Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association.

The IkappaB kinase-related kinases, TBK1 and IKKi, were recently shown to be responsible for the C-terminal phosphorylation of IRF-3. However, the identity of the phosphoacceptor site(s) targeted by these two kinases remains unclear. Using a biological assay based on the IRF-3-mediated production of antiviral cytokines, we demonstrate here that all Ser/Thr clusters of IRF-3 are required for its optimal transactivation capacity. In vitro kinase assays using full-length His-IRF-3 as a substrate combined with mass spectrometry analysis revealed that serine 402 and serine 396 are directly targeted by TBK1. Analysis of Ser/Thr-to-Ala mutants revealed that the S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association, and nuclear accumulation. However, production of antiviral cytokines was still present in IRF-3 S396A-expressing cells. Interestingly, mutation of serine 339, which is involved in IRF-3 stability, also abrogated CBP association and dimerization without affecting gene transactivation as long as serine 396 remained available for phosphorylation. Complementation of IRF-3-knockout mouse embryonic fibroblasts also revealed a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce expression of the interferon-stimulated genes ISG56 and ISG54. These data lead us to reconsider the current model of IRF-3 activation. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough to detect the small fraction of IRF-3 needed to elicit a biological response. Importantly, our study establishes a molecular link between the role of serine 339 in IRF-3 homodimerization, CBP association, and its destabilization.

Roles of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling.

Post-translational protein modifications are involved in all functions of living cells. This includes the ability of cells to recognize pathogens and regulate genes involved in their clearance, a concept known as innate immunity. While phosphorylation mechanisms play essential roles in regulating different aspects of the innate immune response, ubiquitination is now recognized as another post-translational modification that works in parallel with phosphorylation to orchestrate the final proper innate immune response against invading pathogens. More precisely, this review will discuss the most recent advances that address the role of ubiquitination in pattern-recognition receptors and type I interferon receptor signaling.

Ubiquitin-regulated recruitment of IkappaB kinase epsilon to the MAVS interferon signaling adapter.

Induction of the antiviral interferon response is initiated upon recognition of viral RNA structures by the RIG-I or Mda-5 DEX(D/H) helicases. A complex signaling cascade then converges at the mitochondrial adapter MAVS, culminating in the activation of the IRF and NF-kappaB transcription factors and the induction of interferon gene expression. We have previously shown that MAVS recruits IkappaB kinase epsilon (IKKepsilon) but not TBK-1 to the mitochondria following viral infection. Here we map the interaction of MAVS and IKKepsilon to the C-terminal region of MAVS and demonstrate that this interaction is ubiquitin dependent. MAVS is ubiquitinated following Sendai virus infection, and K63-linked ubiquitination of lysine 500 (K500) of MAVS mediates recruitment of IKKepsilon to the mitochondria. Real-time PCR analysis reveals that a K500R mutant of MAVS increases the mRNA level of several interferon-stimulated genes and correlates with increased NF-kappaB activation. Thus, recruitment of IKKepsilon to the mitochondria upon MAVS K500 ubiquitination plays a modulatory role in the cascade leading to NF-kappaB activation and expression of inflammatory and antiviral genes. These results provide further support for the differential role of IKKepsilon and TBK-1 in the RIG-I/Mda5 pathway.

Signaling pathways involved in the cardioprotective effects of cannabinoids.

The aim of the present article is to review the cardioprotective properties of cannabinoids, with an emphasis on the signaling pathways involved. Cannabinoids have been reported to protect against ischemia in rat isolated hearts, as well as in rats and mice in vivo. Although these effects have been observed mostly with a pre-treatment of a cannabinoid, we report that the selective CB(2)-receptor agonist JWH133 is able to reduce infarct size when administered either before ischemia, during the entire ischemic period, or just upon reperfusion. Little is known about the signaling pathways involved in these cardioprotective effects. Likely candidates include protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) since they are activated during ischemia-reperfusion and contribute to the protective effect ischemic preconditioning. The use of pharmacological inhibitors suggests that PKC, p38 MAPK, and p42/p44 MAPK (ERK1/2) contribute to the protective effect of cannabinoids. In addition, perfusion with JWH133 in healthy hearts caused an increase in both p38 MAPK phosphorylation level and activity, whereas the CB(1)-receptor agonist ACEA was associated with an increase in the phosphorylation status of both ERK1 and ERK2 without any change in activity. During ischemia, both agonists doubled p38 MAPK activity, whereas ERK1/2 phosphorylation level and activity during reperfusion were enhanced only by the CB(1)-receptor agonist. Finally, although nitric oxide (NO) was shown to exert both pro and anti-apoptotic effects on cardiomyocytes, with an apparently controversial effect on myocardial survival, our data suggest that NO may contribute to the cardioprotective effect of some cannabinoids.

