Direct binding to ceramide activates protein kinase Cζ before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

We have reported that ceramide mediates binding of atypical protein kinase C (PKC) ζ to its inhibitor protein, PAR-4 (prostate apoptosis response-4), thereby inducing apoptosis in differentiating embryonic stem cells. Using a novel method of lipid vesicle-mediated affinity chromatography, we showed here that endogenous ceramide binds directly to the PKCζ-PAR-4 complex. Ceramide and its analogs activated PKCζ prior to binding to PAR-4, as determined by increased levels of phosphorylated PKCζ and glycogen synthase kinase-3β and emergence of a PAR-4-to-phosphorylated PKCζ fluorescence resonance energy transfer signal that co-localizes with ceramide. Elevated expression and activation of PKCζ increased cell survival, whereas expression of PAR-4 promoted apoptosis. This suggests that PKCζ counteracts apoptosis, unless its ceramide-induced activation is compromised by binding to PAR-4. A luciferase reporter assay showed that ceramide analogs activate nuclear factor (NF)-κB unless PAR-4-dependent inhibition of PKCζ suppresses NF-κB activation. Taken together, our results show that direct physical association with ceramide and PAR-4 regulates the activity of PKCζ. They also indicate that this interaction regulates the activity of glycogen synthase kinase-3β and NF-κB.