PHOSPHORYLATION OF HUMAN I-KAPPA-B-ALPHA ON SERINE-32 AND SERINE-36 CONTROLS I-KAPPA-B-ALPHA PROTEOLYSIS AND NF-KAPPA-B ACTIVATION IN RESPONSE TO DIVERSE STIMULI

Post-translational activation of the higher eukaryotic transcription f actor NF-kappa B requires both phosphorylation and proteolytic degrada tion of the inhibitory subunit I kappa B-alpha, Inhibition of proteaso me activity can stabilize an inducibly phosphorylated form of I kappa B-alpha in intact cells, suggesting that phosphorylation targets the p rotein for degradation, In this study, we have identified serines 32 a nd 36 in human I kappa B-alpha as essential for the control of I kappa B-alpha stability and the activation of NF-kappa B in HeLa cells, A p oint mutant substituting serines 32 and 36 by alanine residues was no longer phosphorylated in response to okadaic acid (OA) stimulation. Th is and various other Ser32 and Ser36 mutants behaved as potent dominan t negative I kappa B proteins attenuating kappa B-dependent transactiv ation in response to OA, phorbol 12-myristate 13-acetate (PMA) and tum or necrosis factor-alpha (TNF), While both endogenous and transiently expressed wild-type I kappa B-alpha were proteolytically degraded in r esponse to PMA and TNF stimulation of cells, the S32/36A mutant of I k appa B-alpha remained largely intact under these conditions, Our data suggest that such diverse stimuli as OA, TNF and PMA use the same kina se system to phosphorylate and thereby destabilize I kappa B-alpha, le ading to NF-kappa B activation.