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-α) 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.

[1]  Andre Levchenko,et al.  High Content Cell Screening in a Microfluidic Device*S , 2009, Molecular & Cellular Proteomics.

[2]  K. Svoboda,et al.  A genetically encoded fluorescent sensor of ERK activity , 2008, Proceedings of the National Academy of Sciences.

[3]  J. R. Pomerening,et al.  Uncovering mechanisms of bistability in biological systems. , 2008, Current opinion in biotechnology.

[4]  E. Cadenas,et al.  c‐Jun N‐terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons , 2007, Journal of neurochemistry.

[5]  C. Weston,et al.  The JNK signal transduction pathway. , 2007, Current opinion in genetics & development.

[6]  Y. Umezawa,et al.  Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells. , 2007, Analytical chemistry.

[7]  Bostjan Kobe,et al.  Uses for JNK: the Many and Varied Substrates of the c-Jun N-Terminal Kinases , 2006, Microbiology and Molecular Biology Reviews.

[8]  Jin Zhang,et al.  Analyzing protein kinase dynamics in living cells with FRET reporters. , 2006, Methods.

[9]  D. Ouyang,et al.  Activation of c-Jun N-terminal kinases by ribotoxic stresses. , 2005, Cellular & molecular immunology.

[10]  Roger Y. Tsien,et al.  Insulin disrupts β-adrenergic signalling to protein kinase A in adipocytes , 2005, Nature.

[11]  T. Herdegen,et al.  JNK2 Translocates to the Mitochondria and Mediates Cytochrome c Release in PC12 Cells in Response to 6-Hydroxydopamine* , 2004, Journal of Biological Chemistry.

[12]  M. White,et al.  Stimulus-Coupled Spatial Restriction of Extracellular Signal-Regulated Kinase 1/2 Activity Contributes to the Specificity of Signal-Response Pathways , 2004, Molecular and Cellular Biology.

[13]  M. Bogoyevitch,et al.  The Critical Features and the Mechanism of Inhibition of a Kinase Interaction Motif-based Peptide Inhibitor of JNK* , 2004, Journal of Biological Chemistry.

[14]  Avraham E Mayo,et al.  Hysteresis vs. Graded Responses: The Connections Make All the Difference , 2004, Science's STKE.

[15]  Ken Jacobson,et al.  JNK phosphorylates paxillin and regulates cell migration , 2003, Nature.

[16]  Eduardo Sontag,et al.  Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2 , 2003, Nature Cell Biology.

[17]  James E. Ferrell,et al.  The JNK Cascade as a Biochemical Switch in Mammalian Cells Ultrasensitive and All-or-None Responses , 2003, Current Biology.

[18]  A. Aronheim,et al.  Differential targeting of the stress mitogen-activated protein kinases to the c-Jun dimerization protein 2. , 2002, The Biochemical journal.

[19]  G. Johnson,et al.  Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases , 2002, Science.

[20]  Susan S. Taylor,et al.  A 15-Residue Bifunctional Element in d-AKAP1 Is Required for Both Endoplasmic Reticulum and Mitochondrial Targeting* , 2002, The Journal of Biological Chemistry.

[21]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[22]  J. Ferrell.  Self-perpetuating states in signal transduction: positive feedback, double-negative feedback and bistability. , 2002, Current opinion in cell biology.

[23]  A. Aronheim,et al.  The AP‐1 repressor, JDP2, is a bona fide substrate for the c‐Jun N‐terminal kinase , 2001, FEBS letters.

[24]  J. Ferrell,et al.  Bistability in the JNK cascade , 2001, Current Biology.

[25]  D. Durocher,et al.  The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms. , 2000, Molecular cell.

[26]  Roger Davis,et al.  The c-Jun NH2-terminal Kinase Promotes Insulin Resistance during Association with Insulin Receptor Substrate-1 and Phosphorylation of Ser307 * , 2000, The Journal of Biological Chemistry.

[27]  M. Karin,et al.  JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension. , 1998, Genes & development.

[28]  J E Ferrell,et al.  The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes. , 1998, Science.

[29]  M. Karin,et al.  JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain , 1994, Cell.

[30]  Jingliu,et al.  Role of JNK activation in apoptosis： A double-edged sword , 2005 .