Drosophila HOPS and AP-3 Complex Genes Are Required for a Deltex-Regulated Activation of Notch in the Endosomal Trafficking Pathway

Abstract

DSL ligands promote proteolysis of the Notch receptor, to release active Notch intracellular domain (N(ICD)). Conversely, the E3 ubiquitin ligase Deltex can activate ligand-independent Notch proteolysis and signaling. Here we show that Deltex effects require endocytic trafficking by HOPS and AP-3 complexes. Our data suggest that Deltex shunts Notch into an endocytic pathway with two possible endpoints. If Notch transits into the lysosome lumen, it is degraded. However, if HOPS and AP-3 deliver Notch to the limiting membrane of the lysosome, degradation of the Notch extracellular domain allows subsequent Presenilin-mediated release of N(ICD). This model accounts for positive and negative regulatory effects of Deltex in vivo. Indeed, we uncover HOPS/AP-3 contributions to Notch signaling during Drosophila midline formation and neurogenesis. We discuss ways in which these endocytic pathways may modulate ligand-dependent and -independent events, as a mechanism that can potentiate Notch signaling or dampen noise in the signaling network.

Drosophila Ndfip is a novel regulator of Notch signaling

“…However, these opposing phenotypes observed for dNdfip expression, in the presence and absence of Nedd4 family E3s, are consistent with a model of Notch trafficking established for Dx-promoted Notch activation and regulation. 5 Dx promotes Notch endocytosis in a HOPS-and AP-3-dependent manner. It promotes targeting of Notch to the limiting membranes of late endosomes and lysosomes.…”

“…8 Once at the lysosome, the extracellular domain of Notch is degraded within the lysosomal lumen, then the Notch intracellular domain can be cleaved by presenilin g-secretase, thereby activating Notch signaling in a non-canonical manner. 5 When dNedd4 or Su(dx) are present, Notch is ubiquitinated and is trafficked to the lysosomal lumen, wherein it is degraded and signaling does not occur. 5 We propose that dNdfip functions in a manner similar to Dx, in that alone it can promote Notch trafficking to the late endosome/lysosome and in the absence of E3, Notch remains at the limiting membrane, whereby it can be activated in a non-canonical manner.…”

“…[1][2][3] Notch receptor turnover in the absence of ligand is an important mechanism to prevent inappropriate signaling in unstimulated cells, requiring AP-2, AP-3 and HOPSdependent endocytic steps. 4,5 Ubiquitination has a key role in regulating the endocytosis and subsequent downregulation of Notch. 6 In the developing Drosophila wing, members of the Nedd4 family of HECT domain ubiquitin ligases (E3s), dNedd4 and Suppressor of deltex (Su(dx)), ubiquitinate and promote the endocytosis and turnover of Notch in a ligandindependent manner.…”

“…7 Dx overexpression can activate Notch in a ligand-independent manner, 8 relying upon the endocytic pathway to traffic Notch to the limiting membrane of the lysosome, permitting g-secretase cleavage and non-canonical activation of Notch. 5 The activity of dNedd4 and Su(dx) overcome the ligandindependent activation of Notch by Dx by promoting the trafficking of Notch to the lysosomal lumen rather than the limiting membrane as promoted by Dx alone. 5,9,10 The Nedd4 family are members of the HECT class of E3s, characterized by two to four WW domains (protein-protein interaction domains) and an N-terminal Ca 2 þ /lipid binding C2 domain.…”

“…5 The activity of dNedd4 and Su(dx) overcome the ligandindependent activation of Notch by Dx by promoting the trafficking of Notch to the lysosomal lumen rather than the limiting membrane as promoted by Dx alone. 5,9,10 The Nedd4 family are members of the HECT class of E3s, characterized by two to four WW domains (protein-protein interaction domains) and an N-terminal Ca 2 þ /lipid binding C2 domain. 11,12 Although many substrates directly bind the WW domains in Nedd4 E3 by means of PY or similar motifs, others rely on accessory/adapter proteins to promote ubiquitination.…”