PI5P4Kγ functions in DTX1-mediated Notch signaling

Abstract

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 PI5P4Kγ, a lipid kinase involved in PI(4,5)P production. Immunolocalization analysis reveals that PI5P4Kγ, 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 PI5P4Kγ. Moreover, loss of PI5P4Kγ activity decreases Notch1 recycling rates and reduces receptor cell-surface levels. Collectively, these findings argue that PI5P4Kγ positively regulates the Notch pathway by promoting receptor recycling. Additionally, they support a model where DTX1 controls Notch1 endosomal sorting decisions by controlling PI5P4Kγ-mediated production of PI(4,5)P.

Genome-wide suppressor screen identifies USP35/USP38 as therapeutic candidates for ciliopathies

“…From a mechanistic, signaling perspective, however, the discovered suppressors potentially link BBS4 to both existing and new links of ciliary biology. The suppressor DTX1 encodes an E3 ubiquitin ligase, which is known to regulate Notch signaling through both ubiquitination-dependent and -independent mechanisms (62)(63)(64). In contrast, Engase is an N-acetylglucosaminidase, supporting the recent observation that regulation of this posttranslational modification might be relevant to ciliary transport and function (65), whereas PITPNM2 is relevant to the function of the retinal degeneration B locus in Drosophila (66).…”

Targeting the PI5P4K lipid kinase family in cancer using novel covalent inhibitors

“…The PI5P4Ks were traditionally thought to mainly be crucial direct regulators of PI-5-P levels Stijf-Bultsma & Sommer et al, 2015;Hasegawa, Strunk, & Weisman, 2017) however, PI5P4Kα was found to synthesize a pool of PI-4,5-P2 that is specifically important in mTORC2 regulation (Bulley et al, 2016) and to play a critical role in intracellular cholesterol transport by modulating PI-4,5-P2 homeostasis on peroxisome membranes (Hu et al, 2018). The low-activity isoform PI5P4Kγ was demonstrated to positively regulate Notch1 signaling by facilitating receptor recycling, suggesting that endosome-localized production of PI(4,5)P2 is involved Notch transport (Zheng & Conner, 2018). PI5P4Kα/β were also shown to be required for autophagosomelysosome fusion during times of metabolic stress, suggesting that they were evolved by multicellular organisms to produce sufficient PI-4,5-P2 in nutrient-deficient conditions (Lundquist et al, 2018).…”

Bone Microenvironment and Osteosarcoma Metastasis

“…Many data show that the Notch pathway is positive in various cancers including OS [ 109 ]. The Notch pathway is activated with the decrease in DTX1 [ 110 ]. At the same time, the depletion of DTX1 results in the improvement of Notch1 in OS cells [ 110 ].…”