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

• Published in: The Journal of biological chemistry 2002-04, Vol.277 (16), p.13840
• Main Authors: Kishore, Nandini, Huynh, Q Khai, Mathialagan, Sumathy, Hall, Troii, Rouw, Sharon, Creely, David, Lange, Gary, Caroll, James, Reitz, Beverley, Donnelly, Ann, Boddupalli, Hymavathi, Combs, Rodney G, Kretzmer, Kuniko, Tripp, Catherine S
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Cell Line; Cloning, Molecular; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; I-kappa B Kinase; Inhibitory Concentration 50; Insecta; Jurkat Cells; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; NF-kappa B - metabolism; Peptides - chemistry; Phosphoric Monoester Hydrolases - metabolism; Phosphorylation; Protein Isoforms; Protein Structure, Tertiary; Protein-Serine-Threonine Kinases - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serine - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M110474200

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.