Role of FLASH in caspase-8-mediated activation of NF-κB: Dominant-negative function of FLASH mutant in NF-κB signaling pathway

Joon Il Jum, Chul Woong Chung, Ho June Lee, Jong Ok Pyo, Kee Nyung Lee, Nam Soon Kim, Yong Sung Kim, Hyang Sook Yoo, Tae Ho Lee, Eunhee Kim, Yong Keun Jung

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23 Citations (Scopus)

Abstract

Caspase-8 is the most receptor-proximal, upstream caspase in the caspase cascade and plays a key role in cell death triggered by various death receptors. Here, we addressed the role of endogenous caspase-8 in tumor necrosis factor (TNF)-α-induced activation of NF-κB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-α-induced activation of NF-κB in NIH3T3, HeLa, and HEK293 cells as determined with luciferase reporter gene and cell fractionation assays. Reconstitution of caspase-8-deficient C33A cells with processing-defective (P/D) mutant of caspase-8 sensitized the cells to TNF-α for NF-κB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-κB and to bind FLICE-associated huge protein (FLASH) in vitro and in vivo. Instead, caspase-8 (D73A) mutant bound to caspase-8 and blocked NF-κB activation triggered by TNF-α and caspase-8. In addition, expression of an NF-κB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-α and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-κB. Further, immunoprecipitation assays showed that caspase-8 formed triple complex with TRAF2 and FLASH. Taken together, these results suggest that endogenous caspase-8 mediates TNF-α-induced activation of NF-κB via FLASH.

Original languageEnglish
Pages (from-to)688-696
Number of pages9
JournalOncogene
Volume24
Issue number4
DOIs
Publication statusPublished - 2005 Jan 20

Bibliographical note

Funding Information:
We thank Dr P Chaudhary (University of Texas, USA) for caspase-8 (D73A) mutant expression plasmid. J Jun was partially supported by the Brain Korea 21 project. This work was supported by National Research Laboratory program (to YK Jung), 21 C Frontier on Functional Genomics and Brain of the Korean Ministry of Science and Technology.

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics
  • Cancer Research

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