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

• Published in: Molecular & cellular proteomics 2017-07, Vol.16 (7), p.1200-1216
• Main Authors: Mohideen, Firaz, Paulo, Joao A., Ordureau, Alban, Gygi, Steve P., Harper, J. Wade
• Format: Article
• Language: English
• Subjects: Apoptosis; Caspase; Caspase 8 - metabolism; Caspase-8; Cell death; Cell Death - drug effects; Cell Line, Tumor; Cell Survival - drug effects; DIABLO protein; FADD protein; Fas-Associated Death Domain Protein - metabolism; Functional analysis; Gene expression; Humans; Inflammation; Jurkat Cells; Kinases; MAP kinase; NF-kappa B - metabolism; NF-κB protein; Peptides; Phosphoproteins - analysis; Phosphorylation; Phosphorylation - drug effects; Proteins; Proteomics; Proteomics - methods; Receptor-Interacting Protein Serine-Threonine Kinases - metabolism; Serine - metabolism; Signal transduction; Signal Transduction - drug effects; Signalling systems; TAK1 protein; Tumor necrosis factor receptors; Tumor Necrosis Factor-alpha - metabolism; Tumor Necrosis Factor-alpha - pharmacology; Tumor necrosis factor-α
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M117.068189

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.