Abstract
Ubiquitination and protein degradation regulate cell cycle progression in all eukaryotes. During mitosis, ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C) triggers sister chromatid separation and mitotic exit. The APC/C is tightly regulated by phosphorylation, ubiquitination, association of activators or inhibitors, and competitive binding of substrates. Much of our understanding of the mechanism of APC/C-dependent ubiquitination has been obtained from studies using extracts of Xenopus laevis eggs or synchronized human tissue culture cells. Here, we describe protocols to prepare extracts of synchronized human cells, and discuss experiments to use extracts for the biochemical analysis of APC/C-dependent ubiquitination.
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References
Sullivan, M., and Morgan, D.O. (2007) Finishing mitosis, one step at a time. Nat Rev. Mol. Cell Biol. 8, 894–903.
Peters, J. M. (2006) The anaphase promoting complex/cyclosome: a machine designed to destroy. Nature Rev. Mol. Cell Biol. 7, 644–656.
Sotillo, R., Hernando, E., Díaz-Rodríguez, E., Teruya-Feldstein, J., Cordón-Cardo, C., Lowe, S.W., and Benezra, R. (2007) Mad2 overexpression promotes aneuploidy and tumorigenesis in mice. Cancer Cell 11, 9–23.
Dye, B.T., and Schulman, B.A. (2007) Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36, 131–50.
Rape, M., Reddy, S.K., and Kirschner, M.W. (2006) The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124, 89–103.
Rape, M., and Kirschner, M.W. (2004) Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry. Nature 432, 588–95.
Yu, H. (2002) Regulation of APC-Cdc20 by the spindle checkpoint. Curr. Opin. Cell Biol. 14, 706–14.
Musacchio, A., and Hardwick, K. G. (2002) The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell Biol. 3, 731–41.
Reddy, S.K., Rape, M., Marganski, W.A., and Kirschner, M.W. (2007). Mutual regulation between the spindle checkpoint and the anaphase-promoting complex ensures timely progression to anaphase. Nature 446, 921–5.
Xia, G., Luo, X., Habu, T., Rizo, J., Matsumoto, T., and Yu, H. (2004). Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint. EMBO J. 23, 3133–43.
Stegmeier, F., et al. (2007) Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities. Nature 446, 876–81.
Brito, D.A., and Rieder, C.L. (2006). Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint. Curr. Biol. 16, 1194–200.
di Fiore, B., and Pines, J. (2007). Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C. J. Cell Biol. 177, 425–37.
Hagting, A., Den Elzen, N., Vodermaier, H.C., Waizenegger, I.C., Peters, J.M., and Pines, J. (2002). Human securin proteolysis is controlled by the spindle checkpoint and reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. 157, 1125–37.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Williamson, A., Jin, L., Rape, M. (2009). Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_19
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DOI: https://doi.org/10.1007/978-1-60327-993-2_19
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