The role of lysines of the intracellular domain of Notch during regulation of the Notch pathway

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Transfer of the polypeptide ubiquitin to the lysine side chains of intracellular domains performed by substrate specific E3 Ligases is an important signal for endocytosis and subsequent degradation of several transmembrane proteins (TMPs) in the lysosome. Moreover, it is assumed that endocytosis, initiated through ubiquitylation, is essential for the activation of the evolutionary conserved Notch signalling receptor by its ligands. This assumption is based on the identification of several E3 ligase required for the full activity of Notch. Notch is a TMP that contains 27 lysines in its intracellular domain. In order to test the importance of lysines/ ubiquitylation for the Notch receptor activity, we have exchanged all lysines (Ks) by asparagine (Rs) and tested the activity of the resulting NK2R variant in Drosophila melanogaster. We found that the variant is more active than the corresponding control and that it can activate the Notch pathway probably in a ligand-independent manner. In this application, we will characterise the NK2R variant further and ask how it is activated. We will use this variant to determine which lysines are important for the regulation of the activity of the receptor. We will further determine the role of the identified E3 ligases in the regulation of the receptor. We will achieve these goals by a combination of microscope analysis (electron and confocal microscope, Live imaging), biochemical assays and molecular and genetic experiments.The analysis will reveal the meaning of Lysines of Notch and the function of E3 ligases during the regulation of the activity of the Notch signalling pathway and is of importance also for Notch signalling in mammals.