title: A Src-family-tyrosine kinase, Lyn, is required for efficient IFN-β expression in pattern recognition receptor, RIG-I, signal pathway by interacting with IPS-1.
abstract: Retinoic acid-inducible gene I (RIG-I) plays an important role in antiviral immunity as a cytosolic receptor recognizing invading viruses. The activation of downstream signaling pathways led by IFN-β promoter stimulator-1 (IPS-1), an adaptor, is known to culminate in the activation of IRFs and the expression of type I interferons. However, the role of Src-family-tyrosine kinases (STKs) in the RIG-I signaling pathway has not been fully evaluated. Through a combined approach of immunoprecipitation and micro reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis, we established that Lyn, one of the STKs, is associated with RIG-I in macrophages. The association of Lyn and RIG-I was confirmed by co-immunoprecipitation study with 293T cells overexpressing Lyn and RIG-I. Suppression of Lyn by siRNA knockdown or a pharmacological inhibitor (PP2) resulted in the attenuation of IRF3 activation and IFN-β expression induced by short poly I:C, a RIG-I agonist, in macrophages. Lyn activation, as determined by phosphorylation of Tyr396 residue, was observed upon short poly I:C stimulation in the mitochondria of macrophages. Short poly I:C induced the formation of speckle-like aggregates of Lyn, which are prominent in mitochondria. Lyn associated with IPS-1, an adaptor protein of RIG-I, which resides on mitochondria membrane. Helicase domain of RIG-I and CARD of IPS-1 are responsible for the interaction with Lyn while SH3 and SH2 domains in Lyn are required for the association with RIG-I and IPS-1. Collectively, our results indicate that Lyn plays a positive regulatory role in RIG-I-mediated interferon expression as a downstream component of IPS-1. They provide further information as to how tyrosine kinases such as STKs play a role in the regulation of antiviral immunity.
authors.affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Young Ju
authors.initials: YJ
authors.lastname: Lim
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Koo
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea.
authors.forename: Eun-Hee
authors.initials: EH
authors.lastname: Hong
authors.affiliation: School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea.
authors.forename: Zee-Yong
authors.initials: ZY
authors.lastname: Park
authors.affiliation: College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-808, Republic of Korea.
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Lim
authors.affiliation: College of Pharmacy, Institute of Pharmaceutical Sciences, Hanyang University, Ansan, Gyeonggido 426-791, Republic of Korea.
authors.forename: Ok-Nam
authors.initials: ON
authors.lastname: Bae
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Republic of Korea. Electronic address: joolee@catholic.ac.kr.
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Lee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D056148:Chromatography, Reverse-Phase; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D010766:Phosphorylation; D011070:Poly I-C / Q000494:pharmacology; D054730:Protein Interaction Domains and Motifs; D034741:RNA, Small Interfering / Q000235:genetics; D051192:Receptors, Pattern Recognition; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D050838:Interferon Regulatory Factor-3; D016899:Interferon-beta; D011070:Poly I-C; D034741:RNA, Small Interfering; D051192:Receptors, Pattern Recognition; D019061:src-Family Kinases; C512612:MAVS protein, mouse; C494233:Irf3 protein, mouse; C505371:MAVS protein, human; C060902:lyn protein-tyrosine kinase; C503356:Ddx58 protein, mouse
keywords: Anti-viral signaling; IRF3; Innate immunity; Src-family-tyrosine kinases; Type I interferon