Interrogating Kinase-Substrate Relationships with Proximity Labeling and Phosphorylation Enrichment

Area of Science:

• Molecular Biology
• Cellular Signaling
• Biochemistry

Background:

• Kinases regulate cellular processes via phosphorylation.
• Identifying specific kinase-substrate interactions remains a significant challenge in molecular biology.
• Proximity labeling coupled with mass spectrometry aids in identifying proteins near a target.

Purpose of the Study:

• To develop and validate a novel method for efficient kinase substrate identification.
• To adapt proximity labeling for simultaneous detection of protein proximity and phosphorylation status.
• To identify novel substrates for specific kinases like MAPK1 and PKA.

Main Methods:

• Phospho-APEX (pAPEX) integrates ascorbate peroxidase (APEX) proximity labeling with phosphoproteomic enrichment.
• The method quantifies changes in protein and phosphorylation site proximity under kinase-active and inactive states.
• Applied to identify substrates of MAPK1 and PKA in human cell lines (HEK293T, HCT116).

Main Results:

• pAPEX successfully identified known and novel candidate kinase substrates.
• Demonstrated identification of MAPK1 substrates in HEK293T and HCT116 cells.
• Confirmed C15orf39 as a novel substrate of MAPK1, validating the pAPEX approach.

Conclusions:

• The pAPEX strategy provides a powerful platform for kinase substrate discovery.
• This method enhances the identification of direct kinase targets by analyzing proximity and phosphorylation.
• The approach is adaptable for broad applications in kinase signaling research.