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

Abstract

Activation of the transcription factor NF-kappaB depends on the specific dual phosphorylation of its inhibitor protein IkappaB by the homologous cytokine-inducible IkappaB kinases 1 and 2 (IKK1/2). Various IkappaB isoforms exist: IkappaBalpha, IkappaBbeta1/2 (two alternative splice variants), and IkappaBepsilon. However, the individual relevance and the specific regulation of these isoforms is not well-understood. We have studied the direct interaction of recombinant IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon with the recombinant homodimeric IKK2. Fluorescence-based active site titration revealed that each IKK2 dimer contains two binding sites for IkappaB. By using surface plasmon resonance analysis, we found that all IkappaB proteins interact with the IKK2 dimer following a noncooperative binding mechanism. Further, the four IkappaB proteins bind to the kinase with equilibrium dissociation constants (KD) in the range of 50-300 nM; the association rate constants for all IkappaB isoforms with IKK2 were between 6.0 x 10(3) and 22.5 x 10(3) M-1 s-1, and the dissociation rate constants were between 1.25 x 10(-3) and 1.75 x 10(-3) s-1. This high-affinity binding suggests that the previously observed preassociation of all analyzed IkappaB proteins with the biochemically purified 700 kDa IkappaB kinase (IKK) complex is based on a direct enzyme-substrate association between the various IkappaB isoforms and the IKK proteins. The apparent catalytic efficiencies (kcat/KM) of IKK2 for IkappaBalpha, IkappaBbeta1, IkappaBbeta2, and IkappaBepsilon were 22 x 10(3), 10 x 10(3), 5.4 x 10(3), and 8.5 x 10(3) s-1 M-1, respectively, with KM values ranging between 1.7 x 10(-6) and 3.2 x 10(-6) M and kcat values ranging between 1.5 x 10(-2) and 3.7 x 10(-2) s-1. The relative affinities and catalytic efficiencies of IKK2 for the IkappaB isoforms were also reflected by the kinetics observed for the TNF-induced, phosphorylation-dependent degradation of the alpha, beta1, beta2, and epsilon isoforms of IkappaB in human umbilical vein endothelial cells. Therefore, differential regulation of the IkappaB isoforms in some cell types is not a direct result of the IKK activity, but appears to be due to parallel events.

Inhibiting IκBβ–NFκB signaling attenuates the expression of select pro-inflammatory genes

“…Previous reports indicate that the kinetics of IκBβ degradation is unique and slower than that of IκBα following LPS stimulation (Thompson et al, 1995). Additionally, IKK is known to have a lower affinity for IκBβ when compared to IκBα (Heilker et al, 1999;Mercurio et al, 1997). It is possible that at lower doses, both parthenolide and BAY 11-7085 inhibit IKK activity enough to prevent phosphorylation of proteins that interact with IKK with lower affinity, including IκBβ.…”

Ubiquitination and Degradation of the Inhibitors of NF- B

“…Mathematical models and experimental data indicated that NF-kB dynamics are sensitive to the timing and duration of the IKK activity (Cheong et al 2008). Indeed, the affinity between IKK and the different IkBs correlates with their degradation rate, implicating IKK as the main factor that shapes the differential kinetics (Heilker et al 1999).…”

Characterization of the Recombinant IKK1/IKK2 Heterodimer

“…The IKK activity has been isolated and characterized from mammalian cells as well as from expression of recombinant IKK1 and IKK2 homodimers in baculovirus systems (9,12,15,(23)(24)(25)(26)(27). Both the isolated IKK complex from mammalian cells and the recombinant IKKs utilize all three isoforms of IBs, ␣, ␤, and ␥, as substrates equally well.…”