Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response
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Protein & Cell
RESEARCH ARTICLE Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response Department of Infectious Diseases, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, College of Life Sciences, Wuhan University, Wuhan 430071, China & Correspondence: [email protected] (H.-B. Shu) Received March 29, 2020 Accepted April 24, 2020
ABSTRACT The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGASmediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6Cdeficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6Cmediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.
KEYWORDS DNA virus, PPP6C, cGAS, innate immune response, phosphorylation, substrate binding
INTRODUCTION The innate immune system represents the first line of host defense against viral infection. Upon viral infection, © The Author(s) 2020
structurally conserved viral components called pathogen associated molecular patterns (PAMPs) are detected by host pattern recognition receptors (PRRs), which triggers intracellular signaling events that lead to induction of type I interferons (IFNs), proinflammatory cytokines and other downstream effectors. These downstream effectors inhibit viral replication, facilitate clearance of virus-infected cells, and promote adaptive immune response (Janeway and Medzhitov, 2002; Akira et al., 2006; Hu and Shu, 2018). Viral nucleic acids are major PAMPs recognized by host PRRs. Upon DNA virus infection, viral DNA in the cytoplasm is sensed by cyclic GMP-AMP (cGAMP) synthase (cGAS). Importantly, cGAS also detects mitochondrial DNA and nuclear DNA mis-located in the cytosol under certain conditions, which plays a key role in certain autoimmune diseases and inflammatory responses during radiotherapy for cancer (West et al., 2015; Fang et al., 2016; Harding et al., 2017; Mackenzie et al., 2017). Upon binding to DNA, cGAS utilizes GTP and ATP as substrates to synthesize the second messenger 2’3’-cGAMP (Sun et al., 2013; Wu et al., 201
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