Dataset Information

Analysis of RIPK1 phosphorylation in vitro

ABSTRACT: This study aims at determining which RIPK1 residues are phosphorylated.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Silvia Surinova

LAB HEAD: Silvia Surinova

PROVIDER: PXD008518 | Pride | 2018-09-25

REPOSITORIES: Pride

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			Action	DRS
	andromeda.7z	Other
	experimentalDesignTemplate.txt	Txt
	experimentalDesignTemplate_annotated.txt	Txt
	regmab2_QEP171124_214.raw	Raw
	regmab2_QEP171124_226.raw	Raw

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Project description:Type 1 diabetes (T1D) is characterized by autoimmune destruction of pancreatic β-cells, insulin insufficiency, and hyperglycemia. Receptor interacting protein kinase 1 (RIPK1) is a multifunctional regulator of cell fate with kinase and scaffolding functions. We previously identified RIPKs as regulators of beta-cell cytotoxicity in vitro, and Ripk1 expression is increased in islets from aged non-obese diabetic (NOD) mice and β-cells from T1D donors. We hypothesized that RIPK1 regulates cytokine- and autoimmune-mediated β-cell demise in a mouse model of T1D. Using NIT-1 beta-cells derived from NOD mice, we tested control (CTL) and Ripk1 gene-edited (Ripk1delta, CRISPR/Cas9 KO) beta-cells treated with TNFα+IFNγ or co-cultured with self-reactive splenocytes from diabetic NOD mice. We performed live cell imaging, RNAseq, kinome profiling, and flow cytometry, tested small molecule RIPK1 inhibitors, and evaluated resistance to beta-cell autoimmunity in vivo. We found TNF alpha +IFN gamma increase RIPK1 phosphorylation, caspase 3/7 activity, and cell death in NIT-1 CTL cells. In contrast, cytotoxicity was blocked with small molecule RIPK1 inhibition or in NIT-1 Ripk1delta cells. Co-labeling of caspase 3/7 activation and cell death in single cells revealed protection from caspase-dependent and -independent death in Ripk1Δ cells. RNAseq uncovered differential cell death-, immune-, and identity-related gene expression between genotypes, and kinome profiling identified changes in MAPK, Eph, JAK, and other kinase activity that were associated with protection from cell death. Furthermore, NIT-1 Ripk1delta cells were protected from destruction following co-culture with self-reactive splenocytes in vitro, and NIT-1 implant graft luminescence was 399-fold greater in Ripk1delta versus CTL cells 19 days post splenocyte administration in vivo. Collectively, our findings indicate RIPK1 regulates beta-cell fate in a model of autoimmune diabetes via actions on gene expression and kinase signaling. Additional studies are needed to characterize RIPK1 signaling in primary islets and human beta-cells, and to determine whether RIPK1 can be targeted to protect beta-cells in T1D. In this data set, NIT-1 cells (control and Ripk1 KO), vehicle treated or treated with IFN-gamma and TNF alpha were used for kinome profiling.

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Analysis of RIPK1 phosphorylation in vitro

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