Searching for biomarkers of Aurora-A kinase activity: identification of in vitro substrates through a modified KESTREL approach.
Aurora-A, -B, and -C are members of a small family of mitotic serine/threonine kinases that regulate centrosome maturation, chromosome segregation, and cytokinesis. They are often overexpressed in different human tumor types and have been identified as attractive targets for anticancer drug development. As specific inhibitors of the Aurora kinases are entering phase I clinical trials, there is a high need for appropriate Aurora-A biomarkers to follow mechanism of action or response. To identify novel Aurora-A substrates potentially useful as specific biomarkers we applied several modifications to the original KESTREL (Kinase Substrate Tracking and Elucidation) method in conjunction with gel electrophoresis and MALDI-MS and LC-MS/MS. The major modifications to the method included the introduction of a heating step to inactivate endogenous kinases after cell lysis and the execution of the in vitro kinase reaction in the presence of 5 mM Mg(2+) and at high (1 mM) ATP concentration. Total and fractionated extracts from nocodazole-treated HeLa cells were used as a source of Aurora-A substrates. Using this approach, we were able to detect a number of Aurora-A specific phospholabeled signals and to identify vimentin as a putative Aurora-A substrate. Vimentin was then confirmed as an in vitro substrate of Aurora-A by the phosphorylation of the recombinant protein followed by MS and antibody detection.
Troiani
Sonia
S
Dept of Biology, Nerviano Medical Sciences, Viale Pasteur 10, 20014 Nerviano (MI), Italy. sonia.troiani@nervianoms.com
Uggeri
Mauro
M
Moll
Jürgen
J
Isacchi
Antonella
A
Kalisz
Henryk M
HM
Rusconi
Luisa
L
Valsasina
Barbara
B
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000378:metabolism / Q000627:therapeutic use; D064127:Aurora Kinases; D015415:Biomarkers; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002458:Cell Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D015739:Nocodazole / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000970:Antineoplastic Agents; D015415:Biomarkers; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D014746:Vimentin; D029867:Xenopus Proteins; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D015739:Nocodazole