Monitoring RIPK1 Phosphorylation in the TNFR1 Signaling Complex

Methods Mol Biol. 2018:1857:171-179. doi: 10.1007/978-1-4939-8754-2_17.

Abstract

Receptor-interacting protein kinase 1 (RIPK1) is a component of the TNFR1 signaling complex (also known as complex I or TNFR-SC), where its ubiquitylation by cIAP1/2 and LUBAC serves to initiate prosurvival and proinflammatory responses through activation of the MAPK and NF-κB pathways. IKKα/β-mediated phosphorylation of RIPK1 in complex I was shown to maintain RIPK1 in a prosurvival modus. Consequently, conditions affecting proper IKKα/β activation perturb IKKα/β-phosphorylation of RIPK1 and switch the TNF response toward RIPK1 kinase-dependent cell death. Methods to study the posttranslational modifications of RIPK1 in complex I are therefore of great value. Here, we describe a detailed protocol to isolate complex I-associated RIPK1 from cells and provide different tools to study the phosphorylation status of RIPK1 in TNFR1 complex I.

Keywords: Apoptosis; Cell death; Complex I; Immunoblotting; Immunoprecipitation; Necroptosis; Phosphorylation; RIPK1; TNF; TNFR1; Ubiquitylation.

MeSH terms

  • Animals
  • Cell Death
  • Cells, Cultured
  • Embryo, Mammalian / metabolism
  • Embryo, Mammalian / pathology*
  • Fibroblasts / metabolism
  • Fibroblasts / pathology*
  • Mice
  • Multiprotein Complexes / metabolism*
  • Phosphorylation
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Receptors, Tumor Necrosis Factor, Type I / metabolism*
  • Signal Transduction

Substances

  • Multiprotein Complexes
  • Receptors, Tumor Necrosis Factor, Type I
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk1 protein, mouse