Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor
- 8 March 2010
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 107 (12), 5459-5464
- https://doi.org/10.1073/pnas.0909671107
Abstract
The signaling pathway mediated by JNK transduces different types of signals, such as stress stimuli and cytokines, into functional responses that mediate apoptosis, as well as proliferation, differentiation, and inflammation. To better characterize the dynamic information flow and signal processing of this pathway in the cellular context, a genetically encoded, fluorescent protein-based biosensor was engineered to detect endogenous JNK activity. This biosensor, named JNKAR1 (for JNK activity reporter), specifically detects stress- (ribotoxic and osmotic) and cytokine- (TNF-alpha) induced JNK activity in living cells with a 15 to 30% increase in the yellow-to-cyan emission ratio because of a phosphorylation-dependent increase in FRET between two fluorescent proteins. JNK activity was detected not only in the cytoplasm, but also in the nucleus, mitochondria, and plasma membrane with similar kinetics after induction of ribotoxic stress by anisomycin, suggesting relatively rapid signal propagation to the nuclear, mitochondrial, and plasma membrane compartments. Furthermore, quantitative single-cell analysis revealed that anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, features that are consistent with behaviors of bistable systems. The development of JNKAR1, therefore, laid a foundation for evaluating the signaling properties and behaviors of the JNK cascade in single living cells.Keywords
This publication has 30 references indexed in Scilit:
- High Content Cell Screening in a Microfluidic DeviceMolecular & Cellular Proteomics, 2009
- A genetically encoded fluorescent sensor of ERK activityProceedings of the National Academy of Sciences of the United States of America, 2008
- c‐Jun N‐terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neuronsJournal of Neurochemistry, 2007
- The JNK signal transduction pathwayCurrent Opinion in Cell Biology, 2007
- Genetically Encoded Fluorescent Indicators To Visualize Protein Phosphorylation by Extracellular Signal-Regulated Kinase in Single Living CellsAnalytical Chemistry, 2007
- Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal KinasesMicrobiology and Molecular Biology Reviews, 2006
- Analyzing protein kinase dynamics in living cells with FRET reportersMethods, 2006
- Insulin disrupts β-adrenergic signalling to protein kinase A in adipocytesNature, 2005
- JNK phosphorylates paxillin and regulates cell migrationNature, 2003
- Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live CellsScience, 2002