Notch
FIG 22.3 Structure of the Notch regulatory region (NRR).
(a) A ribbon representation of the Notch protein. A, B, and C indicate the three Ca2 -binding Lin-12 repeats and the S1 site. Notch is cleaved in the cis-Golgi by a furin-like convertase at the S1-site and arrives at the cell surface as a heterodimer reassembled from the fragments. (b) The Lin-12 repeats mask the S2-cleavage site. The Notch ligand renders this site accessible to the ADAM family of proteases. Cleavage at S1 is ligand-independent, whereas S2 cleaved by ADAM, and S3/S4 cleaved by -secretase, are dependent on ligand interaction. LNR, lin-12/Notch repeat; HD, heterodimerization domain. (c) Lin-12 repeats (spheres) covering the S2 site. (2004).
preventing protein misfolding.21 Fringe, which acts in the Golgi apparatus, generally has the opposite effect, limiting signal output by O-fucose elongation of Notch and its ligands.22,23 Regulation of OFUT1 and Fringe may contribute to the pattern of Notch activation during development.
Activation of Notch
Signalling through Notch occurs only between cells that are in direct contact with each other.24 Activation entails proteolytic processing at two sites. First, ligand binding induces cleavage at the S2 site by members of the ADAM family of metalloproteases (Fig 22.3). Then a second cut by the -secretase complex, cleaves the S3 site in the transmembrane region.25,26 There is a further cut by -secretase at S4, in the centre of the transmembrane domain,
-Secretase is a transmembrane multiprotein protease complex. The eight-pass presenilin comprises the catalytic subunit. This is surrounded and stabilized by nicastrin and APH1. PEN2, a two-pass membrane protein, induces endoproteolysis of the catalytic subunit, required for activity.29 Loss
of function of presenilin correlates with accumulation of plaques in Alzheimer patients (extracellular deposition of amyloid42 polypeptide-aggregates).30 It is currently a focus of interest in terms of pharmacological intervention (see Gilbert2 and Wolfe29), in the hope that it may be possible to define new targets in cancers in which mutations of Notch contribute to the pathology (such
as T cell acute lymphoblastic leukaemia) or in which Notch acts to prevent progenitor cell differentiation (such as in intestinal adenomas).
Members of ADAM/ TACE cleave the juxtamembrane region of transmembrane proteins, causing shedding of
the ectodomains (for instance liberation of TNF- (Figure 16.3, page 490) or EGF Figure 12.25, page 356). The family comprises some 29 members. Of these ADAM10 and ADAM17 (TACE) are able to cleave Notch, whereas ADAM12 cleaves its ligand Delta.
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Signal Transduction
FIG 22.4 Activation of the Notch pathway.
Notch appears at the plasma membrane in a glycosylated (1) and cleaved (2) form. It binds its partner Sanpodo. Binding of ligand (Delta or Jagged) exposes the S2 site, leading to cleavage by ADAM17 (3). This renders Notch susceptible to cleavage at S3 by -secretase (4). Endocytosis of the receptor seems a prerequisite for this. The intracellular segment, Nicd, translocates to the nucleus where it binds CSL and causes the recruitment of mastermind (Mam). This leads to a loss of CSL-associated repressors, which are replaced by Mam-associated activators of transcription (5). Important target genes are members of the Enhancer of Split family, E(spl) in Drosophila, of which Hes and Hey are the equivalent families in mammals.
but the functional significance of the resulting Notch fragment has not been investigated. We limit our description to the signalling role of the S3 Notchintracellular domain (Notch-icd or Nicd) (Figure 22.4).
What exactly renders Notch sensitive to proteolytic cleavage following ligand binding remains unclear, but the removal of the inhibitory Lin-repeats, which mask the S2-cleavage site, seems to be a prerequisite. Why the truncated version then becomes susceptible to the action of -secretase also remains enigmatic. Curiously, the number of EGF repeats seems to determine the efficiency of proteolysis. Long chains are less efficient, and truncated versions, such as those that occur in the human oncogenes TAN1 (Notch1) and INT3 (Notch4), are constitutively active receptors.27 Both ADAM and -secretase are subject to regulation by post-translational modifications that affect their activity. In which subcellular compartment -secretase cuts Notch remains to be resolved. Although clearly evident in early endosomal vesicles, it does not necessarily follow that this is where it acts (see page 713).28
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Notch
Regulation of transcription by the Notch intracellular domain
The untethered intracellular segment of Notch (Nicd) migrates into the nucleus and binds to the transcription factor CSL (Figure 22.4). This interaction first involves the RAM region, which binds the -trefoil domain of CSL,
and then the ankyrin repeats, which bind the C-terminal Rel-homology region (Figure 22.5).31–33 Together, Nicd and CSL create a binding groove that accommodates the helical structure of Mam, which creates a platform for attachment of further components such as GCNS and the nucleosome acetylation factors CBP or p300. It is also involved in the recruitment of SKIP and, importantly, the cell cycle kinase CDK8 (see page 712).34 Numerous copies of these complexes form and they are readily visualized as distinct nuclear foci (Figure 22.5).
