Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

Veit Hornung,Matthias Mann,Isabell Bludau,Maria C Tanzer,Che A Stafford

doi:10.1038/s41467-021-26289-6

Abstract

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/.

Highlights

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases
TNF binds to its receptor (TNFR1), which leads to the recruitment of the adaptor proteins TNF receptor-associated factor 2 (TRAF2), tumor necrosis factor receptor type 1-associated death-domain protein (TRADD), and the receptor-interacting serine/threonineprotein kinase 1 (RIPK1), which play critical roles in the decision between cell death, survival, and inflammation[9,10]
Our findings offer a comprehensive resource of phosphorylation events regulated upon TNF stimulation and TNFinduced cell death, which is available to the community at http:// tnfviewer.biochem.mpg.de/

Summary

Introduction

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/. Both pathways are crucial for the upregulation of many target genes, including a range of cytokines and prosurvival factors[13] One such prosurvival factor is the cellular FLICE-like inhibitory protein (cFLIP), which is required to inhibit caspase-8 activity and prevent cell death[14]. We provide evidence for TNFmediated cross-talk with other innate immune-signaling pathways and identify a role for CDK-kinase activity in TNF-induced cell death

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