The DNA Sensor cGAS is Decorated by Acetylation and Phosphorylation Modifications in the Context of Immune Signaling. Author Bokai Song, Todd Greco, Krystal Lum, Caroline Taber, Ileana Cristea Publication Year 2020 Type Journal Article Abstract The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared with the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses-HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection. Keywords Humans, Signal Transduction, Mutation, Phosphorylation, Protein Processing, Post-Translational, Chromatography, Liquid, HEK293 Cells, Tandem Mass Spectrometry, Cytomegalovirus Infections, Fibroblasts, Immunity, Innate, Acetylation, Interferon-beta, Nucleotidyltransferases, Apoptosis, Host Microbial Interactions, Herpesviridae Infections Journal Mol Cell Proteomics Volume 19 Issue 7 Pages 1193-1208 Date Published 2020 Jul ISSN Number 1535-9484 DOI 10.1074/mcp.RA120.001981 Alternate Journal Mol Cell Proteomics PMCID PMC7338091 PMID 32345711 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML