K63-linked ubiquitination regulates RIPK1 kinase activity to prevent cell death during embryogenesis and inflammation

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of cell death through its kinase activity. However, how its kinase activity is regulated remains poorly understood. Here, we generate Ripk1K376R/K376R knock-in mice in which the Lys(K)63-linked ubiquitination of RIPK1 is impaired. The knock-in mice display an early embryonic lethality due to massive cell death that is resulted from reduced TAK1-mediated suppression on RIPK1 kinase activity and forming more TNFR1 complex II in Ripk1K376R/K376R cells in response to TNFα. Although TNFR1 deficiency delays the lethality, concomitant deletion of RIPK3 and Caspase8 fully prevents embryonic lethality of Ripk1K376R/K376R mice. Notably, Ripk1K376R/- mice are viable but develop severe systemic inflammation that is mainly driven by RIPK3-dependent signaling pathway, indicating that K63-linked ubiquitination on Lys376 residue of RIPK1 also contributes to inflammation process. Together, our study reveals the mechanism by which K63-linked ubiquitination on K376 regulates RIPK1 kinase activity to control cell death programs.


Statistical parameters
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Data
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Sample size
The cell samples were collected and performed with at least three independent experiments. The samples from animal were collected from three mice every group and performed with three independent experiments. The human samples were independently collected for at least three times and measured with two duplicates. The exact size was described in the figure or the legend.

Data exclusions N/A Replication
The findings were reliably reproduced, and data shown are representative of three independent experiments.
Randomization N/A. Experimental animals were grouped according to their genotypes and no randomization was necessary. Antibodies used in this study were described in the methods.

Validation
All antibodies used in this study were validated accrording to the manufacturer's instruction and all worked well.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) The source of HEK293T cells were from ATCC, and the source of immortalized MEFs were described in the methods.

Authentication
The HEK293T cells were purchased from ATCC and authenticated by the vendor, and the immortalized MEFs were authenticated according to the genotypes of embryos.

Mycoplasma contamination
All cell lines were tested to be negative for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.

April 2018
Animals and other organisms Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals Research animals were described in the methods.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field.

Flow Cytometry
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Sample preparation
For mice samples, the spleens from mice were perfused and digested into single-cell suspensions. After RBC lysis buffer treatment, the whole cells were washed with PBS and then stained with corresponding fluorescent antibodies and left at 4 until use. All mouse experiments were performed in compliance with institutional guidelines and according to the protocol approved by the Institutional Animal Care and Use Committee of Tsinghua University.

Instrument
For cell analysis, LSRFortessa (BD Biosciences) was used.

Software
Flow cytometry data were analyzed with FlowJo (Tree Star).
Cell population abundance Without using sorting strategy.
Gating strategy

Characterization of Spleen T cells, B cells and Neutrophils.
In the spleen of mice, the living cell fractions gated from preliminary FSC/SSC gates could be further divided into four cell types: CD4+ T cells, CD8+ T cells, CD19+B220+ B cells and CD11b+Ly6G+ neutrophils. The antibodies and fluorochrome used described as "Antibodies and reagents" in the methods.
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