RelA/p65 Regulation of IκBβ

Abstract

IκB inhibitor proteins are the primary regulators of NF-κB. In contrast to the defined regulatory interplay between NF-κB and IκBα, much less is known regarding the regulation of IκBβ by NF-κB. Here, we describe in detail the regulation of IκBβ by RelA/p65. Using p65^−/− fibroblasts, we show that IκBβ is profoundly reduced in these cells, but not in other NF-κB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65^−/− cells and tissues, IκBα is also reduced, but not nearly to the same extent as IκBβ, thus highlighting the degree to which IκBβ is dependent on p65. This dependence is based on the ability of p65 to stabilize IκBβ protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IκBβ was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65^−/− fibroblasts, expression of a proteolysis-resistant form of IκBβ, but not IκBα, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IκBβ protein by p65 is necessary for the maintenance of cellular homeostasis.

ACKNOWLEDGMENTS

We thank U. Siebenlist and F. Weih for MEF knockout cell lines, A. Beg for RelA/p65 mice, S. Ghosh for κB-Ras antibody, W. Miller for β-arrestin antibody, M. Karin for cell lines and expression plasmids, and A. Baldwin for plasmids and helpful discussion. We also thank S. Acharyya and M. Carathers for technical assistance and the rest of the Guttridge laboratory for support and insight throughout the course of this study.

This work was supported by NIH grant CA97953 and startup funds to D.C.G. and the OSU Up on the Roof Fellowship to J.W.