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Review
. 2012 Sep;13(9):654-66.
doi: 10.1038/nrg3272. Epub 2012 Aug 7.

The Notch signalling system: recent insights into the complexity of a conserved pathway

K G Guruharsha  1 , Mark W Kankel, Spyros Artavanis-Tsakonas
Affiliations
Review

The Notch signalling system: recent insights into the complexity of a conserved pathway

K G Guruharsha et al. Nat Rev Genet. 2012 Sep.

Abstract

Notch signalling links the fate of one cell to that of an immediate neighbour and consequently controls differentiation, proliferation and apoptotic events in multiple metazoan tissues. Perturbations in this pathway activity have been linked to several human genetic disorders and cancers. Recent genome-scale studies in Drosophila melanogaster have revealed an extraordinarily complex network of genes that can affect Notch activity. This highly interconnected network contrasts our traditional view of the Notch pathway as a simple linear sequence of events. Although we now have an unprecedented insight into the way in which such a fundamental signalling mechanism is controlled by the genome, we are faced with serious challenges in analysing the underlying molecular mechanisms of Notch signal control.

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Figures

Figure 1
Figure 1. Summary of the main features of Notch signalling
Three proteolytic cleavage steps are required for canonical Notch receptor signalling. The first proteolytic cleavage step (S1 cleavage) is mediated by Furin, occurs in the trans-Golgi and produces a heterodimer composed of a ligand-binding Notch extracellular domain (NECD) and a single-pass transmembrane signalling domain referred to as the Notch intracellular domain (NICD). The functional importance of this cleavage is still somewhat unclear,. The association of NECD and the transmembrane portion of the receptor heterodimer is dependent on non-covalent interactions. Pathway activation occurs when the NECD binds to Delta– Serrate–LAG2 (DSL) ligands that are expressed on the membrane of neighbouring cells. This trans interaction results in the second proteolytic event (S2 cleavage) of the Notch receptor, which clears most of the NECD from the outer portion of the membrane, a process mediated by the TACE (also known as ADAM17) metalloproteinase. The NECD is subsequently released and internalized through endocytosis by the ligand-expressing cell, where it undergoes lysosomal degradation. Subsequently, γ-secretase cleaves the tethered receptor near the inner leaflet of the membrane (S3 cleavage) in the Notch-expressing cell, producing the transcriptionally active NICD, which translocates to the nucleus through a poorly understood process. In the nucleus, the NICD interacts with Drosophila melanogaster Suppressor of Hairless (SU(H)) and the transcriptional co-activator, Mastermind (MAM) — the mammalian orthologues of which are CBF1–SU(H)–LAG1 (CSL) and Mastermind-like (MAML) proteins, respectively — thereby inducing transcription of target genes, by converting CSL into a transcription activator through the exchange of co-repressors for co-activators. Many Notch target genes encode transcriptional regulators, which influence cell-fate decisions through the regulation of basic helix–loop–helix hairy and enhancer of split (HES) proteins: Hairy and Enhancer of split (E(SPL)) in D. melanogaster and their mammalian orthologues HES1 and HES5. The HES proteins subsequently regulate the expression of genes involved in Notch-dependent cell-fate determination, such as apoptosis, proliferation or differentiation. By contrast, expression of ligands and the Notch receptor on the same cells results in cis inhibition of Notch signals and receptor degradation. Recycling the receptor through the endocytic pathway has been shown to be important for receptor and ligand maturation, non-canonical signalling and degradation (Box 2). Notch activity is regulated by ubiquitylation of nuclear NICD by the E3 ubiquitin ligases, SEL-10 in Caenorhabditis elegans and Suppressor of Deltex (SU(DX)) in D. melanogaster, leading to NICD degradation, thus allowing the cell to become ligand-competent once again. The ubiquitylation status of the receptor (by Kurtz, Deltex and Shrub) in the multi-vesicular bodies can also determine whether Notch continues to signal or undergoes proteasomal degradation. Additionally, signal attenuation is achieved through lysosomal degradation of the NICD.
Figure 2
Figure 2. Overlap of Notch genetic modifiers from different genome-wide screens in Drosophila melanogaster
