The Kinetics of Association and Phosphorylation of IκB Isoforms by IκB Kinase 2 Correlate with Their Cellular Regulation in Human Endothelial Cells

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Abstract
Activation of the transcription factor NF-κB depends on the specific dual phosphorylation of its inhibitor protein IκB by the homologous cytokine-inducible IκB kinases 1 and 2 (IKK1/2). Various IκB isoforms exist: IκBα, IκBβ1/2 (two alternative splice variants), and IκBε. However, the individual relevance and the specific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant IκBα, IκBβ1, IκBβ2, and IκBε with the recombinant homodimeric IKK2. Fluorescence-based active site titration revealed that each IKK2 dimer contains two binding sites for IκB. By using surface plasmon resonance analysis, we found that all IκB proteins interact with the IKK2 dimer following a noncooperative binding mechanism. Further, the four IκB proteins bind to the kinase with equilibrium dissociation constants (KD) in the range of 50−300 nM; the association rate constants for all IκB isoforms with IKK2 were between 6.0 × 103 and 22.5 × 103 M-1 s-1, and the dissociation rate constants were between 1.25 × 10-3 and 1.75 × 10-3 s-1. This high-affinity binding suggests that the previously observed preassociation of all analyzed IκB proteins with the biochemically purified 700 kDa IκB kinase (IKK) complex is based on a direct enzyme−substrate association between the various IκB isoforms and the IKK proteins. The apparent catalytic efficiencies (kcat/KM) of IKK2 for IκBα, IκBβ1, IκBβ2, and IκBε were 22 × 103, 10 × 103, 5.4 × 103, and 8.5 × 103 s-1 M-1, respectively, with KM values ranging between 1.7 × 10-6 and 3.2 × 10-6 M and kcat values ranging between 1.5 × 10-2 and 3.7 × 10-2 s-1. The relative affinities and catalytic efficiencies of IKK2 for the IκB isoforms were also reflected by the kinetics observed for the TNF-induced, phosphorylation-dependent degradation of the α, β1, β2, and ε isoforms of IκB in human umbilical vein endothelial cells. Therefore, differential regulation of the IκB isoforms in some cell types is not a direct result of the IKK activity, but appears to be due to parallel events.