A role for NF-kappaB essential modifier/IkappaB kinase-gamma (NEMO/IKKgamma) ubiquitination in the activation of the IkappaB kinase complex by tumor necrosis factor-alpha

NEMO (NF-kappaB essential modifier)/IKKgamma (IkappaB kinase-gamma) is required for the activation of the IkappaB kinase complex (IKK) by inflammatory stimuli such as tumor necrosis factor (TNF-alpha). Here we show that TNF-alpha stimulates the ubiquitination of NEMO in a manner that does not appear to target it for degradation and that is impaired by mutations in the NEMO zinc finger. Mutations of the zinc finger are found in patients with hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID) and lead to the impairment of TNF-alpha-stimulated IKK phosphorylation and activation. In addition, the ubiquitination of NEMO is mediated by c-IAP1, an inhibitor of apoptosis protein that is a component of the TNF receptor signaling complex. Thus, the ubiquitination of NEMO mediated by c-IAP1 likely plays an important role in the activation of IKK by TNF-alpha. Also, defective NEMO ubiquitination may be responsible for the impaired cellular NF-kappaB signaling found in patients with HED-ID.     

Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation

Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.     

Cutting Edge: Pivotal function of Ubc13 in thymocyte TCR signaling

The Ubc13 E2 ubiquitin-conjugating enzyme is essential for BCR-, TLR-, and IL-1 receptor (IL-1R)-mediated immune responses. Although Ubc13-deficient mice show defects in BCR-, TLR/IL-1R-, or CD40-mediated activation of mitogen-activated protein kinases, the function of Ubc13 in TCR-mediated signaling and responses remains uncertain. To address this, we here generated T cell-specific conditional Ubc13-deficient mice. The frequency of T lymphocytes was severely reduced in spleens from Ubc13-deficient mice. Moreover, Ubc13-deficient thymocytes displayed defective proliferation in response to anti-CD3/CD28 or PMA/ionophore stimulation. Regarding the signal transduction, although NF-kappaB activation was modestly affected, PMA/ionophore-induced activation of Jnk and p38 was profoundly impaired in Ubc13-deficient thymocytes. In addition, PMA/ionophore-mediated ubiquitination of NF-kappaB essential modulator (NEMO)/IkappaB kinase gamma (IKKgamma) and phosphorylation of TGF-beta-activated kinase 1 (TAK1) were nearly abolished in Ubc13-deficient thymocytes. Thus, Ubc13 plays an important role in thymocyte TCR-mediated signaling and immune responses.     

Lymphotoxin-beta receptor mediates NEMO-independent NF-kappaB activation

Lymphotoxin-beta receptor (LTbetaR) is a member of the tumor necrosis factor receptor (TNFR) superfamily that activates nuclear factor-kappaB (NF-kappaB) through the IkappaB kinase (IKK) complex, the core of which is comprised of IKK1, IKK2 and NF-kappaB essential modulator (NEMO). We demonstrate here that the LTbetaR signaling to NF-kappaB activation does not necessarily require NEMO, which is essential for TNFR signaling. In the absence of NEMO, the p50 and RelB, but not RelA subunits of NF-kappaB are found in the nuclear DNA binding complexes induced by the LTbetaR signaling. Our results thus disclose NEMO-independent NF-kappaB activation by LTbetaR.     

The IkappaB kinase (IKK) inhibitor, NEMO-binding domain peptide, blocks osteoclastogenesis and bone erosion in inflammatory arthritis

Activation of NF-kappaB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IkappaB kinase (IKK) complex that phosphorylates IkappaB and leads to its dissociation from the NF-kappaB complex, thus permitting activation of NF-kappaB. The IKK complex contains primarily IKKalpha, IKKbeta, and the regulatory kinase IKKgamma, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKalpha and IKKbeta, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-kappaB. Given the pivotal role of cytokine-induced NF-kappaB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-kappaB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-kappaB in osteoinflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis.     

Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways

The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.     

