TUMOR-NECROSIS-FACTOR (TNF)-ALPHA ACTIVATES C-RAF-1 KINASE VIA THE P55 TNF RECEPTOR ENGAGING NEUTRAL SPHINGOMYELINASE

TNF-alpha mediates proliferation, functional activation and apoptotic death of cells depending upon its concentration and target cell type. The signaling pathways used by TNF-alpha to mount these responses are, at present, not completely understood. We report here that TNF-alpha promotes dose- and time-dependent phosphorylation and activation of th e c-raf-1 kinase engaging the type I p55 TNF receptor (TNF-R), c-raf-k inase activation was duplicated by an agonistic monoclonal antibody di rected against the p55 TNF-R. Moreover, ectopic expression of the huma n p55 TNF-R in murine pre-B 70Z/3 cells was sufficient to confer c-raf -1-kinase activation by human TNF-alpha. By inhibiting intracellular a ctivation of acidic sphingomyelinase (SMase) and by using deleted form s of the type I TNF-R it was shown that the neutral, but not the acidi c SMase, participated in TNF-alpha-mediated phoshorylation and activat ion of the c-raf kinase. TNF-alpha-induced transcriptional activation of a heterologous promoter construct harboring the AP-1 binding site w as also mediated by the type I p55 TNF-R. In this case the initiation of transcription required the same cytoplasmic domain as that responsi ble for activation of c-raf-1 kinase and was liberated in the presence of a dominant negative mutant of c-raf-1.