TI - Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B SUBstrate . AB - The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation . Aurora B function is dependent on the chromosome passenger , INCENP . INCENP , in turn , requires sister chromatid cohesion for its appropriate behaviour . Relatively few SUBstrates have been identified for Aurora B , so that the precise role it plays in controlling mitosis remains to be elucidated . To identify potential novel mitotic SUBstrates of Aurora B , extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP . Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A , CENP-B , CENP-C , ScII and INCENP . Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction . Cloning , fluorescent tagging and expression in HeLa cells of the putative GTP -binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein , with a perichromosomal localisation . Identi fication of the protein bands corresponding to those phosphorylaTED by Aurora B revealed topoisomerase II alpha ( topo IIalpha ) as a potential Aurora B SUBstrate . Purified recombinant human topo IIalpha was phosphorylaTED by Aurora B in vitro , confirming this proteomic approach as a valid method for the initial definition of candidate SUBstrates of key mitotic kinases .