Regulation of the deubiquitinating enzyme CYLD by IkappaB kinase gamma-dependent phosphorylation

Tumor suppressor CYLD is a deubiquitinating enzyme (DUB) that inhibits the ubiquitination of key signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). However, how the function of CYLD is regulated remains unknown. Here we provide evidence that inducible phosphorylation of CYLD is an important mechanism of its regulation. Under normal conditions, CYLD dominantly suppresses the ubiquitination of TRAF2. In response to cellular stimuli, CYLD undergoes rapid and transient phosphorylation, which is required for signal-induced TRAF2 ubiquitination and activation of downstream signaling events. Interestingly, the CYLD phosphorylation requires IκB kinase gamma (IKKγ) and can be induced by IKK catalytic subunits. These findings suggest that CYLD serves as a novel target of IKK and that the site-specific phosphorylation of CYLD regulates its signaling function.     

Regulation of IkappaB kinase-related kinases and antiviral responses by tumor suppressor CYLD

The IkappaB kinase (IKK)-related kinases, IKKepsilon and TBK1, participate in the induction of type I interferons (IFNs) during viral infections. Deregulated activation of IKKepsilon and TBK1 also contributes to the abnormal cell survival and transformation. However, how these kinases are negatively regulated remains unclear. We show here that the tumor suppressor CYLD has an essential role in preventing aberrant activation of IKKepsilon/TBK1. CYLD deficiency causes constitutive activation of IKKepsilon/TBK1, which is associated with hyper-induction of IFNs in virus-infected cells. We further show that CYLD targets a cytoplasmic RNA sensor, RIG-I, and inhibits the ubiquitination of this IKKepsilon/TBK1 stimulator. Consistent with the requirement of ubiquitination in RIG-I function, CYLD potently inhibits RIG-I-mediated activation of the IFN-beta promoter. These findings establish CYLD as a key negative regulator of IKKepsilon/TBK1 and suggest a role for CYLD in the control of RIG-I ubiquitination.     

Control of death receptor and mitochondrial-dependent apoptosis by c-Jun N-terminal kinase in hippocampal CA1 neurones following global transient ischaemia

c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress-induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor-dependent apoptosis (caspase-8 activation 2 days after ischaemia) and a biphasic activation of caspase-3 and caspase-9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5-7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial-dependent apoptosis after transient ischaemia.     

An emerging role of deubiquitinating enzyme cylindromatosis (CYLD) in the tubulointerstitial inflammation of IgA nephropathy

Immunoglobulin A (IgA) nephropathy is an important cause of end-stage kidney disease (ESKD). Tubulointerstitial inflammation and subsequent fibrosis appear to be a major contributor of the disease progression to ESKD; however, the underlying mechanism is poorly understood. Herein, we report that a unique feature of CYLD expression in kidneys of patients with IgA nephropathy and a CYLD-mediated negative regulation of inflammatory responses in human tubular epithelial cells. Immunochemical staining revealed that CYLD was predominantly expressed in renal tubular epithelial cells in 81% of the patients (37 cases) with proteinuric IgA nephropathy. Patients with positive CYLD had significantly less tubulointerstitial lesions and higher estimated glomerular filtration rate (eGFR) levels when compared with those negative. Logistic regression analysis indicated that eGFR was a predictor for the CYLD expression. In cultured human tubular epithelial HK-2 cells, tumor necrosis factor-alpha (TNFα) up-regulated CYLD expression. Adenoviral knockdown of CYLD did not affect albumin-, hydrogen peroxide (H₂O₂)-, tunicamycin- or thapsigargin-induced cell death; however, it enhanced TNFα-induced expression of intracellular adhesion molecule (ICAM)-1 as well as activation of c-Jun N-terminal kinase (JNK). Moreover, monocyte adhesion to the TNFα-inflamed HK-2 cells was significantly increased by the CYLD shRNA approach. Taken together, our results suggest that CYLD negatively regulates tubulointertitial inflammatory responses via suppressing activation of JNK in tubular epithelial cells, putatively attenuating the progressive tubulointerstitial lesions in IgA nephropathy.     

