AFFINITY LABELING DISPLAYS THE STEPWISE ACTIVATION OF ICE-RELATED PROTEASES BY FAS, STAUROSPORINE, AND CRMA-SENSITIVE CASPASE-8

The activation of multiple interleukin-1 beta converting enzyme-relate d proteases (caspases) in apoptotic mammalian cells raises questions a s to whether the multiple active caspases have distinct roles in apopt otic execution as well as how these proteases are organized in apoptot ic signaling pathways. Here we used an affinity-labeling agent, YV(bio )KD-aomk, to investigate the caspases activated during apoptotic cell death, YV(bio))KD-aomk identified sis distinct polypeptides correspond ing to active caspases in Fas-stimulated Jurkat T cells, On staurospor ine treatment, four polypeptides were detected, Competition experiment s showed that the labelled caspases have distinct substrate preference s, Stepwise appearance of the labelled caspases in each cell death eve nt was consistent with the view that the activated caspases are organi zed into protease cascades, Moreover, me found that stepwise activatio n of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/M ch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apop tosis, inhibited the protease activity of caspase-8, Overall, these fi ndings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspase s in Fas-stimulated cells.