Characterization of signaling pathways involved in Fas-mediated apoptosis

Fas plays an important role in the immune system. Crosslinking of the receptor Fas (APO-1/CD95) with either Fas ligand or agonistic antibody results in apoptosis. We took a biochemical and a genetic approach to identify signaling events involved in Fas-mediated apoptosis.; We showed that Fas activated the stress-responsive MAP kinases, p38 and JNK/SAPK, in Jurkat T lymphocytes. Fas-activation of p38 and JNK correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3(glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the p38 pathway in Fas-mediated apoptosis. Fas-induced death is mediated by caspase (ICE/CED-3) family proteases. We showed that the viral caspase inhibitor CrmA antagonized and two peptide caspase inhibitors, YVAD-CMK and Z-VAD-FMK, completely inhibited Fas-activation of p38 kinase activity, demonstrating that Fas-activation of p38 requires caspase family members. These results indicate that stress kinase signaling pathways are activated downstream of caspases.; In order to identify essential components of the Fas-induced apoptotic signaling pathway, we undertook a genetic screen in mammalian cells. Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced death. We obtained cell lines deficient in the receptor Fas, the adaptor FADD or the cysteine protease caspase-8. These mutant cell lines were completely resistant to Fas-induced death. Complementation of the FADD or caspase-8 mutant cell lines with wildtype FADD or caspase-8, respectively, restored Fas-mediated apoptosis. Fas-activation of multiple caspases, and of the stress kinases, p38 and JNK, was completely blocked in the FADD and caspase-8 mutant cell lines. In addition, the generation of ceramide in response to Fas ligation, was blocked in the FADD and caspase-8 mutant cell lines. Furthermore, the caspase-8 mutant was severely deficient in TNF-a-induced death, and was partially deficient in DNA damage-induced death. These results indicate that FADD and caspase-8 are essential for multiple signaling events downstream of Fas.