Abstract
Dynamic protein phosphorylation, a major cellular regulatory system, is tightly controlled by coordinating the reversible action of protein kinases and phosphatases. Recent evidence is consistent with sophisticated mechanisms that regulate both kinases and phosphatases in the cell. Dual specificity phosphatases, which act on phosphorylated serine, threonine, and tyrosine residues in proteins, are valid targets for drug discovery. Chemical complementation combines genetic manipulations with chemical biology and high-content multiparametric analyses and was developed as a screening approach to discover protein phosphatase inhibitors. Using a dual specificity mitogen-activated protein kinase phosphatase as an example, a detailed protocol, discussion of issues relating to data analysis, and high-throughput implementation of the chemical complementation approach to drug discovery is presented.
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References
Zolnierowicz, S. and Bollen, M. (2000) Protein phosphorylation and protein phosphatases. De Panne, Belgium, 19–24 September 1999. EMBO J. 19, 483–488.
Tonks, N. K. and Neel, B. G. (2001) Combinatorial control of the specificity of protein tyrosine phosphatases. Curr. Opin. Cell Biol. 13, 182–195.
Alonso, A., Sasin, J., Bottini, N., et al. (2004) Protein tyrosine phosphatases in the human genome. Cell 117, 699–711.
Zou, X., Tsutsui, T., Ray, D., et al. (2001) The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1. Mol. Cell Biol. 21, 4818–4828.
Chen, P., Hutter, D., Liu, P., and Liu, Y. (2002) A mammalian expression system for rapid production and purification of active MAP kinase phosphatases. Protein Expr. Purif. 24, 481–488.
Slack, D. N., Seternes, O. M., Gabrielsen, M., and Keyse, S. M. (2001) Distinct binding determinants for ERK2/p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1. J. Biol. Chem. 276, 16,491–16,500.
Camps, M., Nichols, A., Gillieron, C., et al. (1998) Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262–1265.
Balis, F. M. (2002) Evolution of anticancer drug discovery and the role of cell-based screening. J. Natl Cancer Inst. 94, 78–79.
Vogt, A., Cooley, K. A., Brisson, M., Tarpley, M. G., Wipf, P., and Lazo, J. S. (2003) Cell-active dual specificity phosphatase inhibitors identified by high content screening. Chem. Biol. 10, 733–742.
Vogt, A., Takahito, A., Ducruet, A. P., et al. (2001) Spatial analysis of key signaling proteins by high-content solid-phase cytometry in Hep3B cells treated with an inhibitor of Cdc25 dual-specificity phosphatases. J. Biol. Chem. 276, 20,544–20,550.
Vogt, A. and Lazo, J. S. (2005) Chemical complementation: a definitive phenotypic strategy for identifying small molecule inhibitors of elusive cellular targets. Pharmacol. Ther. 107, 212–215.
Vogt, A., Tamewitz, A., Skoko, J., Sikorski, R. P., Giuliano, K. A., and Lazo, J. S. (2005) The benzo (C) phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1. J. Biol. Chem. 280, 19,078–19,086.
Young, I. T. (1977) Proof without prejudice: use of the Kolmogorov-Smirnov test for the analysis of histograms from flow systems and other sources. J. Histochem. Cytochem. 25, 935–941.
Lampariello, F. (2000) On the use of the Kolmogorov-Smirnov statistical test for immunofluorescence histogram comparison. Cytometry 39, 179–188.
Watson, J. V. (2001) Proof without prejudice revisited: immunofluorescence histogram analysis using cumulative frequency subtraction plus ratio analysis of means. Cytometry 43, 55–68.
Cox, C., Reeder, J. E., Robinson, R. D., Suppes, S. B., and Wheeless, L. L. (1988) Comparison of frequency distributions in flow cytometry. Cytometry 9, 291–298.
Giuliano, K. A., Chen, Y. T., and Taylor, D. L. (2004) High content screening with siRNA optimizes a cell biological approach to drug discovery: defining the role of P53 activation in the cellular response to anticancer drugs. J. Biomol. Screen. 9, 557–568.
Peacock, J. A. (1983) Two-dimensional goodness-of-fit testing in astronomy. Mon. Not. R. Astron. Soc. 202, 615–627.
Fasano, G. and Francescini, A. (1987) A multidimensional version of the Kolmogorov-Smirnov test. Mon. Not. R. Astron. Soc. 225, 155–170.
Lazo, J. S., Aslan, D. C., Southwick, E. C., et al. (2001) Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25. J. Med. Chem. 44, 4042–4049.
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Vogt, A., Lazo, J.S. (2007). Discovery of Protein Kinase Phosphatase Inhibitors. In: Taylor, D.L., Haskins, J.R., Giuliano, K.A. (eds) High Content Screening. Methods in Molecular Biology, vol 356. Humana Press. https://doi.org/10.1385/1-59745-217-3:389
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DOI: https://doi.org/10.1385/1-59745-217-3:389
Publisher Name: Humana Press
Print ISBN: 978-1-58829-731-0
Online ISBN: 978-1-59745-217-5
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