Related Experiment Video
Updated: Apr 30, 2026

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells
Published on: January 12, 2020
S-nitrosylation of FLICE inhibitory protein determines its interaction with RIP1 and activation of NF-κB
Siera Jo Talbott1, Sudjit Luanpitpong1, Christian Stehlik2
1Department of Pharmaceutical Sciences; West Virginia University; Morgantown, WV USA; Mary Babb Randolph Cancer Center; West Virginia University; Morgantown, WV USA.
Abstract:
Death receptor (DR) ligation can lead to divergent signaling pathways causing either caspase-mediated cell death or cell proliferation and inflammation. These variations in cellular fate are determined by adaptor proteins that are recruited to the DR signaling complex. FLICE inhibitory protein (FLIP) is an established inhibitor of caspase-8-mediated apoptosis, and it is also involved in NF-κB activation. However, the molecular mechanism that regulates FLIP within this complex is unknown. In this study, we provide new evidence for the regulation of NF-κB by FLIP through S-nitrosylation, which involves covalent modification of the protein's cysteine thiol by nitric oxide to form S-nitrosothiol. Point mutations of FLIP at cysteine residues 254 and 259 prevent FLIP S-nitrosylation and its ability to activate NF-κB. The mechanism by which FLIP nitrosylation regulates NF-κB activity involves RIP1 binding and redistribution, whereas TRAF2 binding and distribution are unaffected. We further show that FLIP processing and cleavage is dependent on its nitrosylation status. Collectively, our study reveals a novel pathway for FLIP regulation of NF-κB through protein S-nitrosylation, which is a key posttranslational mechanism controlling DR-mediated cell death and survival. Since increased expression of FLIP and nitric oxide are frequently observed in chemotherapy-resistant tumors, S-nitrosylation of FLIP could be a key mechanism of chemoresistance and tumor growth.
Insights
This study reveals how S-nitrosylation regulates FLIP protein, impacting NF-κB activation and cell fate. This finding is crucial for understanding chemoresistance and tumor growth in cancers.
Area of Science:
- Cellular signaling pathways
- Molecular mechanisms of cell death and survival
- Post-translational modifications
Background:
- Death receptor (DR) ligation triggers diverse cellular outcomes, including apoptosis or proliferation.
- FLICE inhibitory protein (FLIP) modulates both apoptosis and NF-κB activation.
- The precise molecular regulation of FLIP in these pathways remains unclear.
Purpose of the Study:
- To elucidate the regulatory mechanism of FLIP in NF-κB activation.
- To investigate the role of S-nitrosylation in FLIP-mediated signaling.
- To understand the implications of FLIP S-nitrosylation in cancer biology.
Main Methods:
- Investigated FLIP regulation via S-nitrosylation, a modification by nitric oxide.
- Utilized point mutations at cysteine residues 254 and 259 of FLIP.
- Assessed the impact of S-nitrosylation on FLIP binding to RIP1 and TRAF2.
- Examined the effect of nitrosylation status on FLIP processing and cleavage.
Main Results:
- S-nitrosylation of FLIP was identified as a key regulator of NF-κB activation.
- Mutations preventing S-nitrosylation abolished FLIP's ability to activate NF-κB.
- FLIP S-nitrosylation influences RIP1 binding and redistribution, but not TRAF2.
- FLIP processing and cleavage are dependent on its S-nitrosylation status.
Conclusions:
- A novel pathway for FLIP regulation of NF-κB via S-nitrosylation was discovered.
- Protein S-nitrosylation is a critical post-translational mechanism controlling DR-mediated cell death and survival.
- FLIP S-nitrosylation may represent a key mechanism underlying chemoresistance and tumor growth in cancers with elevated FLIP and nitric oxide.
Related Concept Videos
NF-κB-dependent Signaling Pathway
NF-κB-dependent Signaling Mechanism
The...
NF-kB-dependent Signaling Pathway
Regulation of Nuclear Protein Sorting
Co-activators and Co-repressors
Regulation of the Unfolded Protein Response
Nitric Oxide Signaling Pathway

