Characterization of IκB Kinases

The NF-κB transcription factor is activated by a wide variety of stimuli, including phorbol esters such as 12- O -tetradecanoylphorbol-13-acetate. In its inactive state, NF-κB is sequestered in the cytoplasm tethered to an inhibitor protein, IκB. Activation comprises the rapid phosphorylation of IκB-α at N-terminal sites, which presumably marks IκB-α for proteolytic degradation and leads to release of NF-κB into the nucleus. In addition, IκB-α is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IκB function. Protein kinase C (PKC) is activated by 12- O -tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IκB-α in vitro . As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IκB-α phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IκB-α kinases, highly purified PKC isozymes completely failed to phosphorylate IκB-α. Biochemical separation of porcine spleen yielded at least two fractions with IκB-α kinase activity, both of which were devoid of detectable PKC isozymes. One peak contained both Raf-1 and casein kinase II (CKII). Purified Raf-1 does not phosphorylate IκB-α directly, but associates with CKII, which efficiently phosphorylates the C terminus of IκB-α. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IκB-α kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes α, β, γ, δ, ϵ, η, and ζ as well as Raf-1 are not IκB-α kinases. They furthermore demonstrate that IκB-α is targeted by several kinases, one of which appears to be CKII.