BCL-2 ACTIVATES THE TRANSCRIPTION FACTOR NF-KAPPA-B THROUGH THE DEGRADATION OF THE CYTOPLASMIC INHIBITOR I-KAPPA-B-ALPHA

Nuclear factor kappa B (NF kappa B) is a ubiquitously expressed transc ription factor that is regulated by the cytoplasmic inhibitor protein I kappa B alpha. Biological agents such as tumor necrosis factor alpha (TNF alpha), which activate NF kappa B, result in the rapid degradati on of I kappa B alpha Adenoviral-mediated gene transfer of Bcl-2 preve nts apoptosis of neonatal ventricular myocytes induced by TMF alpha, I n view of the growing evidence that NF kappa B may play an important r ole in regulating apoptosis, we determined whether TNF alpha! and Bcl- 2 could modulate the activity of NF kappa B in ventricular myocytes. S timulation of myocytes with TNFa: resulted in a 2.1-fold increase (p < 0.001) in NF kappa B-dependent gene transcription and nuclear DNA bin ding Similarly, a 1.9-fold increase (p < 0.0002) ire NF kappa B-depend ent gene transcription was observed in myocytes expressing Bcl-2. Nucl ear DNA binding activity of NF kappa B was significantly increased in myocytes expressing Bcl-2, with a concomitant reduction in I kappa B a lpha protein level. The BcI-2-mediated loss of I kappa B alpha could b e prevented by the proteasome inhibitor lactacystin, consistent with t he notion that the targeted degradation of I kappa B alpha consequent to overexpression of BcI-2 utilizes the ubiquitin-proteasome pathway. This was further tested in human 293 cells in which the N-terminal reg ion of I kappa B alpha was identified to be an important regulatory si te for Bcl-2. Deletion of this region or a serine to alanine substitut ion mutant at amino acids 32 and 36, which are defective for both phos phorylation and degradation were more resistant than wild type I kappa B alpha to the inhibitory effects of Bcl-2. To our knowledge, this pr ovides the first evidence for the regulation of I kappa B alpha by Eel -2 and suggests a link between Eel-2 and the NF kappa B signaling path way in the suppression of apoptosis.