SPATA2 links CYLD to the TNF-α receptor signaling complex and modulates the receptor signaling outcomes

TNF-alpha is a key regulator of innate immune and proinflammatory responses. However, the composition of the TNF-alpha receptor-associated signaling complexes (TNF-RSC) and the architecture of the downstream signaling networks are incompletely understood. We employed quantitative mass spectrometry to demonstrate that TNF-alpha stimulation induces widespread protein phosphorylation and that the scope of phosphorylation expands in a temporal manner. TNF-alpha stimulation also induces rapid ubiquitylation of components of the TNF-RSC. Temporal analysis of the TNF-RSC composition identified SPATA2 as a novel component of the TNF-RSC. The predicted PUB domain in the N-terminus of SPATA2 interacts with the USP domain of CYLD, whereas the C-terminus of SPATA2 interacts with HOIP. SPATA2 is required for recruitment of CYLD to the TNF-RSC. Downregulation of SPATA2 augments transcriptional activation of NF-kappa B and inhibits TNF-alpha-induced necroptosis, pointing to an important function of SPATA2 in modulating the outcomes of TNF-alpha signaling. Taken together, our study draws a detailed map of TNF-alpha signaling, identifies SPATA2 as a novel component of TNF-beta signaling, and provides a rich resource for further functional investigations.