Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.10 (IKK)
 4,900 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The multisubunit IkappaB kinase (IKK) catalyzes the signal-inducible phosphorylation of N-terminal serines of IkappaB. This phosphorylation is the key step in regulating the subsequent ubiquitination and proteolysis of IkappaB, which then releases NF-kappaB to promote gene transcription. As measured by 33P incorporation into a GST-IkappaB alpha fusion protein, varying both the concentration of GST-IkappaB alpha and [gamma-33P]ATP resulted in a kinetic pattern consistent with a random, sequential binding mechanism. Values of 55 nM and 7 microM were obtained for the dissociation constants of GST-IkappaB alpha and ATP, respectively. The value of alpha, a factor by which binding of one substrate changes the dissociation constant for the other substrate, was determined to be 0.11. This indicates that the two substrates bind in a cooperative fashion. Peptides corresponding to either amino acids 26-42 (N-terminal peptide) or amino acids 279-303 (C-terminal peptide) of IkappaB alpha inhibited the IKK-catalyzed phosphorylation of GST-IkappaB alpha; the C-terminal peptide, unexpectedly, was more potent. The inhibition by the C-terminal peptide was competitive with respect to GST-IkappaB alpha and mixed with respect to ATP, which verified the sequential binding mechanism. The C-terminal peptide was also a substrate for the enzyme, and a dissociation constant of 2.9-6.2 microM was obtained. Additionally, the N-terminal peptide was a substrate (Km = 140 microM). Competitive inhibition of the IKK-catalyzed phosphorylation of the C-terminal peptide by the N-terminal peptide indicated that the peptides are phosphorylated by the same active site. Surprisingly, the presence of the C-terminal peptide greatly accelerated the rate of phosphorylation of the N-terminal peptide as represented by a 160-fold increase in the apparent second-order rate constant (kcat/Km). These results are consistent with an allosteric site present within IKK that recognizes the C terminus of IkappaB alpha and activates the enzyme. This previously unobserved interaction with the C terminus may represent an important mechanism by which the enzyme recognizes and phosphorylates IkappaB.
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PMID:The multisubunit IkappaB kinase complex shows random sequential kinetics and is activated by the C-terminal domain of IkappaB alpha. 957 45

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.
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PMID:HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation. 963 Feb 30

A critical step in the signal-induced activation of the transcription factor NF-kappaB is the site-specific phosphorylation of its inhibitor, IkappaB, that targets the latter for degradation by the ubiquitin-proteasome pathway. We have previously shown that mitogen-activated protein kinase/ERK kinase kinase 1 (MEKK1) can induce both this site-specific phosphorylation of IkappaB alpha at Ser-32 and Ser-36 in vivo and the activity of a high molecular weight IkappaB kinase complex in vitro. Subsequently, others have identified two proteins, IkappaB kinase alpha (IKK-alpha) and IkappaB kinase beta (IKK-beta), that are present in a tumor necrosis factor alpha-inducible, high molecular weight IkappaB kinase complex. These kinases are believed to directly phosphorylate IkappaB based on the examination of the kinase activities of IKK immunoprecipitates, but more rigorous proof of this has yet to be demonstrated. We show herein that recombinant IKK-alpha and IKK-beta can, in fact, directly phosphorylate IkappaB alpha at Ser-32 and Ser-36, as well as homologous residues in IkappaB beta in vitro, and thus are bona fide IkappaB kinases. We also show that MEKK1 can induce the activation of both IKK-alpha and IKK-beta in vivo. Finally, we show that IKK-alpha is present in the MEKK1-inducible, high molecular weight IkappaB kinase complex and treatment of this complex with MEKK1 induces phosphorylation of IKK-alpha in vitro. We conclude that IKK-alpha and IKK-beta can mediate the NF-kappaB-inducing activity of MEKK1.
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PMID:MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. 968 78

The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-kappaB. Here we show that Tax triggers activation of cellular protein kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, which phosphorylate the NF-kappaB inhibitory protein IkappaB alpha, resulting in its degradation and NF-kappaB activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-kappaB signaling also requires the NF-kappaB-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-kappaB activation. Therefore, Tax activates NF-kappaB by targeting cellular signaling molecules, including both IKKs and NIK.
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PMID:NF-kappaB-inducing kinase and IkappaB kinase participate in human T-cell leukemia virus I Tax-mediated NF-kappaB activation. 969 68

