Nuclear factor (NF) kappa B is a ubiquitously expressed transcription factor whose function is regulated by the cytoplasmic inhibitor protein, I kappa B alpha. We have previously shown that I kappa B alpha 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 I kappa B alpha activity. Transfection of human embryonic 293 cells with full length Bcl-2 resulted in a significant 1.9-fold reduction in I kappa B alpha activity (p < 0.006) with a concomitant increase in DNA binding and 3.4-fold increase in NF kappa B-dependent gene transcription (p < 0.022) compared with vector transfected control cells. In contrast, no significant change in I kappa B alpha 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 I kappa B alpha activity was noted with the BH4 mutant I19G, suggesting that this residue may not be critical for I kappa B alpha regulation. Furthermore, adenovirus-mediated delivery of an I kappa B alpha mutant to prevent NF kappa B activation impaired the ability of Bcl-2 to suppress apoptosis provoked by TNF alpha plus cycloheximide in ventricular myocytes, The data provide the first evidence for the regulation of I kappa B alpha by Bcl-2 through a mechanism that requires the conserved BH4 domain that links Bcl-2 to the NF kappa B signaling pathway for suppression of apoptosis.
