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.
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 synthetic glucocorticoid receptor (GR) ligand with the efficacy of a glucocorticoid, but without the accompanying side effects, would meet an unmet medical need for the treatment of inflammatory diseases. It was hypothesized that a GR ligand that shifted helix 12 in a manner distinct from an agonist and an antagonist would confer a distinct GR conformation, resulting in differential gene expression and, ultimately, dissociation of antiinflammatory activity from side effects. A structural feature expected to interfere with helix 12 was incorporated into a nonsteroidal, tricyclic scaffold to create novel, high-affinity, and selective GR ligands that manifested a dual function in cellular assays, partial but robust agonist activity for inflammatory cytokine inhibition, and full antagonist activity for reporter gene activation. In contrast, analogs not likely to hinder helix 12 exhibited partial agonist activity for reporter gene activation. The requirement of full antagonist activity for substantial side effect dissociation was demonstrated in primary human preadipocytes, hepatocytes, and osteoblasts in which effects on adipogenesis, key genes involved in gluconeogenesis, and genes important for bone formation were examined, respectively. The dissociated GR ligands, despite lacking significant reporter gene activation, weakly recruit a limited number of coactivators such as peroxisomal proliferator-activated receptor-γ coactivator 1α. Transcriptional activation was sensitive to both peroxisomal proliferator-activated receptor-γ coactivator 1α and GR levels, providing a basis for cell-selective modulation of gene expression. The antiinflammatory activity of the dissociated ligands was further demonstrated in mouse models of inflammation. Together these results suggest that these ligands are promising candidates with robust antiinflammatory activity and likely dissociation against glucocorticoid-induced side effects.
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