Characterization of the Recombinant IKK1/IKK2 Heterodimer

• Published in: The Journal of biological chemistry Vol. 275; no. 34; pp. 25883 - 25891
• Main Authors: Huynh, Q. Khai, Boddupalli, Hymavathi, Rouw, Sharon A., Koboldt, Carol M., Hall, Troii, Sommers, Cindy, Hauser, Scott D., Pierce, Jennifer L., Combs, Rodney G., Reitz, Beverly A., Diaz-Collier, Judy A., Weinberg, Robin A., Hood, Becky L., Kilpatrick, Bryan F., Tripp, Catherine S.
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 01.08.2000
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M000296200

Nuclear factor kappa B (NF-κB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-κB activation depends on phosphorylation and degradation of its inhibitor protein, IκB, initiated by an IκB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IκB kinase 1 (IKK1) and IκB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-κB essential modulator. To better understand the role of IKKs in NF-κB activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K m values for ATP and IκBα peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 μ m , respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency ( k cat / K m ) of 47.50 h −1 μ m −1 using an IκBα peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h −1 μ m −1 ), its mutant rhIKK2 (S177E, S181E, 1.18 h −1 μ m −1 ), or rhIKK1 (0.02 h −1 μ m −1 ). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IκBα peptide, exhibited competitive inhibitory kinetics, only ADP with the low K i of 0.77 μ m may play a physiological role in regulation of the enzyme activity.