CONSTITUTIVE PHOSPHORYLATION OF I-KAPPA-B-ALPHA BY CASEIN KINASE-II OCCURS PREFERENTIALLY AT SERINE-293 - REQUIREMENT FOR DEGRADATION OF FREE I-KAPPA-B-ALPHA

I kappa B alpha is a phosphoprotein that sequesters the NF-kappa B/Rel transcription factors in the cytoplasm by physical association. Follo wing induction by a wide variety of agents, I kappa B alpha is further phosphorylated and degraded, allowing NF-kappa B/Rel proteins to tran slocate to the nucleus and induce transcription. We have previously re ported that the constitutive phosphorylation site resides in the C-ter minal PEST region of I kappa B alpha and is phosphorylated by casein k inase II (CKII). Here we show that serine 293 is the preferred CKII ph osphorylation 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 regio n of I kappa B alpha are converted to alanine (MutF), is constitutive phosphorylation abolished. Finally, we show that constitutive phosphor ylation is required for efficient degradation of free I kappa B alpha, in that unassociated MutF has a half-life two times longer than wild- type I kappa B alpha. A serine residue alone at position 293, as well as aspartic acid at this position, can revert the MutF phenotype. Ther efore, the constitutive CKII phosphorylation site is an integral part of the PEST region of I kappa B alpha, and this phosphorylation is req uired for rapid proteolysis of the unassociated protein.