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.