Constitutive Phosphorylation of IκBα by Casein Kinase II Occurs Preferentially at Serine 293: Requirement for Degradation of Free IκBα

Abstract

IκBα is a phosphoprotein that sequesters the NF-κB/Rel transcription factors in the cytoplasm by physical association. Following induction by a wide variety of agents, IκBα is further phosphorylated and degraded, allowing IκBα/Rel proteins to translocate to the nucleus and induce transcription. We have previously reported that the constitutive phosphorylation site resides in the C-terminal PEST region of IκBα and is phosphorylated by casein kinase II (CKII). Here we show that serine 293 is the preferred CKII phosphorylation site. Additionally, we show compensatory phosphorylation by CKII at neighboring serine and threonine residues. Thus, only when all five of the serine and threonine residues in the C-terminal region of IκBα are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphorylation is required for efficient degradation of free IκBα, in that unassociated MutF has a half-life two times longer than wild-type IκBα. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the MutF phenotype. Therefore, the constitutive CKII phosphorylation site is an integral part of the PEST region of IκBα, and this phosphorylation is required for rapid proteolysis of the unassociated protein.