Ceramide inhibition of NF-kappaB activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response.
Protein kinase C (PKC) is known to activate NF-kappaB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF-7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC-mediated activation of NF-kappaB by reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCbeta2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC-DA) was resistant to ceramide. A kinase-inactive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC-DA prevented, at least partly, the ability of ceramide to inhibit activation of NF-kappaB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophosphorylation sites.
Signorelli
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Department of Biochemistry and Molecular Biology; Medical University of South Carolina; Charleston, South Carolina 29425, USA.
Luberto
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C
Hannun
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YA
D001692:Biological Transport / Q000187:drug effects; D002518:Ceramides / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002518:Ceramides; D007527:Isoenzymes; D008164:Luminescent Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase; D013755:Tetradecanoylphorbol Acetate