ABSTRACT
The cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin, especially after nuclear envelope breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. Here, we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis in human cell lines and uncovered two parallel mechanisms underlying this suppression. First, cGAS was hyperphosphorylated at the N terminus by mitotic kinases, including Aurora kinase B. The N terminus of cGAS was critical for sensing nuclear chromatin but not mitochondrial DNA. Chromatin sensing was blocked by hyperphosphorylation. Second, oligomerization of chromatin-bound cGAS, which is required for its activation, was prevented. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction.
Subject(s)
Chromatin/metabolism , Mitosis , Nucleotidyltransferases/metabolism , Aurora Kinase B/metabolism , Cell Cycle , Cell Line , DNA/metabolism , DNA, Mitochondrial/metabolism , Enzyme Activation , Humans , Nucleotides, Cyclic/metabolism , Nucleotidyltransferases/chemistry , Phosphorylation , Protein MultimerizationABSTRACT
Cellular senescence is a natural barrier to tumorigenesis and it contributes to the antitumor effects of several therapies, including radiation and chemotherapeutic drugs. Senescence also plays an important role in aging, fibrosis, and tissue repair. The DNA damage response is a key event leading to senescence, which is characterized by the senescence-associated secretory phenotype (SASP) that includes expression of inflammatory cytokines. Here we show that cGMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor that activates innate immunity, is essential for senescence. Deletion of cGAS accelerated the spontaneous immortalization of mouse embryonic fibroblasts. cGAS deletion also abrogated SASP induced by spontaneous immortalization or DNA damaging agents, including radiation and etoposide. cGAS is localized in the cytoplasm of nondividing cells but enters the nucleus and associates with chromatin DNA during mitosis in proliferating cells. DNA damage leads to accumulation of damaged DNA in cytoplasmic foci that contain cGAS. In human lung adenocarcinoma patients, low expression of cGAS is correlated with poor survival. These results indicate that cGAS mediates cellular senescence and retards immortalization. This is distinct from, and complementary to, the role of cGAS in activating antitumor immunity.
Subject(s)
Cellular Senescence/physiology , Nucleotidyltransferases/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma of Lung , Animals , Cells, Cultured , Cellular Senescence/immunology , Cytosol/metabolism , DNA Damage , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , HEK293 Cells , Humans , Immunity, Innate/physiology , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Nucleotidyltransferases/deficiency , Nucleotidyltransferases/genetics , Phenotype , PrognosisABSTRACT
During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Interferon Regulatory Factor-3/metabolism , Membrane Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Sequence , Animals , Cell Line , Humans , I-kappa B Kinase/metabolism , Interferon Regulatory Factor-3/chemistry , Interferon-alpha/biosynthesis , Interferon-beta/biosynthesis , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Multimerization , Protein Serine-Threonine Kinases/metabolism , Recombinant Proteins/metabolism , Sendai virus/physiology , Serine/metabolism , Signal Transduction , Ubiquitination , Vesiculovirus/physiologyABSTRACT
The transcription factor interferon regulatory factor 5 (IRF5) is essential for the induction of inflammatory cytokines, but the mechanism by which IRF5 is activated is not well understood. Here we present evidence that the kinase IKKß phosphorylates and activates IRF5 in response to stimulation in several inflammatory pathways, including those emanated from Toll-like receptors and retinoic acid-inducible gene I-like receptors. IKKß phosphorylates mouse IRF5 at specific residues, including serine 445 (S446 in human IRF5 isoform 1), as evidenced by mass spectrometry analysis and detection with a phosphospecific antibody. Recombinant IKKß phosphorylated IRF5 at Ser-445 in vitro, and a point mutation of this serine abolished IRF5 activation and cytokine production. Depletion or pharmacologic inhibition of IKKß prevented IRF5 phosphorylation. These results indicate that IKKß is an IRF5 kinase that instigates inflammation.
