title: IKK-i and TBK-1 are enzymatically distinct from the homologous enzyme IKK-2: comparative analysis of recombinant human IKK-i, TBK-1, and IKK-2.
abstract: NF-kappaB is sequestered in the cytoplasm by the inhibitory IkappaB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IkappaBs leading to their degradation that results in NF-kappaB activation. IKK-1 and IKK-2 are two direct IkappaB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IkappaBalpha peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.
authors.affiliation: Department of Arthritis and Inflammation Pharmacology, Discovery Research, Pharmacia Corporation, St. Louis, Missouri 63167, USA. N.Kishore@pharmacia.com
authors.forename: Nandini
authors.initials: N
authors.lastname: Kishore
authors.forename: Q Khai
authors.initials: QK
authors.lastname: Huynh
authors.forename: Sumathy
authors.initials: S
authors.lastname: Mathialagan
authors.forename: Troii
authors.initials: T
authors.lastname: Hall
authors.forename: Sharon
authors.initials: S
authors.lastname: Rouw
authors.forename: David
authors.initials: D
authors.lastname: Creely
authors.forename: Gary
authors.initials: G
authors.lastname: Lange
authors.forename: James
authors.initials: J
authors.lastname: Caroll
authors.forename: Beverley
authors.initials: B
authors.lastname: Reitz
authors.forename: Ann
authors.initials: A
authors.lastname: Donnelly
authors.forename: Hymavathi
authors.initials: H
authors.lastname: Boddupalli
authors.forename: Rodney G
authors.initials: RG
authors.lastname: Combs
authors.forename: Kuniko
authors.initials: K
authors.lastname: Kretzmer
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Tripp
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D051550:I-kappa B Kinase; D020128:Inhibitory Concentration 50; D007313:Insecta; D019169:Jurkat Cells; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D016328:NF-kappa B; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012694:Serine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D010744:Phosphoric Monoester Hydrolases