Glycogen synthase kinase 3β inhibition enhances Notch1 recycling

The Notch signaling pathway is essential throughout development and remains active into adulthood, where it performs a critical role in tissue homeostasis. The fact that defects in signaling can lead to malignancy illustrates the need to control Notch activity tightly. GSK3 beta is an established regulator of the Notch signaling pathway, although its mechanism of action remains unclear. Given the emerging role for GSK3 beta in receptor trafficking, we tested the idea that GSK3 beta controls signaling by regulating Notch transport. Consistent with published reports, we find that GSK3 beta inhibition enhances Notch1 signaling activity. Immunolocalization analysis reveals that Notch1 localization within a tubulovesicular compartment is altered when GSK3 beta activity is disrupted. We also find that receptor cell surface levels increase following acute GSK3 beta inhibition. This is followed by elevated Notch intra-cellular domain (NICD) production and a corresponding increase in signaling activity. Moreover, Notch transport assays reveal that receptor recycling rates increase when GSK3 beta activity is inhibited. Collectively, results presented here support a model where GSK3 beta regulates signaling by controlling postendocytic transport of Notch1. Given that GSK3 beta activity is suppressed following stimulation by multiple signal transduction pathways, our findings also suggest that cells can modulate Notch1 activity in response to extracellular signals by mobilizing Notch1 from endosomal stores.