Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.10 (IKK)
4,900 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB 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 IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, 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) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.
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PMID:Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity. 1082 18

p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKgamma, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.
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PMID:Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha. 1115 90

Human T-cell leukemia virus type 1 oncoprotein Tax activates NF-kappaB through direct binding to IKK-gamma, the regulatory component of the IkappaB kinase complex. Mechanisms by which IKK-gamma adapts the Tax signal to the IkappaB kinase are poorly understood. Here we demonstrate that IKK-gamma forms homodimer and homotrimer both in vitro and in yeast or mammalian cells through a C-terminal domain comprising amino acids 251-419. In contrast, Tax protein targets a central region of IKK-gamma, which consists of amino acids 201-250. Interestingly, Tax stimulates the oligomerization of IKK-gamma, likely through direct binding. Taken together, our findings suggest a new model of Tax activation of NF-kappaB, in which Tax interacts with IKK-gamma to stimulate its oligomerization.
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PMID:Stimulation of IKK-gamma oligomerization by the human T-cell leukemia virus oncoprotein Tax. 1243 99

NF-kappa B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I kappa B kinase (IKK) is the converging point for the activation of NF-kappa B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-kappa B-dependent genes in IL-1 beta-induced rheumatoid arthritis-derived synovial fibroblasts in a dose-dependent manner. When the mechanism of NF-kappa B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I kappa B alpha phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I kappa B alpha and p65 were comparable substrates for IKK-2, with similar Km and Kcat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I kappa B alpha phosphorylation/degradation, affecting NF-kappa B nuclear import/export as well as the phosphorylation and transactivation of p65.
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PMID:A selective IKK-2 inhibitor blocks NF-kappa B-dependent gene expression in interleukin-1 beta-stimulated synovial fibroblasts. 1281 46

Nuclear factor kappa B (NF-kappaB) is a transcription factor pivotal for the development of inflammation. A dysregulation of NF-kappaB has been shown to play an important role in many chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Although classical NF-kappaB, a heterodimer composed of the p50 and p65 subunits, has been well studied, little is known about gene regulation by other hetero- and homodimeric forms of NF-kappaB. While p65 possesses a transactivation domain, p50 does not. Indeed, p50/p50 homodimers have been shown to inhibit transcriptional activity. We have recently shown that Interleukin-10 exerts its anti-inflammatory activity in part through the inhibition of NF-kappaB by blocking IkappaB kinase activity and by inhibiting NF-kappaB already found in the nucleus. Since the inhibition of nuclear NF-kappaB could not be explained by an increase of nuclear IkappaB, we sought to further investigate the mechanisms involved in the inhibition of NF-kappaB by IL-10. We show here that IL-10 selectively induced nuclear translocation and DNA-binding of p50/p50 homodimers in human monocytic cells. TNF-alpha treatment led to a strong translocation of p65 and p50, whereas pretreatment with IL-10 followed by TNF-alpha blocked p65 translocation but did not alter the strong translocation of p50. Furthermore, macrophages of p105/p50-deficient mice exhibited a significantly decreased constitutive production of MIP-2alpha and IL-6 in comparison to wild type controls. Surprisingly, IL-10 inhibited high constitutive levels of these cytokines in wt macrophages but not in p105/p50 deficient cells. Our findings suggest that the selective induction of nuclear translocation and DNA-binding of the repressive p50/p50 homodimer is an important anti-inflammatory mechanism utilized by IL-10 to repress inflammatory gene transcription.
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PMID:Molecular mechanisms of interleukin-10-mediated inhibition of NF-kappaB activity: a role for p50. 1467 66

