Proteomic analysis reveals novel molecules involved in insulin signaling pathway

J Proteome Res. 2006 Apr;5(4):846-55. doi: 10.1021/pr050391m.


The binding of insulin to its receptor triggers a signaling cascade regulated by protein complexes via tyrosine phosphorylation events on a multitude of associated proteins. To search novel phosphotyrosine proteins or associated proteins involved in insulin signaling pathway, we employed a method in which Rat1 cells stably expressing the human insulin receptor were stimulated with or without insulin and sub-fractionated prior to enrichment of phosphotyrosine proteins by immunoprecipitation and analysis by LC-MS/MS. Bioinformatic analysis and manual confirmation of peptide phosphorylation site assignments led to identification of 35 phosphotyrosine sites derived from 31 protein groups. Over 50% of these proteins were reported for the first time as tyrosine phosphorylated, including gigaxonin, XIAP and CDK10. In addition, we also found that calcium/calmodulin-dependent protein serine kinase (CASK), a key protein in protein-targeting and vesicle transport in neurons, forms a complex with two unidentified phosphotyrosine proteins pp100 and pp95 in response to insulin-stimulation, though CASK is not itself tyrosine phosphorylated. Furthermore, insulin was able to decrease CASK nuclear location, as well as down-regulate the expression of CASK targeted genes. Our results imply CASK as a novel joint knot connecting CASK-mediated pathways with the insulin signaling. Our data provide a wealth of information potentially paving the way to identify new components in the insulin signaling network.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Fractionation
  • Cell Line
  • Centrifugation
  • Chromatography, Liquid
  • Computational Biology
  • Fibroblasts / drug effects
  • Fluorescein-5-isothiocyanate
  • Fluorescent Antibody Technique, Indirect
  • Fluorescent Dyes
  • Genes, Reporter
  • Humans
  • Insulin / metabolism*
  • Insulin / pharmacology
  • Luciferases / metabolism
  • Mass Spectrometry
  • Microscopy, Fluorescence
  • Phosphorylation
  • Precipitin Tests
  • Proteome / analysis*
  • Proteomics / methods*
  • Rats
  • Receptor, Insulin / metabolism
  • Signal Transduction*
  • Subcellular Fractions
  • Tyrosine / chemistry


  • Fluorescent Dyes
  • Insulin
  • Proteome
  • Tyrosine
  • Luciferases
  • Receptor, Insulin
  • Fluorescein-5-isothiocyanate