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J. Biol. Chem., Vol. 275, Issue 34, 25883-25891, August 25, 2000

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Characterization of the Recombinant IKK1/IKK2 Heterodimer
MECHANISMS REGULATING KINASE ACTIVITY^*

Q. Khai Huynh, Hymavathi Boddupalli, Sharon A. Rouw, Carol M. Koboldt, Troii Hall, Cindy Sommers, Scott D. Hauser, Jennifer L. Pierce, Rodney G. Combs, Beverly A. Reitz, Judy A. Diaz-Collier, Robin A. Weinberg, Becky L. Hood, Bryan F. Kilpatrick, and Catherine S. Tripp

From Discovery Research, G. D. Searle and Company, the Monsanto Life Science Company, St. Louis, Missouri 63167

Nuclear factor kappa B (NF-B) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-B activation depends on phosphorylation and degradation of its inhibitor protein, IB, initiated by an IB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IB kinase 1 (IKK1) and IB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-B essential modulator. To better understand the role of IKKs in NF-B 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 IB peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 µM, 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 h¹ µM¹ using an IB peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h¹ µM¹), its mutant rhIKK2 (S177E, S181E, 1.18 h¹ µM¹), or rhIKK1 (0.02 h¹ µM¹). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IB peptide, exhibited competitive inhibitory kinetics, only ADP with the low K_i of 0.77 µM may play a physiological role in regulation of the enzyme activity.

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