A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

Abstract Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activate...

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Autores principales:	Kanoko Takahashi, Takahiro Niki, Emari Ogawa, Kiku Fumika, Yu Nishioka, Masaaki Sawa, Hiroyuki Arai, Kojiro Mukai, Tomohiko Taguchi
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d0e2578f1c324ddc8a46d5d697abf0e7
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spelling	oai:doaj.org-article:d0e2578f1c324ddc8a46d5d697abf0e72021-12-02T17:34:31ZA cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation10.1038/s41598-021-91562-z2045-2322https://doaj.org/article/d0e2578f1c324ddc8a46d5d697abf0e72021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91562-zhttps://doaj.org/toc/2045-2322Abstract Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.Kanoko TakahashiTakahiro NikiEmari OgawaKiku FumikaYu NishiokaMasaaki SawaHiroyuki AraiKojiro MukaiTomohiko TaguchiNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kanoko Takahashi Takahiro Niki Emari Ogawa Kiku Fumika Yu Nishioka Masaaki Sawa Hiroyuki Arai Kojiro Mukai Tomohiko Taguchi A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
description	Abstract Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.
format	article
author	Kanoko Takahashi Takahiro Niki Emari Ogawa Kiku Fumika Yu Nishioka Masaaki Sawa Hiroyuki Arai Kojiro Mukai Tomohiko Taguchi
author_facet	Kanoko Takahashi Takahiro Niki Emari Ogawa Kiku Fumika Yu Nishioka Masaaki Sawa Hiroyuki Arai Kojiro Mukai Tomohiko Taguchi
author_sort	Kanoko Takahashi
title	A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
title_short	A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
title_full	A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
title_fullStr	A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
title_full_unstemmed	A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation
title_sort	cell-free assay implicates a role of sphingomyelin and cholesterol in sting phosphorylation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/d0e2578f1c324ddc8a46d5d697abf0e7
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A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

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