Abstract
The checkpoint protein Chfr delays entry into mitosis in the presence of mitotic stress. We have analyzed the Chfr checkpoint pathway in the Xenopus cell-free system. We showed that Chfr is a ubiquitin ligase that targets polo-like kinase (Plk1) for degradation, leading to delayed activation of the Cdc25C phosphatase and prolonged inhibitory phosphorylation of Cdc2 at the G2/M transition. In this chapter, we will describe biochemical methods we developed to analyze the Chfr auto-ubiquitination activity and the ubiquitination of its substrate Plk1, as well as functional assays to investigate the Chfr pathway in Xenopus extracts.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scolnick, D. M. and Halazonetis, T. D. (2000) Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature 406, 430–435.
Kang, D., Chen J., Wong, J., and Fang, G. (2002) The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J. Cell Biol. 156, 249–259.
Jha, M. N., Bamburg, J. R., and Bedford, J. S. (1994) Cell cycle arrest by Colcemid differs in human normal and tumor cells. Cancer Res. 54, 5011–5015.
Mizuno, K., Osada, H., Konishi, H., et al. (2002) Aberrant hypermethylation of the CHFR prophase checkpoint gene in human lung cancers. Oncogene 21, 2328–2333.
Shibata, Y., Haruki, N., Kuwabara, Y., et al. (2002) Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer. Carcinogenesis 23, 1695–1699.
Corn, P. G., Fogt, S. M., Virmani, F., Gazdar, A. K., Halazonetis, T. D., and El-Deiry, W. S. (2003) Frequent hypermethylation of the 5′ CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer. Carcinogenesis 24, 47–51.
Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138.
Murray, A. W. (1991) Cell cycle extracts. Methods in Cell Biology 36, 581–605.
Parker, C. W. (1990) Radiolabeling of proteins. Meth. Enzymol. 182, 721–737.
King, R. W., Jackson, P. K., and Kirschner, M. W. (1994) Mitosis in transition. Cell 79, 563–571.
Izumi, T., Walker, D. H., and Maller, J. L. (1992) Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. Mol. Biol. Cell 3, 927–939.
Izumi, T. and Maller, J. L. (1993) Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase. Mol. Biol. Cell 4, 1337–1350.
Kumagai, A. and Dunphy, W. G. (1992) Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell 70, 139–151.
Kumagai, A. and Dunphy, W. G. (1996) Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273, 1377-1380.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Kang, D., Wong, J., Fang, G. (2004). A Xenopus Cell-Free System for Functional Analysis of the Chfr Ubiquitin Ligase Involved in Control of Mitotic Entry. In: Schönthal, A.H. (eds) Checkpoint Controls and Cancer. Methods in Molecular Biology™, vol 280. Humana Press. https://doi.org/10.1385/1-59259-788-2:229
Download citation
DOI: https://doi.org/10.1385/1-59259-788-2:229
Publisher Name: Humana Press
Print ISBN: 978-1-58829-214-8
Online ISBN: 978-1-59259-788-8
eBook Packages: Springer Protocols