Abstract
Signaling pathways transduce extracellular stimuli from the membrane to the nucleus. Constitutive and thus inappropriate stimulation of these kinase cascades is associated with and observed in a majority of tumors. The transduction of signals in these pathways is achieved through protein-protein interactions regulated by changes in the phosphorylation status of key members. Therefore, the analysis of the interactions formed or broken in response to mitogenic stimulation is an important step toward understanding the molecular mechanisms of carcinogenesis. Today, mass spectrometry-based proteomics is one of the most widely used methods to unravel the molecular protein interaction networks that underlie these signaling cascades. This approach is powerful, but usually results in long lists of binding partners that may contain many false-positive hits and no information about the physiological role of the interacting proteins. Functional information can be derived by mapping changes in the interactome in response to specific stimuli or by comparing the interactome of related proteins with overlapping and different biological functions. As paradigms for these experimental approaches and the associated methodology, we describe here the functional proteomic analysis of the interactome of two distinct members of the mitogen-activated protein kinase (MAPK) cascade. The first is the analysis of interaction partners of the extracellular signal-regulated kinase (ERK) regulated by growth factor stimulation. The second is the differential analysis of binding partners of the C-terminal SH3 domain of the two small adaptor proteins Grb2 and GRAP.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Kolch, W. (2000) Meaningful relationships: The regulation of the Ras/Raf/ MEK/ERK pathway by protein interactions. Biochem. J. 351(Pt. 2), 289–305.
Vogelstein, B. and Kinzler, K. W. (2004) Cancer genes and the pathways they control. Nat. Med. 10(8), 789–799.
Hahn, W. C. and Weinberg, R. A. (2002) Rules for making human tumor cells. N. Engl. J. Med. 347(20), 1593–1603.
Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J., and Mann, M. (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21(3), 315–318.
Cho, S., Park, S. G., Lee, D. H., and Park, B. C. (2004) Protein-protein interaction networks: from interactions to networks. J. Biochem. Mol. Biol. 37(1), 45–52.
von Kriegsheim, A., Pitt, A., Grindlay, G. J., Kolch, W., and Dhillon, A. S. (2006) Regulation of the Raf-MEK-ERK pathway by protein phosphatase 5. Nat. Cell Biol. 8(9), 1011–1106.
Pawson, T. (1994) SH2 and SH3 domains in signal transduction. Adv. Cancer Res. 64, 87–110.
Pawson, T. and Nash, P. (2000) Protein-protein interactions define specificity in signal transduction. Genes Dev. 14(9), 1027–1047.
Schlessinger, J. (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110(6), 669–672.
Pawson, T. (2004) Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116(2), 191–203.
Wellbrock, C., Karasarides, M., and Marais, R. (2004) The RAF proteins take centre stage. Nat. Rev. Mol. Cell Biol. 5(11), 875–885.
Yoon, S. and Seger, R. (2006) The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24(1), 21–44.
Short, B., Preisinger, C., Schaletzky, J., Kopajtich, R., and Barr, F. A. (2002) The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes. Curr. Biol. 12(20), 1792–1795.
Ren, S. Y., Bolton, E., Mohi, M. G., Morrione, A., Neel, B. G., and Skorski, T. (2005) Phosphatidylinositol 3-kinase p85 {alpha subunit-dependent interaction with BCR/ABL-related fusion tyrosine kinases: molecular mechanisms and biological consequences}. Mol. Cell. Biol.} 25(18), 8001–80
Trinkle-Mulcahy, L., Andersen, J., Lam, Y. W., Moorhead, G., Mann, M., and Lamond, A. I. (2006) Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability. J. Cell Biol. 172(5), 679–692.
Puig, O., Caspary, F., Rigaut, G., et al. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3), 218–229.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68(5), 850–858.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, Totowa, NJ
About this protocol
Cite this protocol
von Kriegsheim, A., Preisinger, C., Kolch, W. (2008). Mapping of Signaling Pathways by Functional Interaction Proteomics. In: Thompson, J.D., Ueffing, M., Schaeffer-Reiss, C. (eds) Functional Proteomics. Methods in Molecular Biology, vol 484. Humana Press. https://doi.org/10.1007/978-1-59745-398-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-398-1_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-971-0
Online ISBN: 978-1-59745-398-1
eBook Packages: Springer Protocols