Ceramide inactivates cellular protein kinase C alpha

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 PKC alpha and beta(II) isoforms of PKC, To determine the effects of ceramide on PKC, we developed an immunoprecipitation assay for PKC alpha activity. Exposure of Molt-4 cells to C-6-ceramide resulted in a concentration and time-dependent inhibition of immunoprecipitated protein kinase C alpha (PKC alpha). Initial inhibition was observed as early as 4.5 h after treatment of cells with C-6-ceramide, and the activity was completely lost by 13 h, Inhibition of PKC alpha activity was seen at concentrations of ceramide as low as 5 mu M with maximal effects occurring at a concentration of 15 mu M. Both C-2 and C-6-ceramide were inhibitory, but C-2 and C-6 dihydroceramides were not, Ceramide did not directly inhibit PKC alpha in vitro or modulate the levels of PKC alpha protein, suggesting that ceramide acted indirectly, Moreover, ceramide did not inhibit PMA-induced translocation of PKC alpha. Taken together, these results suggested that ceramide caused inactivation of PKC alpha. Since PKC requires phosphorylation for activity, we determined the effects of ceramide on phosphorylation of PKC alpha. C-6-ceramide inhibited basal and PMA-induced phosphorylation of PKC alpha. In addition, okadaic acid, a potent phosphatase inhibitor, slightly stimulated PKC activity and blocked the effects of ceramide on PKC alpha inhibition. These results demonstrate that ceramide causes inhibition/inactivation of PKC alpha and suggest these effects of ceramide may be mediated by a protein phosphatase.