Activation mechanisms of endothelial NF-kappaB, IKK, and MAP kinase by tert-butyl hydroperoxide

Lipid peroxidation plays a major role in vascular dysfunction and age-related cardiovascular diseases. A major product of lipid peroxidation, tert-butyl hydroperoxide (t-BHP), has been reported to modulate vascular reactivity and cellular signaling. To better understand vascular abnormality, we set out to delineate the activation mechanism of nuclear factor kappa B (NF-kappaB) by t-BHP and the regulation of MAPK in endothelial cells. The results showed that t-BHP induces NF-kappaB activation by an inhibitor of kappaB (IkappaB) phosphorylation through IkappaB kinase (IKK) activation. Our data from this t-BHP study also showed increased p38 MAP kinase and ERK activity; however, interestingly, t-BHP showed no influence on JNK. Pretreatment with the p38 MAP kinase inhibitor, SB203580 and the ERK1/2 inhibitor, PD98059, prevented t-BHP-induced increases in p65 translocation, NF-kappaB luciferase activity, and phospho-IKKalpha/beta. Data suggested that t-BHP induces NF-kappaB activation through the IKK pathway, which involves p38 MAPK and ERK activation. This study illustrates a role of t-BHP in NF-kappaB activation and MAPK related-signaling pathways. The t-BHP-induced activation of NF-kappaB and MAPK could be a major player in vascular dysfunctions, as seen in oxidative stressed responses and the vascular inflammatory process.     

IKKgamma /NEMO facilitates the recruitment of the IkappaB proteins into the IkappaB kinase complex

IKKgamma/NEMO is an essential regulatory component of the IkappaB kinase complex that is required for NF-kappaB activation in response to various stimuli including tumor necrosis factor-alpha and interleukin-1beta. To investigate the mechanism by which IKKgamma/NEMO regulates the IKK complex, we examined the ability of IKKgamma/NEMO to recruit the IkappaB proteins into this complex. IKKgamma/NEMO binding to wild-type, but not to a kinase-deficient IKKbeta protein, facilitated the association of IkappaBalpha and IkappaBbeta with the high molecular weight IKK complex. Following tumor necrosis factor-alpha treatment of HeLa cells, the majority of the phosphorylated form of endogenous IkappaBalpha was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKKgamma/NEMO-deficient cells. Finally, we demonstrate that IKKgamma/NEMO facilitates the association of the IkappaB proteins and IKKbeta and leads to increases in IKKbeta kinase activity. These results suggest that an important function of IKKgamma/NEMO is to facilitate the association of both IKKbeta and IkappaB in the high molecular weight IKK complex to increase IkappaB phosphorylation.     

Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation

Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.     

Yersinia outer protein P suppresses TGF-beta-activated kinase-1 activity to impair innate immune signaling in Yersinia enterocolitica-infected cells

Pathogenic Yersinia spp. use a panel of virulence proteins that antagonize signal transduction processes in infected cells to undermine host defense mechanisms. One of these proteins, Yersinia enterocolitica outer protein P (YopP), down-regulates the NF-kappaB and MAPK signaling pathways, which suppresses the proinflammatory host immune response. In this study, we explored the mechanism by which YopP succeeds to simultaneously disrupt several of these key signaling pathways of innate immunity. Our data show that YopP operates upstream of its characterized eukaryotic binding partner IkappaB kinase-beta to shut down the NF-kappaB signaling cascade. Accordingly, YopP efficiently impaired the activities of TGF-beta-activated kinase-1 (TAK1) in infected cells. TAK1 is an important activator of the IkappaB kinase complex in the TLR signaling cascade. The repression of TAK1 activities correlated with reduced activation of NF-kappaB- as well as AP-1-dependent reporter gene expression in Yersinia-infected murine macrophages. This suggests that the impairment of the TAK1 enzymatic activities by Yersinia critically contributes to down-regulate activation of NF-kappaB and of MAPK members in infected host cells. The inhibition of TAK1 potentially results from the blockade of signaling events that control TAK1 induction. This process could involve the attenuation of ubiquitination of the upstream signal transmitter TNFR-associated factor-6. Together, these results indicate that, by silencing the TAK1 signaling complex, Yersinia counteracts the induction of several conserved signaling pathways of innate immunity, which aids the bacterium in subverting the host immune response.     

Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappaB activation: TAO2 regulates TAK1 pathways

Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IkappaB kinase (IKK) that leads to degradation of IkappaB and subsequent NF-kappaB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IkappaB or activation of NF-kappaB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-kappaB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.     

Coordinated regulation of Toll-like receptor and NOD2 signaling by K63-linked polyubiquitin chains

K63 polyubiquitin chains spatially and temporally link innate immune signaling effectors such that cytokine release can be coordinated. Crohn's disease is a prototypical inflammatory disorder in which this process may be faulty as the major Crohn's disease-associated protein, NOD2 (nucleotide oligomerization domain 2), regulates the formation of K63-linked polyubiquitin chains on the I kappa kinase (IKK) scaffolding protein, NEMO (NF-kappaB essential modifier). In this work, we study these K63-linked ubiquitin networks to begin to understand the biochemical basis for the signaling cross talk between extracellular pathogen Toll-like receptors (TLRs) and intracellular pathogen NOD receptors. This work shows that TLR signaling requires the same ubiquitination event on NEMO to properly signal through NF-kappaB. This ubiquitination is partially accomplished through the E3 ubiquitin ligase TRAF6. TRAF6 is activated by NOD2, and this activation is lost with a major Crohn's disease-associated NOD2 allele, L1007insC. We further show that TRAF6 and NOD2/RIP2 share the same biochemical machinery (transforming growth factor beta-activated kinase 1 [TAK1]/TAB/Ubc13) to activate NF-kappaB, allowing TLR signaling and NOD2 signaling to synergistically augment cytokine release. These findings suggest a biochemical mechanism for the faulty cytokine balance seen in Crohn's disease.     

A point mutation in NEMO associated with anhidrotic ectodermal dysplasia with immunodeficiency pathology results in destabilization of the oligomer and reduces lipopolysaccharide- and tumor necrosis factor-mediated NF-kappa B activation

The NEMO (NF-kappaB essential modulator) protein plays a crucial role in the canonical NF-kappaB pathway as the regulatory component of the IKK (IkappaB kinase) complex. The human disease anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID) has been recently linked to mutations in NEMO. We investigated the effect of an alanine to glycine substitution found in the NEMO polypeptide of an EDA-ID patient. This pathogenic mutation is located within the minimal oligomerization domain of the protein, which is required for the IKK activation in response to diverse stimuli. The mutation does not dramatically change the native-like state of the trimer, but temperature-induced unfolding studied by circular dichroism showed that it leads to an important loss in the oligomer stability. Furthermore, fluorescence studies showed that the tyrosine located in the adjacent zinc finger domain, which is possibly required for NEMO ubiquitination, exhibits an alteration in its spectral properties. This is probably due to a conformational change of this domain, providing evidence for a close interaction between the oligomerization domain and the zinc finger. In addition, functional complementation assays using NEMO-deficient pre-B and T lymphocytes showed that the pathogenic mutation reduced TNF-alpha and LPS-induced NF-kappaB activation by altering the assembly of the IKK complex. Altogether, our findings provide understanding as to how a single point mutation in NEMO leads to the observed EDA-ID phenotype in relation to the NEMO-dependent mechanism of IKK activation.     

Phosphatidylinositol 3-kinase-dependent mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 and NF-kappa B signaling pathways are required for B cell antigen receptor-mediated cyclin D2 induction in mature B cells

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class I(A) PI-3K p85alpha regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85alpha regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85alpha(-/-) splenic B cells are defective in BCR-induced IkappaB kinase beta and IkappaBalpha phosphorylation. We demonstrate that NF-kappaB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IkappaB kinase beta and IkappaBalpha phosphorylation in p85alpha(-/-) splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-kappaB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.