Nuclear factor-kappaB (NF-kappaB) has a central role in coordinating the expression of a wide variety of genes that control cerebral ischemia. Although there has been intense research on NF-kappaB, its mechanisms in the ischemic brain have not been clearly elucidated. We investigated the temporal profile of NF-kappaB-related genes using a complementary DNA array method in wild-type mice and human copper/zinc-superoxide dismutase transgenic (SOD 1 Tg) mice that had low-level reactive oxygen species (ROS) by scavenging superoxide. Our DNA array showed that IkappaB kinase (IKK) complex (IKKalpha, beta, and gamma) mRNA in the wild-type mice was decreased as early as 1 h after reperfusion, after 30 mins of transient focal cerebral ischemia (tFCI). In contrast, tFCI in the SOD1 Tg mice caused an increase in the IKK complex. The IKK complex protein levels were also drastically decreased at 1 h in the wild-type mice, but did not change in the SOD 1 Tg mice throughout the 7 days. Electrophoretic mobility shift assay revealed activation of NF-kappaB DNA binding after tFCI in the wild-type mice. Nuclear factor-kappaB activation occurred at the same time, as did the phosphorylation and degradation of the inhibitory protein IkappaBalpha. However, SOD 1 prevented NF-kappaB activation, and phosphorylation and degradation of IkappaBalpha after tFCI. Superoxide production and ubiquitinated protein in the SOD 1 Tg mice were also lower than in the wild-type mice after tFCI. These results suggest that ROS are implicated in transient downregulation of IKKalpha, beta, and gamma in cerebral ischemia.
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PMID:Oxidative stress transiently decreases the IKK complex (IKKalpha, beta, and gamma), an upstream component of NF-kappaB signaling, after transient focal cerebral ischemia in mice. 1582 15

Transcription factor nuclear factor kappa B (NF-kappaB) plays a central role in the pathogenesis of classical Hodgkin lymphoma (cHL). In anaplastic large-cell lymphomas (ALCLs), which share molecular lesions with cHL, the NF-kappaB system has not been equivalently investigated. Here we describe constitutive NF-kappaB p50 homodimer [(p50)2] activity in ALCL cells in the absence of constitutive activation of the IkappaB kinase (IKK) complex. Furthermore, (p50)2 contributes to the NF-kappaB activity in Hodgkin/Reed-Sternberg (HRS) cells. Bcl-3, which is an inducer of nuclear (p50)2 and is associated with (p50)2 in ALCL and HRS cell lines, is abundantly expressed in ALCL and HRS cells. Notably, a selective overexpression of Bcl-3 target genes is found in ALCL cells. By immunohistochemical screening of 288 lymphoma cases, a strong Bcl-3 expression in cHL and in peripheral T-cell non-Hodgkin lymphoma (T-NHL) including ALCL was found. In 3 of 6 HRS cell lines and 25% of primary ALCL, a copy number increase of the BCL3 gene locus was identified. Together, these data suggest that elevated Bcl-3 expression has an important function in cHL and peripheral T-NHL, in particular ALCL.
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PMID:Elevated NF-kappaB p50 complex formation and Bcl-3 expression in classical Hodgkin, anaplastic large-cell, and other peripheral T-cell lymphomas. 1679 May 85

Over the last several years, significant progress has been made in identifying chromatin-regulated events that govern NF-kappaB transcription. Using either laminin attachment or tumor necrosis factor alpha as a physiological stimulus of NF-kappaB activation, we demonstrate that IkappaB kinase alpha (IKKalpha) is recruited to chromatin in distinct phases. In the initial phase, IKKalpha is responsible for derepressing the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT)-histone deacetylase 3 (HDAC3) corepressor complex from the p50 homodimer. However, in the latter phase, chromatin-bound IKKalpha coordinates the simultaneous phosphorylation of RelA/p65(S536) and SMRT(S2410) as detected by chromatin immunoprecipitation (ChIP) assays. Although phosphorylated SMRT remains bound to the active p50-RelA/p65 heterodimer of NF-kappaB, derepression of SMRT is evidenced by the loss of chromatin-associated HDAC3 activity. ChIP and re-ChIP analysis demonstrates that phosphorylation of RelA/p65(S536) and SMRT(S2410) occurs prior to acetylation of RelA/p65 at K310. Moreover, IKKalpha-induced phosphorylation of RelA/p65(S536) displaces corepressor activity, allowing p300-mediated acetylation of RelA/p65. Introduction of nonphosphorylatable mutants of RelA/p65 and SMRT proteins or the inhibition of IKK activity results in active repression of NF-kappaB promoters by tethering the SMRT-HDAC3 complex. Similar to phosphorylation within the Rel homology domain of RelA/p65, which governs an exchange of HDAC1 for CBP/p300 acetyltransferases, we demonstrate that phosphorylation within the transactivation domain of RelA/p65(S536) displaces SMRT-HDAC3 repressor activity, allowing p300 to acetylate RelA/p65.
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PMID:IkappaB kinase alpha-mediated derepression of SMRT potentiates acetylation of RelA/p65 by p300. 1638 38

