Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Identification of Small GTPases That Phosphorylate IRF3 through TBK1 Activation Using an Active Mutant Library Screen

  • Jae-Hyun, Yu (Department of Pharmacology and Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea) ;
  • Eun-Yi, Moon (Department of Bioscience and Biotechnology, Sejong University) ;
  • Jiyoon, Kim (Department of Pharmacology and Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea) ;
  • Ja Hyun, Koo (College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University)
  • Received : 2022.09.06
  • Accepted : 2022.09.26
  • Published : 2023.01.01
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Abstract

Interferon regulatory factor 3 (IRF3) integrates both immunological and non-immunological inputs to control cell survival and death. Small GTPases are versatile functional switches that lie on the very upstream in signal transduction pathways, of which duration of activation is very transient. The large number of homologous proteins and the requirement for site-directed mutagenesis have hindered attempts to investigate the link between small GTPases and IRF3. Here, we constructed a constitutively active mutant expression library for small GTPase expression using Gibson assembly cloning. Small-scale screening identified multiple GTPases capable of promoting IRF3 phosphorylation. Intriguingly, 27 of 152 GTPases, including ARF1, RHEB, RHEBL1, and RAN, were found to increase IRF3 phosphorylation. Unbiased screening enabled us to investigate the sequence-activity relationship between the GTPases and IRF3. We found that the regulation of IRF3 by small GTPases was dependent on TBK1. Our work reveals the significant contribution of GTPases in IRF3 signaling and the potential role of IRF3 in GTPase function, providing a novel therapeutic approach against diseases with GTPase overexpression or active mutations, such as cancer.

Keywords

Acknowledgement

This work was supported by National Research Foundation of Korea grants funded by the Korea government (MSIP) (2021R1C1C1013323, 2021R1A4A5033289) as well as by the Creative-Pioneering Researchers Program and New Faculty Startup Fund from Seoul National University.

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