Abstract
Many cancers have been associated with the deregulation of kinases, and thus, kinases have become a prime target for the development of cancer treatments. This focus on kinases has resulted in the approval of several small-molecule kinase inhibitors for cancer treatments. Further, the use of these inhibitors as tools to study cancer has provided valuable information about biological mechanisms. However, to date, not much is known about the global effects of kinases on the proteome or phosphoproteome. In this protocol, we describe methodology to study the impact of kinase inhibitors on the proteome and phosphoproteome using mass spectrometry-based quantitative proteomics. More specifically, we focus on the effects of Aurora B kinase inhibitors on the proteome, cytoskeleton proteome, the phosphoproteome, and the cytoskeleton phosphoproteome during cell cycle. This methodology is easily extended to other biological studies whose aim is to study the global proteomic effects of a kinase inhibitor.
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References
Taylor, S., and Peters, J. M. (2008) Polo and Aurora kinases: lessons derived from chemical biology. Curr. Opin. Cell. Biol. 20, 77–84.
Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9, 28–39.
Lapenna, S., and Giordano, A. (2009) Cell cycle kinases as therapeutic targets for cancer. Nat. Rev. Drug Discov. 8, 547–566.
Lemeer, S., and Heck, A. J. (2009) The phosphoproteomics data explosion. Curr. Opin. Chem. Biol. 13, 414–420.
Feng, Y., Mitchison, T. J., Bender, A., Young, D. W., and Tallarico, J. A. (2009) Multi-parameter phenotypic profiling: using cellular effects to characterize small-molecule compounds. Nat. Rev. Drug Discov. 8, 567–578.
Bantscheff, M., Eberhard, D., Abraham, Y., Bastuck, S., Boesche, M., Hobson, S., Mathieson, T., Perrin, J., Raida, M., Rau, C., Reader, V., Sweetman, G., Bauer, A., Bouwmeester, T., Hopf, C., Kruse, U., Neubauer, G., Ramsden, N., Rick, J., Kuster, B., and Drewes, G. (2007) Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25, 1035–1044.
Kelly, A. E., and Funabiki, H. (2009) Correcting aberrant kinetochore microtubule attachments: an Aurora B-centric view. Curr. Opin. Cell Biol. 21, 51–58.
Keen, N., and Taylor, S. (2004) Aurora-kinase inhibitors as anticancer agents, Nat. Rev. Cancer 4, 927–936.
Harrington, E. A., Bebbington, D., Moore, J., Rasmussen, R. K., Ajose-Adeogun, A. O., Nakayama, T., Graham, J. A., Demur, C., Hercend, T., Diu-Hercend, A., Su, M., Golec, J. M., and Miller, K. M. (2004) VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med. 10, 262–267.
Fang, G., Yu, H., and Kirschner, M. W. (1998) Direct binding of CDC20 protein family members activates the anaphase-promoting complex in mitosis and G1. Mol. Cell 2, 163–171.
Ozlu, N., Monigatti, F., Renard, B. Y., Field, C. M., Steen, H., Mitchison, T. J., and Steen, J. J. Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition. Mol. Cell Proteomics 9, 336–350.
Steen, J. A., Steen, H., Georgi, A., Parker, K., Springer, M., Kirchner, M., Hamprecht, F., and Kirschner, M. W. (2008) Different phosphorylation states of the anaphase promoting complex in response to antimitotic drugs: a quantitative proteomic analysis. Proc. Natl. Acad. Sci. USA 105, 6069–6074.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Budde, P. P., Desai, A., and Heald, R. (2006) Analysis of microtubule polymerization in vitro and during the cell cycle in Xenopus egg extracts. Methods 38, 29–34.
Cox, J., and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372.
Cox, J., Matic, I., Hilger, M., Nagaraj, N., Selbach, M., Olsen, J. V., and Mann, M. (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705.
Graumann, J., Hubner, N. C., Kim, J. B., Ko, K., Moser, M., Kumar, C., Cox, J., Scholer, H., and Mann, M. (2008) Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins. Mol. Cell Proteomics 7, 672–683.
Sawin, K. E., and Mitchison, T. J. (1991) Poleward microtubule flux mitotic spindles assembled in vitro. J. Cell Biol. 112, 941–954.
Molina, H., Horn, D. M., Tang, N., Mathivanan, S., and Pandey, A. (2007) Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2199–2204.
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Özlü, N., Kirchner, M., Steen, J.J. (2012). Measuring Phosphorylation-Specific Changes in Response to Kinase Inhibitors in Mammalian Cells Using Quantitative Proteomics. In: Kuster, B. (eds) Kinase Inhibitors. Methods in Molecular Biology, vol 795. Humana Press. https://doi.org/10.1007/978-1-61779-337-0_15
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DOI: https://doi.org/10.1007/978-1-61779-337-0_15
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