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)

Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.
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PMID:All three IkappaB isoforms and most Rel family members are stably associated with the IkappaB kinase 1/2 complex. 991

IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.
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PMID:IKK1-deficient mice exhibit abnormal development of skin and skeleton. 1034 20

Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.
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PMID:IkappaB kinases phosphorylate NF-kappaB p65 subunit on serine 536 in the transactivation domain. 1052 9

We investigated the role of protein kinase C theta (PKCtheta) in the activation of the NF-kappaB cascade in primary human CD4(+) lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCtheta participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IkappaB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca(2+)-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCtheta physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4(+) T cells. The same set of stimuli also induced relocation of endogenous PKCtheta and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IkappaBalpha sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCtheta and IKKss colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCtheta activated IKKbeta in Jurkat T cells. Expression of dominant-active PKCtheta also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCtheta by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.
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PMID:The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28. 1112 Aug 19

Phosphatidylinositol 3'-kinase (PI3K) and the serine/threonine kinase AKT have critical roles in phosphorylating and transactivating the p65 subunit of nuclear factor kappaB (NF-kappaB) in response to the pro-inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). Mouse embryo fibroblasts (MEFs) lacking either the alpha or beta subunit of IkappaB kinase (IKK) were deficient in NF-kappaB-dependent transcription following treatment with IL-1 or TNF. However, in contrast to IKKbeta-null MEFs, IKKalpha-null MEFs were not substantially defective in the cytokine-stimulated degradation of Ikappabetaalpha or in the nuclear translocation of NF-kappaB. The IKK complexes from IKKalpha- or IKKbeta-null MEFs were both deficient in PI3K-mediated phosphorylation of the transactivation domain of the p65 subunit of NF-kappaB in response to IL-1 and TNF, and constitutively activated forms of PI3K or AKT did not potentiate cytokine-stimulated activation of NF-kappaB in either IKKalpha- or IKKbeta-null MEFs. Collectively, these data indicate that, in contrast to IKKbeta, which is required for both NF-kappaB liberation and p65 phosphorylation, IKKalpha is required solely for the cytokine-induced phosphorylation and activation of the p65 subunit of NF-kappaB that are mediated by the PI3K/AKT pathway.
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PMID:Distinct roles of the Ikappa B kinase alpha and beta subunits in liberating nuclear factor kappa B (NF-kappa B) from Ikappa B and in phosphorylating the p65 subunit of NF-kappa B. 1173 37

The translocation of the transcription factor NF-kappaB into the nucleus plays a critical role in many physiological events. In unstimulated cells, NF-kappaB is sequestered in the cytosol, bound to its inhibitor IkappaB. Activation primarily occurs via the IkappaB kinase (IKK) complex which phosphorylates IkappaBalpha at serines 32 and 36, creating a recognition site for IkappaB ubiquitination which then targets IkappaB for degradation. Often it is useful to measure IKK activity to assess upstream signaling events leading to NF-kappaB activation. Current methods of assessing IKK activity are limited to IKK isoforms which are recognized by available IKK antibodies. Here, we describe a procedure to qualitatively assess the overall IKK activity in a cell lysate which can be used on any IKK isoform capable of phosphorylating human IkappaBalpha. This nonradioactive assay is based on measurement of the ability of the cell lysate to phosphorylate GST-IkappaBalpha, as measured by Western blotting, using an anti-phospho-IkappaBalpha antibody. We have used this assay to observe the kinetics of TCR-mediated activation of IKK as compared to PMA/ionomycin in primary rat T cells. PMA/ionomycin induces maximal IKK activity within 1 min of stimulation and this activity remains elevated for over 20 min. In comparison, TCR ligation induces maximal IKK activity after 5 min of stimulation and this activity rapidly diminishes to background levels. These data indicate that different stimuli can activate and inactivate IKK with different kinetics and suggest that TCR-mediated activation of IKK is closely linked to the rapid phosphorylation and dephosphorylation, respectively, of TCR-associated kinases.
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PMID:Measurement of IKK activity in primary rat T cells: rapid activation and inactivation. 1213 32

