Kinase TAK1 Research Articles

Abstract C155: Inhibition of TAK1 promotes apoptosis in KRAS and Wnt pathway codependent colon cancers.

Abstract Colorectal cancers frequently harbor KRAS mutations but KRAS-mutant cancers are refractory to conventional and targeted therapeutics. The primary aim of this study was to identify novel therapeutic strategies to target KRAS-mutant colon cancers. Firstly, we found that a subset of KRAS-mutant colorectal cancer cell lines are dependent upon KRAS signaling for survival. Secondly, we found that KRAS-dependent colorectal cancer cells require the TAK1 kinase (MAP3K7) for sustained viability. TAK1 depletion or the small molecule TAK1 kinase inhibitor 5Z-7-oxozeaenol results in profound apoptotic cell death in KRAS-dependent cells. Lastly, we noted that sensitivity to TAK1 disruption is associated with enhanced Wnt signaling resulting from APC mutations combined with KRAS hyperactivation. This effect can be attributed in part to a KRAS-mediated activation of BMP signaling, leading to TAK1 activation. TAK1 then directly regulates Wnt signaling specifically in KRAS-dependent cells. In primary human colorectal cancers, expression of a gene signature derived from TAK1-dependent cell lines is correlated with APC and KRAS mutational status. Thus, our studies reveal TAK1 inhibition as a potential therapeutic strategy for the generally treatment-refractory subset of colorectal cancers that are driven by the KRAS and Wnt pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C155.

The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells.

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic β-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.

The Deubiquitinase CYLD Targets Smad7 Protein to Regulate Transforming Growth Factor β (TGF-β) Signaling and the Development of Regulatory T Cells

CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-β led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4(+) cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-β signaling function in T cells and the development of Tregs through deubiquitination of Smad7.

Direct, Noncatalytic Mechanism of IKK Inhibition by A20

A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20's seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.

FOXO3a-Dependent Mechanism of E1A-Induced Chemosensitization

Gene therapy trials in human breast, ovarian, and head and neck tumors indicate that adenovirus E1A can sensitize cancer cells to the cytotoxic effects of paclitaxel in vitro and in vivo. Resistance to paclitaxel has been reported to occur in cells expressing low levels of the Forkhead transcription factor FOXO3a. In this article, we report that FOXO3a is critical for E1A-mediated chemosensitization to paclitaxel. RNA interference-mediated knockdown of FOXO3a abolished E1A-induced sensitivity to paclitaxel. Mechanistic investigations indicated that E1A indirectly stabilized FOXO3a by acting at an intermediate step to inhibit a ubiquitin-dependent proteolysis pathway involving the E3 ligase βTrCP and the FOXO3a inhibitory kinase IKKβ. E1A derepressed this inhibitory pathway by stimulating expression of the protein phosphatase 2A (PP2A)/C protein phosphatases, which by binding to the TGF-β-activated kinase TAK1, inhibited its ability to activate IKKβ and, thereby, to suppress βTrCP-mediated degradation of FOXO3a. Thus, by stimulating PP2A/C expression, E1A triggers a signaling cascade that stabilizes FOXO3a and mediates chemosensitization. Our findings provide a leap forward in understanding paclitaxel chemosensitization by E1A, and offer a mechanistic rational to apply E1A gene therapy as an adjuvant for improving therapeutic outcomes in patients receiving paclitaxel treatment.

RNAi Screening Identifies TAK1 as a Potential Target for the Enhanced Efficacy of Topoisomerase Inhibitors

In an effort to develop strategies that improve the efficacy of existing anticancer agents, we have conducted a siRNA-based RNAi screen to identify genes that, when targeted by siRNA, improve the activity of the topoisomerase I (Top1) poison camptothecin (CPT). Screening was conducted using a set of siRNAs corresponding to over 400 apoptosisrelated genes in MDA-MB-231 breast cancer cells. During the course of these studies, we identified the silencing of MAP3K7 as a significant enhancer of CPT activity. Follow-up analysis of caspase activity and caspase-dependent phosphorylation of histone H2AX demonstrated that the silencing of MAP3K7 enhanced CPT-associated apoptosis. Silencing MAP3K7 also sensitized cells to additional compounds, including CPT clinical analogs. This activity was not restricted to MDA-MB-231 cells, as the silencing of MAP3K7 also sensitized the breast cancer cell line MDA-MB-468 and HCT-116 colon cancer cells. However, MAP3K7 silencing did not affect compound activity in the comparatively normal mammary epithelial cell line MCF10A, as well as some additional tumorigenic lines. MAP3K7 encodes the TAK1 kinase, an enzyme that is central to the regulation of many processes associated with the growth of cancer cells (e.g. NF- κB, JNK, and p38 signaling). An analysis of TAK1 signaling pathway members revealed that the silencing of TAB2 also sensitizes MDA-MB-231 and HCT-116 cells towards CPT. These findings may offer avenues towards lowering the effective doses of Top1 inhibitors in cancer cells and, in doing so, broaden their application.

