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 <i>Drosophila </i>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.

Photocrosslinking with p-azidophenylalanine used to identify the location of Cic binding on ERK

249th National Meeting (2015)

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