Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

Nat Struct Mol Biol. 2016 Jun;23(6):608-18. doi: 10.1038/nsmb.3218. Epub 2016 May 2.

Abstract

A fascinating conundrum in cell signaling is how stimulation of the same receptor tyrosine kinase with distinct ligands generates specific outcomes. To decipher the functional selectivity of EGF and TGF-α, which induce epidermal growth factor receptor (EGFR) degradation and recycling, respectively, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling.

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • Epidermal Growth Factor / metabolism*
  • ErbB Receptors / metabolism*
  • HeLa Cells
  • Humans
  • Membrane Proteins / metabolism
  • Phosphorylation
  • Protein Interaction Maps
  • Protein Transport
  • Proteolysis
  • Proteomics
  • Signal Transduction
  • Transforming Growth Factor alpha / metabolism*
  • rab GTP-Binding Proteins / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Membrane Proteins
  • RAB11FIP1 protein, human
  • Transforming Growth Factor alpha
  • rab7 protein
  • Epidermal Growth Factor
  • EGFR protein, human
  • ErbB Receptors
  • rab GTP-Binding Proteins