IKK-i and TBK-1 are Enzymatically Distinct from the Homologous Enzyme IKK-2

• Published in: The Journal of biological chemistry 2002-04, Vol.277 (16), p.13840-13847
• 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
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110474200

NF-κB is sequestered in the cytoplasm by the inhibitory IκB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IκBs leading to their degradation that results in NF-κB activation. IKK-1 and IKK-2 are two direct IκB 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 IκBα 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.