Endosomal regulation of Notch by Nedd4 proteins

For cells to function properly and develop into the right type of cell in the correct location, then signals have to pass between them. These signals are most often detected by receptors at the cell surface, which respond to the signal by altering their structure. This conformational change initiates a series of events inside the cell, ultimately changing the number and level of genes expressed. When such signals go wrong there can be serious consequences resulting in diseases like cancer for example. It will be evident that the more we understand these signals, the better we will be able to exploit the potential to manipulate them to our advantage. The aim of this proposal is to investigate the regulation of one such signal that mediated by a receptor called Notch. One way in which a signal can be regulated is to alter the transport of receptor within different locations in the cell. We have found that if the Notch receptor is trafficked to a particular cellular compartment then it is activated. We have found intracellular regulatory proteins that bind to Notch, modify it in a way as to send Notch to or divert it from this cellular location. This controls whether Notch is activated or not once it has been internalised into the cell. Notch is also thought to be activated at the cell surface by binding to ligands on an adjacent cell. We do not understand yet how these two modes of activation are related. Are they two different mechanisms of activating the receptor, or are they two different steps in the same molecular pathway? The work for this project aims to understand, at the biochemical level, the different types of modification of Notch that regulate its intracellular location and activation. We will also determine how the endosomal activation route is related to the normal mechanism of ligand-dependant Notch signalling. This work will be carried out within the Faculty of Life Sciences at the University of Manchester. Our experimental organism is the fruit fly, which is a commonly used organism because many of the signals controlling its development are also found in humans. The fly has obvious advantages in that it is easy to experiment with and because it breeds fast it is easy to identify mutations that affect the signals that we wish to study. It is by looking at what happens when things go wrong that we can find out how they work normally. This grants seeks support for a post-doctoral research scientist who has already performed important work in this area.

Notch is a developmental signalling receptor that acts in pattern formation and to specify cell fate. Misregulation of Notch is associated with disease states including cancer, dementia and age-related reduction in muscle repair. It is important to understand how Notch is normally regulated to underpin efforts to control it. In the current model, ligand binding by Delta or Serrate leads to two proteolytic cleavages of Notch, which release the soluble intracellular domain, which migrates to the nucleus. There it binds to and activates the transcription factor Suppressor of Hairless. It has been thought these cleavages occur only at the cell surface, but now we have shown that some signalling by Notch is dependent on its endocytosis. We have also shown that redundant Nedd4-related ubiquitin ligases (DNedd4 and Su(dx)) ubiquitinate Notch and divert it from an endosomal compartment in which it is activated. When Nedd4 activity is disrupted, endosomal accumulation of Notch can lead to strong ectopic signalling. In contrast Deltex, a ring finger ubiquitin ligase which also ubiquitinates Notch, acts to drive the latter's endosytosis and ubiquitination. How do two different ubiquitin ligase interactions produce such contrasting effects on receptor activity? This project will investigate at the biochemical level the reasons for the different activities of Deltex and Nedd4 proteins on Notch. At present we do not know the relationship between endosomal-dependent activation of Notch and the normal signalling pathway used in development. Endosomal-dependent activation might be an alternative mechanism using some, but not necessarily all, of the components of the canonical Notch pathway to boost the levels of signalling, or offer control of the extent and duration of it. Alternatively all Notch signalling may require a prior endocytic step. Therefore this project will also investigate the relationship between endosomal-dependant Notch signalling and the canonical Notch pathway.