Characterization of I kappa B kinases - I kappa B-alpha is not phosphorylated by Raf-1 or protein kinase C isozymes, but is a casein kinase II substrate

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