RelA/p65 regulation of IκBβ

I kappa B inhibitor proteins are the primary regulators of NF-kappa B. In contrast to the defined regulatory interplay between NF-kappa B and I kappa B kappa, much less is known regarding the regulation of I kappa B beta by NF-kappa B. Here, we describe in detail the regulation of I kappa B beta by RelA/p65. Using p65(-/-) fibroblasts, we show that I kappa B beta is profoundly reduced in these cells, but not in other NF-kappa B subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, I kappa B alpha is also reduced, but not nearly to the same extent as I kappa B beta, thus highlighting the degree to which I kappa B beta is dependent on p65. This dependence is based on the ability of p65 to stabilize I kappa B beta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, I kappa B beta 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 I kappa B beta, but not I kappa B alpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of I kappa B beta protein by p65 is necessary for the maintenance of cellular homeostasis.