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

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

Stimulator of interferon genes (STING) is crucial for the kind I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs within the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and prompts TANK-binding kinase 1 (TBK1) on the trans-Golgi community (TGN).
Activated TBK1 then phosphorylates STING at Ser365, producing an interferon regulatory issue 3-docking website on STING. How this response proceeds particularly on the TGN stays poorly understood. Right here we report a cell-free response by which endogenous STING is phosphorylated by TBK1.
The response makes use of microsomal membrane fraction ready from TBK1-knockout cells and recombinant TBK1. We noticed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the character of STING phosphorylation in vivo.
Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract ldl cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and ldl cholesterol within the TGN, these outcomes could present the molecular foundation underlying the precise phosphorylation response of STING on the TGN.

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