RESUMO
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.
Assuntos
Biomarcadores , Proteínas de Ciclo Celular/metabolismo , Espectrometria de Massas/métodos , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Aurora Quinases , Proteínas de Ciclo Celular/antagonistas & inibidores , Fracionamento Celular , Eletroforese em Gel Bidimensional , Células HeLa , Temperatura Alta , Humanos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Nocodazol/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vimentina/genética , Vimentina/metabolismo , Proteínas de Xenopus/antagonistas & inibidoresRESUMO
An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor-protein interactions as a complementary approach to two-dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin-dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat-shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.
