Phosphorylation of IRF-3 on Ser 339 Generates a Hyperactive Form of IRF-3 Through Regulation of Dimerization and CBP Association

Faculty of Pharmacy, Institut de Recherche en Immunologie et Cancérologie, Departments of Pharmacology and Molecular Biology, Centre Hospitalier de l'Université de Montréal and Department of Biochemistry, University of Montreal, Montreal, H3C 3J7, Canada

^* To whom correspondence should be addressed. Email: marc.servant{at}umontreal.ca.

	Abstract

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. Using a biological assay based on the production of IRF-3-mediated anti-viral cytokines production, 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-IRF3 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 S396A mutation, located in cluster II, abolished IRF-3 homodimerization, CBP association and nuclear accumulation. However, production of anti-viral 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 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. We propose that conventional biochemical assays used to measure IRF-3 activation are not sensitive enough do 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.