Viral infection triggers host innate immune responses through activation of the transcription factors NF-kappaB and IRF 3, which coordinately regulate the expression of type-I interferons such as interferon-beta (IFN-beta). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-kappaB and IRF 3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-kappaB and IRF 3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-beta through activation of NF-kappaB and IRF 3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF 3 and IkappaB and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity.

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.

The activation of NF-kappaB and IKK requires an upstream kinase complex consisting of TAK1 and adaptor proteins such as TAB1, TAB2, or TAB3. TAK1 is in turn activated by TRAF6, a RING domain ubiquitin ligase that facilitates the synthesis of lysine 63-linked polyubiquitin chains. Here we present evidence that TAB2 and TAB3 are receptors that bind preferentially to lysine 63-linked polyubiquitin chains through a highly conserved zinc finger (ZnF) domain. Mutations of the ZnF domain abolish the ability of TAB2 and TAB3 to bind polyubiquitin chains, as well as their ability to activate TAK1 and IKK. Significantly, replacement of the ZnF domain with a heterologous ubiquitin binding domain restored the ability of TAB2 and TAB3 to activate TAK1 and IKK. We also show that TAB2 binds to polyubiquitinated RIP following TNFalpha stimulation. These results indicate that polyubiquitin binding domains represent a new class of signaling domains that regulate protein kinase activity through a nonproteolytic mechanism.

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.

By suppressing expression of TRAF6 and IRAK1, miR-146a regulates NF-κB activation in T cells through a negative feedback loop and controls the resolution of T cell responses in mice.

NF-B is a key activator of inflammatory and immune responses with important pathological roles in cancer, heart disease, and autoimmune diseases. Transcriptional activity of NF-B is regulated by different posttranslational modifications. Here, we report a novel mechanism of NF-B regulation through lysine monomethylation by SET9 methyltransferase. Set9 specifically methylates p65 at lysine 37. Both TNF␣ and IL-1␤ treatments induced methylation of p65. Methylated p65 is restricted to the nucleus and this modification regulates the promoter binding of p65. Moreover, Set9 mediated methylation of p65 is required for the expression of a subset of NF-B target genes in response to TNF␣ stimulation.N F-B is a transcription factor that plays a pivotal role in regulating multiple biological functions including inflammation, immunity, cell proliferation, and apoptosis. NF-B represents a group of evolutionarily conserved and structurally related proteins. The 5 members of the mammalian NF-B, p65 (RelA), RelB, cRel, p50/p105 (NF-B1), and p52/p100 (NF-B2), form homo-or heterodimers that bind to I B family proteins in unstimulated cells (1). NF-B is sequestered in the cytoplasm through its interaction with the I B in resting cells. Stimulation of cells with a variety of ligands, such as tumor necrosis factor-␣ (TNF␣), interleukin-1␤ (IL-1␤), or pathogenassociated molecular patterns (PAMPs), leads to the rapid phosphorylation of I B by the I B kinase complex (IKK). The IKK kinase complex contains 2 catalytic subunits, IKK␣ and IKK␤, and the regulatory subunit, IKK␥. IKK catalyzes the phosphorylation of I B at 2 serine residues in the N terminus. The phosphorylated I B becomes ubiquitinated and subsequently degraded by 26S proteasome, thereby allowing NF-B to enter the nucleus to turn on a large array of target genes (1, 2).Although the activity of NF-B is regulated by nuclear translocation, covalent modifications of the protein by various events including phosphorylation, ubiquitination, nitrosylation, and acetylation can affect its activity (3). These regulatory modifications have distinct functional consequences. For example, acetylation of p65 at K218 and K221 inhibits I B␣ binding and enhances DNA-binding (4) whereas acetylation of p65 at K122 and K123 inhibits its transcriptional activating activity (5).Proteins can be posttranslationally methylated at lysine, arginine, histidine, and dicarboxylic amino acids by highly specific methyltransferases (6). In the process of protein-lysine methylation, the addition of methyl groups to the -amine of a lysine residue results in the formation of mono-, di-, or trimethyllysine. This process can be reversed by demethylases. Histone is one of the best studied proteins that undergoes methylation (7). Specific sites of methylation on histones correlate with either activation or repression of transcription. Recently, several transcription factors, including p53 (8, 9), STAT1 (10), RAR␣ (11), and ER␣ (12), have been shown to be methylated and their biological activity modified by the modif...

TRAF6 (tumor necrosis factor receptor-associated factor 6) is a RING (really interesting new gene) domain ubiquitin (Ub) ligase that mediates the activation of protein kinases, such as transforming growth factor ␤-activated kinase (TAK1) and IB kinase (IKK), by catalyzing the formation of a unique polyubiquitin chain linked through Lys-63 of Ub. Here, we present evidence that TIFA (TRAFinteracting protein with a forkhead-associated domain, also known as T2BP) activates IKK by promoting the oligomerization and Ub ligase activity of TRAF6. We show that recombinant TIFA protein, but not TRAF6-binding-defective mutant, can activate IKK in crude cytosolic extracts. Furthermore, TIFA activates IKK in an in vitro reconstitution system consisting of purified proteins, including TRAF6, the TAK1 kinase complex, and Ub-conjugating enzyme complex Ubc13-Uev1A. Interestingly, a fraction of recombinant TIFA protein exists as high-molecular-weight oligomers, and only these oligomeric forms of TIFA can activate IKK. Importantly, TIFA induces the oligomerization and polyubiquitination of TRAF6, which leads to the activation of TAK1 and IKK through a proteasome-independent mechanism.

Circular RNAs (circRNAs) constitute a large class of RNA species formed by the back-splicing of co-linear exons, often within protein-coding transcripts. Despite much progress in the field, it remains elusive whether the majority of circRNAs are merely aberrant splicing by-products with unknown functions, or their production is spatially and temporally regulated to carry out specific biological functions. To date, the majority of circRNAs have been cataloged in resting cells. Here, we identify an LPS-inducible circRNA: mcircRasGEF1B, which is predominantly localized in cytoplasm, shows cell-type specific expression, and has a human homolog with similar properties, hcircRasGEF1B. We show that knockdown of the expression of mcircRasGEF1B reduces LPS-induced ICAM-1 expression. Additionally, we demonstrate that mcircRasGEF1B regulates the stability of mature ICAM-1 mRNAs. These findings expand the inventory of functionally characterized circRNAs with a novel RNA species that may play a critical role in fine-tuning immune responses and protecting cells against microbial infection.