Caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain.
Caspase activation resulting from cytochrome c release from the mitochondria is an essential component of the mechanism of apoptosis initiated by a range of factors. The activation of Bid by caspase-8 in this pathway promotes further cytochrome c release, thereby completing a positive feedback loop of caspase activation. Although the identity of the caspases necessary for caspase-8 activation in this pathway are known, it is still unclear which protease directly cleaves caspase-8. In order to identify the factor responsible we undertook a biochemical purification of caspase-8 cleaving activity in cytosolic extracts to which cytochrome c had been added. Here we report that caspase-6 is the only soluble protease in cytochrome c activated Jurkat cell extracts that has significant caspase-8 cleaving activity. Furthermore the caspase-6 that we purified was sufficient to induce Bid dependent cytochrome c releasing activity in cell extracts. Inhibition of caspase-6 activity in cells significantly inhibited caspase-8 cleavage and apoptosis, therefore establishing caspase-6 as a major activator of caspase-8 in vivo and confirming that this pathway can have a critical role in promotion of apoptosis. We also show that caspase-6 is inactive until the short prodomain is removed. We suggest that the requirement for two distinct cleavage steps to activate an effector caspase may represent an effective mechanism for restriction of spontaneous caspase activation and aberrant entry into apoptosis.
Cowling
V
V
Signal Transduction Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
Downward
J
J
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019556:COS Cells / Q000201:enzymology; D002352:Carrier Proteins / Q000378:metabolism; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism; D005057:Eukaryotic Cells / Q000201:enzymology*; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D019169:Jurkat Cells; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D003574:Cytochrome c Group; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D046911:Macromolecular Substances; D010450:Endopeptidases; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases