Ceramide enables fas to cap and kill.
Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into "caps" on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15-30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase -/- hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C(16)-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C(16)-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.
Cremesti
A
A
Laboratory of Signal Transduction and Department of Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Paris
F
F
Grassmé
H
H
Holler
N
N
Tschopp
J
J
Fuks
Z
Z
Gulbins
E
E
Kolesnick
R
R
D000818:Animals; D000906:Antibodies / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D002518:Ceramides / Q000096:biosynthesis / Q000494:pharmacology / Q000502:physiology*; D053222:Fas Ligand Protein; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects; D051379:Mice; D018345:Mice, Knockout; D011940:Receptor Aggregation; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D019014:fas Receptor / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*
D000906:Antibodies; D002518:Ceramides; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D019014:fas Receptor; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase