Mechanism of Topoisomerase II Inhibition by Staurosporine and Other Protein Kinase Inhibitors

Piotr Lassota,Guyanand Singh,Robert Kramer

doi:10.1074/jbc.271.42.26418

Piotr Lassota, Guyanand Singh + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.42.26418

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Abstract

Topoisomerase II is an essential enzyme for proliferation of eukaryotic cells. It is also a target for many antineoplastic drugs that promote stabilization of covalent complexes between topoisomerase II and DNA. Topoisomerase II and protein kinases both catalyze the transfer of phosphoester bonds from nucleotides to proteins. This similarity suggests that inhibitors may affect both classes of enzymes. In the present study, we have examined the mechanism of topoisomerase II inhibition by three different classes of protein kinase inhibitors. We report that staurosporine inhibited the catalytic activity of topoisomerase II by blocking the transfer of phosphodiester bonds from DNA to the active tyrosine site, a mechanism of inhibition not previously reported for this enzyme. In contrast, other kinase inhibitors, such as methyl 2,5-dihydroxycinnamate, most likely inactivated topoisomerase II by alkylation of essential amino acids, whereas the mechanism of inhibition of bis-indolylmaleimide possibly involved a direct interaction with DNA.

Highlights

The catalytic cycle of topoisomerase II can be separated into six discrete steps as reviewed by Osheroff et al [17]: 1) noncovalent binding of topoisomerase II to DNA; 2) establishing pre-strand passage cleavage/religation equilibrium; 3) DNA strand passage upon binding of ATP; 4) establishing cleavage/ religation equilibrium following strand passage; and 5) ATP hydrolysis that results in the 6) dissociation of the enzyme from the DNA
Inhibition of the Topoisomerase II-mediated Decatenation by Protein Kinase Inhibitors—The catalytic activity of topoisomerase II was determined using a [3H]KDNA decatenation assay that quantitates the formation of the final products of this multistep reaction
Binding of ATP is required for activity of both protein kinases and topoisomerase II, and it was initially suggested that genistein inhibits both enzymes by interfering with their ATP cassettes [18]

Summary

EXPERIMENTAL PROCEDURES

Enzymes and Chemicals—Topoisomerase II (from Drosophila melanogaster) was purchased from Amersham Corp. Topoisomerase II (2.5 ng) and [3H]thymidine-labeled KDNA (0.4 ␮g) were incubated for 1 h at 30 °C in 40 ␮l of total volume of the reaction buffer (10 mM Tris/HCl, pH 7.8, 5 mM MgCl2, 0.1 mM EDTA, and 15 ␮g/ml BSA) containing 150 mM NaCl, 200 ␮M ATP, and various concentrations of inhibitors, ranging from 0.1 to 200 ␮M. Inhibition of Etoposide-stabilized Pre-strand Passage Topoisomerase II-cleaved DNA Complex—Topoisomerase II (0.5 ␮g) was incubated with 1 ␮g KDNA in 20 ␮l of the reaction buffer (10 mM Tris/HCl, pH 7.8, 5 mM MgCl2, 0.1 mM EDTA, and 15 ␮g/ml BSA) that contained 25 mM. The amount of inorganic phosphate generated by topoisomerase II in the absence of DNA was small and was subtracted from the total to give the DNA-dependent ATP hydrolysis

RESULTS

TABLE I

Relative amount of complex formed

DISCUSSION