title: IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.
abstract: Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.
authors.affiliation: Signal Pharmaceuticals, Inc., San Diego, California 92121, USA. fmercuri@signalpharm.com
authors.forename: F
authors.initials: F
authors.lastname: Mercurio
authors.forename: B W
authors.initials: BW
authors.lastname: Murray
authors.forename: A
authors.initials: A
authors.lastname: Shevchenko
authors.forename: B L
authors.initials: BL
authors.lastname: Bennett
authors.forename: D B
authors.initials: DB
authors.lastname: Young
authors.forename: J W
authors.initials: JW
authors.lastname: Li
authors.forename: G
authors.initials: G
authors.lastname: Pascual
authors.forename: A
authors.initials: A
authors.lastname: Motiwala
authors.forename: H
authors.initials: H
authors.lastname: Zhu
authors.forename: M
authors.initials: M
authors.lastname: Mann
authors.forename: A M
authors.initials: AM
authors.lastname: Manning
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes; D016328:NF-kappa B / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D035602:Transcriptional Elongation Factors
chemical_list: D002352:Carrier Proteins; C114720:Elp1 protein, human; C491968:Ikbkap protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D046912:Multiprotein Complexes; D016328:NF-kappa B; D011994:Recombinant Proteins; D035602:Transcriptional Elongation Factors; 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