Phosphorylation Of Human Interferon Regulatory Factor 9 During Type I Interferon Response
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Date
2021-10
Authors
Alvin Dickson Anak Paul Joko
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Type I interferon response describes the cellular innate antiviral response whereby hundreds of interferon-stimulated genes (ISGs) are upregulated following virus infection. This massive induction of ISGs is mediated by the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, where interferon regulatory factor 9 (IRF9), together with phosphorylated STAT1 and STAT2, forms the transcription activator, ISGF3. Post-translational modification by phosphorylation is an essential regulatory mechanism within the JAK-STAT pathway. Furthermore, studies have shown ubiquitous phosphorylation regulation for other human IRF family members. However, none have been described for IRF9. In this study, IRF9 was shown to be phosphorylated in response to type I interferon (e.g. IFNβ). Total protein lysate subjected to selective phosphoprotein enrichment column showed enrichment of IRF9 in the phosphoprotein fraction. Furthermore, Phos-Tag™ polyacrylamide assay showed the presence of two IRF9 protein phosphorylation states. Both of the observations indicated that IRF9 is a phosphoprotein. To determine the specific phosphorylated amino acid residue(s), IRF9 was immunoprecipitated and then subjected to tandem mass spectrometry analysis. The phosphorylation of IRF9 occurred at serine 252 (S252) and serine 253 (S253) under IFNβ-induced conditions in human embryonic kidney 293T (HEK293T) cells. These sites lie within the IRF-associated domain of IRF9. Site-directed mutagenesis was carried out on S252 and S253 to infer the physiological importance of both phosphorylation sites
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Keywords
Phosphorylation , Human Interferon Regulatory Factor 9 , Type I Interferon Response , Phosphorylation Of Human Interferon Regulatory Factor 9