Differential phosphoproteomics of fibroblast growth factor signaling: identification of Src family kinase-mediated phosphorylation events.
Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein-protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent.
School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
Debbie L
DL
Cunningham
Steve M M
SM
Sweet
Helen J
HJ
Cooper
John K
JK
Heath
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; D010455:Peptides; D010750:Phosphoproteins; D020543:Proteome; D016601:RNA-Binding Proteins; D016222:Fibroblast Growth Factor 2; D019061:src-Family Kinases