32 AG00029 (to J. Y. L.) and K08AG00520 (to L. M. O.) from the NIA, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- CAPP
-
ceramide-activated protein phosphatase
- PKC
-
protein kinase C
- DiC8
-
dioctanoylglycerol
- FBS
-
fetal bovine serum
- PBS
-
phosphate-buffered saline
- Rb
-
retinoblastoma
- PMA
-
phorbol 12-myristate 13-acetate
- PS
-
phosphatidylserine
- PP2A
-
protein phosphatase 2A
- PAGE
-
polyacrylamide gel electrophoresis.
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↵2 M. E. Venable and L. M. Obeid, unpublished results.
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- Received January 22, 1996.
- Revision received March 22, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
