Interrogating Kinase–Substrate Relationships with Proximity Labeling and Phosphorylation Enrichment

Kinases govern many cellular responses
through the reversible 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
ascorbate peroxidase (APEX) proximity labeling and a phosphorylation
enrichment-based workflow, Phospho-APEX (pAPEX), to rapidly identify
phosphorylated and biotinylated neighbor proteins which can be considered
for candidate substrates. The pAPEX strategy enriches and quantifies
differences in proximity for proteins and phosphorylation sites proximal
to an APEX2-tagged kinase under the kinase “ON” and
kinase “OFF” conditions. As a proof of concept, we identified
candidate substrates of MAPK1 in HEK293T and HCT116 cells and candidate
substrates of PKA in HEK293T cells. In addition to many known substrates,
C15orf39 was identified and confirmed as a novel MAPK1 substrate.
In all, we adapted the proximity labeling-based platform to accommodate
phosphorylation analysis for kinase substrate identification.