Noula Shembade

The ubiquitin‐editing enzyme A20 requires RNF11 to downregulate NF‐κB signalling

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor‐beta (TGF‐β) signalling. RNF11 has been proposed to regulate TGF‐β signalling by interacting with HECT‐ and SCF‐type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF‐κB and jun N‐terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)‐ and lipopolysaccharide (LPS)‐mediated NF‐κB and JNK signalling. RNF11 interacted with the NF‐κB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus‐dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF‐mediated NF‐κB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin‐editing protein complex that ensures transient activation of inflammatory signalling pathways.

Essential role for TAX1BP1 in the termination of TNF-α-, IL-1- and LPS-mediated NF-κB and JNK signaling

The NF‐κB transcription factor is normally transiently activated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS); however, persistent NF‐κB activation is commonly observed in inflammatory disease and malignancy. The ubiquitin editing enzyme A20 serves an essential role in the termination of TNF‐α‐ and LPS‐mediated NF‐κB signaling by inactivating key signaling molecules. However, little is known about how A20 is regulated and if other molecules play a role in the termination of NF‐κB signaling. Here we demonstrate that Tax1‐binding protein 1 (TAX1BP1) is essential for the termination of NF‐κB and JNK activation in response to TNF‐α, IL‐1 and LPS stimulation. In TAX1BP1‐deficient mouse fibroblasts, TNF‐α‐, IL‐1‐ and LPS‐mediated IKK and JNK activation is elevated and persistent owing to enhanced ubiquitination of RIP1 and TRAF6. Furthermore, in the absence of TAX1BP1, A20 is impaired in RIP1 binding, deubiquitination of TRAF6 and inhibition of NF‐κB activation. Thus, TAX1BP1 is pivotal for the termination of NF‐κB and JNK signaling by functioning as an essential regulator of A20.

The Human T-Cell Leukemia Virus Type 1 Tax Oncoprotein Requires the Ubiquitin-Conjugating Enzyme Ubc13 for NF-κB Activation

ABSTRACT Ubiquitination of the human T-cell leukemia virus 1 Tax oncoprotein provides an important regulatory mechanism that promotes the Tax-mediated activation of NF-κB. However, the type of polyubiquitin chain linkages and the host factors that are required for Tax ubiquitination have not been identified. Here, we demonstrate that Tax polyubiquitin chains are composed predominantly of lysine 63-linked chains. Furthermore, the ubiquitination of Tax is critically dependent on the E2 ubiquitin-conjugating enzyme Ubc13. Tax interacts with Ubc13, and small interfering RNA-mediated knockdown of Ubc13 expression abrogates Tax ubiquitination and the activation of NF-κB. Mouse fibroblasts lacking Ubc13 exhibit impaired Tax activation of NF-κB despite normal tumor necrosis factor- and interleukin-1-mediated NF-κB activation. Finally, the interaction of Tax with NEMO is disrupted in the absence of Tax ubiquitination and Ubc13 expression, suggesting that Tax ubiquitination is critical for NEMO binding. Collectively, our results reveal that Ubc13 is essential for Tax ubiquitination, its interaction with NEMO, and Tax-mediated NF-κB activation.

Regulation of NF-κB signaling by the A20 deubiquitinase

The NF-κB transcription factor is a central mediator of inflammatory and innate immune signaling pathways. Activation of NF-κB is achieved by K63-linked polyubiquitination of key signaling molecules which recruit kinase complexes that in turn activate the IκB kinase (IKK). Ubiquitination is a highly dynamic process and is balanced by deubiquitinases that cleave polyubiquitin chains and terminate downstream signaling events. The A20 deubiquitinase is a critical negative regulator of NF-κB and inflammation, since A20-deficient mice develop uncontrolled and spontaneous multi-organ inflammation. Furthermore, specific polymorphisms in the A20 genomic locus predispose humans to autoimmune disease. Recent studies also indicate that A20 is an important tumor suppressor that is inactivated in B-cell lymphomas. Therefore, targeting A20 may form the basis of novel therapies for autoimmune disease and lymphomas.