CYLD deubiquitinase as a recurrent target in oncogenic processes

CYLD deubiquitinase has been originally defined as a tumor suppressor based exclusively on genetic findings. Indeed, inactivation of CYLD in humans results in familial cylindromatosis and multiple trichoepithelioma, two pathologies characterized by the development of tumors originating specifically from the skin appendages. A set of recent publications has revealed that recurrent inactivation of CYLD occurs through diverse mechanisms in several forms of cancer, unequivocally confirming its tumor suppressor function. This property is associated with the critical role played by CYLD in negatively regulating several signaling pathway, among them the NF-κB signaling pathway.     

The Rac1/JNK pathway is critical for EGFR-dependent barrier formation in human airway epithelial cells

The airway epithelial barrier provides defenses against inhaled antigens and pathogens, and alterations of epithelial barrier function have been proposed to play a significant role in the pathogenesis of chronic airway diseases. Although the epidermal growth factor receptor (EGFR) plays roles in various physiological and pathological processes on the airway epithelium, the role of EGFR on barrier function in the airway remains largely unknown. In the present study, we assessed the effects of EGFR activation on paracellular permeability in airway epithelial cells (AECs). EGFR activation induced by the addition of EGF increased transepithelial electrical resistance (TER) in AECs. An EGFR-blocking antibody eradicated the development of TER, paracellular influx of dextran, and spatial organization of tight junction. Moreover, the effects of EGFR activation on paracellular permeability were eradicated by knockdown of occludin. To identify the EGFR signaling pathway that regulates permeability barrier development, we investigated the effects of several MAP kinase inhibitors on permeability barrier function. Pretreatment with a JNK-specific inhibitor, but not an ERK- or p38-specific inhibitor, attenuated the development of TER induced by EGFR activation. Rac1 is one of the upstream activators for JNK in EGFR signaling. Rac1 knockdown attenuated the phosphorylation of JNK activation and EGFR-mediated TER development. These results suggest that EGFR positively regulates permeability barrier development through the Rac1/JNK-dependent pathway.     

Drosophila Smt3 negatively regulates JNK signaling through sequestering Hipk in the nucleus

Post-translational modification by the small ubiquitin-related modifier (SUMO) is important for a variety of cellular and developmental processes. However, the precise mechanism(s) that connects sumoylation to specific developmental signaling pathways remains relatively less clear. Here, we show that Smt3 knockdown in Drosophila wing discs causes phenotypes resembling JNK gain of function, including ectopic apoptosis and apoptosis-induced compensatory growth. Smt3 depletion leads to an increased expression of JNK target genes Mmp1 and puckered. We show that, although knockdown of the homeodomain-interacting protein kinase (Hipk) suppresses Smt3 depletion-induced activation of JNK, Hipk overexpression synergistically enhances this type of JNK activation. We further demonstrate that Hipk is sumolylated in vivo, and its nuclear localization is dependent on the sumoylation pathway. Our results thus establish a mechanistic connection between the sumoylation pathway and the JNK pathway through the action of Hipk. We propose that the sumoylation-controlled balance between cytoplasmic and nuclear Hipk plays a crucial role in regulating JNK signaling.     

Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis

Two key features of atherosclerotic plaques that precipitate acute atherothrombotic vascular occlusion ("vulnerable plaques") are abundant inflammatory mediators and macrophages with excess unesterified, or "free," cholesterol (FC). Herein we show that FC accumulation in macrophages leads to the induction and secretion of two inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The increases in TNF-alpha and IL-6 mRNA and protein were mediated by FC-induced activation of the IkappaB kinase/NF-kappaB pathway as well as activation of MKK3/p38, Erk1/2, and JNK1/2 mitogen-activated protein kinases (MAPK). Activation of IkappaB kinase and JNK1/2 was needed for the induction of both cytokines. However, MKK3/p38 signaling was specifically involved in TNF-alpha induction, and Erk1/2 signaling was required for IL-6. Most interestingly, activation of all of the signaling pathways and induction of both cytokines required cholesterol trafficking to the endoplasmic reticulum (ER). The CHOP branch of the unfolded protein response, an ER stress pathway, was required for Erk1/2 activation and IL-6 induction. In contrast, one or more other ER-related pathways were responsible for activation of p38, JNK1/2, and IkappaB kinase/NF-kappaB and for the induction of TNF-alpha. These data suggest a novel scenario in which cytokines are induced in macrophages by endogenous cellular events triggered by excess ER cholesterol rather than by exogenous immune cell mediators. Moreover, this model may help explain the relationship between FC accumulation and inflammation in vulnerable plaques.     