IKKi: a novel regulator of Act1, IL-17 signaling and pulmonary inflammation

IKKi: a novel regulator of Act1, IL-17 signaling and pulmonary inflammation

Tissue-Specific Alternative Splicing of Tak1 Is Conserved in Deuterostomes

Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific isoforms of a target gene that expedites a highly conserved signaling pathway.

TRIM5 Acts as More Than a Retroviral Restriction Factor

The retrovirus restriction factor TRIM5α blocks post-entry infection of retroviruses in a species-specific manner. As a cellular E3 ubiquitin ligase, TRIM5α binds to the retroviral capsid lattice in the cytoplasm of an infected cell and accelerates the uncoating process of retroviral capsid, thus providing a potent restriction to HIV-1 and other retrovirus infections. The precise mechanism by which this restriction is imposed remains under scrutiny, and evidence is lacking to link the E3 ubiquitin ligase activity of TRIM5α to its ability to restrict retrovirus infection. In a recent study, Pertel and colleagues have uncovered the link between the two, providing compelling evidence to suggest that following the interaction with the retroviral capsid, TRIM5 triggers an antiviral innate immune response by functioning as a pattern recognition receptor [1]. This unique function of TRIM5 is dependent on its association with the E2 ubiquitin-conjugating enzyme complex UBC13-UEV1A and subsequent activation of the TAK1 kinase complex and downstream genes involved in innate immune responses. These findings have defined a novel function for TRIM5 as a pattern recognition receptor in innate immune recognition and provided valuable mechanistic insight into its role as a retroviral restriction factor. Here we discuss the significance of these new findings in understanding TRIM5-mediated HIV restriction.

The Pleckstrin Homology Domain in the SKAP55 Adapter Protein Defines the Ability of the Adapter Protein ADAP To Regulate Integrin Function and NF-κB Activation

Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter Src kinase-associated phosphoprotein of 55 kDa (SKAP55). In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP-ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP-ADAP chimera associated with the LFA-1 integrin after TCR stimulation. Although the SKAP-ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP(-/-) T cells. A single point mutation in the pleckstrin homology domain of SKAP55 (R131M) blocks the ability of the SKAP-ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.

A class IA PI3K controls inflammatory cytokine production in human neutrophils

Neutrophils are generally the first leukocytes to arrive at sites of inflammation or injury, where they release a variety of inflammatory mediators, which contribute to shaping the ensuing immune response. Here, we show that in neutrophils exposed to physiological stimuli (i.e. LPS and TNF-α), inhibition of the PI3K signaling pathway impairs the synthesis and secretion of IL-8, Mip-1α, and Mip-1β. Further investigation showed that Mip-1α and Mip-1β gene transcription was similarly decreased, whereas IL-8 transcription and steady-state mRNA levels were unaffected. Accordingly, PI3K inhibition had no impact on NF-κB or C/EBP activation, which are essential for IL-8 transcription, but the basis for this selective inhibition of chemokine transcription remains elusive. We nevertheless identified translational targets of the PI3K pathway (S6, S6 kinase, 4E-BP1). Inhibitor studies and overexpression experiments further established that the various effects of PI3K on chemokine production can be ascribed to p85α and p110δ subunits. Finally, we show that in LPS- and TNF-activated neutrophils, PI3K acts downstream of the kinases p38 MAPK and TAK1. Given the importance of neutrophils and their products in numerous chronic inflammatory disorders, the PI3K pathway could represent an attractive therapeutic target.

TRIM5 is an innate immune sensor for the retrovirus capsid lattice

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by HIV-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm1,2. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice3,4 and several reports indicate that TRIM5 plays a role in signal transduction5–7, but the precise mechanism of restriction is unknown8. Here we demonstrate that TRIM5 promotes innate immune signaling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13/UEV1A, TRIM5 catalyzes the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (MAP3K7) kinase complex and stimulate AP-1 and NFκB signaling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13/UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NFκB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13/UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signaling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Toll-like receptor-4 signalling is specifically tak1-independent in synovial fibroblasts

Background and objectiveActivated synovial fibroblasts are key players in the pathogenesis of rheumatoid arthritis (RA) by driving inflammation and joint destruction. Numerous molecules including cytokines and toll-like receptor (TLR) ligands...

RNA-Binding Protein AUF1 Regulates Lipopolysaccharide-Induced IL10 Expression by Activating IκB Kinase Complex in Monocytes

ABSTRACT Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40 AUF1 cDNA restores both signaling pathways. To define the molecular mechanisms by which p40 AUF1 controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40 AUF1 serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40 AUF1 does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40 AUF1 regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.

