Kinetic Mechanisms of IκB-related Kinases (IKK) Inducible IKK and TBK-1 Differ from IKK-1/IKK-2 Heterodimer

• Published in: The Journal of biological chemistry Vol. 277; no. 15; p. 12550
• Main Authors: Q. Khai Huynh, Nandini Kishore, Sumathy Mathialagan, Ann M. Donnelly, Catherine S. Tripp
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 12-04-2002
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111526200

Nuclear factor-κB activation depends on phosphorylation and degradation of its inhibitor protein, IκB. The phosphorylation
of IκBα on Ser 32 and Ser 36 is initiated by an IκB kinase (IKK) complex that includes a catalytic heterodimer composed of IκB kinase 1 (IKK-1) and IκB
kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-κB essential modulator. Recently, two related IκB kinases, TBK-1
and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i
phosphorylate Ser 36 of IκBα. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins,
rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random
sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly
different. ATP has no effect on the binding of an IκBα peptide for the rhIKK-1/rhIKK-2 heterodimer (α = 0.99), whereas the
binding of ATP decreased the affinity of the IκBα peptide for both rhTBK-1 (α = 10.16) and rhIKK-i (α = 62.28). Furthermore,
the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation
constants of the IκBα peptide for each IKK isoforms is unique with rhTBK-1 being the highest ( K
IκBα = 69.87 μ m ), followed by rhIKK-i ( K
IκBα = 5.47 μ m ) and rhIKK-1/rhIKK-2 heterodimers ( K
IκBα = 0.12 μ m ). Thus this family of IκB kinases has very unique kinetic properties.