Application of TurboID-mediated proximity labeling and phosphoproteomics for mapping a GSK3 kinase signaling network in Arabidopsis

Transient protein-protein interactions (PPIs), such as those between posttranslational modifying enzymes and their substrates, play key roles in cellular regulation, but are difficult to identify. Here we demonstrate the application of enzyme-catalyzed proximity labeling (PL), using the engineered promiscuous biotin ligase TurboID, as a sensitive method for characterizing PPIs in signaling networks. We show that TurboID fused with the GSK3-like kinase BIN2 or a PP2A phosphatase biotinylate their known substrate, the BZR1 transcription factor, with high specifically and efficiency. We optimized the protocol of biotin labeling and affinity purification in transgenic Arabidopsis expressing a BIN2-TurboID fusion protein. Subsequent quantitative mass spectrometry (MS) analysis identified about three hundred proteins biotinylated by BIN2-TurboID more efficiently than the YFP-TurboID control. These include a significant subset of previously proven BIN2 interactors and a large number of new BIN2 interactors that uncover a broad BIN2 signaling network. Our study illustrates that PL-MS using TurboID is a powerful tool for mapping signaling networks in plants, and reveals broad roles of this GSK3-like kinase in cellular signaling and regulation.