Tax corresponds to a 40-kDa transforming protein from the pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that activates nuclear expression of the NF-kappaB/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of IkappaB alpha, a principal cytoplasmic inhibitor of NF-kappaB. Our studies now demonstrate that HTLV-1 Tax activates the recently identified cellular kinases IkappaB kinase alpha (IKKalpha) and IKKbeta, which normally phosphorylate IkappaB alpha on both of its N-terminal regulatory serines in response to tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) stimulation. In contrast, a mutant of Tax termed M22, which does not induce NF-kappaB, fails to activate either IKKalpha or IKKbeta. Furthermore, endogenous IKK enzymatic activity was significantly elevated in HTLV-1-infected and Tax-expressing T-cell lines. Transfection of kinase-deficient mutants of IKKalpha and IKKbeta into either human Jurkat T or 293 cells also inhibits NF-kappaB-dependent reporter gene expression induced by Tax. Similarly, a kinase-deficient mutant of NIK (NF-kappaB-inducing kinase), which represents an upstream kinase in the TNF-alpha and IL-1 signaling pathways leading to IKKalpha and IKKbeta activation, blocks Tax induction of NF-kappaB. However, plasma membrane-proximal elements in these proinflammatory cytokine pathways are apparently not involved since dominant negative mutants of the TRAF2 and TRAF6 adaptors, which effectively block signaling through the cytoplasmic tails of the TNF-alpha and IL-1 receptors, respectively, do not inhibit Tax induction of NF-kappaB. Together, these studies demonstrate that HTLV-1 Tax exploits a distal part of the proinflammatory cytokine signaling cascade leading to induction of NF-kappaB. The pathological alteration of this cytokine pathway leading to NF-kappaB activation by Tax may play a central role in HTLV-1-mediated transformation of human T cells, clinically manifested as the adult T-cell leukemia.
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PMID:Human T-cell leukemia virus type 1 Tax induction of NF-kappaB involves activation of the IkappaB kinase alpha (IKKalpha) and IKKbeta cellular kinases. 971 Jun

Optimal T cell activation and interleukin-2 production requires a second signal in addition to antigen-mediated T cell receptor (TCR) signaling. The CD28 molecule has been demonstrated to act as an effective costimulatory molecule upon binding by B7.1 or B7.2 present on antigen-presenting cells. The CD28 signal acts in concert with the TCR signal to significantly augment activation of the NF-kappaB family of transcription factors. The interleukin-2 gene is regulated by NF-kappaB among other transcription factors, in part, via a CD28 responsive element (CD28RE) present in the IL-2 promoter. Enhanced activation of NF-kappaB by CD28 is mediated by rapid phosphorylation and proteasome-mediated degradation of the NF-kappaB inhibitory proteins IkappaB alpha and IkappaB beta, which allows for accelerated nuclear expression of the liberated NF-kappaB. Herein, we provide evidence that the catalytic activities of two recently identified IkappaB kinases, IKKalpha and IKKbeta, are significantly elevated when T cells are stimulated through CD28 in addition to mitogen treatment. Catalytically inactive forms of IKKs are able to block the in vivo phosphorylation of IkappaB alpha induced by mitogen and CD28. Furthermore, CD28-mediated reporter gene transactivation of the CD28RE/AP-1 composite element is consistently attenuated by the IKK mutants. These findings suggest that cellular signaling pathways initiated at the TCR and CD28 converge at or upstream of IKK, resulting in more robust kinase activity and enhanced and prolonged NF-kappaB activation.
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PMID:IkappaB kinases serve as a target of CD28 signaling. 973 79

Nuclear factor-kappa B (NF-kappaB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kappaB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkappaB alpha and IkappaB beta. On activation, IkappaB is phosphorylated by IkappaB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kappaB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kappaB in the brain is unclear. In vitro, NF-kappaB activation can be either protective or deleterious. The role of NF-kappaB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kappaB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kappaB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kappaB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kappaB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kappaB activation in the MCAO model was evaluated. No significant activation of NF-kappaB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kappaB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kappaB.
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PMID:Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia. 1072 23

