Research ArticleNotch Signaling

Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing

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Sci. Signal.  13 Jun 2017:
Vol. 10, Issue 483, eaag2989
DOI: 10.1126/scisignal.aag2989

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Ordering the steps to Notch activation

Signaling by the receptor Notch enables cell position to influence cell fate through information conveyed between neighboring cells. Binding of ligand, which is a transmembrane protein on a cell that neighbors the Notch-bearing cell, stimulates Notch cleavage in multiple stages. One cleavage event is mediated by an ADAM family protease and is followed by the cleavage event that generates the biologically active Notch receptor component in the receptor-bearing cell. Chastagner et al. found that ubiquitylation of Notch1 by the E3 ubiquitin ligase DTX4 promoted its internalization in response to ligand binding. Furthermore, ubiquitylation preceded the processing of Notch1 by ADAM10 in an endosomal compartment, and not at the cell surface as is usual for ADAM10 substrates. These results show that the endocytosis of the ligand-bound Notch1 by the ligand-bearing cell is not necessary for the endocytosis of Notch1 by the receptor-bearing cell, as had been previously thought, but rather that both of these endocytosis events are triggered by Notch1 ubiquitylation in the receptor-bearing cell. Because these results also show that the processing of Notch1 by ADAM10 occurs intracellularly, they suggest that it may be necessary for ADAM10 inhibitors to be cell-penetrant to be effective in treating cancers associated with constitutively activated Notch.

Abstract

The Notch signaling pathway, which is activated by cell-cell contact, is a major regulator of cell fate decisions. Mammalian Notch1 is present at the cell surface as a heterodimer of the Notch extracellular domain associated with the transmembrane and intracellular domains. After ligand binding, Notch undergoes proteolysis, releasing the Notch intracellular domain (NICD) that regulates gene expression. We monitored the early steps of activation with biochemical analysis, immunofluorescence analysis, and live-cell imaging of Notch1-expressing cells. We found that, upon ligand binding, Notch1 at the cell surface was ubiquitylated by the E3 ubiquitin ligase DTX4. This ubiquitylation event led to the internalization of the Notch1 extracellular domain by the ligand-expressing cell and the internalization of the membrane-anchored fragment of Notch1 and DTX4 by the Notch1-expressing cell, which we referred to as bilateral endocytosis. ADAM10 generates a cleavage product of Notch that is necessary for the formation of the NICD, which has been thought to occur at the cell surface. However, we found that blocking dynamin-mediated endocytosis of Notch1 and DTX4 reduced the colocalization of Notch1 with ADAM10 and the formation of the ADAM10-generated cleavage product of Notch1, suggesting that ADAM10 functions in an intracellular compartment to process Notch. Thus, this study suggests that a specific pool of ADAM10 acts on Notch in an endocytic compartment, rather than at the cell surface.

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