All three IκB isoforms and most Rel family members are stably associated with the IκB kinase 1/2 complex

Nuclear factor kappa B (NF-kappa B) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappa B is present in the cytosol where it is complexed to its inhibitor I kappa B. Activation of NF-kappa B depends on the signal-induced phosphorylation of I kappa B by specific I kappa B kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in I kappa B alpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known I kappa B kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90(rsk1)), the I kappa B kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappa B-related and I kappa B-related proteins: RelA, RelB, cRel, p100, p105, I kappa B alpha, I kappa B beta and I kappa B epsilon. The existence of stable kinase-substrate complexes, the presence of all three known I kappa B isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating I kappa B alpha-, I kappa B beta- and I kappa B epsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappa B species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of I kappa B alpha, we observed efficient phosphorylation of serine 32 in I kappa B alpha by the purified endogenous CK II complex. Therefore, both p90(rsk1) and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.