Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7.
Interferon regulatory factor (IRF)7 is a key transcription factor required for establishment of antiviral resistance. In response to infection, IRF7 is activated by phosphorylation through the action of the non-canonical IkappaB kinases, IkappaB kinase-epsilon and TANK-binding kinase 1. Activation leads to nuclear retention, DNA binding, and derepression of transactivation ability. Clusters of serine residues located in the carboxyl-terminal regulatory domain of IRF7 are putative targets of virus-activated kinases. However, the exact sites of phosphorylation have not yet been established. Here, we report a comprehensive structure-activity examination of potential IRF7 phosphorylation sites through analysis of mutant proteins in which specific serine residues were altered to alanine or aspartate. Phosphorylation patterns of these mutants were analyzed by two-dimensional gel electrophoresis, and their transcriptional activity was monitored by reporter assays. Essential phosphorylation events were mapped to amino acids 437-438 and a redundant set of sites at either amino acids 429-431 or 441. IRF7 recovered from infected cells was heterogeneously phosphorylated at these sites, and greater phosphorylation correlated with increased transactivation. Interestingly, a distinct serine cluster conserved in the related protein IRF3 was also essential for IRF7 activation and distal phosphorylation. However, the essential role of this motif did not appear to be fulfilled by phosphorylation. Rather, these serine residues and an adjacent leucine were required for phosphorylation at distal sites and may determine a conformational element required for function.
Caillaud
Alexandre
A
Unité de Virologie et Immunologie Cellulaire, Institut Pasteur, 75724 Paris, France.
Hovanessian
Ara G
AG
Levy
David E
DE
Marié
Isabelle J
IJ
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D050839:Interferon Regulatory Factor-7; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D012694:Serine / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; C494234:IRF7 protein, human; D050839:Interferon Regulatory Factor-7; C494235:Irf7 protein, mouse; D012694:Serine
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