Cell Death Differ. 2026 Feb 03.
Shene Chiou,
Christopher R Horne,
Komal M Patel,
Adele Preaudet,
James A Rickard,
Samuel N Young,
Asha Jois,
Sarah E Garnish,
Anne Hempel,
Cathrine Hall,
Joanne M Hildebrand,
Andrew J Kueh,
John Silke,
Tracy L Putoczki,
Edwin D Hawkins,
Andre L Samson,
James M Murphy.
The pro-inflammatory programmed cell death pathway, necroptosis, relies on phosphorylation of the terminal effector, MLKL, by RIPK3. RIPK3-deficient mice or those harboring the kinase-inactivating mutation, RIPK3K51A, are ostensibly normal in the absence of challenge, indicating that RIPK3 and its kinase activity are dispensable for development. However, another kinase-inactivating mutation, RIPK3D161N, results in embryonic lethality in mice due to widespread apoptosis. As a result, the RIPK3D161N mutation is thought to confer a toxic gain-of-function. Here, to further explore the impacts of RIPK3 inactivation, we compared the stability and cellular interactions of RIPK3D161N and RIPK3K51A to a third previously-uncharacterized kinase-dead variant, RIPK3D143N. We show that RIPK3K51A was unstable and did not associate with RIPK1, RIPK3D161N was unstable but interacted with RIPK1, whereas RIPK3D143N was stable and bound RIPK1 in a manner comparable to wild-type RIPK3. Thus, all three variants scaffold differently, suggesting that the assembly of cell death machinery by RIPK3 is finely tuned, not just by its kinase activity, but also by the conformation of its kinase domain. Physiologically, Ripk3D143N/D143N mice exhibited a partially penetrant lethality in utero. However, once born, Ripk3D143N/D143N mice were fertile and phenotypically indistinguishable from wild-type mice in the absence of challenge. Full blockade of necroptotic signaling was shown in cells from Ripk3D143N/D143N mice, with the RIPK3D143N mutation also protecting Casp8-/- mice from lethal necroptosis during embryogenesis and preventing necroptotic ileitis in mice that lacked intestinal epithelial caspase-8 expression. Our studies support the idea that RIPK3 is a nexus between apoptotic and necroptotic signaling, and highlight the importance of considering kinase domain conformation in RIPK3 inhibitor development.