Characterization of the Recombinant IKK1/IKK2 Heterodimer

Abstract

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 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 M ؊1 using an IB␣ peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h ؊1 M ؊1 ), its mutant rhIKK2 (S177E, S181E, 1.18 h ؊1 M ؊1 ), or rhIKK1 (0.02 h ؊1 M ؊1 ). 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 M may play a physiological role in regulation of the enzyme activity.

A Selective IKK-2 Inhibitor Blocks NF-κB-dependent Gene Expression in Interleukin-1β-stimulated Synovial Fibroblasts

“…Binding of ␥-35 S-ATP also occurred only with the active, phosphorylated form of rhIKK-2. We have shown previously that the rhIKK-2 expressed in a baculovirus system is phosphorylated and active (36,37). As demonstrated previously, phosphatase-treated rhIKK-2 had no kinase activity, and Western blot analysis confirmed that equal amounts of rhIKK-2 were immunoprecipitated and assayed (Fig.…”

“…Antibody complexes were pelleted by centrifugation and washed 3 times with 1 ml of cold whole-cell lysis buffer followed by 2 washes in kinase buffer (25 mM HEPES, pH 7.6, 2 mM MgCl 2 , 2 mM MnCl 2 , 10 mM NaF, 5 mM DTT, and 1 mM phenylmethylsulfonyl fluoride). 100 -200 g of immunoprecipitated IKK was analyzed for kinase activity in a reaction containing 10 M biotinylated I B␣ peptide as substrate and 1 M [␥-33 P]ATP (2500 Ci/mmol) as described previously (36,37). After incubation at room temperature for 30 min, 25 l of the reaction mixture was withdrawn and added to a SAM TM 96 biotin capture plate.…”

“…Incorporation of [␥-33 P]ATP was measured using a Top-Count NXT (Packard Instrument Co.). K m determinations of rhIKK-2 have been described previously (36,37). Briefly, various concentrations of [␥-33 P]ATP or peptide substrates were used in the assay at a fixed (3ϫ K m ) concentration of the second substrate and 100 ng of rhIKK-2 in a final volume of 50 l. Following incubation for 30 min at 25°C, the reaction was stopped by the addition of 150 l of AG1XB resin in 900 mM sodium formate buffer, pH 3 (the resin is in slurry of 1 volume resin to 2 volume of sodium formate buffer).…”

BMS-345541 Is a Highly Selective Inhibitor of IκB Kinase That Binds at an Allosteric Site of the Enzyme and Blocks NF-κB-dependent Transcription in Mice

“…Not surprisingly, ADP has been shown to be a competitive inhibitor with respect to the ATP substrate (24), and the peptide inhibitor is a competitive inhibitor with respect to peptide substrate (20). As shown in Fig.…”

Compensatory IKKα activation of classical NF-κB signaling during IKKβ inhibition identified by an RNA interference sensitization screen

“…First, because IKK␤ and IKK␣ are in the same high-molecular-weight complex in the cytoplasm, it is conceivable that by inhibiting the ability of IKK␤ to phosphorylate I B␣, I B␣ may be more available to IKK␣ as a substrate. In this regard, it is notable that IKK␣ is considerably less active than IKK␤ as an I B␣ kinase in vitro (33), and thus if I B␣ is brought in proximity to the IKK␣␤␥ complex during cellular signaling, it may be preferentially phosphorylated by IKK␤. During antigen receptor stimulation, BCL10 is an additional substrate of IKK␤ (34,35).…”