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.
Clément
Jean-François
JF
Faculté de Pharmacie, Université de Montréal, C.P. 6128, succursale Centre-Ville, Montréal, Québec, Canada. marc.servant@umontreal.ca
Bibeau-Poirier
Annie
A
Gravel
Simon-Pierre
SP
Grandvaux
Nathalie
N
Bonneil
Eric
E
Thibault
Pierre
P
Meloche
Sylvain
S
Servant
Marc J
MJ
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000096:biosynthesis; D019281:Dimerization; D005347:Fibroblasts; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis; D050880:p300-CBP Transcription Factors / Q000378:metabolism*
D000998:Antiviral Agents; D016207:Cytokines; C522692:ISG54 protein, mouse; C522693:ISG56 protein, mouse; D050838:Interferon Regulatory Factor-3; D014157:Transcription Factors; D012694:Serine; D050880:p300-CBP Transcription Factors; C498588:Tbk1 protein, mouse; D017346:Protein Serine-Threonine Kinases