Characterization of the recombinant IKK1/IKK2 heterodimer - Mechanisms regulating kinase activity

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