Detection of kinase substrates using quantitative phosphoproteomics
Boris Maček, Proteome Center Tuebingen
Protein phosphorylation is a key posttranslational modification, which reversibly regulates almost all processes in the living cell. Deregulated signaling is a hallmark of cancer and other diseases, and protein kinases are prominent drug targets. Phosphorylation events are commonly probed in a targeted manner by phosphorylation-specific antibodies.
In contrast, advances in proteomics technology, including phosphopeptide enrichment, high-accuracy mass spectrometry, and associated bioinformatics now make it possible to analyze entire phosphoproteomes. Consequently, the challenge and the emphasis in the fields of proteomics and cell biology have now shifted to the next stage – global detection of kinase and phosphatase substrates and their integration into regulatory networks in the cell. We have developed an elaborate (phospho)proteomic workflow based on quantitative MS and phosphopeptide enrichment to identify substrates of several eukaryotic and prokaryotic kinases, such as Aurora1 and PKD2.
The workflow generally involves SILAC- or label-free based phosphoproteome analysis of cell lines/tissues where the kinase of interest is inhibited and compared with the unstimulated kinase. After biological treatment, cells are harvested, proteomes mixed, digested with a protease and resulting peptides are subjected to two-stage phosphopeptide enrichment, LC-MS measurement and bioinformatic integration of proteome and phosphoproteome data. We are currently pursuing substrate mapping of several key kinases in various organisms; the initial results of these analyses will be presented and discussed.
References
- Koch, A., Krug, K., Pengelley, S., Macek, B., Hauf, S. 2011. Mitotic substrates of the kinase aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast.. Science Signaling 4(179): rs6
- Franz-Wachtel, M., Eisler, S., Krug, K., Wahl, S., Carpy, A., Nordheim, A., Pfizenmaier, K., Hausser, A., Macek, B. 2012. Global detection of Protein Kinase D-dependent phosphorylation events in nocodazole-treated human cells. Mol Cell Proteomics 11(5):160-70.