The kinase IKKα inhibits activation of the transcription factor NF-κB by phosphorylating the regulatory molecule TAX1BP1

In response to stimulation with proinflammatory cytokines, the deubiquitinase A20 inducibly interacts with the regulatory molecules TAX1BP1, Itch and RNF11 to form the A20 ubiquitin-editing complex. However, the molecular signal that coordinates the assembly of this complex has remained elusive. Here we demonstrate that TAX1BP1 was inducibly phosphorylated on Ser593 and Ser624 in response to proinflammatory stimuli. The kinase IKKα, but not IKKβ, was required for phosphorylation of TAX1BP1 and directly phosphorylated TAX1BP1 in response to stimulation with tumor necrosis factor (TNF) or interleukin 1 (IL-1). TAX1BP1 phosphorylation was pivotal for cytokine-dependent interactions among TAX1BP1, A20, Itch and RNF11 and downregulation of signaling by the transcription factor NF-κB. IKKα therefore serves a key role in the negative feedback of NF-κB canonical signaling by orchestrating assembly of the A20 ubiquitin-editing complex to limit inflammatory gene activation.

Human T Cell Leukemia Virus Type 1 Tax Inhibits Innate Antiviral Signaling via NF-κB-Dependent Induction of SOCS1

ABSTRACT Human T cell leukemia virus type 1 (HTLV-1) inhibits host antiviral signaling pathways although the underlying mechanisms are unclear. Here we found that the HTLV-1 Tax oncoprotein induced the expression of SOCS1, an inhibitor of interferon signaling. Tax required NF-κB, but not CREB, to induce the expression of SOCS1 in T cells. Furthermore, Tax interacted with SOCS1 in both transfected cells and in HTLV-1-transformed cell lines. Although SOCS1 is normally a short-lived protein, in the presence of Tax, the stability of SOCS1 was greatly increased. Accordingly, Tax enhanced the replication of a heterologous virus, vesicular stomatitis virus (VSV), in a SOCS1-dependent manner. Surprisingly, Tax required SOCS1 to inhibit RIG-I-dependent antiviral signaling, but not the interferon-induced JAK/STAT pathway. Inhibition of SOCS1 by RNA-mediated interference in the HTLV-1-transformed cell line MT-2 resulted in increased IFN-β expression accompanied by reduced HTLV-1 replication and p19Gag levels. Taken together, our results reveal that Tax inhibits antiviral signaling, in part, by hijacking an interferon regulatory protein.

TNF pretreatment interferes with mitochondrial apoptosis in the mouse liver by A20-mediated down-regulation of Bax.

Pretreatment with low doses of the proinflammatory cytokine TNF has been shown to prevent hepatocellular apoptosis and liver damage in inflammatory as well as in ischemia/reperfusion-induced liver injury. The underlying mechanisms of protection have not been elucidated so far. In this study, these mechanisms were investigated in murine hepatocyte cultures as well as in a mouse model of TNF-dependent apoptotic liver damage (galactosamine/TNF model). Our results show that pretreatment with TNF, or application of small-interfering RNA directed against the proapoptotic Bcl2 family member Bax, interfered with the onset of mitochondrial apoptosis in vivo. Knockdown of TNF-α-induced-protein 3 (A20) restored mitochondrial apoptosis, Bax expression, and liver damage. The underlying mechanism of protection seems to involve a cascade of events, where TNF induces the expression of A20 in hepatocytes, A20 down-modulates Bax expression by interference with transcriptional activation, and the reduced availability of Bax interferes with the onset of mitochondrial apoptosis and the ensuing apoptotic liver damage. In conclusion, we identified Bax and A20 as key players in TNF-induced protection from apoptotic liver damage. Because treatment with TNF itself might be a risk factor for patients, we propose that overexpression of A20 might represent an alternative approach for protection from inflammation related apoptotic liver damage, as well as for TNF preconditioning during transplantation.

Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Requires CADM1/TSLC1 for Inactivation of the NF-κB Inhibitor A20 and Constitutive NF-κB Signaling

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation.

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.

A20 inhibition of NFκB and inflammation: targeting E2:E3 ubiquitin enzyme complexes.

Comment on: Shembade N, Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science 2010; 327:1135-9.