Detection of IKKβ-IKKγ Subcomplexes in Monocytic Cells and Characterization of Associated Signaling

Quirling, Martina; Page, Sharon; Jilg, Nikolaus; Plenagl, Katharina; Péus, Dominik; Grubmüller, Christine; Weingärtner, Monika; Neumeier, D.; Brand, Korbinian

doi:10.1074/jbc.m312119200

Cited by 9 publications

(12 citation statements)

References 48 publications

(87 reference statements)

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“…In any event, the rapid fall in IKK␣ levels observed upon neutrophil stimulation indicates that to a great extent, the activated and functional IKK complex of neutrophils must mainly comprise IKK␤ and IKK␥. Such a scenario is consistent with the recent demonstration that endogenous IKK␤-IKK␥ subcomplexes exist in the cytosplasm of human monocytic THP-1 cells, and display increased IKK activity following cell stimulation with TNF (59). Similarly, immunodepletion studies showed the presence of IKK␤-IKK␥ complexes activated by anti-CD3/CD28 in primary human T cells (60).…”

Section: Discussionsupporting

confidence: 75%

Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

Ear

Cloutier

McDonald

2005

The Journal of Immunology

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A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-κB/Rel proteins and IκB-α in the nucleus. In this study, we show that in these cells, IκB kinase α (IKKα), IKKβ, and IKKγ also partially localize to the nucleus, whereas IKK-related kinases (IKKε, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-κB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKβ and IKKγ become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKα transiently vanishes from both compartments in what appears to be an IKKβ-dependent process. These responses are paralleled by the degradation of IκB-α, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IκB-α phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (α, β, γ) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-κB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IκB signaling cascade within the cell nucleus.

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Section: Discussionsupporting

confidence: 75%

Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

Ear

Cloutier

McDonald

2005

The Journal of Immunology

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“…Importantly, the differential effect of the Ser 68 phosphorylation on the IKK␣ or IKK␤ interaction could have variable effects on IKK complexes depending on whether they contain IKK␣-IKK␤ heterodimers or homodimers of IKK␣ or IKK␤. Indeed, differences in the activity and regulation of IKK␣-IKK␤ heterodimers and IKK␤-homodimers have been observed (15). Furthermore, the recently described IKK␣-NEMO and IKK␤-NEMO heterodimers suggest the existence of different subpools of IKK complexes with a different composition regarding IKK␣-homodimers, IKK␤-homodimers, and/or IKK␣-IKK␤-heterodimers (22).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity

Palkowitsch

Leidner

Ghosh

et al. 2008

Journal of Biological Chemistry

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The transcription factor NF-B plays a crucial role in the initiation of innate and adaptive immune responses, in inflammation and tumorigenesis (1-3). In its inactive state NF-B is bound to small cytoplasmic proteins, the IB proteins. Stimulation with a wide variety of agonists, for example pro-inflammatory cytokines like TNF-␣, 2 bacterial components like lipopolysaccharide or by antigen receptors, funnels in the activation of a multisubunit IB-kinase complex, which phosphorylates the IB proteins at two specific serine residues. This phosphorylation marks the IB proteins for proteasomal degradation setting NF-B free, which then translocates into the nucleus and supports the expression of various pro-inflammatory or anti-apoptotic gene products. The IB kinase (IKK) complex is composed of two catalytically active subunits, termed IKK␣ (IKK1) and IKK␤ (IKK2), as well as NEMO/IKK␥, a subunit with regulatory and adaptor functions (4, 5). Analysis of mice deficient for either IKK␣ or IKK␤ suggested that the IKK␤ subunit is the major IKK regulating the canonical NF-B pathway, and the IKK␣ subunit is crucial for a second, alternative NF-B pathway leading to the activation of RelB-p52 heterodimers (6, 7). In contrast to the canonical NF-B pathway, which depends on the presence of NEMO, the alternative pathway is NEMO-independent but requires the protein kinase NIK. However, recent data suggest that, instead of a clear assignment of IKK␣ and IKK␤ to the alternative and the canonical NF-B pathway, respectively, both kinases contribute to the activation of NF-B by the canonical pathway with only gradual differences (8,9). Although a precise model regarding the molecular mechanism underlying IKK activation is still missing, it became clear that various post-translational modifications at the different IKK subunits are involved in this process. Besides the phosphorylation of IKK␣ and IKK␤ at two serine residues in their T-loop, the ubiquitination and occasionally the phosphorylation at a specific serine residue of NEMO seem to be required (10 -12). This serine residue at position 85 of NEMO has been identified as a protein kinase C␣, and more recently as an ATM target-site crucial for the IKK activation induced by genotoxic stress (12, 13). In addition, overexpression of the TNF-␣-receptor I or the human T-cell lymphotrophic virus I Tax protein induces the IKK␤-mediated NEMO phosphorylation at several

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“…33,34 Recently, it was shown that the main function of IKKa binding to IKKb and IKKg is to increase the activity of IKK complex and to accelerate downstream signaling pathways such as proteolysis of IjBa. 35 Earlier investigations in our laboratory have shown that, although prostate cancer cells respond to TRAIL treatment, some cell lines, such as LNCaP, are resistant. Therefore, we have been exploring combination therapies to sensitize these cells to TRAIL and to understand the mechanism by which these cells are sensitized.…”

mentioning

confidence: 99%

SAHA‐sensitized prostate cancer cells to TNFα‐related apoptosis‐inducing ligand (TRAIL): Mechanisms leading to synergistic apoptosis

Lakshmikanthan

Kaddour‐Djebbar

Lewis

et al. 2006

Intl Journal of Cancer

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Treatment of cancer cells with histone deacetylase inhibitors (HDACi) such as suberolylanilide hydroxamic acid (SAHA) activates genes that promote apoptosis. To enhance proapoptotic efficiency, SAHA has been used in combination with radiation, kinase inhibitors and cytotoxic drugs. Although several prostate cells respond to TNFα‐Related Apoptosis‐Inducing Ligand (TRAIL), LNCaP are resistant. This model system was utilized to examine the advantages of combined treatment with SAHA and TRAIL. In LNCaP cells, TRAIL induced synergistic apoptosis when combined with even with the lowest dose SAHA. Treatment with caspase inhibitor confirmed that SAHA‐induced apoptosis was mediated through caspases. In addition to induction of apoptosis, SAHA and TRAIL decreased the levels of proapoptotic proteins IKKα, IKKβ and IKKγ, suggesting that SAHA treatment may reduce the activity of NFκB. However, assay for NFκB luciferase reporter activity showed highly significant increase in SAHA‐treated cells, supporting earlier suggestions that HDACi promotes NFκB transcriptional activity. Further analyses to determine the mechanisms by which the combination of SAHA and TRAIL led to synergistic apoptosis indicated that the apoptotic response of LNCaP is due to a complex regulation of death receptor pathway and alterations of NFκB activity at several regulatory steps. © 2006 Wiley‐Liss, Inc.

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Detection of IKKβ-IKKγ Subcomplexes in Monocytic Cells and Characterization of Associated Signaling

Cited by 9 publications

References 48 publications

Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

Constitutive Nuclear Expression of the IκB Kinase Complex and Its Activation in Human Neutrophils

Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity

SAHA‐sensitized prostate cancer cells to TNFα‐related apoptosis‐inducing ligand (TRAIL): Mechanisms leading to synergistic apoptosis

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