High-sensitivity measurements of multiple kinase activities in live single cells
- PMID: 24949979
- PMCID: PMC4097317
- DOI: 10.1016/j.cell.2014.04.039
High-sensitivity measurements of multiple kinase activities in live single cells
Abstract
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
Copyright © 2014 Elsevier Inc. All rights reserved.
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Comment in
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Live reporting of kinase dynamics.Nat Methods. 2014 Aug;11(8):788-9. doi: 10.1038/nmeth.3058. Nat Methods. 2014. PMID: 25229097 No abstract available.
References
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- Bagowski CP, Ferrell JE., Jr Bistability in the JNK cascade. Current biology : CB. 2001;11:1176–1182. - PubMed
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