Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking

Proc Natl Acad Sci U S A. 2015 Jul 14;112(28):8590-5. doi: 10.1073/pnas.1501373112. Epub 2015 Jun 29.


Extracellular signal-regulated kinase (ERK) coordinates cellular responses to a range of stimuli by phosphorylating its numerous substrates. One of these substrates, Capicua (Cic), is a transcriptional repressor that was first identified in Drosophila and has been implicated in a number of human diseases. Here we use a chemical biology approach to map the binding interface of ERK and Cic. The noncanonical amino acid p-azidophenylalanine (AzF) was introduced into the ERK-binding region of Drosophila Cic, and photocrosslinking and tandem mass spectrometry were used to pinpoint its binding site on ERK. We also identified the ERK-binding region of human Cic and showed that it binds to the same site on ERK despite lacking conservation with the Drosophila Cic binding region. Finally, we mapped the amino acids involved in human Cic binding to ERK using AzF-labeled ERK. These results reveal the molecular details of the ERK-Cic interaction and demonstrate that the photocrosslinking approach is complementary to existing methods for mapping kinase-substrate binding interfaces.

Keywords: photocrosslinking; protein–protein interactions; signal transduction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Drosophila
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / metabolism*
  • Extracellular Signal-Regulated MAP Kinases / metabolism*
  • HMGB Proteins / chemistry
  • HMGB Proteins / metabolism*
  • Humans
  • Mass Spectrometry
  • Molecular Sequence Data
  • Photochemical Processes
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism*


  • Drosophila Proteins
  • HMGB Proteins
  • Repressor Proteins
  • cic protein, Drosophila
  • Extracellular Signal-Regulated MAP Kinases