Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation.
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 IIalpha, 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 IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha 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 IIalpha 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.
Shapiro
P S
PS
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA. shapirop@stripe.colorado.edu
Whalen
A M
AM
Tolwinski
N S
NS
Wilsbacher
J
J
Froelich-Ammon
S J
SJ
Garcia
M
M
Osheroff
N
N
Ahn
N G
NG
D000818:Animals; D000951:Antigens, Neoplasm; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002843:Chromatin / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins; D019281:Dimerization; D004330:Drosophila / Q000201:enzymology; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D019950:Mitogen-Activated Protein Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D014162:Transfection
D000951:Antigens, Neoplasm; D002843:Chromatin; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010750:Phosphoproteins; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D004250:DNA Topoisomerases, Type II