Photocrosslinking with p-Azidophenylalanine Used to Identify the Location of Cic Binding on ERK

Extracellular signal-regulated kinase (ERK) is responsible for integrating cellular responses to a wide range of stimuli. ERK-mediated phosphorylation of its many substrates regulates critical cellular processes including proliferation, differentiation and death. The transcriptional repressor Capicua (Cic) is one such ERK substrate that was first identified in Drosophila and has been implicated in a number of human diseases including cancer and neurodegenerative disorders. The details of Cic recognition by ERK remain largely unknown. We employed a novel strategy using biophysical and chemical biological assays to dissect the interaction between ERK and Cic. Using pull-down assays and Biolayer Interferometry, we identified residues in Cic that are required for its interaction with ERK. We then incorporated the unnatural amino acid p-azidophenylalanine into Cic positions adjacent to these critical ERK-interacting residues. This enabled the covalent crosslinking of ERK and Cic at the binding interface by UV irradiation. Analysis of the proteolytically digested ERK/Cic crosslink product by LC-MS/MS enabled the identification of the precise location on ERK used to bind to Cic, providing a detailed picture of the protein-protein interaction between Cic and ERK.