SUMMARY RIG-I detects invading viral RNA and activates the transcription factors NF-κB and IRF3 through the mitochondrial protein MAVS. Here we show that RNA bearing 5′-triphosphate strongly activates the RIG-I–IRF3 signaling cascade in a reconstituted system composed of RIG-I, mitochondria and cytosol. Activation of RIG-I requires not only RNA, but also polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. RIG-I binds specifically to K63 polyubiquitin chains through its tandem CARD domains in a manner that depends on RNA and ATP. Mutations in the CARD domains that abrogate ubiquitin binding also impair RIG-I activation. Remarkably, unanchored K63 ubiquitin chains, which are not conjugated to any target protein, potently activate RIG-I. These ubiquitin chains function as an endogenous ligand of RIG-I in human cells. Our results delineate the mechanism of RIG-I activation, identify CARD domains as a new ubiquitin sensor, and demonstrate that unanchored K63 polyubiquitin chains are signaling molecules in antiviral innate immunity.

TRAF6 is a ubiquitin ligase essential for the activation of NF-κB and MAP kinases in multiple signaling pathways including those emanating from the interleukin-1 and Toll-like receptors (IL-1R/TLR)1-3. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of Ubc13 and Uev1A to catalyze Lys-63 (K63)-linked polyubiquitination, which activates the TAK1 kinase complex4,5. TAK1 in turn phosphorylates and activates IκB kinase (IKK), leading to activation of NF-κB. Although several proteins are known to be polyubiquitinated in the IL-1R/TLR pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. Herein, we reconstituted TAK1 activation in vitro using purified proteins and found that free K63 polyubiquitin chains, which are not conjugated to any target protein, directly activated TAK1 through binding to the ubiquitin receptor TAB2. This binding leads to autophosphorylation and activation of TAK1. We also found that unanchored polyubiquitin chains synthesized by TRAF6 and Ubc5 activated the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a novel mechanism of protein kinase regulation.

Transforming growth factor b activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, has emerged as a key regulator of signal transduction cascades leading to the activation of the transcription factors nuclear factor-kappa B (NF-jB) and activator protein-1 (AP-1). Stimulation of cells with cytokines and microbial pathogens results in the activation of TAK1, which subsequently activates the I-kappa B kinase complex (IKK) and mitogen-activated protein (MAP) kinases, culminating in the activation of NF-jB and AP-1, respectively. Recent studies have shown that polyubiquitination of signalling proteins through lysine (Lys)-63-linked polyubiquitin chains plays an important role in the activation of TAK1 and IKK. Unlike Lys-48-linked polyubiquitination, which normally targets proteins for degradation by the proteasome, Lys-63-linked polyubiquitin chains act as scaffolds to assemble protein kinase complexes and mediate their activation through proteasome-independent mechanisms. The concept of ubiquitin-mediated activation of protein kinases is supported by the discoveries of ubiquitination and deubiquitination enzymes as well as ubiquitin-binding proteins that function upstream of TAK1 and IKK. Recent biochemical and genetic studies provide further insights into the mechanism and function of ubiquitin signalling and these advances will be the focus of this review.

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including G␣ subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various G␣⅐GDP subunits and forms a stable nucleotide-free Ric-8A:G␣ complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes G␣ subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for G␣⅐GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:G␣ il and that of Ric-8A with the AGS3-C:G␣ il ⅐GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:G␣ i1 ⅐GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:G␣ i1 ⅐GDP. Subsequent dissociation of AGS3-C and GDP from G␣ i1 yields a stable nucleotide free Ric-8A⅐G␣ i1 complex that, in the presence of GTP, dissociates to yield Ric-8A and G␣ i1 ⅐GTP. AGS3-C does not induce dissociation of the Ric-8A⅐G␣ i1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:G␣ i1 ⅐GDP ensures unidirectional activation of G␣ subunits that cannot be reversed by AGS3.Canonical G-protein signaling pathways are activated when agonist-bound heptahelical receptors, acting as guanine nucleotide exchange factors (GEFs), 2 promote the exchange of GDP for GTP on G␣ subunits present in G␣⅐GDP:G␤␥ heterotrimers (1-3). Upon binding GTP, conformational changes in the switch regions of G␣ subunits destabilize the heterotrimer and allow G␣⅐GTP to dissociate from G␤␥ subunits (4, 5). Downstream regulatory molecules such as the regulators of G-protein signaling (RGS) accelerate G␣-catalyzed GTP hydrolysis, allowing the G␣ subunits to revert to their resting GDP-bound conformation and priming them for the next receptor-induced G-protein cycle (6 -8). Receptor-mediated signaling accounts for the majority of G-protein-regulated cellular control mechanisms. However, during the past few years evidence has emerged that, in both lower and higher eukaryotes, multicomponent G-protein signaling systems, operating outside the realm of membrane-bound receptors, play significant roles in various biological processes (9). These include control of the generation of microtubule pulling forces during cell division (10 -16), synaptic signaling processes (17), and cardiovascular function (18). A receptor-independent G-protein-mediated signaling pathway, regulating a fundamental event such as asymmetric cell division, may involve proteins that can modulate G-protein nucleotide exchange in a manner that resembles the action of agonist-bound receptors and G␤␥ subunits. In nematodes, asymmetric cell division is a result of eccentr...

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G␣ i1 and inhibited the binding of GTP by stabilizing the GDP-bound conformation of G␣ i1 . The stoichiometry, free energy, enthalpy, and dissociation constant for binding of AGS3-C to G␣ i1 were determined using isothermal titration calorimetry. AGS3-C possesses two apparent high affinity (K d ϳ 20 nM) and two apparent low affinity (K d ϳ 300 nM) binding sites for G␣ i1 . Upon deletion of the Cterminal GPR motif from AGS3-C, the remaining sites were approximately equivalent with respect to their affinity (K d ϳ 400 nM) for G␣ i1 . Peptides corresponding to each of the four GPR motifs of AGS3 (referred to as GPR1, GPR2, GPR3, and GPR4, respectively, going from N to C terminus) bound to G␣ i1 with K d values in the range of 1-8 M. Although GPR1, GPR2, and GPR4 inhibited the binding of the fluorescent GTP analog BODIPY-FL-guanosine 5-3-O-(thio)triphosphate to G␣ i1 , GPR3 did not. However, addition of N-and C-terminal flanking residues to the GPR3 GoLoco core increased its affinity for G␣ i1 and conferred GDI activity similar to that of AGS3-C itself. Similar increases were observed for extended GPR2 and extended GPR1 peptides. Thus, while the tertiary structure of AGS3 may affect the affinity and activity of the GPR motifs contained within its sequence, residues outside of the GPR motifs strongly potentiate their binding and GDI activity toward G␣ i1 even though the amino acid sequences of these residues are not conserved among the GPR repeats.