This Article Full Text Full Text (PDF) Other Versions of this Article: JVI.02526-07v1 82/8/3984    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Google Scholar Articles by Clément, J.-F. Articles by Servant, M. J. PubMed PubMed Citation Articles by Clément, J.-F. Articles by Servant, M. J.

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

Faculty of Pharmacy,¹ Institut de Recherche en Immunologie et Cancérologie,² Departments of Pharmacology and Molecular Biology,³ Centre Hospitalier de l'Université de Montréal and Department of Biochemistry, University of Montreal, Montreal H3C 3J7, Canada⁴

Received 26 November 2007/ Accepted 30 January 2008

The IB 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.

Published ahead of print on 13 February 2008.