FLASH coordinates NF-kappa B activity via TRAF2.
FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-alpha-induced activation of NF-kappaB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-kappaB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-kappaB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKalpha, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856-1191 of FLASH activated NF-kappaB as efficiently as the full-length and could directly bind to TRAF2 in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-kappaB activity via a TRAF2-dependent pathway in the TNF-alpha signaling.
Choi
Y H
YH
Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500-712, Korea.
Kim
K B
KB
Kim
H H
HH
Hong
G S
GS
Kwon
Y K
YK
Chung
C W
CW
Park
Y M
YM
Shen
Z J
ZJ
Kim
B J
BJ
Lee
S Y
SY
Jung
Y K
YK
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D019169:Jurkat Cells; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000097782:NF-kappaB-Inducing Kinase
D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D051550:I-kappa B Kinase; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C485604:CASP8AP2 protein, human; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human