Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate.
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.
Morrison
Ciaran
C
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. ciaran.morrison@nuigalway.ie
Henzing
Alexander J
AJ
Jensen
Ole Nørregaard
ON
Osheroff
Neil
N
Dodson
Helen
H
Kandels-Lewis
Stefanie E
SE
Adams
Richard R
RR
Earnshaw
William C
WC
D000951:Antigens, Neoplasm; D064107:Aurora Kinase B; D064127:Aurora Kinases; D002877:Chromosomes, Human / Q000737:chemistry / Q000201:enzymology*; D004250:DNA Topoisomerases, Type II / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D008677:Metaphase; D009687:Nuclear Proteins / Q000032:analysis / Q000145:classification; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D019204:GTP-Binding Proteins; C498988:GTPBP4 protein, human; D004250:DNA Topoisomerases, Type II