Dissection of MKK6 and p38 Signaling Using Light-Activated Protein Kinases

Stress-activated signaling pathways orchestrate cellular behaviors and fates. Studying the precise role(s) of stress-activated protein kinases is challenging, because stress conditions induce adaptation and impose selection pressure. To meet this challenge, we have applied an optogenetic system with a single plasmid to express light-activated p38 alpha or its upstream activator, MKK6, in conjunction with live-cell fluorescence microscopy. In starved cells, decaging of constitutively active p38 alpha or MKK6 by brief exposure to UV light elicits rapid p38-mediated signaling, release of cytochrome c from mitochondria, and apoptosis with different kinetics. In parallel, light activation of p38 alpha also suppresses autophagosome formation, similarly to stimulation with growth factors that activate PI3K/Akt/mTORC1 signaling. Active MKK6 negatively regulates serum-induced ERK activity, which is p38-independent as previously reported. Here, we reproduce that result with the one plasmid system and show that although decaging active p38 alpha does not reduce basal ERK activity in our cells, it can block growth factor-stimulated ERK signaling in serum-starved cells. These results clarify the roles of MKK6 and p38 alpha in dynamic signaling programs, which act in concert to actuate apoptotic death while suppressing cell survival mechanisms. Studying stress-activated signaling is challenging, because stress conditions induce adaptation and impose selection pressure. Applying an optogenetic system to express light-activated p38 alpha or its upstream activator, MKK6, the functional roles of these kinases in dynamic signaling programs are clarified.image

Automated summary

Studying stress-activated signaling is challenging due to induced adaptation and imposed selection pressure. By using an optogenetic system to express light-activated p38 alpha or its upstream activator MKK6, the functional roles of these kinases in dynamic signaling programs are clarified.