Phosphorylation of human IκB-α on serines 32 and 36 controls IκB-α proteolysis and NF-κB activation in response to diverse stimuli

Abstract

Post-translational activation of the higher eukaryotic transcription factor NF-κB requires both phosphorylation and proteolytic degradation of the inhibitory subunit IκB-α. Inhibition of proteasome activity can stabilize an inducibly phosphorylated form of IκB-α in intact cells, suggesting that phosphorylation targets the protein for degradation. In this study, me have identified serines 32 and 36 in human IκB-α as essential for the control of IκB-α stability and the activation of NF-κB in HeLa cells. A point mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. This and various other Ser32 and Ser36 mutants behaved as potent dominant negative IκB proteins attenuating κB-dependent transactivation in response to OA, phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-a (TNF). While both endogenous and transiently expressed wild-type IκB-α were proteolytically degraded in response to PMA and TNF stimulation of cells, the S32/36A mutant of IκB-α remained largely intact under these conditions. Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kinase system to phosphorylate and thereby destabilize IκB-α, leading to NF-κB activation.