title: IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain.
abstract: Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.
authors.affiliation: Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., 16-89 Kashima 3-chome, Yodogawa-ku, Osaka 532-8505, Japan. hsakurai@tanabe.co.jp
authors.forename: H
authors.initials: H
authors.lastname: Sakurai
authors.forename: H
authors.initials: H
authors.lastname: Chiba
authors.forename: H
authors.initials: H
authors.lastname: Miyoshi
authors.forename: T
authors.initials: T
authors.lastname: Sugita
authors.forename: W
authors.initials: W
authors.lastname: Toriumi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013379:Substrate Specificity; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014981:Xenopus
chemical_list: D046911:Macromolecular Substances; D016328:NF-kappa B; D010710:Phosphates; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse