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KMID : 0578320080260020165
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Molecules and Cells
2008 Volume.26 No. 2 p.165 ~ p.170
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In vitro Activation of Procaspase-8 by Forming the Cytoplasmic Component of the Death-inducing Signaling Complex (cDISC)
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Roy Ankoor
Lee Bong-Jin
Hong Jong-Hui
Lee Jin-Hee
Lee Young-Tae
Kim Key-Sun
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Abstract
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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 (?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.
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KEYWORD
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DISC, activation, procaspase-8
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