Mechanisms of insect limb development

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Ligand-induced proteolytic cleavage and internalization of the plasma membrane receptor Notch leads to its activation. Ligand-independent, steady state internalization of Notch, however, does not lead to activation. The mechanism by which downstream effectors discriminate these bipartite modes of Notch internalization is not understood. Nedd4 is a HECT domain-containing E3 ubiquitin ligase that targets transmembrane receptors containing the PPSY motif for endocytosis. Deltex is a positive regulator of Notch signaling and encodes a putative ubiquitin ligase of the ring finger type. We show that Notch is ubiquitinated, and destabilized by Nedd4 in a manner requiring the PPSY motif in the Notch intracellular domain. Loss of Nedd4 function dominantly suppresses the Notch and Deltex mutant phenotypes and its hyper activation attenuates Notch activity. In tissue culture cells, dominant-negative form of Nedd4 blocks steady-state Notch internalization and activates Notch signaling independently of ligand binding, and this effect was further potentiated by Deltex. Nedd4 destines Deltex to degradation in a Notch-dependent manner. Nedd4 antagonizes Notch signaling by promoting internalization of Notch and degradation of Notch and Deltex. Those functions of Nedd4 may be important for the protection of unstimulated cells from sporadic activation of Notch signaling.