Live-Cell Characterization of the JNK Signaling Cascade

Abstract

Signaling cascades process extracellular information about environmental changes into spatiotemporally regulated signaling events, through a series of signal relays as well as feedback loops and crosstalk mechanisms. The JNK signaling cascade is a major mediator of vital cellular decision making processes, including cellular differentiation and inflammatory response. The cascade transduces a variety of extracellular signals into these functional responses, but its response to TNF, an important cytokine in immune system communications, has not been well characterized, especially at the single mammalian cell level. To characterize JNK quantitatively in single living cells, we used a FRET-based JNK activity reporter to visualize JNK activity in HEK cells. We found that JNK produces a graded response to varying concentrations of TNF, suggesting that JNK relays a graded input signal to its downstream components, which is subsequently converted into a digital response for executing important cellular decisions such as programmed cell death. Using the reporter targeted to the plasma membrane, we also discovered that JNK activation kinetics is slower at the plasma membrane compared to the cytosol, suggesting that JNK activities are regulated spatially. Moreover, JNK in HEK cells exhibited increased sensitivity to TNF when prestimulated with a low dose of the stimulus