1 Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet2 Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet3 Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry4 Proteomics Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, Københavns Universitet
Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus. Our data set revealed a consensus motif for PI(3,4,5)P3-interacting pleckstrin homology (PH) domains, which enabled in silico identification of phosphoinositide interactors. Members of the dedicator of cytokinesis family C exhibited specificity toward both PI(3,4,5)P3 and PI(4,5)P2. Structurally, this dual specificity is explained by a decreased number of positively charged residues in the L1 subdomain compared with DOCK1. The presented PIP-binding proteome and its specificity toward individual PIPs should be a valuable resource for the community.