Kinases govern many cellular responses through the transfer of a phosphate moiety to their substrates. However, pairing a substrate with a kinase is challenging. In proximity labeling experiments, proteins proximal to a target protein are marked by biotinylation, and mass spectrometry can be used for their identification. Here, we combine ascorbic proximity labeling (APEX) and a phosphorylation enrichment-based workflow, Phospho-APEX (pAPEX), to rapidly identify phosphorylated and biotinylated neighbor proteins which are potential substrates. The pAPEX strategy enriches and quantifies proteins (APEX-Protein) and phosphorylation sites (APEX-Phos.) proximal to an APEX2-tagged kinase under the kinase “ON” and kinase “OFF” conditions. Compared to the traditional phospho-proteome analysis for kinase substrates identification, pAPEX captures subtle changes and enriches low abundance proteins through proximity labeling, providing more candidate substrates for further validation. Using pAPEX, we identified candidate substrates of MAPK1 in HEK293T and HCT116 cells and candidate substrates of PKA in HEK293T cells. Besides many known substrates, C15orf39 was identified and confirmed as a novel MAPK1 substrate. In all, we created a proximity labeling-based platform for kinase substrate identification.

Interrogating kinase-substrate relationships with proximity labeling and phosphorylation enrichment

ACS Spring 2022

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