CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis.
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 diacylglycerol 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.
Grullich
C
C
Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Sullards
M C
MC
Fuks
Z
Z
Merrill
A H
AH
Kolesnick
R
R
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000378:metabolism; D015843:Serpins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014764:Viral Proteins; D019014:fas Receptor / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002518:Ceramides; D004792:Enzyme Precursors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; D019014:fas Receptor; C074641:interleukin-1beta-converting enzyme inhibitor; D003513:Cycloheximide; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases