Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas. Evidence for caspase-8-ganglioside interaction in T cells |
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Authors: | Garofalo Tina Misasi Roberta Mattei Vincenzo Giammarioli Anna Maria Malorni Walter Pontieri Giuseppe M Pavan Antonio Sorice Maurizio |
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Institution: | Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Roma, viale Regina Elena 324, Italy. |
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Abstract: | 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. |
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