Vascular endothelial growth factor (VEGF) inhibits of the activation of transcription factor nuclear factor-kappaB (NF-kappaB) in hematopoietic progenitor cells (HPCs), and this is associated with alterations in the development of multiple lineages of hematopoietic cells and defective immune induction in tumor-bearing animals. Antibodies to VEGF have been shown to abrogate this effect. The mechanism by which VEGF antagonizes the induction of NF-kappaB was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor alpha (TNF-alpha) induced the nuclear translocation and DNA binding of p65-containing complexes, VEGF alone induced nuclear translocation and DNA binding of the complexes containing RelB. These results were confirmed by immunofluorescence confocal microscopy. VEGF effectively blocked TNF-alpha-induced NF-kappaB activation in HPCs from RelB-/- mice, however, similar to the effect observed in HPCs obtained from RelB+/- and RelB+/+ mice. This suggests that RelB is not required for VEGF to inhibit NF-kappaB activation. However, although TNF-alpha induced rapid activation of IkappaB kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of IkappaB alpha phosphorylation and degradation of IkappaB alpha and IkappaB epsilon in HPCs. VEGF alone, however, did not have any effect on phosphorylation of IkappaB alpha or degradation of IkappaB alpha and other inhibitory molecules IkappaB beta, IkappaB epsilon, or Bcl-3. SU5416, a potent inhibitor of the VEGF receptor I (VEGFR1) and VEGFR2 receptor tyrosine kinases, did not abolish the inhibitory effect of VEGF, indicating that the VEGF effect is mediated by a mechanism unrelated to VEGFR1 or VEGFR2 tyrosine kinase activity. Thus, VEGF appears to inhibit TNF-alpha-induced NF-kappaB activation by VEGFR kinase-independent inhibition of IKK. Therapeutic strategies aimed at overcoming VEGF-mediated defects in immune induction in tumor-bearing hosts will need to target this kinase-independent pathway.
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PMID:Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells. 1128 Jul 61

Hyperoxia and tumor necrosis factor-alpha (TNFalpha) are two canonical signals centrally involved in the pathophysiology of acute lung injury. We have attempted to elucidate the effects of these two stimuli on the signal transduction pathways of lung parenchymal cells. In cultured human lung epithelial cells, exposure to hyperoxia alone (95% oxygen) did not affect NF-kappaB activation or degradation of the NF-kappaB inhibitory protein, IkappaB alpha. Stimulation with TNFalpha alone increased NF-kappaB activation within 1 h and induced IkappaB alpha degradation within 0.5 h. After TNFalpha alone, NF-kappaB activation returned to baseline within 2 h and this corresponded with near complete IkappaB alpha resynthesis within 1 h of stimulation. In contrast, simultaneous exposure to hyperoxia and TNFalpha prolonged NF-kappaB activation up to 4 h, and IkappaB alpha degradation up to 2 h after stimulation. Hyperoxia did not affect TNFalpha-mediated resynthesis of IkappaB alpha mRNA. Hyperoxia alone did not induce IkappaB kinase (IKK) activity, but significantly prolonged TNFalpha-mediated activation of IKK activity. Hyperoxia alone did not activate the intercellular adhesion molecule-1 (ICAM-1) promoter, but augmented TNFalpha-mediated activation of the ICAM-1 promoter. These data demonstrate that while hyperoxia alone does not affect activation of NF-kappaB, hyperoxia prolongs TNFalpha-mediated activation of NF-kappaB. The mechanism of this effect involves, in part, prolonged degradation of IkappaB alpha resulting from prolonged activation of IKK.
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PMID:Hyperoxia prolongs tumor necrosis factor-alpha-mediated activation of NF-kappaB: role of IkappaB kinase. 1195 26

Interleukin-8 (IL-8) is a principle neutrophil chemoattractant and activator in humans. There is interest in developing novel pharmacological inhibitors of IL-8 gene expression as a means for modulating inflammation in disease states such as acute lung injury. Herein we determined the effects of epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, on tumor necrosis factor-alpha (TNF-alpha)-mediated expression of the IL-8 gene in A549 cells. EGCG inhibited TNF-alpha-mediated IL-8 gene expression in a dose response manner, as measured by ELISA and Northern blot analysis. This effect appears to primarily involve inhibition of IL-8 transcription because EGCG inhibited TNF-alpha-mediated activation of the IL-8 promoter in cells transiently transfected with an IL-8 promoter-luciferase reporter plasmid. In addition, EGCG inhibited TNF-alpha-mediated activation of IkappaB kinase and subsequent activation of the IkappaB alpha/NF-kappaB pathway. We conclude that EGCG is a potent inhibitor of IL-8 gene expression in vitro. The proximal mechanism of this effect involves, in part, inhibition of IkappaB kinase activation.
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PMID:A green tea-derived polyphenol, epigallocatechin-3-gallate, inhibits IkappaB kinase activation and IL-8 gene expression in respiratory epithelium. 1223 66


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