Ceramide inhibition of NF-kappa B 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 med iator ceramide was recently shown to inhibit activation of this transcripti on 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-me diated activation of NF-kappaB by reverting PKC translocation to the membra ne. Next, confocal and immunofluorescence studies were performed to evaluat e the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)-PKCb2 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-inact ive mutant (PKC-KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpressi on 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 phosphoryl ation of the autophosphorylation sites.