부산대학교 도서관

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post‐transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP‐APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS‐based quantification. We report the time‐resolved interplay of more than 50 previously undescribed modification and hundreds of protein–protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site‐specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll‐like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost‐effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells. SYNOPSIS: A robust proteomics strategy is used to elucidate the time‐resolved interplay of protein‐protein interactions (PPIs) and post‐translational modifications (PTMs) in human immune cells with a focus on TRAF2, MAPK14, and MAP3K7. An experimental strategy to reveal dynamic signaling checkpoints in primary cells is developed.Crosstalk between protein modifications and interactions in signaling complexes is identified.TRAF2 ISGylation, phosphorylation, and its ARHGEF18 interaction in monocytes are discovered.Differential drug modes of action for MAPK14 inhibitors are dissected. [ABSTRACT FROM AUTHOR]