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Nucleic Acids Research, 2002, Vol. 30, No. 23 5318-5327
© 2002 Oxford University Press

Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate

Ciaran Morrison*, Alexander J. Henzing, Ole Nørregaard Jensen1, Neil Osheroff2, Helen Dodson, Stefanie E. Kandels-Lewis, Richard R. Adams and William C. Earnshaw

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, Swann Building, King’s Buildings, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK, 1 Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark and 2 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA

*To whom correspondence should be addressed at present address: Department of Biochemistry, National University of Ireland, Galway, Ireland. Tel: +353 91 512 334; Fax: +353 91 512 504; Email: ciaran.morrison{at}nuigalway.ie

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 II{alpha}) as a potential Aurora B substrate. Purified recombinant human topo II{alpha} 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.


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