Casein kinase II catalyzes a mitotic phosphorylation on threonine 1342 of human DNA topoisomerase IIα, which is recognized by the 3F3/2 phosphoepitope antibody

Abstract

The 3F3/2 antibody recognizes a phosphoepitope that is implicated in the mitotic checkpoint regulating the metaphase-to-anaphase transition. Immunoprecipitation and Western blotting revealed that the 3F3/2 antibody binds to human DNA topoisomerase IIα (HsTIIα) from mitotic but not interphase HeLa cells. Extracts from mitotic cells efficiently catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα expressed in bacteria. Expression and site-directed mutagenesis of various HsTIIα protein fragments mapped the 3F3/2 phosphoepitope to the region of HsTIIα containing phosphorylated threonine 1342. This threonine lies within a consensus sequence for phosphorylation by casein kinase II (CKII). CKII is present in cellular extracts and is associated with isolated mitotic chromosomes. The 3F3/2 phosphoepitope kinase present in mitotic cell extracts was able to create the epitope using GTP and was inhibited by heparin. A kinase associated with the isolated chromosomes also generated the 3F3/2 phosphoepitope on HsTKIIα. Recombinant CKII catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIα containing threonine 1342. These results indicate that the mitotic 3F3/2 phosphoepitope kinase activity is attributable to CKII. We suggest that the 3F3/2 phosphoepitope reflects a CKII-catalyzed phosphorylation of threonine 1342 that may regulate mitotic functions of HsTIIα.