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

J Biol Chem. 1996 Jun 7;271(23):13868-74. doi: 10.1074/jbc.271.23.13868.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Casein Kinase II
  • Cell Line
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • I-kappa B Kinase
  • I-kappa B Proteins*
  • In Vitro Techniques
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Molecular Sequence Data
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism
  • Phosphorylation
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-raf
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spodoptera
  • Substrate Specificity
  • Swine

Substances

  • DNA-Binding Proteins
  • I-kappa B Proteins
  • Isoenzymes
  • NF-kappa B
  • Proto-Oncogene Proteins
  • Recombinant Proteins
  • NF-KappaB Inhibitor alpha
  • Casein Kinase II
  • Protein Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-raf
  • I-kappa B Kinase
  • Protein Kinase C