Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase.
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 Bcdc13. 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 of 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.
Baratte
B
B
CNRS, Station Biologique, Roscoff, France.
Meijer
L
L
Galaktionov
K
K
Beach
D
D
D000595:Amino Acid Sequence; D000906:Antibodies; D000970:Antineoplastic Agents / Q000494:pharmacology*; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008938:Mitosis / Q000187:drug effects; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D006656:Protamine Kinase / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D020687:cdc25 Phosphatases
D000906:Antibodies; D000970:Antineoplastic Agents; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014638:Vanadates; D005982:Glutathione Transferase; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases; D004229:Dithiothreitol