Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.
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.
Fosbrink
Matthew
M
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Aye-Han
Nwe-Nwe
NN
Cheong
Raymond
R
Levchenko
Andre
A
Zhang
Jin
J
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D020935:MAP Kinase Signaling System; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013312:Stress, Physiological; D013347:Subcellular Fractions / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases