Quantitative Phospho-proteomic Analysis of TNF?/NF?B Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

ABSTRACT: TNF? is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NF?B pathway. Moreover, in some contexts, TNF? 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, IKK?/IKK?, I?B?, and NF?B. However, immediate downstream phosphorylation events occurring in response to TNF? signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNF?-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, NF?B, 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 TNF? stimulation. This study provides a resource for further elucidation of TNF?-dependent signaling pathways.

SUBMITTER: Mohideen F 

PROVIDER: S-EPMC5500755 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature