S-Nitrosylation of the Death Receptor Fas Promotes Fas Ligand–Mediated Apoptosis in Cancer Cells

Abstract

BACKGROUND & AIMS: Fas belongs to the family of tumor necrosis factor receptors which induce apoptosis. Many cancer cells express Fas but do not undergo Fasmediated apoptosis. Nitric oxide reverses this resistance by increasing levels of Fas at the plasma membrane. We studied the mechanisms by which NO affects Fas function. METHODS: Colon a nd mammary cancer cell lines were incubated with the NO donor glyceryl trinitrate or lipid A; S-nitrosylation of Fas was monitored using the biotin switch assay. Fas constructs that contained mutations at cysteine residues that prevent S-nitrosylation were used to investigate the involvement of S-nitrosylation in Fas-mediated cell death. Apoptosis was monitored according to morphologic criteria. RESULTS: NO induced S-nitrosylation of cysteine residues 199 and 304 in the cytoplasmic part of Fas. In cancer cells that overexpressed wild-type Fas, S-nitrosylation induced Fas recruitment to lipid rafts and sensitized the cells to Fas ligand. In cells that expressed a mutant form of Fas in which cysteine 304 was replaced by valine residue, NOmediated translocation of Fas to lipid rafts was affected and the death-inducing signal complex and synergistic effect of glyceryl trinitrate–Fas ligand were inhibited significantly. These effects were not observed in cells that expressed Fas with a mutation at cysteine 199. CONCLUSIONS: We identified post-translational modifications (S-nitrosylation of cysteine residues 199 and 304) in the cytoplasmic domain of Fas. S-nitrosylation at cysteine 304 promotes redistribution of Fas to lipid rafts, formation of the death-inducing signal complex, and induction of cell death.