The high affinity IgE Fc receptor (FcepsilonRI) beta chain functions as a signal amplifier and has been linked to atopy, asthma, and allergy. Herein, we report on a previously unrecognized negative regulatory role for the nonconventional beta chain immunoreceptor tyrosine-based activation motif that contains three tyrosine residues (YX5YX3Y). Degranulation and leukotriene production was found to be impaired in cells expressing the mutated FcepsilonRIbeta immunoreceptor tyrosine-based activation motifs FYY, YYF, FYF, and FFF. In contrast, cytokine synthesis and secretion were enhanced in the YFY and FFF mutants. FcepsilonRI phosphorylation and Lyn kinase co-immunoprecipitation was intact in the YFY mutant but was lost in the FYF and FFF mutants. The phosphorylation of Syk, LAT, phospholipase gamma1/2, and Srchomology 2 domain-containing protein phosphatase 2 was intact, whereas the phosphorylation of SHIP-1 was significantly reduced in the YFY mutant cells. The FYF and FFF mutants were defective in phosphorylating all of these molecules. In contrast, the phosphorylation of ERK, p38 MAPK, IkappaB kinase beta (IKKbeta), and nuclear NFkappaB activity was enhanced in the YFY and FFF mutants. These findings show that the FcepsilonRIbeta functions to both selectively amplify (degranulation and leukotriene secretion) and dampen (lymphokine) mast cell effector responses.
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PMID:The FcepsilonRIbeta immunoreceptor tyrosine-based activation motif exerts inhibitory control on MAPK and IkappaB kinase phosphorylation and mast cell cytokine production. 1535 79

Phosphorylation of NF-kappaB p65(RelA) serine 536 is physiologically induced in response to a variety of proinflammatory stimuli, but the responsible pathways have not been conclusively unraveled, and the function of this phosphorylation is largely elusive. In contrast to previous studies, we found no evidence for a role of c-Jun N-terminal kinase, p38 kinase, extracellular signal-regulated kinase, or phosphatidylinositol 3-kinase in interleukin-1- or tumor necrosis factor-induced Ser-536 phosphorylation, as revealed by pharmacological inhibitors. We were not able to suppress Ser-536 phosphorylation by either RNA interference directed at IkappaB kinase (IKK)-alpha/beta (the best characterized Ser-536 kinases so far) or the IKKbeta inhibitor SC-514 or dominant negative mutants of either IKK. A green fluorescent protein p65 fusion protein was phosphorylated at Ser-536 in the absence of IKK activation, suggesting the existence of IKKalpha/beta-independent Ser-536 kinases. Chromatographic fractionation of cell extracts allowed the identification of two distinct enzymatic activities phosphorylating Ser-536. Peak 1 represents an unknown kinase, whereas peak 2 contained IKKalpha, IKKbeta, IKKepsilon, and TBK1. Overexpressed IKKepsilon and TBK1 phosphorylate Ser-536 in vivo and in vitro. Reconstitution of mutant p65 proteins in p65-deficient fibroblasts that either mimicked phosphorylation (S536D) or preserved a predicted hydrogen bond between Ser-536 and Asp-533 (S536N) revealed that phosphorylation of Ser-536 favors interleukin-8 transcription mediated by TATA-binding protein-associated factor II31, a component of TFIID. In the absence of phosphorylation, the hydrogen bond favors binding of the corepressor amino-terminal enhancer of split to the p65 terminal transactivation domain. Collectively, our results provide evidence for at least five kinases that converge on Ser-536 of p65 and a novel function for this phosphorylation site in the recruitment of components of the basal transcriptional machinery to the interleukin-8 promoter.
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PMID:Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. 1548 27

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.
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PMID:Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha. 1567 38

The development and growth of the rat heart implies hyperplasia, which stops at birth, and hypertrophy, allowing cardiac mass to grow in response to programmed genetic events along with to haemodynamic overload. Moreover, hypertrophy is accomplished to apoptosis which controls the final number of myocardial cells, deletes vestigial structures, and takes part in remodelling the organ. Since at the basis of all these processes, which lead to the complete development of the heart, the activation of specific signalling pathways underlies, attention has been addressed to the role played in vivo by Protein Kinase C zeta (PKC zeta) in regulating NF-kB signalling system and intrinsic mitochondrial apoptotic route at days 1, 4, 10 and 22 of rat life. In fact, a role has been assigned to PKC zeta in indirectly phosphorylating IKBa, which peaks between 10 and 22 days, through a IKK determining, in turn, NF-kB activation, concomitantly to cytochrome c/Apaf 1 co-localization in the cytoplasm and caspase-9/caspase-3 activation, which leads to the occurrence of apoptosis. Thus a key role for PKC zeta in regulating the hypertrophic and apoptotic events leading to establishment of complete function in rat neonatal heart is here suggested.
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PMID:Protein kinase C zeta regulation of hypertrophic and apoptotic events occurring during rat neonatal heart development and growth. 1569 10


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