A novel proteomic screen for peptide-protein interactions.
Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in "active" (e.g. phosphorylated) and "control" (e.g. non-phosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, B., Kratchmarova, I., Ong, S.-E., Nielsen, M., Foster, L. J., and Mann, M. (2003) Nat. Biotechnol. 21, 315-318). A tyrosine-phosphorylated peptide of the epidermal growth factor receptor specifically retrieved the Src homology domain (SH) 2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins as specific binding partners. Two of these, Pacsin 3 and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline-SH3 domain switch.
Schulze
Waltraud X
WX
Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense.
Mann
Matthias
M
D000199:Actins / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D004705:Endocytosis; D066246:ErbB Receptors / Q000737:chemistry; D051380:GRB2 Adaptor Protein; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D013058:Mass Spectrometry; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry; D017354:Point Mutation; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D058305:Sorting Nexins; D014443:Tyrosine / Q000737:chemistry; D033921:Vesicular Transport Proteins; D018909:src Homology Domains
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C421762:PACSIN3 protein, human; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; C400533:SNX9 protein, human; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D014443:Tyrosine; D011392:Proline; D066246:ErbB Receptors