Analyses of Virus-Induced Homomeric and Heteromeric Protein Associations between IRF-3 and Coactivator CBP/p300

Abstract

Cellular genes including the type I interferon genes are activated in response to viral infection. We previously reported that IRF-3 (interferon regulatory factor 3) is specifically phosphorylated on serine residues and directly transmits a virus-induced signal from the cytoplasm to the nucleus, and then participates in the primary phase of gene induction. In this study, we analyzed the molecular mechanism of IRF-3 activation further. The formation of a stable homomeric complex of IRF-3 between the specifically phosphorylated IRF-3 molecules occurred. While virus-induced IRF-7 did not bind to p300, the phosphorylated IRF-3 complex formed a stable multimeric complex with p300 (active holocomplex). Competition using a synthetic phosphopeptide corresponding to the activated IRF-3 demonstrated that p300 directly recognizes the structure in the vicinity of the phosphorylated residues of IRF-3. These results indicated that the phosphorylation of serine residues at positions 385 and 386 is critical for the formation of the holocomplex, presumably through a conformational switch facilitating homodimer formation and the generation of the interaction interface with CBP/p300.

Regulation of the Transcriptional Activity of the IRF7 Promoter by a Pathway Independent of Interferon Signaling

“…In cells transfected with IRF7 alone or IRF3 alone, we sometimes detected two specific bands in EMSA, one of which ran faster than VAF⅐DNA complex (data not shown), suggesting that IRF7 or IRF3 homodimers may be involved in IRF7 regulation. Also, previous reports have shown that IRF3 or IRF7 homodimers are involved in the activation of their target genes (36,54). IRF7 homodimers form in the absence of phosphorylation (55).…”

Identification of Ser-386 of Interferon Regulatory Factor 3 as Critical Target for Inducible Phosphorylation That Determines Activation

“…According to their model, the inhibition is reversed by the phosphorylation of the 5ST site, which confers negative charges to the inhibitory domain, resulting in a dramatic conformational change (12 However, the model is inconsistent with several observations. First, the deletion mutant IRF-3 (1-394), originally described as constitutively active (12), was not active even after viral stimulation (13). The 5D mutant was active as reported when produced in human 293T cells (12); however, it was much less active in mouse L929 cells (13) and was produced as an inactive monomer in Escherichia coli.…”

“…Recently, IKK-i (also known as IKK-⑀) and TBK-1 were shown to function as IRF-3 kinases (10,11). The phosphorylated IRF-3 undergoes homodimerization (12,13) and associates with coactivators CBP/ p300, and then a holocomplex of IRF-3 results (5,12). The holocomplex is conferred an ability to specifically recognize the target DNA sequence and accumulate in the nucleus (5,14,15).…”

“…The exact phosphorylation site of IRF-3 is the subject of some controversy (5,9,12,13,18). However, extensive mutagenesis has identified critical Ser residues.…”

“…The mutation of Ser-385 or -386 to Ala totally abolished the activity of IRF-3 (5,12,13,18), whereas the substitution of Ser/Thr (5ST site) residues at 396, 398, 402, 404, and 405 with Ala (5A mutant) resulted in virtually a normal phenotype, i.e. 5A forms a homodimer, associates with CBP/p300 (13), and retains transactivation potential (18). These results strongly suggest that the 2S site is the critical phosphorylation target and the 5ST site may have an accessory role.…”