REGULATION OF RAF-1 KINASE BY TNF VIA ITS 2ND-MESSENGER CERAMIDE AND CROSS-TALK WITH MITOGENIC SIGNALING

Raf-1 kinase is a central regulator of mitogenic signal pathways, wher eas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined, We have investigated mechanisms of Raf-1 r egulation by TNF and its messenger ceramide in cell-free assays, insec t and mammalian cell lines, In vitro, ceramide specifically bound to t he purified catalytic domain and enhanced association with activated R as proteins, but did not affect the kinase activity of Raf-1. Cell-per meable ceramides induced a marked increase of Ras-Raf-1 complexes in c ells co-expressing Raf-1 and activated Ras, Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 rhabdomyosarcoma cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation, Failure of T NF or ceramide to induce Raf-1 kinase was observed in several TNF-resp onsive cell lines, Both TNF and exogeneous C-6-ceramide interfered wit h the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity, TNF also indu ced the translocation of Raf-1 from the cytosolic to the particulate f raction, indicating that this negative regulatory cross-talk occurs at the cell membrane, Interference with mitogenic signals at the level o f Raf-1 could be an important initial step in TNF's cytostatic action.