Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4.
Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.
Protein Laboratory, Panum Institute 6.1, Blegdamsvej 3C, University of Copenhagen, DK-2200, Denmark.
Anders M
AM
Hinsby
Jesper V
JV
Olsen
Matthias
M
Mann
D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology; D020816:Amino Acid Motifs; D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D019992:Databases as Topic; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D006801:Humans; D047468:Immunoprecipitation; D055504:Insulin Receptor Substrate Proteins; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D020033:Protein Isoforms; D020543:Proteome; D017468:Receptors, Fibroblast Growth Factor / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D017550:Spectroscopy, Fourier Transform Infrared; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; C107686:IRS4 protein, human; D055504:Insulin Receptor Substrate Proteins; D010455:Peptides; D010750:Phosphoproteins; D020033:Protein Isoforms; D020543:Proteome; D017468:Receptors, Fibroblast Growth Factor; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D016222:Fibroblast Growth Factor 2; D014443:Tyrosine; D001120:Arginine; D019869:Phosphatidylinositol 3-Kinases