PI5P4Kγ functions in DTX1-mediated Notch signaling

Notch signaling is an evolutionarily conserved pathway that is essential for development, where it controls processes ranging from cell differentiation to survival. Transport through endosomes is a critical step in regulating Notch signaling capacity, where the E3 ubiquitin ligase DTX1 is thought to control Notch1 intracellular transport decisions by direct receptor ubiquitination. However, how DTX1 regulates Notch1 transport within endosomes and the consequence of Notch1 ubiquitination by DTX1 remain unresolved. Here we demonstrate that DTX1 colocalizes with Notch1 on tubulovesicular recycling endosomes. We find that DTX1 silencing leads to enhanced Notch1 recycling from this compartment to the cell surface via a rab4a-mediated transport route. This, in turn, increases Notch1 cell-surface levels and enhances signaling. Surprisingly, we discovered that DTX1 depletion also elevates Notch1 activity mediated by a mutant form of the receptor that lacks lysine residues for ubiquitination, suggesting that DTX1 targets additional factors. Using an activity-based screen for ubiquitination targets, we identified multiple DTX1 substrates including PI5P4K gamma, a lipid kinase involved in PI(4,5)P-2 production. Immunolocalization analysis reveals that PI5P4K gamma, like DTX1 and Notch1, is present on tubulovesicular recycling endosomes. However, in contrast to DTX1, Notch1 signaling is inhibited by pharmacological inactivation or siRNA depletion of PI5P4K gamma. Moreover, loss of PI5P4K gamma activity decreases Notch1 recycling rates and reduces receptor cell-surface levels. Collectively, these findings argue that PI5P4K gamma positively regulates the Notch pathway by promoting receptor recycling. Additionally, they support a model where DTX1 controls Notch1 endosomal sorting decisions by controlling PI5P4K gamma-mediated production of PI(4,5) P-2.