Phosphorylation and Polyubiquitination of Transforming Growth Factor β-Activated Kinase 1 Are Necessary for Activation of NF-κB by the Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor

Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) protein has been shown to induce several signaling pathways leading to the modulation of host gene expression. The hijacking of these pathways facilitates the viral life cycle and leads to tumorigenesis. In the present work, we show that transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is an important player in NF-κB activation induced by vGPCR. We observed that the expression of an inactive TAK1 kinase mutant (TAK1M) reduces vGPCR-induced NF-κB nuclear translocation and transcriptional activity. Consequently, the expression of several NF-κB target genes normally induced by vGPCR was blocked by TAK1M expression, including interleukin 8 (IL-8), Gro1, IκBα, COX-2, cIAP2, and Bcl2 genes. Similar results were obtained after downregulation of TAK1 by small interfering RNA (siRNA) technology. The expression of vGPCR recruited TAK1 to the plasma membrane, and vGPCR interacts with TAK1. vGPCR expression also induced TAK1 phosphorylation and lysine 63-linked polyubiquitination, the two markers of the kinase's activation. Finally, inhibition of TAK1 by celastrol inhibited vGPCR-induced NF-κB activation, indicating this natural compound could be used as a potential therapeutic drug against KSHV malignancies involving vGPCR.

Inhibition of Tak-1 by AZ-Tak1 Impairs NF-κB Activation, Downregulates XIAP and Activates Caspase-9 Inducing Apoptosis In Mantle Cell lymphoma

AbstractAbstract 2852TGF-b-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, plays a key role in regulating inflammation, immunity, metabolism, and cell death in a variety of cell types. It is activated in response to a variety of cytokines, including tumor necrosis factor (TNF), TGF-b, and interleukin 1 (IL-1). Upon receptor binding, TAK1 binds to adaptor proteins, and subsequently phosphorylate downstream molecules leading to activation of p38MAPK, JNK, and NF-kB. In this study, we examined the expression pattern of TAK1 and its potential therapeutic role as a target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). In contrast, PBL from healthy donors had no expression of TAK1 protein. TAK1 was also highly expressed in primary lymph node sections of MCL compared with benign reactive lymph nodes. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1–0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when TAK1 expression in MCL cells was downregulated by TAK1- specific SiRNA and when primary mantle cell lymphoma specimens were examined after treatment with AZ-Tak1 for 24h (300nM). Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, nuclear NF-κB p65 levels were decreased, cytosolic levels of SMAC/DIABLO and cytochrome-C were increased in AZ-Tak1 treated cells, which were associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and consequent cells apoptosis. Collectively, our data demonstrate that TAK1 is essential for MAPK and NF-κB activation. Inhibition of TAK1 by the small molecule inhibitor AZ-Tak1 or TAK1-SiRNA induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma. Disclosures:No relevant conflicts of interest to declare.

Ablation of TAK1 Upregulates Reactive Oxygen Species and Selectively Kills Tumor Cells

TAK1 kinase activates multiple transcription factors and regulates the level of reactive oxygen species (ROS). We have previously reported that ablation of TAK1 in keratinocytes causes hypersensitivity to ROS-induced cell apoptosis. It is known that some tumor cells produce ROS at higher levels compared with normal cells. We used inducible epidermal-specific TAK1 knockout mice and examined whether ablation of TAK1 in preexisting skin tumors could cause an increase in ROS and result in tumor cell death. Deletion of tak1 gene in skin tumors caused the accumulation of ROS and increased apoptosis, and skin tumors totally regressed within 5 to 10 days. Normal skin did not exhibit any significant abnormality on tak1 gene deletion. Thus, TAK1 kinase could be a new and effective molecular target for ROS-based tumor killing.

ATM- and NEMO-Dependent ELKS Ubiquitination Coordinates TAK1-Mediated IKK Activation in Response to Genotoxic Stress

Activation of the transcription factor NF-κB by multiple genotoxic stimuli modulates cancer cell survival. This response is mediated by a conserved pathway involving the nuclear ATM kinase and cytoplasmic IκB kinase (IKK); however, the molecular link between them remains incompletely understood. Here we show that ATM activates the IKK kinase TAK1 in a manner dependent on IKKγ/NEMO and ELKS (a protein rich in glutamate, leucine, lysine, and serine). K63-linked polyubiquitination of ELKS, dependent on the ubiquitin ligase XIAP and the conjugating enzyme UBC13, allows ELKS association with TAK1 via its ubiquitin-binding subunits TAB2/3. Although NEMO mutants defective in ubiquitin binding permit ATM-dependent TAK1 activation, they block NEMO association with ELKS and IKK activation. Thus, ATM- and NEMO-dependent ubiquitination of ELKS leads to the ubiquitin-dependent assembly of TAK1/TAB2/3 and NEMO/IKK complexes, resulting in IKK and NF-κB activation following genotoxic stimuli.

Mechanism of Hypoxia-Induced NF-κB

ABSTRACT NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.