Reactive astrogliosis is the gliotic response to brain injury with activated astrocytes and microglia being the major effector cells. These cells secrete inflammatory cytokines, proteinases, and proteinase inhibitors that influence extracellular matrix (ECM) remodeling. In astrocytes, the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) is up-regulated by interleukin-1 (IL-1), which is a major neuroinflammatory cytokine. We report that IL-1 activates TIMP-1 expression via both the IKK/NF-kappaB and MEK3/6/p38/ATF-2 pathways in astrocytes. The activation of the TIMP-1 gene can be blocked by using pharmacological inhibitors, including BAY11-7082 and SB202190, overexpression of the dominant-negative inhibitor of NF-kappaB (IkappaBalphaSR), or by the knock-down of p65 subunit of NF-kappaB. Binding of activated NF-kappaB (p50/p65 heterodimer) and ATF-2 (homodimer) to two novel regulatory elements located -2.7 and -2.2 kb upstream of the TIMP-1 transcription start site, respectively, is required for full IL-1-responsiveness. Mutational analysis of these regulatory elements and their weak activity when linked to the minimal tk promoter suggest that cooperative binding is required to activate transcription. In contrast to astrocytes, we observed that TIMP-1 is expressed at lower levels in gliomas and is not regulated by IL-1. We provide evidence that the lack of TIMP-1 activation in gliomas results from either dysfunctional IKK/NF-kappaB or MEK3/6/p38/ATF-2 activation by IL-1. In summary, we propose a novel mechanism of TIMP-1 regulation, which ensures an increased supply of the inhibitor after brain injury, and limits ECM degradation. This mechanism does not function in gliomas, and may in part explain the increased invasiveness of glioma cells.
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PMID:A novel mechanism of tissue inhibitor of metalloproteinases-1 activation by interleukin-1 in primary human astrocytes. 1701 36

The tertiary interactions between amide-I vibrators on the separate helices of transmembrane helix dimers were probed by ultrafast 2D vibrational photon echo spectroscopy. The 2D IR approach proves to be a useful structural method for the study of membrane-bound structures. The 27-residue human erythrocyte protein Glycophorin A transmembrane peptide sequence: KKITLIIFG(79)VMAGVIGTILLISWG(94)IKK was labeled at G(79) and G(94) with (13)C=(16)O or (13)C=(18)O. The isotopomers and their 50:50 mixtures formed helical dimers in SDS micelles whose 2D IR spectra showed components from homodimers when both helices had either (13)C=(16)O or (13)C=(18)O substitution and a heterodimer when one had (13)C=(16)O substitution and the other had (13)C=(18)O substitution. The cross-peaks in the pure heterodimer 2D IR difference spectrum and the splitting of the homodimer peaks in the linear IR spectrum show that the amide-I mode is delocalized across a pair of helices. The excitation exchange coupling in the range 4.3-6.3 cm(-1) arises from through-space interactions between amide units on different helices. The angle between the two Gly(79) amide-I transition dipoles, estimated at 103 degrees from linear IR spectroscopy and 110 degrees from 2D IR spectroscopy, combined with the coupling led to a structural picture of the hydrophobic interface that is remarkably consistent with results from NMR on helix dimers. The helix crossing angle in SDS is estimated at 45 degrees. Two-dimensional IR spectroscopy also sets limits on the range of geometrical parameters for the helix dimers from an analysis of the coupling constant distribution.
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PMID:Amide vibrations are delocalized across the hydrophobic interface of a transmembrane helix dimer. 1707 37


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