Discovering Protein-Protein Interactions within the Programmed Cell Death Network Using a Protein-Fragment Complementation Screen

•The cell death network is surveyed by an unbiased protein interaction screen•Interactions within and between autophagy and apoptosis modules are discovered•The autophagy regulator DAPK2 interacts with 14-3-3 via a Ser/Thr rich domain•The interaction inhibits DAPK2’s dimerization, cellular, and kinase activities

SummaryApoptosis and autophagy are distinct biological processes, each driven by a different set of protein-protein interactions, with significant crosstalk via direct interactions among apoptotic and autophagic proteins. To measure the global profile of these interactions, we adapted the Gaussia luciferase protein-fragment complementation assay (GLuc PCA), which monitors binding between proteins fused to complementary fragments of a luciferase reporter. A library encompassing 63 apoptotic and autophagic proteins was constructed for the analysis of ∼3,600 protein-pair combinations. This generated a detailed landscape of the apoptotic and autophagic modules and points of interface between them, identifying 46 previously unknown interactions. One of these interactions, between DAPK2, a Ser/Thr kinase that promotes autophagy, and 14-3-3τ, was further investigated. We mapped the region responsible for 14-3-3τ binding and proved that this interaction inhibits DAPK2 dimerization and activity. This proof of concept underscores the power of the GLuc PCA platform for the discovery of biochemical pathways within the cell death network.

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