| Over the past four years, the field of the innate immune response has been highly influenced by the discovery of the IκB kinase (IKK)-related kinases, TBK1 and IKKi, which regulate the activity of IRF-3/IRF-7 and NF-κB transcription factors. The IKK-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. By combining mass spectrometry analysis to in vitro kinase assays using full length His-IRF3 as a substrate, we have demonstrated that serine 402 and serine 396 were directly targeted by TBK1. Analysis of Ser/Thr to Ala mutants revealed that S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association and nuclear accumulation. 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 MEFs IRF-3 KO also reveals a compensatory mechanism of serine 339 and serine 396 in the ability of IRF-3 to induce IFN-stimulated genes (ISGs) ISG56 and ISG54 expression. These data lead us to reconsider the current model of IRF-3 activation.;TRAF3 is also a central mediator that is important for inducing type I interferon production in response to intracellular double-stranded RNA. By combining Flag-Affinity purification using Flag-TRAF3 as a bait to mass spectrometry, we have identified Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel TRAF3 interactors. We found that TRAF3 localizes to the ER-to-Golgi vesicular pathway and behaves like a cis-Golgi protein. Depletion of p115 or Sec16A disrupts the cis-Golgi cellular localization of TRAF3 and affects type I Interferon response following double-stranded RNA treatment. Furthermore, we demonstrate that TRAF3 colocalizes with TRADD at the cis-Golgi and also interacts with the translocon protein Sec61β in a Sec5 dependent manner. Together, our data suggest that the cellular localization of TRAF3 to the ER-to-Golgi transport compartments is required for an optimal RIG-I-like Helicases (RLH)-Cardif-dependent antiviral immune response. Our findings also highlight the potential role of the exocyst in the innate immune response.;Keywords : Interferon, antiviral state, IRF-3, TRAF3, phosphorylation. |
