IKK-i and TBK-1 are Enzymatically Distinct from the Homologous Enzyme IKK-2

Abstract

NF-B is sequestered in the cytoplasm by the inhibitory IB proteins. Stimulation of cells by agonists leads to the rapid phosphorylation of IBs leading to their degradation that results in NF-B activation. IKK-1 and IKK-2 are two direct IB kinases. Two recently identified novel IKKs are IKK-i and TBK-1. We have cloned, expressed, and purified to homogeneity recombinant human (rh)IKK-i and rhTBK-1 and compared their enzymatic properties with those of rhIKK-2. We show that rhIKK-i and rhTBK-1 are enzymatically similar to each other. We demonstrate by phosphopeptide mapping and site-specific mutagenesis that rhIKK-i and rhTBK-1 are phosphorylated on serine 172 in the mitogen-activated protein kinase kinase activation loop and that this phosphorylation is necessary for kinase activity. Also, rhIKK-i and rhTBK-1 have differential peptide substrate specificities compared with rhIKK-2, the mitogen-activated protein kinase kinase activation loop of IKK-2 being a more favorable substrate than the IB␣ peptide. Finally, using analogs of ATP, we demonstrate unique differences in the ATP-binding sites of rhIKK-i, rhTBK-1, and rhIKK-2. Thus, although these IKKs are structurally similar, their enzymatic properties may provide insights into their unique functions.

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 After incubation at room temperature for 30 min, 25 l of the reaction mixture was withdrawn and added to a SAM TM 96 biotin capture plate.…”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).…”

“…For p65 FL-GST, 0.043-2.72 M concentrations were used. Once K m graphs were fitted using Grafit TM Phosphatase Treatment of IKK-2-rhIKK-2 and native IKK kinase complexes were subjected to phosphatase treatment as described previously (36). Briefly, 4 -8 g of rhIKK-2 or 0.5 mg of native IKK complex were immunoprecipitated as described above, washed, and resuspended in 50 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, and 2 mM MnCl 2 .…”

IKK-i/IKKϵ Controls Constitutive, Cancer Cell-associated NF-κB Activity via Regulation of Ser-536 p65/RelA Phosphorylation

“…IKK⑀ and TBK1 are enzymatically distinct from the homologous enzymes IKK␣ and IKK␤ (9) and have been shown to play important roles in the innate immune response. These kinases function as critical components of the interferon regulatory factor 3 (IRF3) and IRF7 signaling pathways involved in responses to viral infection or dsRNA treatment (10,11).…”