Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas. Evidence for caspase-8-ganglioside interaction in T cells.
In this investigation we show that the death-inducing signaling complex (DISC) associates with glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement. We primarily analyzed the ganglioside pattern and composition of GEM after triggering through CD95/Fas and observed that GM3 is the main ganglioside constituent of GEM. Stimulation with anti-CD95/Fas did not cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon CD95/Fas engagement, as revealed by the analysis of linear sucrose gradient fractions. It indicates that the DISC associates with GEM; no changes were observed in the distribution of caspase-9. The disruption of GEM by methyl-beta-cyclodextrin prevented DNA fragmentation, as well as CD95/Fas clustering on the cell surface, demonstrating a role for GEM in initiating of Fas signaling. These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway.
Garofalo
Tina
T
Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Roma, viale Regina Elena 324, Italy.
Misasi
Roberta
R
Mattei
Vincenzo
V
Giammarioli
Anna Maria
AM
Malorni
Walter
W
Pontieri
Giuseppe M
GM
Pavan
Antonio
A
Sorice
Maurizio
M
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002855:Chromatography, Thin Layer; D053200:Fas-Associated Death Domain Protein; D005679:G(M3) Ganglioside / Q000378:metabolism*; D006801:Humans; D013601:T-Lymphocytes / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D005679:G(M3) Ganglioside; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases