Contribution of Ser386 and Ser396 to Activation of Interferon Regulatory Factor 3

• Published in: Journal of molecular biology 2008-05, Vol.379 (2), p.251-260
• Main Authors: Chen, Weijun, Srinath, Hema, Lam, Suvana S., Schiffer, Celia A., Royer, William E., Lin, Kai
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; CREB-binding protein; Humans; interferon regulatory factor-3; Interferon Regulatory Factor-3 - chemistry; Interferon Regulatory Factor-3 - genetics; Interferon Regulatory Factor-3 - metabolism; Models, Molecular; Molecular Sequence Data; Mutation; oligomerization; p300-CBP Transcription Factors - genetics; p300-CBP Transcription Factors - metabolism; phosphomimetic form; Phosphorylation; Protein Binding; Protein Conformation; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Serine - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.03.050

IRF-3, a member of the interferon regulatory factor (IRF) family of transcription factors, functions in innate immune defense against viral infection. Upon infection, host cell IRF-3 is activated by phosphorylation at its seven C-terminal Ser/Thr residues: 385 SSLENTVDLHI SN SHPL SL TS 405. This phosphoactivation triggers IRF-3 to react with the coactivators, CREB-binding protein (CBP)/p300, to form a complex that activates target genes in the nucleus. However, the role of each phosphorylation site for IRF-3 phosphoactivation remains unresolved. To address this issue, all seven Ser/Thr potential phosphorylation sites were screened by mutational studies, size-exclusion chromatography, and isothermal titration calorimetry. Using purified proteins, we show that CBP (amino acid residues 2067–2112) interacts directly with IRF-3 (173–427) and six of its single-site mutants to form heterodimers, but when CBP interacts with IRF-3 S396D, oligomerization is evident. CBP also interacts in vitro with IRF-3 double-site mutants to form different levels of oligomerization. Among all the single-site mutants, IRF-3 S396D showed the strongest binding to CBP. Although IRF-3 S386D alone did not interact as strongly with CBP as did other mutants, it strengthened the interaction and oligomerization of IRF-3 S396D with CBP. In contrast, IRF-3 S385D weakened the interaction and oligomerization of IRF-3 S396D and S386/396D with CBP. Thus, it appears that Ser385 and Ser386 serve antagonistic functions in regulating IRF-3 phosphoactivation. These results indicate that Ser386 and Ser396 are critical for IRF-3 activation, and support a phosphorylation-oligomerization model for IRF-3 activation.