UC San Diego

The Notch pathway is a cell-cell signaling pathway conserved in metazoans and utilized extensively during development to mediate cell fate decisions. Signaling is initiated by a functional interaction between the transmembrane Notch (N) receptor on the surface of one cell with a transmembrane ligand on a neighboring cell. Stimulation of the N receptor leads to transcriptional activation of N-target genes in signal-receiving cells. Many N-dependent processes also rely on the activity of the E3 ubiquitin ligase, Neuralized (Neur), in the signal- sending cell. In the fruit fly, Drosophila melanogaster, Neur activates the signaling capability of the N ligands Delta (Dl) and Serrate (Ser) by directing their ubiquitination-mediated endocytosis. The activity of Neur is inhibited by a family of N-target genes known as the Bearded (Brd) family. In Chapter 1, we demonstrate that Brd proteins and the N ligand Dl use a common protein motif, which we call the NXXN motif, to directly interact with Neur. We show that the NXXN motifs of Dl are required in vivo for its Neur-dependent endocytosis. In Brd proteins, we find that NXXN motifs are required to disrupt an in vitro Neur-Dl interaction and inhibit Neur activity in vivo. In Chapter 2, we find that the basic amphipathic [alpha]-helix, or B domain, of Brd proteins mediates an in vitro interaction with PI(3)P. The B domain is necessary for Brd proteins to colocalize with Neur in PI(3)P- positive endosomes in vivo. Using additional markers of endocytic compartments we observe that Brd protein/Neur endosomes also colocalize with the late endosomal and lysosomal proteins Rab7 and LAMP1, suggesting Brd proteins mediate the targeting of Neur to lysosomes for degradation. In Chapter 3, we describe several generated null alleles of Brd family genes. Cells mutant for Brd genes are found to be sensitized to levels of Neur activity. We show that Brd genes act redundantly to inhibit Neur activity in non-SOP cells of the proneural cluster during lateral inhibition