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.
Wang
Yiguo
Y
Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
Li
Rongxia
R
Du
Dan
D
Zhang
Chenyi
C
Yuan
Haixin
H
Zeng
Rong
R
Chen
Zhengjun
Z
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002498:Centrifugation; D002853:Chromatography, Liquid; D019295:Computational Biology; D005347:Fibroblasts / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D014443:Tyrosine / Q000737:chemistry
D005456:Fluorescent Dyes; D007328:Insulin; D020543:Proteome; D014443:Tyrosine; D008156:Luciferases; D011972:Receptor, Insulin; D016650:Fluorescein-5-isothiocyanate