Abstract
Protein phosphorylation is almost certainly the most important posttranslational mechanism of enzyme regulation in eukaryotic cells (reviewed in 1). The equilibrium between phosphorylation by protein kinases and dephosphorylation by protein phosphatases modulates the activity, subcellular localization, or DNA/RNA/protein binding properties of numerous proteins. Indeed, it appears that the majority of intracellular proteins in human cells are phosphorylated to some degree under certain conditions of cell growth. However, interest in protein phosphorylation is more generally directed toward a study of the alterations in phosphorylation status that either accompany a change in cell physiology or are invoked by exposure to an extracellular stimulus (reviewed in 2).
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
References
Edelman, A. M., Blumenthal, D. K., and Krebs, E. G. (1987) Protein serine/threonine kinases. Annu. Rev. Biochem. 56, 567–613.
Hunter, T. (1995) Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signalling. Cell 80, 225–236.
van der Geer, P., Luo, K., Sefton, B. M., and Hunter, T. (1993) Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates, in Protein Phosphorylation (Hardie, D. G., ed.), Oxford University Press, Oxford, UK, pp 31–58.
Woodgate, J. R. (1992) Analysis of protein phosphorylation in cell lines, in Neuronal Cell Lines: A Practical Approach (Wood, J., ed.), IRL, Oxford, UK, pp. 133–159.
Lane, D. P. and Crawford, L. V. (1979) T antigen is bound to a host protein in SV40-transformed cells. Nature 278, 261–263
Zhang, H., Hannon, G. J., and Beach, D. (1994) p21-containing cyclin kinases exist in both active and inactive states. Genes Dev. 8, 1750–1758.
Glisson, B., Gupta, R., Smallwood-Kentro, S., and Ross, W. (1986) Characterization of acquired epipodophyllotoxin resistance in a chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity. Cancer Res. 46, 1934–1938.
Wells, N. J., Addison, C. M., Fry, A. M., Ganapathi, R., and Hickson, I. D. (1994) Serine-1524 is a major site of phosphorylation on human topoisomerase IIα protein in vivo and is a substrate for casein kinase II in vitro. J. Biol. Chem. 269, 29,746–29,751.
Morgan, D. O., Kaplan, J. M., Bishop, J. M., and Varmus, H. E. (1989) Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell 57, 775–786.
Taagepera, S., Rao, P. N., Drake, F. H., and Gorbsky, G. J. (1993) DNA topoisomerase IIα is the major chromosome protein recognised by the mitotic phosphoprotein antibody MPM2. Proc. Natl. Acad. Sci. USA 90, 8407–8411.
Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase. J. Biol. Chem. 261, 10,489–10,492.
Tsai-Pflugfelder, M. T., Liu, L. F., Liu, A. A., Tewey, K. M., Whong-Peng, J., Knutsen, T., Huebner, K., Croce, C. M., and Wang, J. C. (1988) Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localisation of the gene to chromosome 17q21–21. Proc. Natl. Acad. Sci. USA 85, 7177–7181.
Wells, N. J., Fry, A. M., Guano, F., Norbury, C., and Hickson, I. D. (1995) Cell cycle phase-specific phosphorylation of human topoisomerase IIα—evidence of a role for protein kinase C. J. Biol. Chem. 270, 28,357–28,363.
Wells, N. J. and Hickson, I. D. (1995) Human topoisomerase IIα is phosphorylated in a cell cycle phase-dependent manner by a proline-directed kinase. Eur. J. Biochem. 231, 491–497.
Hardie, D. G., Campbell, D. G., Caudwell, F. B., and Haystead, T. A. J. (1993) Analysis of sites phosphorylated in vivo and in vitro, in Protein Phosphorylation: A Practical Approach, vol. 123 (Hardie, D. G., ed.), Oxford University Press, Oxford, UK, pp. 61–84.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc.
About this protocol
Cite this protocol
Wells, N.J., Hickson, I.D. (1999). Metabolic Labeling, Immunoprecipitation, and Two-Dimensional Tryptic Phosphopeptide Mapping of Human Topoisomerase II. In: Bjornsti, MA., Osheroff, N. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology, vol 94. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-259-7:243
Download citation
DOI: https://doi.org/10.1385/1-59259-259-7:243
Published:
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-0-89603-444-0
Online ISBN: 978-1-59259-259-3
eBook Packages: Springer Protocols