title: Characterization of IkappaB kinases. IkappaB-alpha is not phosphorylated by Raf-1 or protein kinase C isozymes, but is a casein kinase II substrate.
abstract: The NF-kappaB 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-kappaB is sequestered in the cytoplasm tethered to an inhibitor protein, IkappaB. Activation comprises the rapid phosphorylation of IkappaB-alpha at N-terminal sites, which presumably marks IkappaB-alpha for proteolytic degradation and leads to release of NF-kappaB into the nucleus. In addition, IkappaB-alpha is constitutively phosphorylated at the C terminus, which may be a prerequisite for proper IkappaB function. Protein kinase C (PKC) is activated by 12-O-tetradecanoylphorbol-13-acetate and has been previously reported to phosphorylate IkappaB-alpha in vitro. As PKC has turned out to constitute a multigene family encoding isozymes with different biological functions, we have reinvestigated IkappaB-alpha phosphorylation by PKC using recombinant PKC isozymes expressed in insect cells. While crude PKC preparations were efficient IkappaB-alpha kinases, highly purified PKC isozymes completely failed to phosphorylate IkappaB-alpha. Biochemical separation of porcine spleen yielded at least two fractions with IkappaB-alpha 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 IkappaB-alpha directly, but associates with CKII, which efficiently phosphorylates the C terminus of IkappaB-alpha. Two-dimensional phosphopeptide mapping and high pressure liquid chromatography-mass spectroscopy analysis showed that all IkappaB-alpha kinases induced phosphorylation at the same prominent sites in the C terminus. Our results clearly indicate that PKC isozymes alpha, beta, gamma, delta, epsilon, eta, and zeta as well as Raf-1 are not IkappaB-alpha kinases. They furthermore demonstrate that IkappaB-alpha is targeted by several kinases, one of which appears to be CKII.
authors.affiliation: Institut für Klinische Molekularbiologie und Tumorgenetik, Forschungszentrum für Umwelt und Gesundheit, Marchioninistrasse 25, D-81377 München, Federal Republic of Germany.
authors.forename: P
authors.initials: P
authors.lastname: Janosch
authors.forename: M
authors.initials: M
authors.lastname: Schellerer
authors.forename: T
authors.initials: T
authors.lastname: Seitz
authors.forename: P
authors.initials: P
authors.lastname: Reim
authors.forename: M
authors.initials: M
authors.lastname: Eulitz
authors.forename: M
authors.initials: M
authors.lastname: Brielmeier
authors.forename: W
authors.initials: W
authors.lastname: Kölch
authors.forename: J M
authors.initials: JM
authors.lastname: Sedivy
authors.forename: H
authors.initials: H
authors.lastname: Mischak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D047390:Casein Kinase II; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D013552:Swine
chemical_list: D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007527:Isoenzymes; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000072000:NF-KappaB Inhibitor alpha; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D051550:I-kappa B Kinase; D011493:Protein Kinase C