Role of FLASH in caspase-8-mediated activation of NF-kappaB: dominant-negative function of FLASH mutant in NF-kappaB signaling pathway.
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)-alpha-induced activation of NF-kappaB. Direct targeting of caspase-8 with siRNA and antisense (AS) approaches abolished TNF-alpha-induced activation of NF-kappaB 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-alpha for NF-kappaB activation. In contrast to wild-type caspase-8, death effector domain mutant replacing Asp73 with Ala (caspase-8 (D73A)) failed to activate NF-kappaB 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-kappaB activation triggered by TNF-alpha and caspase-8. In addition, expression of an NF-kappaB-activating domain-deletion mutant of FLASH or transfection of FLASH AS oligonucleotides abolished TNF-alpha and caspase-8, but not phorbol 12-myristate 13-acetate, -induced activation of NF-kappaB. 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-alpha-induced activation of NF-kappaB via FLASH.
Jun
Joon-Il
JI
Department of Life Science, Gwangju Institute of Science and Technology, 1-Oryong-dong, Buk-gu, Gwangju 500-712, Korea.
Chung
Chul-Woong
CW
Lee
Ho-June
HJ
Pyo
Jong-Ok
JO
Lee
Kee Nyung
KN
Kim
Nam-Soon
NS
Kim
Yong Sung
YS
Yoo
Hyang-Sook
HS
Lee
Tae-Ho
TH
Kim
Eunhee
E
Jung
Yong-Keun
YK
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D053181:Caspase 8; D061945:Caspase Inhibitors; D020169:Caspases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; C485604:CASP8AP2 protein, human; D002135:Calcium-Binding Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D001224:Aspartic Acid; C505530:CASP8 protein, human; C505526:Casp8 protein, mouse; D053181:Caspase 8; D020169:Caspases