Kinase-Catalyzed Biotinylation to Map Cell Signaling Pathways: Application to Epidermal Growth Factor Signaling

Cell signaling involves a network of protein-protein interactions and post-translational modifications that govern cellular responses to environmental cues. To understand and ultimately modulate these signaling pathways to confront disease, the complex web of proteins that becomes phosphorylated after extracellular stimulation has been studied using mass spectrometry-based proteomics methods. To complement prior work and fully characterize all phosphorylated proteins after the stimulation of cell signaling, we developed K-BMAPS (kinasecatalyzed biotinylation to map signaling), which utilizes ATP-biotin as a kinase cosubstrate to biotin label substrates. As a first application of the K-BMAPS method, the well-characterized epidermal growth factor receptor (EGFR) kinase signaling pathway was monitored by treating epidermal growth factor (EGF)-stimulated HeLa lysates with ATP-biotin, followed by streptavidin enrichment and quantitative mass spectrometry analysis. On the basis of the dynamic phosphoproteins identified, a pathway map was developed considering functional categories and known interactors of EGFR. Remarkably, 94% of the K-BMAPS hit proteins were included in the EGFR pathway map. With many proteins involved in transcription, translation, cell adhesion, and GTPase signaling, K-BMAPS identified phosphoproteins were associated with late and continuous signaling events. In summary, the K-BMAPS method is a powerful tool to map the dynamic phosphorylation governing cell signaling pathways.

Automated summary

Cell signaling involves a complex network of protein interactions and post-translational modifications. The K-BMAPS method, utilizing ATP-biotin to label substrates, is a powerful tool for mapping dynamic phosphorylation in cell signaling pathways. This method has been used successfully to study the EGFR pathway and identify key phosphoproteins associated with late and continuous signaling events.