CYLD: A Multifunctional Deubiquitinase

The nuclear factor-kappaB (NF-κB) and c-Jun NH2-terminal kinase (JNK) signaling pathways regulate diverse biological processes, including the immune and inflammatory response, cell growth, apoptosis, and tumor formation. Not surprisingly therefore defects to either pathway contribute to the progression of numerous human disorders. Enhancing our understanding of the mechanisms that control signaling through these pathways is therefore significant as it may enable development of specific treatments. In this regard, CYLD was recently identified as a negative regulator of NF-κB and JNK signaling. CYLD has a C-terminal catalytic domain characteristic of deubiquitinating enzymes, and this is essential for CYLD to remove ubiquitin from certain proteins that positively mediate signaling through the NF-κB and JNK pathways. Recent studies have revealed a requirement for CYLD in many different processes and have provided some insight into the underlying mechanisms.     

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.     

Axin forms a complex with MEKK1 and activates c-Jun NH(2)-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling

Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i. e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade.     

Sh3rf2/POSHER protein promotes cell survival by ring-mediated proteasomal degradation of the c-Jun N-terminal kinase scaffold POSH (Plenty of SH3s) protein

We report that Sh3rf2, a homologue of the pro-apoptotic scaffold POSH (Plenty of SH3s), acts as an anti-apoptotic regulator for the c-Jun N-terminal kinase (JNK) pathway. siRNA-mediated knockdown of Sh3rf2 promotes apoptosis of neuronal PC12 cells, cultured cortical neurons, and C6 glioma cells. This death appears to result from activation of JNK signaling. Loss of Sh3rf2 triggers activation of JNK and its target c-Jun. Also, apoptosis promoted by Sh3rf2 knockdown is inhibited by dominant-negative c-Jun as well as by a JNK inhibitor. Investigation of the mechanism by which Sh3rf2 regulates cell survival implicates POSH, a scaffold required for activation of pro-apoptotic JNK/c-Jun signaling. In cells lacking POSH, Sh3rf2 knockdown is unable to activate JNK. We further find that Sh3rf2 binds POSH to reduce its levels by a mechanism that requires the RING domains of both proteins and that appears to involve proteasomal POSH degradation. Conversely, knockdown of Sh3rf2 promotes the stabilization of POSH protein and activation of JNK signaling. Finally, we show that endogenous Sh3rf2 protein rapidly decreases following several different apoptotic stimuli and that knockdown of Sh3rf2 activates the pro-apoptotic JNK pathway in neuronal cells. These findings support a model in which Sh3rf2 promotes proteasomal degradation of pro-apoptotic POSH in healthy cells and in which apoptotic stimuli lead to rapid loss of Sh3rf2 expression, and consequently to stabilization of POSH and JNK activation and cell death. On the basis of these observations, we propose the alternative name POSHER (POSH-eliminating RING protein) for the Sh3rf2 protein.     

Casein kinase Iepsilon modulates the signaling specificities of dishevelled

Wnt signaling is critical to many aspects of development, and aberrant activation of the Wnt signaling pathway can cause cancer. Dishevelled (Dvl) protein plays a central role in this pathway by transducing the signal from the Wnt receptor complex to the beta-catenin destruction complex. Dvl also plays a pivotal role in the planar cell polarity pathway that involves the c-Jun N-terminal kinase (JNK). How functions of Dvl are regulated in these two distinct pathways is not clear. We show that deleting the C-terminal two-thirds of Dvl, which includes the PDZ and DEP domains and is essential for Dvl-induced JNK activation, rendered the molecule a much more potent activator of the beta-catenin pathway. We also found that casein kinase Iepsilon (CKIepsilon), a previously identified positive regulator of Wnt signaling, stimulated Dvl activity in the Wnt pathway, but dramatically inhibited Dvl activity in the JNK pathway. Consistent with this, overexpression of CKIepsilon in Drosophila melanogaster stimulated Wnt signaling and disrupted planar cell polarity. We also observed a correlation between the localization and the signaling activity of Dvl in the beta-catenin pathway and the JNK pathway. Furthermore, by using RNA interference, we demonstrate that the Drosophila CKIepsilon homologue Double time positively regulates the beta-catenin pathway through Dvl and negatively regulates the Dvl-induced JNK pathway. We suggest that CKIepsilon functions as a molecular switch to direct Dvl from the JNK pathway to the beta-catenin pathway, possibly by altering the conformation of the C terminus of Dvl.     

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.