Second-Generation JNK Activity Reporter Illuminates the Mechanism of AT1R-Induced JNK Activation.

Abstract

c-Jun N-terminal kinases (JNKs) are central integrators of cell stress and are responsible for a multitude of signaling and gene expression changes in response to various environmental cues. Here, we describe the development of JNKAR2, a second-generation FRET-based JNK biosensor. JNKAR2 exhibits a ∼3.9-fold higher response amplitude over JNKAR1 while maintaining robust response dynamics and selectivity. Using JNKAR2 to illuminate JNK activation driven by angiotensin II (Ang II)/Ang II receptor type 1 (AT1R) signaling in HEK293 cells, we found that AT1R-mediated JNK activation is independent of β-arrestin but requires Gαq/11. Supporting a mechanism of Gαq/11-mediated JNK activation downstream of G protein-coupled receptors, stimulating the Gαq/11 coupling muscarinic acetylcholine M5 and M3 as well as endothelin type B receptors also induced robust JNKAR2 responses. On the other hand, neither the Gαi/o-preferring μ-opioid receptor nor the Gαs-preferring β2-adrenergic receptor was able to support JNK activation. The improved dynamic range of JNKAR2 further enabled us to probe Ang II-induced JNK signals in primary vascular smooth muscle cells, which were abolished by Gαq/11 inhibition. Targeting JNKAR2 to various subcellular compartments revealed that anisomycin induced global JNK signals, while AT1R-mediated JNK activation was absent from the plasma membrane and early endosomes. Together, we report on a second-generation JNK biosensor with substantially improved response amplitude that enables sensitive and selective visualization of JNK activation in real time and with subcellular resolution. JNKAR2 facilitates the dissection of the molecular mechanisms underlying JNK signaling.