Abstract
Mass spectrometry–based proteomics1 can reveal protein-protein interactions on a large scale2,3, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway4,5,6, we employ stable isotopic amino acids in cell culture (SILAC)7 to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.
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References
Pandey, A. & Mann, M. Proteomics to study genes and genomes. Nature 405, 837–846 (2000).
Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).
Gavin, A.C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002).
Hunter, T. Signaling—2000 and beyond. Cell 100, 113–127 (2000).
Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 103, 211–225 (2000).
Pawson, T. & Nash, P. Protein-protein interactions define specificity in signal transduction. Genes Dev. 14, 1027–1047 (2000).
Ong, S.E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).
Pandey, A. et al. Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. USA 97, 179–184 (2000).
Moores, S.L. et al. Vav family proteins couple to diverse cell surface receptors. Mol. Cell. Biol. 20, 6364–6373 (2000).
Erickson, J.W., Cerione, R.A. & Hart, M.J. Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase. J. Biol. Chem. 272, 24443–24447 (1997).
Kawashima, T. et al. MgcRacGAP is involved in the control of growth and differentiation of hematopoietic cells. Blood 96, 2116–2124 (2000).
Sotillos, S. & Campuzano, S. DRacGAP, a novel Drosophila gene, inhibits EGFR/Ras signalling in the developing imaginal wing disc. Development 127, 5427–5438 (2000).
Robinson, M.S. & Bonifacino, J.S. Adaptor-related proteins. Curr. Opin. Cell Biol. 13, 444–453 (2001).
Feng, G.S., Hui, C.C. & Pawson, T. SH2-containing phosphotyrosine phosphatase as a target of protein-tyrosine kinases. Science 259, 1607–1611 (1993).
Vogel, W., Lammers, R., Huang, J. & Ullrich, A. Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. Science 259, 1611–1614 (1993).
Thomas, D., Patterson, S.D. & Bradshaw, R.A. Src homologous and collagen (Shc) protein binds to F-actin and translocates to the cytoskeleton upon nerve growth factor stimulation in PC12 cells. J. Biol. Chem. 270, 28924–28931 (1995).
Leung, C.L., Green, K.J. & Liem, R.K. Plakins: a family of versatile cytolinker proteins. Trends Cell Biol. 12, 37–45 (2002).
Spike, C.A., Shaw, J.E. & Herman, R.K. Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans. Mol. Cell. Biol. 21, 4985–4995 (2001).
Di Benedetto, A.J., Klick Stoddard, J. & Glavan, B.J. Cloning and molecular characterization of a novel gene encoding a WD-repeat protein expressed in restricted areas of adult rat brain. Gene 271, 21–31 (2001).
Lin, S.Y. et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nat. Cell Biol. 3, 802–808 (2001).
Wells, A. & Marti, U. Signalling shortcuts: cell-surface receptors in the nucleus? Nat. Rev. Mol. Cell. Biol. 3, 697–702 (2002).
Cheung, P., Allis, C.D. & Sassone-Corsi, P. Signaling to chromatin through histone modifications. Cell 103, 263–271 (2000).
Korty, P.E., Brando, C. & Shevach, E.M. CD59 functions as a signal-transducing molecule for human T cell activation. J. Immunol. 146, 4092–4098 (1991).
Stefanova, I., Horejsi, V., Ansotegui, I.J., Knapp, W. & Stockinger, H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254, 1016–1019 (1991).
Murray, E.W. & Robbins, S.M. Antibody cross-linking of the glycosylphosphatidylinositol-linked protein CD59 on hematopoietic cells induces signaling pathways resembling activation by complement. J. Biol. Chem. 273, 25279–25284 (1998).
Pandey, A., Andersen, J.S. & Mann, M. Use of mass spectrometry to study signaling pathways. Sci. STKE 2000, PL1 (2000).
Rappsilber, J., Ryder, U., Lamond, A.I. & Mann, M. Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231–1245 (2002).
Kenworthy, A.K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–296 (2001).
Acknowledgements
We thank other members of our laboratory for help and fruitful discussions and comments on the manuscript. Akhilesh Pandey provided the GST-SH2 (Grb2) domain construct. Work in the Center for Experimental BioInformatics (CEBI) is supported by a generous grant by the Danish National Research foundation.
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Blagoev, B., Kratchmarova, I., Ong, SE. et al. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21, 315–318 (2003). https://doi.org/10.1038/nbt790
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DOI: https://doi.org/10.1038/nbt790