Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death

Mol Cell Proteomics. 2017 Jul;16(7):1200-1216. doi: 10.1074/mcp.M117.068189. Epub 2017 May 24.


TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caspase 8 / metabolism
  • Cell Death / drug effects
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Fas-Associated Death Domain Protein / metabolism
  • Humans
  • Jurkat Cells
  • NF-kappa B / metabolism
  • Phosphoproteins / analysis*
  • Phosphorylation / drug effects
  • Proteomics / methods*
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Serine / metabolism
  • Signal Transduction / drug effects
  • Tumor Necrosis Factor-alpha / metabolism*
  • Tumor Necrosis Factor-alpha / pharmacology


  • FADD protein, human
  • Fas-Associated Death Domain Protein
  • NF-kappa B
  • Phosphoproteins
  • TNF protein, human
  • Tumor Necrosis Factor-alpha
  • Serine
  • RIPK1 protein, human
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • CASP8 protein, human
  • Caspase 8