Proteomic Analysis Reveals Novel Molecules Involved in Insulin Signaling Pathway

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.
Keywords: insulin signaling pathway • subcellular fractionation • immuno-affinity purification • phosphotyrosine
sites • CASK • T-element