PI5P4Kγ functions in DTX1-mediated Notch signaling

Significance The Notch signaling pathway performs a vital role in biological processes ranging from stem cell maintenance to cell viability. This highly conserved pathway must be tightly controlled, since defects in signaling can promote disease. The E3 ubiquitin ligase DTX1 has emerged as a key negative regulator of Notch signaling, where Notch ubiquitination by DTX1 is thought to control intracellular sorting decisions of the receptor. Here we show that DTX1 can regulate Notch activity independent of directly ubiquitinating the receptor, suggesting that DTX1 targets other factors involved in Notch transport. Using an activity-based screen for DTX1 substrates, we identify PI5P4Kγ, a lipid kinase, and discover that PI5P4Kγ and DTX1 have opposing activities in regulating Notch transit through recycling endosomes.