The first screen to take advantage of the Exelixis collection of insertional mutations was carried out by Kankel et al., and the authors screened for modifiers of a Drosophila melanogaster wing margin phenotype that resulted from ectopic expression of dominant negative version of Mastermind (MAM). The second screen to use the Exelixis collection was carried out by Shalaby et al. and isolated modifiers of the cell-fate alterations in the D. melanogaster eye that are caused by overexpression of the Notch ligand Delta. Among the genes identified from these two independent genetic screens, 50 genes were observed in both studies. Genome-wide RNAi screens were carried out by several different groups using in vivo developmental phenotypes associated with the Notch pathway or in vitro cell-based Notch-signalling-dependent reporter assays. Mummery-Widmer et al. used external sensory organ development during thoracic bristle formation as a phenotypic parameter to isolate genes that affect Notch signalling, whereas Saj et al. carried out a genome-wide screen in S2 cells for regulators of Notch activity. Nearly half of the genes identified in the primary S2-cell-based screen were subsequently in vivo validated in developing wing imaginal discs. Mourikis et al. measured the transcriptional response of a luciferase-reporter (m3-luc) in Kc-167 cells to identify modifiers of Notch activity. However, the overlap of genes identified in all of the in vivo and in vitro RNAi screens is small (consisting of only two genes). From these screens, a total of 2,208 genes (14.25% of 15,494 genes based on FlyBase release 5.43) were found to affect Notch signalling (Supplementary information S4 (table)), 132 of which were identified in both Exelixis-based and RNAi-based screens. The ‘Wing margin’ image is reproduced, with permission, from REF. 36 © (2007) Genetics Society of America. The ‘Thoracic bristles’ image is reproduced, with permission, from REF. 132. The ‘Wing imaginal discs’ image is reproduced from REF. 38 © (2010) Cell Press.
Figure 3
Figure 3. Summary of crosstalk between Notch and other signalling pathways
a,b | A schematic representation depicting classical and current views of signalling pathways and how these pathways are thought to integrate their signals (crosstalk) within a cell. Panel a portrays signal transduction pathways as quite distinct sets of systems or cascades that transmit information by a step-by-step or linear mechanism with minimal crosstalk. Proteins are shown as circles and their physical interactions as blue lines. Pathways are distinguished by different coloured circles. Panel b displays a more representative and current perspective of signalling crosstalk with different signalling pathways as complex, highly interconnected networks with several shared members (red circles). The network was generated using human signal transduction pathway members from KEGG database and queried in GeneMania for genetic and physical interactions with the Notch pathway, as reported in the literature. Network visualization was created using Cytoscape. c| A summary version of the network in panel b, highlighting the fact that Notch pathway genes (yellow circle) interact genetically or physically with most genes that are associated with other signalling pathways. The number of genes in each category is listed in its corresponding coloured circle, and the number shown on the blue line indicates the number of genetic and physical interactions between categories. Only a small subset (52 genes, pink circle) appears to connect to the Notch pathway indirectly. The common interactors (grey circle) are 100 genes that are not classified as signalling genes by KEGG or GeneMania and may represent potential points of signal integration or crosstalk between Notch and other canonical signalling pathways.
Figure 4
Figure 4. Mapping genetic modifiers on proteome map
The highly interconnected major component of the Drosophila Protein Interaction Map (DPiM), showing Notch signalling modifiers that were identified from Exelixis screens (blue), RNAi screens (red) or both (pink). This overlay of genetic data on a proteomics map helps to combine independent and seemingly disparate data sets into one integrated network while providing potential mechanistic insights into the basic biochemistry of these interactions. Grey lines indicate protein–protein interactions, the thickness being proportional to the interaction score. In many cases, it is clear that several members of a protein complex were identified in independent screens, suggesting that the complex, as a functional unit, is important for Notch signalling. Such integrative analyses provide numerous experimentally testable hypotheses into the links between Notch signalling and these modifiers, as well as their associated functions, as defined in DPiM. Within DPiM, connections between uncharacterized proteins to protein complexes with defined Gene Ontological terms provide a potential function to the uncharacterized proteins. A high-resolution version of this figure is available in Supplementary information S5 (figure).
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