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Activation of death-inducing signaling complex (DISC) by pro-apoptotic C-terminal fragment of RIP
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea.
Abstract:
The two opposite signaling pathways that stimulate NF-kappaB activation and apoptosis are both mediated by tumor necrosis factor receptor 1 (TNFR1) and its cytosolic associated proteins. In this study, we demonstrate that the proteolytic cleavage of receptor interacting protein (RIP) by caspase-8 during TNF-induced apoptosis abrogates the stimulatory role of RIP on TNF-induced NF-kappaB activation. The uncleavable RIPD324A mutant was less apoptotic, but its ability to activate NF-kappaB activation was greater than the wild type counterpart. Ectopic expression of the pro-apoptotic C-terminal fragment of RIP inhibited TNF-induced NF-kappaB activation by suppressing the activity of I-kappaB kinasebeta (IKKbeta) which phosphorylates I-kB, an inhibitor of NF-kappaB, and triggers its ubiquitin-mediated degradation. The C-terminal fragment of RIP also enhanced the association between TNFR1 and death domain proteins including TNFR1 associated death domain (TRADD) and Fas associated death domain (FADD), resulting in the activation of caspase-8 and stimulation of apoptosis. The present study suggest that the C-terminal fragment of RIP produced by caspase-8 activates death-inducing signaling complex (DISC), attenuates NF-kappaB activation, and thereby amplifies the activation of caspase-8 which initiates the downstream apoptotic events. Oncogene (2000) 19, 4491 - 4499.
Insights
Tumor necrosis factor receptor 1 (TNFR1) signaling activates NF-kappaB and apoptosis. Caspase-8 cleavage of receptor interacting protein (RIP) during apoptosis reduces RIP
Area of Science:
- Cellular signaling pathways
- Apoptosis and cell death mechanisms
- Molecular biology
Background:
- Tumor necrosis factor receptor 1 (TNFR1) mediates opposing signals for NF-kappaB activation and apoptosis.
- Receptor interacting protein (RIP) is a key cytosolic protein associated with TNFR1 signaling.
- Caspase-8 plays a critical role in initiating apoptosis.
Purpose of the Study:
- To investigate the role of receptor interacting protein (RIP) cleavage by caspase-8 in TNF-induced apoptosis and NF-kappaB activation.
- To elucidate the mechanism by which the C-terminal fragment of RIP influences TNFR1 signaling.
- To understand how RIP cleavage impacts the balance between apoptosis and NF-kappaB activation.
Main Methods:
- Site-directed mutagenesis to create an uncleavable RIP mutant (RIPD324A).
- Ectopic expression of RIP fragments in cells.
- Analysis of NF-kappaB activation using reporter assays.
- Assessment of apoptosis through cell death assays.
- Co-immunoprecipitation to study protein-protein interactions.
Main Results:
- Proteolytic cleavage of RIP by caspase-8 during TNF-induced apoptosis abrogates RIP's stimulatory role on NF-kappaB activation.
- An uncleavable RIP mutant (RIPD324A) exhibited reduced apoptosis but enhanced NF-kappaB activation compared to wild-type RIP.
- The pro-apoptotic C-terminal fragment of RIP inhibited TNF-induced NF-kappaB activation by suppressing IKKbeta activity.
- The C-terminal fragment of RIP enhanced TNFR1 association with TRADD and FADD, promoting caspase-8 activation and apoptosis.
Conclusions:
- The C-terminal fragment of RIP, generated by caspase-8, activates the death-inducing signaling complex (DISC).
- This fragment attenuates NF-kappaB activation, thereby amplifying caspase-8 activation and downstream apoptotic events.
- RIP cleavage by caspase-8 represents a critical regulatory point controlling the switch between survival (NF-kappaB) and death (apoptosis) signaling pathways initiated by TNFR1.