Abstract

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 necroptosis1,2,3. However, mice expressing catalytically inactive RIPK1 are viable2,4,5, 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. Ripk1RHIM/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 necroptosis6, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses7,8 but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/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 Ripk1RHIM/RHIMMlkl−/− macrophages, but not in wild-type, Mlkl−/− or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.

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Acknowledgements

We thank J. Diaz, M. Long, C. Allen, M. Garcia, A. Verducci and J. Anunciacion for animal husbandry, R. Newman, K. Heger and the Genentech genetic analysis and histology laboratories for technical assistance, and J. Lai and S. Stawicki for antibody project management.

Author information

Affiliations

  1. Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

    • Kim Newton
    • , Katherine E. Wickliffe
    • , Allie Maltzman
    • , Debra L. Dugger
    •  & Vishva M. Dixit
  2. Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia

    • Andreas Strasser
  3. Department of Medical Biology, Melbourne University, Victoria 3010, Australia

    • Andreas Strasser
  4. Department of Proteomics and Biological Resources, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

    • Victoria C. Pham
    •  & Jennie R. Lill
  5. Department of Molecular Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

    • Merone Roose-Girma
    •  & Søren Warming
  6. Department of Pathology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

    • Margaret Solon
    • , Hai Ngu
    •  & Joshua D. Webster

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Contributions

M.R.G. and S.W. generated the Ripk1RHIM/+ and Ripk3RHIM/+ mice, K.N., A.M., D.L.D., K.E.W., and A.S. designed and performed experiments, V.C.P. and J.R.L. characterized the RIPK1 and RIPK3 autophosphorylation sites, M.S. performed immunohistochemistry with quantitative analyses by H.N., J.D.W. analysed histological data, and V.M.D. helped with experimental design.

Competing interests

All authors were employees or visiting scientists of Genentech.

Corresponding authors

Correspondence to Kim Newton or Vishva M. Dixit.

Reviewer Information Nature thanks H. Walczak and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature20559

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