IKKβ is required for Bcl-2-mediated NF-κB activation in ventricular myocytes

Abstract

The transcription factor nuclear factor κB (NF-κB) is regulated by cytoplasmic inhibitor IκBα. An integral step in the activation of NF-κB involves the phosphorylation and degradation of IκBα. We have previously reported that IκBα activity is diminished in ventricular myocytes expressing Bcl-2 (de Moissac, D., Zheng, H., and Kirshenbaum, L. A. (1999) J. Biol. Chem. 274, 29505-29509). The underlying mechanism by which Bcl-2 activates NF-κB is undefined. In view of growing evidence that the IκB kinases (IKKs), notably IKKβ, are involved in signal induced phosphorylation of IκBα, we ascertained whether IKKβ is necessary and sufficient for Bcl-2 mediated NF-κB activation. Here we demonstrate that expression of Bcl-2 in ventricular myocytes resulted in an increase in NF-κB-dependent DNA binding, NF-κB gene transcription and reduced IκBα levels. An increase in the IKKβ kinase activity was observed in cells expressing full-length Bcl-2 but not in cells expressing the BH4 deletion mutant of Bcl-2 (ABH4; residues 10-30). Catalytically inactive mutants of IKKβ, but not IKKα, suppressed Bcl-2-mediated IκBα phosphorylation and NF-κB activation. Transfection of human embryonic 293 cells with a kinase-defective Raf-1 or a kinase-defective mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEKK-1) suppressed Bcl-2-mediated IKKβ activity and NF-κB activation. Further, Bcl-2-mediated NF-κB activity was impaired in nullizygous mouse embryonic fibroblasts deficient for IKKβ. In this report, we provide the first direct evidence that Bcl-2 activates NF-κB by a signaling mechanism that involves Raf-1/MEKK-1 mediated activation of IKKβ.