Quantitative NMR analysis of Erk activity and inhibition by U0126 in a panel of patient-derived colorectal cancer cell lines.
We comparatively analyzed the basal activity of extra-cellular signal-regulated kinase (Erk1/2) in lysates of 10 human colorectal cancer cell lines by semi-quantitative Western blotting and time-resolved NMR spectroscopy. Both methods revealed heterogeneous levels of endogenous Erk1/2 activities in a highly consistent manner. Upon treatment with U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK) acting upstream of Erk1/2, Western-blotting and NMR congruently reported specific modulations of cellular phospho-Erk levels that translated into reduced kinase activities. Results obtained in this study highlight the complementary nature of antibody- and NMR-based phospho-detection techniques. They further exemplify the usefulness of time-resolved NMR measurements in providing fast and quantitative readouts of kinase activities and kinase inhibitor efficacies in native cellular environments. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
In-Cell NMR Laboratory, Department of NMR-supported Structural Biology, Leibniz Institute of Molecular Pharmacology FMP Berlin, Robert-Rössle Strasse 10, 13125 Berlin, Germany.
Honor May
HM
Rose
Marchel
M
Stuiver
Rossukon
R
Thongwichian
Francois-Xavier
FX
Theillet
Stephan M
SM
Feller
Philipp
P
Selenko
D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D045325:HCT116 Cells; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013997:Time Factors
D002070:Butadienes; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3