biostudies-literatureRodriguez DADeutsche ForschungsgemeinschaftNIAID NIH HHSHHS | NIH | National Cancer InstituteFoundation for the National Institutes of HealthNCI NIH HHSU.S National Cancer Institutee2207240119https://www.ebi.ac.uk/biostudies/studies/S-EPMC9565532biostudies-literatureUnknown119(41)The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (<i>Mlkl^FLAG/FLAG</i>), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. <i>Casp8^-/-Mlkl^FLAG/FLAG</i> mice were viable and displayed phosphorylated MLKL in aProceedings of the National Academy of Sciences of the United States of AmericaCaspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis.PMC9565532CA2311620R01 AI135025P30 CA006927AI44828CA0211765CA006927AI144400AI135025P30 CA021765R01 AI144400R01 AI044828R35 CA231620R37 AI044828KA 4830/1-1Kalkavan HPelletier SQuarato GLiedmann STummers BBalachandran SGuy CZhang TChen MJShaw JJPRodriguez DACrawford JCFitzgerald PGreen DRPalacios GfalseCaspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis.The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis in vivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (<i>Mlkl^FLAG/FLAG</i>), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. <i>Casp8^-/-Mlkl^FLAG/FLAG</i> mice were viable and displayed phosphorylated MLKL in a2022-01-01T00:00:00Z2022 Oct2026-07-14T19:05:47.067Z2025-04-07T03:15:37.626ZS-EPMC95655323619121110.1073/pnas.2207240119