Caspase cleavage product lacking amino-terminus of IkappaBalpha sensitizes resistant cells to TNF-alpha and TRAIL-induced apoptosis.
In response to a diverse array of signals, IkappaBalpha is targeted for phosphorylation-dependent degradation by the proteasome, thereby activating NF-kappaB. Here we demonstrate a role of the cleavage product of IkappaBalpha in various death signals. During apoptosis of NIH3T3, Jurkat, Rat-1, and L929 cells exposed to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), Fas, serum deprivation, or TNF-alpha, respectively, IkappaBalpha was cleaved in a caspase-dependent manner. In vitro and in vivo cleavage assays and site-directed mutagenesis showed that caspase-3 cleaved IkappaBalpha between Asp31 and Ser32. Expression of the cleavage product lacking amino-terminus (1-31), DeltaIkappaBalpha, sensitized otherwise resistant NIH3T3 fibroblast cells to apoptosis induced by TNF-alpha or TRAIL, and HeLa tumor cells to TNF-alpha. DeltaIkappaBalpha was more pro-apoptotic compared to wild type or cleavage-resistant (D31E)IkappaBalpha mutant and the sensitization elicited by DeltaIkappaBalpha was as effective as that by the dominant negative mutant, (S32,36A)IkappaBalpha, in NIH3T3 cells. DeltaIkappaBalpha suppressed the transactivation of NF-kappaB induced by TNF-alpha or TRAIL, as reflected by luciferase-reporter activity. Conversely, expression of the p65 subunit of NF-kappaB suppressed TNF-alpha-, TRAIL-, and serum deprivation-induced cell death. On the contrary, DeltaIkappaBalpha was less effective at increasing the death rate of HeLa cells that were already sensitive to death signals including TRAIL, etoposide, or taxol. These results suggest that DeltaIkappaBalpha generated by various death signals sensitizes cells to apoptosis by suppressing NF-kappaB activity.
Kim
Ki-Woo
KW
Department of Life Science, Kwangju Institute of Science and Technology, Puk-Gu, Kwangju 500-712, Korea.
Kim
Byung Ju
BJ
Chung
Chul-Woong
CW
Jo
Dong-Gyu
DG
Kim
In-Ki
IK
Song
You-Hyun
YH
Kwon
Yun-Kyung
YK
Woo
Ha-Na
HN
Jung
Yong-Keun
YK
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001224:Aspartic Acid / Q000378:metabolism; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D020672:I-kappa B Proteins; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000502:physiology; D017239:Paclitaxel / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand; D014407:Tumor Cells, Cultured / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000378:metabolism
D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D008562:Membrane Glycoproteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505655:Tnfsf10 protein, rat; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D001224:Aspartic Acid; D005047:Etoposide; D008156:Luciferases; D001616:beta-Galactosidase; D020169:Caspases; D017239:Paclitaxel