Selective phosphorylation of serine 345 on p47-phox serves as a priming signal of ROS-mediated axonal degeneration.

Oxidative stress is a well-known inducer of two major neurodegenerative pathways, neuronal cell death and neurite degeneration. We previously reported that reactive oxygen species (ROS) generated by NADPH oxidases induces EGFR-dependent phosphorylation and activation of ZNRF1 ubiquitin ligase in neurons, which promotes neuronal cell death and neurite degeneration. While these findings provide a potential therapeutic avenue for neurodegeneration, a deeper understanding of the molecular mechanisms of this pathway have emerged as key points of interest. Here, we show that a NADPH oxidase subunit p47-phox/neutrophil cytosolic factor 1 regulates ZNRF1 activity. Using an in vitro neurite degeneration model, we demonstrate that transection-induced phosphorylation of p47-phox at the 345th serine residue by p38 MAPK serves as an initiating signal to activate ZNRF1. The phosphorylated p47 (pS345) or a phospho-mimetic mutant p47-phox binds directly to ZNRF1 whereas a phosphorylation-resistant mutant p47-phox cannot bind to ZNRF1 and its overexpression in neurites significantly suppresses ZNRF1 activation, AKT ubiquitination, and degeneration after transection, suggesting that pS345 might enhance the EGFR-mediated phosphorylation-dependent activation of ZNRF1. These results suggest that pS345 might represent an important checkpoint to initiate the ZNRF1-mediated neurite degeneration. Our findings provide novel insights into the mechanism of ROS-mediated neurodegeneration.