The Notch transactivation process generally resembles the mechanism of induction of transcription by the Wnt– -catenin pathway (see page 421). Thus, the Notch and Wnt target genes are normally repressed by the association
of corepressors with CSL or with LEF. The Notch transactivation mechanism, however, differs slightly in that translocation of Nicd requires both CSL and MAM in order to initiate transcription on chromatin templates in vitro,
whereas -catenin requires only LEF.34,35 Note that CSL may also repress basal transcription through direct binding to, and inhibition of, TFIIA and TFIID.36
Effector genes of Notch signalling
The Nicd-induced transcriptional complex leads to the expression of genes that are members of the E(spl) class in Drosophila (a complex of at least seven tandem genes) or the Hes class in mammals. (Hes and Hey are the mammalian counterparts of E(spl).) These are transcription factor genes, the so-called Notch-effector genes.37,38 In mouse and rat, seven hes (1–7) and three hey (1, 2, and L) genes have been identified. The Drosophila e(spl) and the hes and hey gene products belong to a large family of small proteins characterized by a helix–loop–helix (HLH) motif.39 Well-known members of this family of DNAbinding transcription factors are MyoD (differentiation of muscle tissue) and c-Myc (oncogene, regulation of expression of components of ribosomes).
The N-terminal DNA-binding basic domain of these proteins is contiguous with one of two -helices separated by a loop (helix–loop–helix) that serves as a dimerization domain and as a platform for additional protein interactions (Figure 22.6). This is followed by two additional -helical stretches making up the Orange domain, which also serves as an interface for protein interactions, including the formation of homoand heterodimers of HLH proteins. It
also acts as a transcriptional repressor. In Hes proteins, a highly conserved C-terminal tetrapeptide motif -WRPW- recruits the corepressors of the TLE family, of which Groucho (Drosophila) is the most familiar (see also page 422). The Hey proteins lack this motif and cannot bind Groucho, but their bHLH
Split and Enhancer of Split
Split is one of the non-lethal alleles of the notch locus. It is a partial loss-of-function mutant that affects formation of the bristles as well as the eyes. The macrochaetae, particularly the dorsocentrals and
the scutellars on the mesonotum of the thorax, appear split, doubled, or absent, and so do some of the microchaetae. The eyes
are of rough, of unvarying texture, and smaller
than the wild type.40 The partial loss of function is associated with an isoleucine to threonine
mutation in the 14th EGFlike repeat.
Enhancer of Split exaggerates the effects of Split, for the worse. In genetic terms, Split and Enhancer of Split are said to interact.
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Signal Transduction
FIG 22.5 The Notch transcriptional complex.
(a) The intracellular Notch segment, Nicd, first binds the -trefoil domain of CSL and then folds over to attach, by its ankyrin repeats, to the C-terminal domain. (b) This creates a binding site for Mam and a trimeric complex is formed. The C-terminal domain of Mam recruits numerous proteins that cause acetylation of histones, so rendering the DNA amenable for transcription. Among the recruited molecules is CDK8, which later phosphorylates Nicd in both its TAD and PEST regions. Phosphorylated PEST is recognized by the receptor subunit of a nuclear E3-ligase complex, resulting in polyubiquitylation of Nicd, followed by proteasome-mediated destruction. (c) Numerous transcriptional complexes thus formed are readily visible by immunostaining of p300 (d) and
(e) Histone acetylation unwinds DNA and transcription follows removal of repressors and recruitment of transcriptional activators.Image (c) from Fryer et al.34
(basic helix–loop–helix) motif interacts with yet another histone deacetylase, SIRT1. The corepressors and associated histone deacetylases render the DNA inaccessible to RNA polymerase and thus silence gene expression. In some cases Hes proteins bind CBP and act as transcription factors. Apart from binding DNA and recruiting transcriptional repressors or activators, bHLH proteins have other means of interfering with transcription. For instance, they may sequester other DNA-binding proteins away from the DNA, or they may bind other transcription factors, thus bringing gene silencing repressors to promoter regions where they themselves do not bind. For more information about Hes and Hey proteins, see Fischer and Gessler.38
The response to Notch is not fixed by expression of a set of transcription factors that endow certain phenotypes; rather, it acts as a switch with the outcome of its action being determined by the context in which it operates (cell type, developmental stage, other first messengers). The situation is similar to that described for Wnt in Chapter 14. In this way it is applied in cell
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