Linkage of the BH4 domain of Bcl-2 and the nuclear factor kappaB signaling pathway for suppression of apoptosis.
Nuclear factor (NF) kappaB is a ubiquitously expressed transcription factor whose function is regulated by the cytoplasmic inhibitor protein, IkappaBalpha. We have previously shown that IkappaBalpha activity is diminished in ventricular myocytes expressing Bcl-2. (de Moissac, D., Mustapha, S., Greenberg, A. H., and Kirshenbaum, L. A. (1998) J. Biol. Chem. 273, 23946-23951). In view of the growing evidence that the conserved N-terminal BH4 domain of Bcl-2 plays a critical role in suppressing apoptosis, we ascertained whether this region accounts for the underlying effects of Bcl-2 on IkappaBalpha activity. Transfection of human embryonic 293 cells with full length Bcl-2 resulted in a significant 1.9-fold reduction in IkappaBalpha activity (p < 0.006) with a concomitant increase in DNA binding and 3.4-fold increase in NFkappaB-dependent gene transcription (p < 0. 022) compared with vector transfected control cells. In contrast, no significant change in IkappaBalpha activity was detected with either a BH4 domain deletion mutant (residues 10-30) or BH4 domain point substitution mutants, I14G, V15G, Y18G, K22G, and L23G (p = 2.77). However, a small 0.60-fold decrease (p < 0.04) in IkappaBalpha activity was noted with the BH4 mutant I19G, suggesting that this residue may not be critical for IkappaBalpha regulation. Furthermore, adenovirus-mediated delivery of an IkappaBalpha mutant to prevent NFkappaB activation impaired the ability of Bcl-2 to suppress apoptosis provoked by TNFalpha plus cycloheximide in ventricular myocytes. The data provide the first evidence for the regulation of IkappaBalpha by Bcl-2 through a mechanism that requires the conserved BH4 domain that links Bcl-2 to the NFkappaB signaling pathway for suppression of apoptosis.
de Moissac
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Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Department of Physiology, University of Manitoba, Winnipeg, Manitoba R2H 2A6, Canada.
Zheng
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Kirshenbaum
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D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D020672:I-kappa B Proteins; D009206:Myocardium; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics*; D009687:Nuclear Proteins / Q000032:analysis; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D013489:Suppression, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D003513:Cycloheximide