Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis

Proc Natl Acad Sci U S A. 2022 Oct 11;119(41):e2207240119. doi: 10.1073/pnas.2207240119. Epub 2022 Oct 3.


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 (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies in vitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation in vivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

Keywords: MLKL; ZBP1; caspase-8; interferon; necroptosis.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Apoptosis / physiology
  • Caspase 8 / genetics
  • Caspase 8 / metabolism
  • DNA-Binding Proteins / metabolism
  • Fas-Associated Death Domain Protein / genetics
  • Interferons / metabolism
  • Mice
  • Necroptosis*
  • Nucleic Acids*
  • Nucleotidyltransferases / metabolism
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism


  • DNA-Binding Proteins
  • Fadd protein, mouse
  • Fas-Associated Death Domain Protein
  • Nucleic Acids
  • RNA-Binding Proteins
  • Zbp1 protein, mouse
  • Interferons
  • Protein Kinases
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Nucleotidyltransferases
  • Casp8 protein, mouse
  • Caspase 8