RIPK1 inhibits ZBP1-driven necroptosis during development

Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis(1-3). However, mice expressing catalytically inactive RIPK1 are viable(2,4,5), and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1(RHIM/RHIM) mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis(6), in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1(RHIM/RHIM) mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses(7,8) but previously had no defined role in development, also prevented perinatal lethality in Ripk1(RHIM/RHIM) mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1(RHIM/RHIM)Mlkl(-/-) macrophages, but not in wild-type, Mlkl(-/-) or Ripk1(RHIM/RHIM)Ripk3(RHIM/RHIM) macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.