Caillaud A, Hovanessian AG, Levy DE, Marie IJ.. Regulatory serine residues mediate phosphorylation-dependent and phosphorylation-independent activation of interferon regulatory factor 7. J Biol Chem 280: 17671-17677

NYU Langone Medical Center, New York, New York, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2005; 280(18):17671-7. DOI: 10.1074/jbc.M411389200
Source: PubMed


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.

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    • "Upon KSHV infection, IRF-7 competes with RTA for viral gene promoters to modulate viral transactivation (Wang et al., 2005). The function of IRF-7 can in turn be regulated by post-translational modifications including phosphorylation (Caillaud et al., 2005; Marie et al., 2000), acetylation (Caillaud et al., 2002), ubiquitination (Kawai et al., 2004; Ning et al., 2008; Yu et al., 2005) and SUMOylation (Chang et al., 2009; Kubota et al., 2008). Lysine residues are potential sites for acetylation, ubiquitination and SUMOylation, and may serve as targets to modulate the functions of IRF-7. "
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    ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) infection goes through latent and lytic phases, which are controlled by the viral replication and transcription activator (RTA). Upon KSHV infection, the host responds by suppressing RTA-activated lytic gene expression through interferon regulatory factor 7 (IRF-7), a key regulator of host innate immune response. Lysine residues are potential sites for post-translational modification of IRF-7, and were suggested to be critical for its activity. In this study, we analysed the 15 lysine residues for their effects on IRF-7 function by site-directed mutagenesis. We found that some mutations affect the ability of IRF-7 to activate interferon (IFN)-α1 and IFN-β promoters, to suppress RTA-mediated lytic gene expression and to repress KSHV reactivation and lytic replication. However, other mutations affect only a subset of these four functions. These findings demonstrate that the lysine residues of IRF-7 play important roles in mediating IFN synthesis and modulating viral lytic replication.
    Journal of General Virology 01/2011; 92(Pt 1):181-7. DOI:10.1099/vir.0.021816-0 · 3.18 Impact Factor
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    • "IKKΣ and TBK1 are critical inducers of interferon signaling in response to viral infection (Fitzgerald et al., 2003; Sharma et al., 2003). Following activation of toll-like receptors (TLR) via viral components, IKKΣ and TBK1 assemble with TRAF3 and TANK to phosphorylate interferon regulatory factors (IRF) 3, 5, and 7 at multiple serine and threonine residues (Caillaud et al., 2005; Cheng et al., 2006; McWhirter et al., 2004; Mori et al., 2004; Pomerantz and Baltimore, 1999). This activity allows for heterodimerization and nuclear translocation of the IRFs and induction of proinflammatory and antiviral genes, including type I interferon (Lin et al., 1998; Sato et al., 2000). "
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    ABSTRACT: The IκB Kinase (IKK)-related kinases TBK1 and IKKɛ have essential roles as regulators of innate immunity by modulating interferon and NF-κB signaling. Recent work has also implicated these non-canonical IKKs in malignant transformation. IKKɛ is amplified in ∼30% of breast cancers and transforms cells through the activation of NF-κB. TBK1 participates in RalB-mediated inflammatory responses and cell survival, and is essential for the survival of non-small cell lung cancers driven by oncogenic KRAS. The delineation of target substrates and downstream activities for TBK1 and IKKɛ has begun to define their role(s) in promoting tumorigenesis. In this review, we will highlight the mechanisms by which IKKɛ and TBK1 orchestrate pathways involved in inflammation and cancer.
    Oncogene 11/2010; 30(6):631-41. DOI:10.1038/onc.2010.493 · 8.46 Impact Factor
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    • "The C-terminal end of IRFs is generally more variable among family members [39] and contains a SMAD/FHA domain, commonly found in transcription factors and responsible for interaction with other phosphorylated molecules [40]. In silico analysis of SmIRF using NetPhos [41] revealed the presence of several serine phosphorylation sites [Additional file 3]; in mammalians, IRF phosphorylation is important for its function and regulation [42]. "
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    ABSTRACT: Schistosoma mansoni is a blood helminth parasite that causes schistosomiasis, a disease that affects 200 million people in the world. Many orthologs of known mammalian genes have been discovered in this parasite and evidence is accumulating that some of these genes encode proteins linked to signaling pathways in the parasite that appear to be involved with growth or development, suggesting a complex co-evolutionary process. In this work we found 427 genes conserved in the Deuterostomia group that have orthologs in S. mansoni and no members in any nematodes and insects so far sequenced. Among these genes we have identified Insulin Induced Gene (INSIG), Interferon Regulatory Factor (IRF) and vasohibin orthologs, known to be involved in mammals in mevalonate metabolism, immune response and angiogenesis control, respectively. We have chosen these three genes for a more detailed characterization, which included extension of their cloned messages to obtain full-length sequences. Interestingly, SmINSIG showed a 10-fold higher expression in adult females as opposed to males, in accordance with its possible role in regulating egg production. SmIRF has a DNA binding domain, a tryptophan-rich N-terminal region and several predicted phosphorylation sites, usually important for IRF activity. Fourteen different alternatively spliced forms of the S. mansoni vasohibin (SmVASL) gene were detected that encode seven different protein isoforms including one with a complete C-terminal end, and other isoforms with shorter C-terminal portions. Using S. mansoni homologs, we have employed a parsimonious rationale to compute the total gene losses/gains in nematodes, arthropods and deuterostomes under either the Coelomata or the Ecdysozoa evolutionary hypotheses; our results show a lower losses/gains number under the latter hypothesis. The genes discussed which are conserved between S. mansoni and deuterostomes, probably have an ancient origin and were lost in Ecdysozoa, being still present in Lophotrochozoa. Given their known functions in Deuterostomia, it is possible that some of them have been co-opted to perform functions related (directly or indirectly) to host adaptation or interaction with host signaling processes.
    BMC Genomics 02/2007; 8(1):407. DOI:10.1186/1471-2164-8-407 · 3.99 Impact Factor
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