The CATERPILLER protein monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor alpha-, and Mycobacterium tuberculosis-induced pro-inflammatory signals

The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) alpha and Mycobacterium tuberculosis. Monarch-1 reduces NFkappaB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFkappaB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFkappaB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFalpha, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation.     

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.     

Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.     

Regulation of p53 and Rb Links the Alternative NF-κB Pathway to EZH2 Expression and Cell Senescence

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis.     

Structure of the Nuclear Factor κB-inducing Kinase (NIK) Kinase Domain Reveals a Constitutively Active Conformation

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5′-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.     

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK^−/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.Subject terms: Cancer metabolism, Energy metabolism, Cancer metabolism     

Tumor Necrosis Factor (TNF) Signaling,but Not TWEAK (TNF-like Weak Inducer of Apoptosis)-triggered cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) Degradation,Requires cIAP1 RING Dimerization and E2 Binding

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-κB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-κB, however, delayed activation of NF-κB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-κB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.     

Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

Background

Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.

Methodology/Principal Findings

Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.

Conclusions/Significance

Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer.     

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Activates the JNK Pathway through Its Extreme C Terminus via a Mechanism Involving TRADD and TRAF2

The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-κB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-κB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-κB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro³⁷⁹, Glu³⁸¹, Ser³⁸³, or Tyr³⁸⁴ diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-κB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-κB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-κB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-κB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.     

RIP4 (DIK/PKK), a novel member of the RIP kinase family,activates NF-kappa B and is processed during apoptosis

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals.     

The distinct nucleotide binding states of the transporter associated with antigen processing (TAP) are regulated by the nonhomologous C-terminal tails of TAP1 and TAP2.

The transporter associated with antigen processing (TAP) delivers peptides into the lumen of the endoplasmic reticulum for binding onto major histocompatibility complex class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each with an N-terminal transmembrane domain and a C-terminal cytosolic nucleotide binding domain (NBD). The two NBDs have distinct intrinsic nucleotide binding properties. In the resting state of TAP, the NBD1 has a much higher binding activity for ATP than the NBD2, while the binding of ADP to the two NBDs is equivalent. To attribute the different nucleotide binding behaviour of NBD1 and NBD2 to specific sequences, we generated chimeric TAP1 and TAP2 polypeptides in which either the nonhomologous C-terminal tails downstream of the Walker B motif, or the core NBDs which are enclosed by the conserved Walker A and B motifs, were reciprocally exchanged. Our biochemical and functional studies on the different TAP chimeras show that the distinct nucleotide binding behaviour of TAP1 and TAP2 is controlled by the nonhomologous C-terminal tails of the two TAP chains. In addition, our data suggest that the C-terminal tail of TAP2 is required for a functional transporter by regulating ATP binding. Further experiments indicate that ATP binding to NBD2 is important because it prevents simultaneous uptake of ATP by TAP1. We propose that the C-terminal tails of TAP1 and TAP2 play a crucial regulatory role in the coordination of nucleotide binding and ATP hydrolysis by TAP.