Affiliations 

  • 1 Department of Biomedical Science, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam 13200, Kepala Batas, Pulau Pinang, Malaysia. alvin.amdi1@gmail.com
  • 2 Analytical Biochemistry Research Centre, Universiti Sains Malaysia, 11900, Bayan Lepas, Pulau Pinang, Malaysia
  • 3 Department of Biomedical Science, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam 13200, Kepala Batas, Pulau Pinang, Malaysia
  • 4 Department of Biomedical Science, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam 13200, Kepala Batas, Pulau Pinang, Malaysia. skng@usm.my
Mol Biol Rep, 2023 Apr;50(4):3909-3917.
PMID: 36662450 DOI: 10.1007/s11033-023-08253-3

Abstract

BACKGROUND: IRF9 is a transcription factor that mediates the expression of interferon-stimulated genes (ISGs) through the Janus kinase-Signal transducer and activator of transcription (JAK-STAT) pathway. The JAK-STAT pathway is regulated through phosphorylation reactions, in which all components of the pathway are known to be phosphorylated except IRF9. The enigma surrounding IRF9 regulation by a phosphorylation event is intriguing. As IRF9 plays a major role in establishing an antiviral state in host cells, the topic of IRF9 regulation warrants deeper investigation.

METHODS: Initially, total lysates of 2fTGH and U2A cells (transfected with recombinant IRF9) were filter-selected and concentrated using phosphoprotein enrichment assay. The phosphoprotein state of IRF9 was further confirmed using Phos-tag™ assay. All protein expression was determined using Western blotting. Tandem mass spectrometry was conducted on immunoprecipitated IRF9 to identify the phosphorylated amino acids. Finally, site-directed mutagenesis was performed and the effects of mutated IRF9 on relevant ISGs (i.e., USP18 and Mx1) was evaluated using qPCR.

RESULTS: IRF9 is phosphorylated at S252 and S253 under IFNβ-induced condition and R242 under non-induced condition. Site-directed mutagenesis of S252 and S253 to either alanine or aspartic acid has a modest effect on the upregulation of USP18 gene-a negative regulator of type I interferon (IFN) response-but not Mx1 gene.

CONCLUSION: Our preliminary study shows that IRF9 is phosphorylated and possibly regulates USP18 gene expression. However, further in vivo studies are needed to determine the significance of IRF9 phosphorylation.

* Title and MeSH Headings from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.