Binding and regulation of the activity of the DSL ligand Serrate by E3 ligases

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The Notch pathway is required in many developmental and homeostatic processes in all metazoans. In addition, it is involved in the pathogenesis of an increasing number of diseases (e. g. cancer) in humans. Hence, it is important to understand the regulation of its activity in detail. The Notch receptor is activated through binding to ligands of the Dl/Ser/Lag2 (DSL) protein family, In the genome of Drosophila two DSL ligands are present: Delta (Dl) and Serrate (Ser). To activate the Notch receptor, the ligands in the signal-sending cells must be endocytosed. The work so far indicates that the membrane-associated E3 ligases Neuralized (Neur) and Mindbomb1 (Mib1) play crucial roles during activation of the ligands and their endocytosis 3. These enzymes mediate the ubiquitylation (ubi) of the intracellular domains (ICDs) of DSL ligands on lysine (K) side chains. A further component required for ligand activation and with a documented role as a cargo adapter in endocytosis is the Epsin homolog Liquid facets (Lqf) 4. Based on these findings, a simple model has been suggested in which the ligases initiate endocytosis of the DSL ligands by ubiquitylating their ICDs. This label is recognised by Lqf to physically link the ligands to the endocytic core machinery.In Drosophila most DSL signalling is mediated by Mib1. We recently found that MIB1 binds to two separate small binding boxes in the intracellular domain ICD of the human Ser ortholog JAGGED1 (JAG1), termed N- and C-box. Both interactions are needed for normal stimulation of Notch signal transduction. We could also show that the Ks are important for Dl signalling. However, it is not clear whether the rules found for MIB1 dependent JAG1 binding apply to other ligands. Moreover, it is not known which Ks in the ICD of Ser or Dl are ubiquitylated by Mib1 and whether there is a common rule underlying ubi. By focussing on the Drosophila ligand Serrate, will answer these questions in this funding period.