Abstract
Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.
Highlights
NF-B1 is an inducible, ubiquitous transcription factor that is primarily involved in immune, inflammatory, and stress responses (1–3)
We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human proteins, rhTBK-1, rhIKK-I, and rhIKK-1/ rhIKK-2 heterodimers
The dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IB␣ peptide for each IKK isoforms is unique with rhTBK-1 being the highest (KIB␣ ؍69.87 M), followed by rhIKK-i (KIB␣ ؍5.47 M) and rhIKK-1/ rhIKK-2 heterodimers (KIB␣ ؍0.12 M)
Summary
SAMTM 96 Biotin capture plates were purchased from Promega. Anti-FLAG affinity resin, Nonidet P-40, BSA, ATP, ADP, and dithiothreitol were obtained from Sigma. The standard reaction mixture for heterodimer rhIKK-1/rhIKK-2 assay contained 5 M biotinylated IB␣ peptide, 1 M [␥-33P]ATP (about 1 ϫ 105 cpm), 1 mM DTT, 50 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 10 mM NaF, 25 mM Hepes buffer, pH 7.6, and enzyme solution (0.02 to 0.2 g) in a final volume of 50 l. Due to their high activity and higher Km for IB␣ peptide, rhTBK-1 or rhIKK-i (0.01 to 0.025 g) was assayed with higher concentrations of IB␣ peptide (100 –500 M) and ATP (5–15 M) In these cases, the reaction mixtures were diluted 20-fold with 25 mM Hepes buffer, pH 7.6, containing 1 mM DTT, 50 mM KCl, 2 mM MgCl2, 2 mM MnCl2, 10 mM NaF prior to adding 25 l of the diluted solution to a 96-well Promega plate. Initial velocity studies were performed with varying ATP concentrations at constant IB␣ concentrations and several fixed inhibitor concentrations
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