Cross-inhibition of JNK by p38 visualized by multiplexed fluorescence imaging

The stress activated protein kinases, Jun N-terminal kinase (JNK) and p38, orchestrate cellular responses to diverse environmental stresses and inflammatory signals. Crosstalk between JNK and p38 is emerging as an important regulatory mechanism in the inflammatory and stress responses. However, it is still unknown how this crosstalk regulates the signaling dynamics, cell-to-cell variabilities, and cell fate decisions at the single cell level. To address these issues, we established a multiplexed live-cell imaging system to simultaneously monitor JNK and p38 activities with high specificity and sensitivity at the single cell resolution. Exposure to various stresses and cytokines resulted in differential dynamics of JNK and p38 activation. In all cases, p38 negatively regulated JNK activity without affecting its own activation. We quantitatively demonstrate that p38 antagonizes JNK through both transcriptional and posttranslational mechanisms with the relative contribution of the pathway depending on the stimulant. This cross-inhibition of JNK appears to generate cellular heterogeneity in JNK activity in response to inflammatory cytokines and stresses.