Baratte, B., Meijer, L., Galaktionov, K., Beach, D. (May 1992) Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase. Anticancer Research, 12 (3). pp. 873-880. ISSN 0250-7005
Abstract
A universal intracellular factor, the <<M phase-promoting factor>> (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin B(cdc13). In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds. The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts in Escherichia coli and easily purified by affinity chromatography on glutathione-agarose. Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined. Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity. However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor. The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | CDC2 CDC25 CYCLIN-B CELL CYCLE MPF M-PHASE PROMOTING FACTOR ANTIMITOTIC COMPOUNDS PHOSPHOTYROSINE TYROSINE PHOSPHATASE PARA-NITROPHENYLPHOSPHATE ANTICANCER DRUGS SCREENING CELL-CYCLE CONTROL FISSION YEAST M-PHASE MITOTIC INDUCER P34CDC2 PHOSPHORYLATION HISTONE-H1 P80CDC25 |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > Cyclins organs, tissues, organelles, cell types and functions > organelles, types and functions > mitosis bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > protein tyrosine phosphatase |
| CSHL Authors: | |
| Communities: | CSHL labs > Beach lab |
| Depositing User: | Matt Covey |
| Date: | May 1992 |
| Date Deposited: | 01 Oct 2015 17:07 |
| Last Modified: | 01 Oct 2015 17:07 |
| URI: | https://repository.cshl.edu/id/eprint/31769 |
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