Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.
The activation of multiple interleukin-1beta converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic 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 six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.
Takahashi
A
A
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Japan.
Hirata
H
H
Yonehara
S
S
Imai
Y
Y
Lee
K K
KK
Moyer
R W
RW
Turner
P C
PC
Mesner
P W
PW
Okazaki
T
T
Sawai
H
H
Kishi
S
S
Yamamoto
K
K
Okuma
M
M
Sasada
M
M
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002645:Chickens; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D019169:Jurkat Cells; D007797:Laminin / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015843:Serpins / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D014764:Viral Proteins; D019014:fas Receptor / Q000494:pharmacology
D000345:Affinity Labels; D015853:Cysteine Proteinase Inhibitors; D007797:Laminin; D009842:Oligopeptides; D015843:Serpins; D014764:Viral Proteins; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; D019014:fas Receptor; C096429:L 709049; C084098:laminin A; C074641:interleukin-1beta-converting enzyme inhibitor; D011065:Poly(ADP-ribose) Polymerases; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases; D019311:Staurosporine