The proinflammatory actions of angiotensin II are dependent on p65 phosphorylation by the IkappaB kinase complex.

The vasoactive hormone angiotensin II (Ang II) probably triggers inflammatory cardiovascular diseases by activating transcription factors such as NF-kappaB. We describe here a novel mode of NF-kappaB activation in cultured vascular smooth muscle cells exposed to Ang II. Ang II treatment resulted in an increase in the phosphotransferase activity of the IKK complex, which was mediated through the AT1 receptor subtype. The typical phosphorylation and proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha were not observed. Rather, Ang II treatment of vascular smooth muscle cells led to the phosphorylation of p65 on serine 536, a signal detected in both the cytoplasm and the nuclear compartments. The use of pharmacological inhibitors that inhibit the activation of MEK by Ang II revealed that phosphorylation of p65 on serine 536 did not require the MEK-ERK-RSK signaling pathway. On the other hand, specifically targeting the IKKbeta subunit of the IKK complex by overexpression of a dominant negative version of IKKbeta (IKKbeta K44A) or silencing RNA technology demonstrated that the IKKbeta subunit of the IKK complex was responsible for the detected phosphoserine 536 signal in Ang II-treated cells. Characterization of the signaling pathway leading to activation of the IKK complex by Ang II revealed that neither epidermal growth factor receptor transactivation nor the phosphatidylinositol 3-kinase-AKT signaling cascade were involved. Collectively, our data demonstrate that the proinflammatory activity of Ang II is independent of the classical pathway leading to IkappaBalpha phosphorylation and degradation but clearly depends on the recruitment of an IKK complex signaling cascade leading to phosphorylation of p65 on serine 536.

Involvement of the IkappaB kinase (IKK)-related kinases tank-binding kinase 1/IKKi and cullin-based ubiquitin ligases in IFN regulatory factor-3 degradation.

Activation of the innate arm of the immune system following pathogen infection relies on the recruitment of latent transcription factors involved in the induction of a subset of genes responsible for viral clearance. One of these transcription factors, IFN regulatory factor 3 (IRF-3), is targeted for proteosomal degradation following virus infection. However, the molecular mechanisms involved in this process are still unknown. In this study, we show that polyubiquitination of IRF-3 increases in response to Sendai virus infection. Using an E1 temperature-sensitive cell line, we demonstrate that polyubiquitination is required for the observed degradation of IRF-3. Inactivation of NEDD8-activating E1 enzyme also results in stabilization of IRF-3 suggesting the NEDDylation also plays a role in IRF-3 degradation following Sendai virus infection. In agreement with this observation, IRF-3 is recruited to Cullin1 following virus infection and overexpression of a dominant-negative mutant of Cullin1 significantly inhibits the degradation of IRF-3 observed in infected cells. We also asked whether the C-terminal cluster of phosphoacceptor sites of IRF-3 could serve as a destabilization signal and we therefore measured the half-life of C-terminal phosphomimetic IRF-3 mutants. Interestingly, we found them to be short-lived in contrast to wild-type IRF-3. In addition, no degradation of IRF-3 was observed in TBK1(-/-) mouse embryonic fibroblasts. All together, these data demonstrate that virus infection stimulates a host cell signaling pathway that modulates the expression level of IRF-3 through its C-terminal phosphorylation by the IkappaB kinase-related kinases followed by its polyubiquitination, which is mediated in part by a Cullin-based ubiquitin ligase.