FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).
Upon activation, the apoptosis-inducing cell membrane receptor CD95 (APO-1/Fas) recruits a set of intracellular signaling proteins (CAP1-4) into a death-inducing signaling complex (DISC). In the DISC, CAP1 and CAP2 represent FADD/MORT1. CAP4 was identified recently as an ICE-like protease, FLICE, with two death effector domains (DED). Here we show that FLICE binds to FADD through its N-terminal DED. This is an obligatory step in CD95 signaling detected in the DISC of all CD95-sensitive cells tested. Upon prolonged triggering of CD95 with agonistic antibodies all cytosolic FLICE gets proteolytically activated. Physiological FLICE cleavage requires association with the DISC and occurs by a two-step mechanism. Initial cleavage generates a p43 and a p12 fragment further processed to a p10 fragment. Subsequent cleavage of the receptor-bound p43 results in formation of the prodomain p26 and the release of the active site-containing fragment p18. Activation of FLICE is blocked by the peptide inhibitors zVAD-fmk, zDEVD-fmk and zIETD-fmk, but not by crmA or Ac-YVAD-CHO. Taken together, our data indicate that FLICE is the first in a cascade of ICE-like proteases activated by CD95 and that this activation requires a functional CD95 DISC.
Medema
J P
JP
Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany.
Scaffidi
C
C
Kischkel
F C
FC
Shevchenko
A
A
Mann
M
M
Krammer
P H
PH
Peter
M E
ME
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D007527:Isoenzymes; D051379:Mice; D008954:Models, Biological; D011233:Precipitin Tests; D011485:Protein Binding; D015843:Serpins / Q000494:pharmacology; D015398:Signal Transduction; D014764:Viral Proteins; D019014:fas Receptor / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; C505533:FADD protein, human; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D007527:Isoenzymes; D015843:Serpins; D014764:Viral Proteins; D019014:fas Receptor; C074641:interleukin-1beta-converting enzyme inhibitor; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases