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

Nuclear factor kappa B (NF-kappa B) is an important transcription factor fo r the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under vari ous 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 b y specific I kappa B kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-s timulated and okadaic-acid-stimulated HeLa cells to purify three biochemica lly distinct kinase activities targeting one or both of the two serines (S3 2 and S36) in I kappa B alpha which induce its rapid degradation upon cytok ine 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 fo und 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 i ndirectly 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 al pha-, I kappa B beta- and I kappa B epsilon-derived peptides at the respect ive degradation-relevant serines suggests that the IKK complex exerts a bro ad regulatory role for the activation of different NF-kappa B species. In contrast to previous studies, which locate CK II phosphorylation sites e xclusively 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 e ndogenous CK II complex. Therefore, both p90(rsk1) and CK II have the same preference for phosphorylating only one of the two serines which are releva nt for inducible degradation.