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)

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.
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PMID:Recombinant IkappaB kinases alpha and beta are direct kinases of Ikappa Balpha. 980 49

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

The p105 precursor protein of NF-kappaB1 acts as an NF-kappaB inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor alpha (TNFalpha) and interleukin-1alpha (IL-1alpha) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the IkappaB kinase (IKK) complex is essential for these pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (Asp-Ser(927)-Gly-Val-Glu-Thr), related to the IKK target sequence in IkappaBalpha, which is conserved between human, mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants in which serine/threonine residues within and adjacent to this motif were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression. This residue is also required for TNFalpha and IL-1alpha to stimulate p105 degradation. By using a specific anti-phosphopeptide antibody, it was confirmed that IKK2 overexpression induces serine 927 phosphorylation of co-transfected p105 and that endogenous p105 is also rapidly phosphorylated on this residue after TNFalpha or IL-1alpha stimulation. In vitro kinase assays with purified proteins demonstrated that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-kappaB1 p105 by direct phosphorylation of serine 927 in its PEST domain.
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PMID:Direct phosphorylation of NF-kappaB1 p105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis. 1129 57

Dbl is a guanine nucleotide exchange factor that activates the Rho family GTPases Cdc42, Rac, and Rho. Dbl and all three GTPases are strong activators of transcription factor NF kappa B, which has been shown to have an important role in Dbl-induced oncogenic transformation. Here we show that although Dbl activation of NF kappa B requires Cdc42, Rac, and Rho, the different GTPases activate NF kappa B by different mechanisms. Whereas Rac stimulates the activity of the I kappa B kinase IKK beta, Cdc42 and Rho activate NF kappa B without activating either IKK alpha or IKK beta. Like Dbl, Rac activation of IKK beta is mediated by the serine/threonine kinases NIK but not MEKK. This differs from Rac activation of the JNK pathway, which was previously shown to be mediated by MEKK. The pathway leading from Rho and Cdc42 to NF kappa B is more elusive, but our results suggest that it involves an IKK alpha/IKK beta-independent mechanism. Finally, we show that the signaling enzymes that mediate NF kappa B activation by Dbl and the Rho GTPases are also necessary for malignant transformation induced by oncogenic Dbl.
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PMID:Dbl and the Rho GTPases activate NF kappa B by I kappa B kinase (IKK)-dependent and IKK-independent pathways. 1133 92

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.
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PMID:Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88. 1197 20

NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
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PMID:betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932. 1248 91

The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of aurora1 and aurora2 were built using 1CDK as the template structure. Molecular dynamics and docking simulations, targeting the ATP binding site of aurora2 with adenylyl imidodiphosphate (AMP-PNP), staurosporine, and six small molecular S/T kinase inhibitors, identified active-site residues that interact with these inhibitors differentially. The docked structures of the aurora2-AMP-PNP and aurora2-staurosporine complexes indicated that the adenine ring of AMP-PNP and the indolocarbazole moiety of staurosporine have similar positions and orientations and provided the basis for the docking of the other S/T kinase inhibitors. Inhibitors with isoquinoline and quinazoline moieties were recognized by aurora2 in which H-89 and 6,7-dimethoxyquinazoline compounds exhibited high binding energies compared with that of staurosporine. The calculated binding energies for the docked small-molecule inhibitors were qualitatively consistent with the IC(50) values generated using an in vitro kinase assay. The aurora2 structural model provides a rational basis for site-directed mutagenesis of the active site; design of novel H-89, staurosporine, and quinazoline analogues; and the screening of the available chemical database for the identification of other novel, small-molecular entities.
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PMID:Targeting aurora2 kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design. 1265 23

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

The global incidence of diabetes is increasing at epidemic rates. Estimates suggest there are currently 150 million people with diabetes and this number is expected to double in the next 20 years. Type 2 diabetes accounts for 95% of all cases and is characterized in part by impaired sensitivity to insulin or 'insulin resistance'. Defects in the insulin signalling pathways underpin this resistance. In the current article we discuss the regulation of Insulin Receptor Substrate-1 (IRS-1), a protein that plays a pivotal role in insulin signalling and whose function is impaired in subjects with insulin resistance. Coordination of IRS-1 function is multi-faceted, involving phosphorylation of IRS-1 at multiple serine/threonine residues. This controls many aspects of IRS-1, including its interaction with the insulin receptor and subsequent tyrosine phosphorylation, as well as its subcellular distribution and targeting for degradation by the proteasome. Such tight control ensures appropriate transduction and attenuation of the insulin signal, thereby regulating insulin action in healthy individuals. Emerging evidence indicates that 'diabetogenic factors' associated with insulin resistance, such as TNFalpha and elevated circulating fatty acids, impact on insulin signalling at the level of IRS-1 serine/threonine phosphorylation. The expression and/or activity of several kinases, such as IkappaB kinase beta (IKKbeta) and salt-induced kinase 2 (SIK2), and the phosphorylation of IRS-1 at key sites, such as Ser307 and Ser789, are increased in states of insulin resistance. Identifying the pathways by which such factors activate these and other kinases, and defining the precise roles of specific serine/ threonine phosphorylation events in IRS-1 regulation, represent important goals which may eventually provide a rationale for therapeutic intervention.
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PMID:IRS-1 regulation in health and disease. 1458 87

The ubiquitously expressed interferon regulatory factor 3 (IRF-3) is directly activated following virus infection and functions as a key activator of the immediate-early Type 1 interferon (IFN) genes. Using DNA microarray analysis (8,556 genes) in Jurkat T cells inducibly expressing constitutively active IRF-3, several target genes directly regulated by IRF-3 were identified. Among the genes upregulated by IRF-3 were transcripts for a subset of known IFN-stimulated genes (ISGs), including ISG56, which functions as an inhibitor of translation initiation. Phosphorylation of C-terminal Ser/Thr residues--(382)GGASSLENTVDLHISNSHPLSLTSDQY(408)-is required for IRF-3 activation. Using C-terminal point mutations and a novel phosphospecific antibody, Ser396 was characterized as the minimal phosphoacceptor site required in vivo for IRF-3 activation following Sendai virus (SeV) infection, expression of viral nucleocapsid, or double-stranded RNA (dsRNA) treatment. The identity of the virus-activated kinase (VAK) activity that targets and activates IRF-3 and IRF-7 has remained a critical missing link in the understanding of interferon signaling. We report that the IKK-related kinases-IKKepsilon/TBK-1-are components of VAK that mediate IRF-3 and IRF-7 phosphorylation and thus functionally link the NF-kappaB and IRF pathways in the development of the antiviral response.
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PMID:Convergence of the NF-kappaB and interferon signaling pathways in the regulation of antiviral defense and apoptosis. 1503 28


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