An overview of the Notch signalling pathway
Introduction
Notch is an essential gene encoding a signalling receptor that is required throughout development to regulate the spatial patterning, timing and outcomes of many different cell fate decisions in both vertebrate and invertebrate species [1], [2]. Notch is a single spanning transmembrane protein, which has a modular architecture (Fig. 1) including many repeats of a protein module resembling epidermal growth factor (EGF) and three membrane-proximal Lin12/Notch/Glp-1 (LNG) repeats. The intracellular domain has four distinct regions, the RAM domain, the ankyrin repeats, a transcriptional activator domain (TAD) and the PEST (proline-, glutamate-, serine-, threonine-rich) sequence. Two nuclear localisation sequences are present prior to and following the ankyrin repeats. Data from several groups have led to a model of Notch activation involving proteolytic cleavages at three sites S1, S2 and S3 [3], [4] (Fig. 1). S1 cleavage occurs within the secretory pathway so that a processed heterodimeric form is transported to the cell surface. S2 cleavage occurs following ligand binding by Delta or Serrate (Jagged in mammals) through their DSL domains (Delta/Serrate/Lag2), and releases a membrane tethered form of the Notch intracellular domain. The latter is a constitutive substrate for the S3 cleavage, which releases the soluble intracellular domain of Notch (NIc). NIc is translocated to the nucleus where it binds via the RAM domain and ankyrin repeats to a transcription factor, Suppressor of Hairless (Su(H)), or CBF1 in vertebrates. In the absence of a Notch signal, Su(H)/CBF1 can repress transcription through the recruitment of a histone deacetylase (HDAC) [5]. Binding of NIc displaces HDAC (Fig. 1) and allows recruitment of histone acetylases and the nuclear protein Mastermind, which together activate transcription [6], [7], [8]. The complex molecular interactions which regulate the molecular switch of Su(H)/CBF1 from a negative to a positive regulator of transcription have been reviewed recently [3].
Alternative mechanisms of Notch signalling involving Su(H)/CBF1-independent events have also been proposed and recently reviewed [4]. The molecular nature of these signals is yet to be fully elucidated but a recent report provides in vivo data that Drosophila Deltex, a cytoplasmic Notch binding protein, is required for a Su(H)-independent signal [9]. Interpretations of the role of Deltex are complicated however, because in some developmental contexts Deltex acts as a positive regulator of Su(H)-dependent Notch activity, upstream of the S3 cleavage [10].
Aberrant Notch function is associated with several developmental disorders, neurodegenerative disease and cancers [11], [12]. In addition, in the light of current interest in developing cell-based therapies, Notch signalling may become an attractive target for manipulating the outcome of cell fate decisions to produce defined cell types in vitro. The involvement of many enzymatic steps in the Notch pathway provides much potential for the design of Notch inhibitors or activators, which may have therapeutic value. This review discusses our current understanding of the central role played by proteolytic and ubiquitin ligase enzymes in the generation and regulation of the Notch signal.
Section snippets
S1 site cleavage: Notch processing within the secretory pathway
It has been shown in mammalian cells that Notch-1 and -2 are cleaved within their extracellular domains [13] whilst in the secretory pathway. The Notch-1 S1 cleavage depends on a Furin-like convertase and occurs at a site matching the Furin consensus sequence (RXR/KR) [14] (Fig. 2). Cleavage at this site yields a 180 kDa fragment containing the majority of the extracellular domain, and a 120 kDa fragment consisting of a membrane tethered intracellular domain with a short extracellular sequence.
S2 site cleavage: ligand-dependent cleavage of Notch
The S2 cleavage of Notch occurs following ligand binding to the Notch extracellular domain and is thought to be dependent on a member of the ADAM metalloprotease family. One candidate is Kuzbanian whose mutation gives phenotypes resembling those of Notch [19], [20]. However, the true role for Kuzbanian is clouded by several conflicting reports. The kuzbanian mutations were found to prevent a cleavage of Drosophila Notch that was originally thought to be at the S1 site [20] which, as discussed
The signal generation step: S3 cleavage
The membrane tethered NIc product of the S2 cleavage is itself a substrate for a proteolytic activity that cleaves Notch at the S3 site [39], within the transmembrane domain (Fig. 2), to release soluble NIc. The latter product is translocated to the nucleus to activate the Su(H)/CBF1-dependent signal. In vivo studies in Caenorhabditis elegans, Drosophila and mice [40], [41], [42] have shown that Presenilin proteins are required for Notch signalling. In mammals, Presenilins (Presenilin-1 and -2)
Switching off the Notch signal
Ubiquitin ligase and proteolytic steps are also involved in down-regulation of the Notch signal. Sel-10 was originally identified in C. elegans as a negative regulator of Notch signalling and is a substrate-targeting component of an SCF class E3 ubiquitin ligase [64]. Mammalian homologues of Sel-10 protein can stimulate phosphorylation-dependent ubiquitination of nuclear NIc and trigger its proteosome-dependent degradation [65], [66]. The latter interaction is dependent on the C-terminal region
Conclusion
Notch is a vitally important signalling receptor, which has a major contribution to proper development, influencing cell fate, proliferation and survival. The contribution of aberrant Notch signalling to human disease is likely to be increasingly recognised in the future. The participation of multiple enzymatic steps in the generation of the signal and its regulation offers many potential points of therapeutic intervention. While considerable progress has been made in elucidating the molecular
Acknowledgements
I thank Keith Brennan, Stephen High and Marian Wilkin for critical comments and Spyros Artavanis-Tsakonas for discussing data ahead of publication. I acknowledge financial support from the Wellcome Trust and MRC.
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