Extracellular Signal-Regulated Kinase Activates Topoisomerase IIα through a Mechanism Independent of Phosphorylation

Abstract

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIα, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIα proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIα and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIα in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.

ACKNOWLEDGMENTS

We are indebted to Daniel Bogenhagen, University of Colorado Health Sciences Center, Denver, for providing kinetoplast DNA and advice on decatenation assays; to Melanie Cobb, University of Texas Southwestern Medical Center, Dallas, for providing ERK2 and PAK constructs; to Roger Davis for providing p38 MAPK constructs; and to Ian Macara for providing green fluorescent protein constructs. We also thank Jocelyn Wright and Edwin Krebs, University of Washington, for sharing results prior to publication.

This study was supported by the Searle Scholars Program (N.G.A.) and by grants RO1 GM48521 (N.G.A.), F32 GM18151 (P.S.), and RO1 GM33944 (N.O.) from the National Institutes of Health.