In vitro activation of procaspase-8 by forming the cytoplasmic component of the death-inducing signaling complex (cDISC).
Procaspase-8 is activated by forming a death-inducing signaling complex (DISC) with the Fas-associated death domain (FADD) and the Fas receptor, but the mechanism of its activation is not well understood. Procaspase-8 devoid of the death effector domain at its N-terminus (delta nprocaspase-8) was reported to be activated by kosmotropic salts, but it has not been induced to form a DISC in vitro because it cannot interact with FADD. Here, we report the production of full-length procaspase-8 and show that it is activated by adding the Fas death domain (Fas-DD) and the FADD forming the cytoplasmic part of the DISC (cDISC). Furthermore, mutations known to affect DISC formation in vivo were shown to have the same effect on procaspase-8 activation in vitro. An antibody that induces Fas-DD association enhanced procaspase-8 activation, suggesting that the Fas ligand is not required for low-level activation of procaspase-8, but that Fas receptor clustering is needed for high-level activation of procaspase-8 leading to cell death. In vitro activation of procaspase-8 by forming a cDISC will be invaluable for investigating activation of ligand-mediated apoptosis and the numerous interactions affecting procaspase-8 activation.
Roy
Ankoor
A
Center for Neural Science, Korea Institute of Science and Technology, Seoul 130-650, Korea.
Hong
Jong Hui
JH
Lee
Jin-Hee
JH
Lee
Young-Tae
YT
Lee
Bong-Jin
BJ
Kim
Key-Sun
KS
D053181:Caspase 8 / Q000378:metabolism*; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000096:biosynthesis*; D004789:Enzyme Activation; D053200:Fas-Associated Death Domain Protein / Q000502:physiology; D006801:Humans; D017510:Protein Folding
D053418:Death Domain Receptor Signaling Adaptor Proteins; D053200:Fas-Associated Death Domain Protein; D053181:Caspase 8