CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis Journal Article
| Authors: | Grullich, C.; Sullards, M. C.; Fuks, Z.; Merrill, A. H. Jr; Kolesnick, R. |
| Article Title: | CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis |
| Abstract: | Although numerous studies document caspase-independent ceramide generation preceding apoptosis upon environmental stress, the molecular ordering of ceramide generation during cytokine-induced apoptosis remains uncertain. Here, we show that CD95-induced ceramide elevation occurs during the initiation phase of apoptosis. We titrated down the amount of FADD transfected into HeLa and 293T cells until it was insufficient for apoptosis, although cycloheximide (CHX) still triggered the effector phase. Even in the absence of CHX, ceramide levels increased rapidly, peaking at 2.7 ± 0.2-fold of control 8 h post-transfection. Dominant negative FADD failed to confer ceramide generation or CHX-mediated apoptosis. Ceramide generation induced by FADD was initiator caspase-dependent, being blocked by crmA. Limited pro- caspase 8 overexpression also increased ceramide levels 2.7 ± 0.2-fold, yet failed, without CHX, to initiate apoptosis. Expression of membrane-targeted oligomerized CD-8 caspase 8 induced apoptosis without CHX, yet elevated ceramide only to a level equivalent to limited pro-caspase 8 transfection. Ceramide elevations were detected concurrently by diacyl-glycerol kinase and electrospray tandem mass spectrometry. These investigations provide evidence that ceramide generation is initiator caspase-dependent and occurs prior to commitment to the effector phase of apoptosis, definitively ordering ceramide as proximal in CD95 signaling. |
| Keywords: | signal transduction; controlled study; human cell; mass spectrometry; apoptosis; fas antigen; serpins; caspase; caspases; hela cells; cytokine; carrier proteins; recombinant proteins; adaptor proteins, signal transducing; caspase 8; caspase 9; enzyme precursors; ceramide; ceramides; viral proteins; cycloheximide; antigens, cd95; fas-associated death domain protein; diacylglycerol kinase; humans; human; priority journal; article |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 12 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-03-24 |
| Start Page: | 8650 |
| End Page: | 8656 |
| Language: | English |
| DOI: | 10.1074/jbc.275.12.8650 |
| PUBMED: | 10722705 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
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