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RIPK3 sequentially recruits MLKL and RIPK1 to induce PANoptosis and chemokine production

Cell Death & Differentiation (2026)Cite this article

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Abstract

Receptor-interacting protein kinase 3 (RIPK3) has emerged as a central player in necroptosis and apoptosis activation in specific scenarios, concurrently modulating inflammatory responses. Here, we reveal that direct activation of RIPK3 concomitantly triggers mixed lineage kinase domain-like (MLKL) phosphorylation, caspase activation, and gasdermin cleavage within individual cells, inducing PANoptotic cell death. This process is orchestrated by the formation of RIPK3-MLKL-RIPK1-FADD-Caspase-8 complexes on progressively polymerized RIPK3 homo-aggregates, achieved through sequential recruitment dictated by the differential affinities of MLKL and Receptor-interacting protein kinase 1 (RIPK1) for distinct oligomeric states of RIPK3. In this process, MLKL- and GSDMD-mediated membrane rupture is respectively inhibited by Caspase-3-dependent cleavage of RIPK3 and GSDMD cleavage, while the pro-necrotic kinase activity of RIPK3 impedes RIPK1 recruitment and attenuates caspase activation. Cross-regulation between pathways results in unique cellular morphology, altered damage-associated molecular patterns (DAMPs) release profiles and distinct chemokine secretion paradigms that differ fundamentally from classical necroptosis, apoptosis and pyroptosis. This work highlights a common mechanism unveiling RIPK3 as a multimolecular platform to modulate and integrate different programmed cell death (PCD) pathways, thus providing a framework for targeting inflammatory cell death in disease.

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Fig. 1: Osmotic stress induces simultaneous RIPK3-dependent MLKL phosphorylation and caspase cleavage in single cells.
Fig. 2: Direct RIPK3 activation orchestrates PANoptotic cell death.
Fig. 3: The RIPK1-FADD axis mediates caspase processing following direct RIPK3 activation.
Fig. 4: Differential binding affinity of MLKL and RIPK1 for RIPK3 orchestrates stepwise assembly of RIPK3-cored death complex.
Fig. 5: RIPK3–RIPK1 structural configuration gates MLKL-mediated cytolytic execution.
Fig. 6: RIPK3-initiated PANoptosis generates unique cytomorphological signatures during cell death execution.
Fig. 7: RIPK1 dominantly coordinates chemokine production and macrophage recruitment via NF-κB during PANoptosis execution.
Fig. 8: A working model of RIPK3-initiated PANoptosis.

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Data availability

All data and materials supporting the conclusions of this paper are presented in the main text, figures, supplementary data figures, and attachment files. The uncropped Western blots are included in the Supplementary Material. Further data can be received from the corresponding author on reasonable request. RNA-sequencing data were deposited into the Gene Expression Omnibus (GEO) database under accession code GSE320510. Source data are provided with this paper.

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Acknowledgements

The authors thank D Xiao, Y Lei, H Pan, S Qiu, and L Chen for reagents. We thank Drs. S He, M Zheng, X Yang, B Han, and H Wang for helpful discussion. We thank X Dong for the identification of GSDMD-deficient clones. The authors thank the Chemical Biology Core Facility at SIBCB for technical assistance in time-lapse imaging.

Funding

This study is supported by the grants from the National Natural Science Foundation of China (82072573, 82273335, 8240113215 and 82403114), Shanghai Municipal Health Commission (2022LJ015, 2022YQ039 and 20224Y0186).

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Author notes

  1. These authors contributed equally: Yu Yang, Yue Wang, Yang Wang, Erpeng Wu.

Authors and Affiliations

  1. Department of Respiratory and Critical Care Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yu Yang, Yue Wang, Erpeng Wu, Chi Zhang, Shuhui Cao, Jingwen Li, Huiping Qiang, Lincheng Zhang, Yuqing Lou, Rong Qiao, Yan Zhou, Runbo Zhong & Hua Zhong

  2. Department of Cell and Developmental Biology at School of Life Sciences, State Key Laboratory of Genetic Engineering, Fudan University, Shanghai, China

    Yang Wang

  3. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Hao Chen

  4. State Key Laboratory of Cell Biology, Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Liming Sun & Shenao Zhou

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Contributions

YY, RZ, and HZ conceptualized the study. YY, YuW, and EW designed the methodology. YY and YuW performed the majority of experiments and data analysis. YaW performed the confocal microscopy and image analysis. LS did the mass spectrometry analysis. HC performed the bioinformatics analysis. CZ, SC, and JL housed the mice and conducted the experiments in the animal facility. LZ and HQ generated all the plasmids. YL, RQ, and SZ discussed the results and provided valuable expertise. YY wrote the manuscript with input from all authors. YZ and HZ acquired the funding.

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Correspondence to Yu Yang, Yan Zhou, Runbo Zhong or Hua Zhong.

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All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University (Ethical Approval No. KS(Y)24008), and tumor dimensions complied with IACUC-specified limits. No human material was used.

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Yang, Y., Wang, Y., Wang, Y. et al. RIPK3 sequentially recruits MLKL and RIPK1 to induce PANoptosis and chemokine production. Cell Death Differ (2026). https://doi.org/10.1038/s41418-026-01737-2

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