CERAMIDE-BINDING AND ACTIVATION DEFINES PROTEIN-KINASE C-RAF AS A CERAMIDE-ACTIVATED PROTEIN-KINASE

Interleukin 1 is the prototype of an inflammatory cytokine, and eviden ce suggests that it uses the sphingomyelin pathway and ceramide produc tion to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinate d photoaffinity labeling analog of ceramide [I-125]3-trifluoromethyl-3 -(m-iodophenyl)diazirine ceramide) was employed. It is observed that c eramide specifically binds to and activates protein kinase c-Raf, lead ing to a subsequent activation of the MARK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protei n kinase C-zeta. These data identify protein kinase c-Raf as a specifi c molecular target for interleukin 1 beta-stimulated ceramide formatio n and demonstrate that ceramide is a lipid cofactor participating in r egulation of c-Raf activity.