The kinase domain of RIPK3 tunes its scaffolding functions
- Author(s)
- Chiou, S; Horne, CR; Patel, KM; Preaudet, A; Rickard, JA; Young, SN; Jois, A; Garnish, SE; Hempel, A; Hall, C; Hildebrand, JM; Kueh, AJ; Silke, J; Putoczki, TL; Hawkins, ED; Samson, AL; Murphy, JM;
- Journal Title
- Cell Death & Differentiation
- Publication Type
- Feb 3
- Abstract
- 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, RIPK3(K51A), are ostensibly normal in the absence of challenge, indicating that RIPK3 and its kinase activity are dispensable for development. However, another kinase-inactivating mutation, RIPK3(D161N), results in embryonic lethality in mice due to widespread apoptosis. As a result, the RIPK3(D161N) 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 RIPK3(D161N) and RIPK3(K51A) to a third previously-uncharacterized kinase-dead variant, RIPK3(D143N). We show that RIPK3(K51A) was unstable and did not associate with RIPK1, RIPK3(D161N) was unstable but interacted with RIPK1, whereas RIPK3(D143N) 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, Ripk3(D143N/D143N) mice exhibited a partially penetrant lethality in utero. However, once born, Ripk3(D143N/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 Ripk3(D143N/D143N) mice, with the RIPK3(D143N) 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.
- Publisher
- Springer Nature
- Research Division(s)
- Inflammation; Personalised Oncology; New Medicines and Diagnostics; Blood Cells and Blood Cancer
- PubMed ID
- 41634369
- Publisher's Version
- https://doi.org/10.1038/s41418-026-01677-x
- Open Access at Publisher's Site
https://doi.org/10.1038/s41418-026-01677-x- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2026-02-09 09:18:21
Last Modified: 2026-02-09 09:23:24