Signal Transduction
gene complexes include the antennapedia, bithorax, and achaete/scute (AS) complexes. The hallmark of all these gene complexes is that within any complex the genes are evolutionarily related and jointly regulated. Genes of the E(spl) and the AS complexes regulate neurogenesis and related differentiation pathways. Whereas transcription factors encoded by the AS complex generally activate transcription of other genes, gene products of the E(spl) complex generally repress transcription. Proteins encoded by AS are proneural, initiating neurogenesis, whereas E(spl)-coded proteins prevent neurogenesis. The E(spl) complex contains three additional Notch-responsive, non-bHLH genes: of these, m4 and m are structurally related, while m2 encodes a novel protein. For the most part, the genes of the E(spl) complex are redundant; only the nominally defining gene of the complex, Enhancer of Split, yields a noticeable phenotype when mutated. For further information, see Simpson.11
Destruction of the Notch-icd, Nicd
The content of Nicd in the nucleus is vanishingly small because of its rapid degradation. In fact, the assembly of a transcriptionally active protein complex is paralleled by an almost immediate phosphorylation of Nicd and subsequent recognition by a nuclear E3-ubiquitin ligase so that it signals its own demise. Recruitment of CDK8 leads to phosphorylation of Nicd
in the transactivation and PEST domain (Figure 22.5). The phosphorylated protein is recognized by the receptor-subunit Fbw7/Sel10 (a WD40 F-box receptor protein) of a nuclear SCF-type E3-ubiquitin ligase complex and the polyubiquitylated Nicd is destroyed by the 26S proteasome.41,42 If signalling is to be maintained, a continuous supply of Nicd must be provided through ligand-mediated cleavage of the intact membrane protein.
The importance of a continuous supply is well illustrated by the observation that severe suppression of Delta expression in the satellite cells of ageing livers leads to impaired regenerative capacity; due to a failure of Notch signalling, the cells can no longer escape terminal differentiation.43 Conversely, loss of the PEST domain renders the protein very stable and this contributes to the development of acute lymphoblastic leukaemia (an excess of undifferentiated cells).15
Both receptor and ligand trafficking are essential for Notch signalling
One might expect that signal-transmitting cells would exhibit high levels of ligands on their surface so as to activate Notch on adjacent receiving cells. Curiously, in Drosophila, most of the Delta is confined to intracellular vesicles and the membrane pool is constantly being removed. Moreover,
only ubiquitylated ligands, linked to clathrin-associated sorting proteins, are competent to activate Notch.
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Compelling evidence for a role of trafficking came from the finding that shibire (shi), a temperature-sensitive mutant of dynamin, causes a Notch-like phenotype in Drosophila. At the restrictive temperature (29°C), Shibire mutants develop an excess of nervous system at the expense of ventral epidermis.44
Notch signalling is often required to prevent the default neurogenic phenotype (thus to obtain an epidermal cell instead). Therefore, it follows that loss of function of dynamin prevents effective Notch signalling, (even though all the related components, receptors, and ligands, etc., are normally expressed). Dynamin is a GTP-binding protein necessary for the pinching-off (fission) of endocytotic vesicles from the plasma membrane.45 Cells that express shibire still form clathrin-coated pits but fail to internalize these vesicles. This correlation prompted the question of whether a block in endocytosis could account for the interruption in the communication necessary for normal epidermal and neural cell differentiation? The answer is yes; trafficking of both ligands and receptor affect signalling, though the full picture still remains unclear.
Trafficking of ligand (Delta, Serrate)
Further important evidence for the role of endocytosis in the regulation of Notch signalling came from genetic screens in Drosophila and zebrafish (Danio rerio). These revealed three key regulators: epsin-1, neur, and mib
(Table 22.1). Epsin is a long, almost linear protein that acts as an adaptor for endocytosis, coupling polyubiquitylated protein to -adaptin (AP-2).46 Neur and mib are E3-ubiquitin ligases that ubiquitylate Delta and Serrate.47 Loss of Neur in Drosophila and Xenopus laevis and of mib in zebrafish results in neurogenic phenotypes resembling those of Notch mutants.48 Two schemes have been proposed to explain why endocytosis is necessary for proper
signalling. The first postulates that uptake of ligand, when bound to the Notch receptor, pulls away the three Lin repeats that normally mask the S2 cleavage site. This ‘pulling’ renders S2 accessible to ADAM/TACE (see Figures 22.3 and 22.7).
The other idea proposes that ligand uptake leads to glycosylation of the EGFlike repeats in the early endosome compartment. This recycling pathway not only renders the ligand competent for receptor binding but may also deliver the ligand to the correct membrane domain in a ‘clustered’ manner (Figure 22.7). This does not excluded a third possibility, that trafficking of Delta and Serrate, after binding to Notch, is required for signalling within the ligand-presenting sending cell, in a similar way as for Notch in the ligand-receiving cell.49
Trafficking of receptor (Notch)
Ubiquitylation and the subsequent endocytosis of Notch has both stimulatory and inhibitory consequences. The inhibitory aspect is rather straightforward; it concerns removal of the unoccupied receptor from the plasma membrane, thereby preventing interaction with ligand. Two ubiquitin E3 ligases stand out in the regulation of endocytosis; Nedd4 and Itch (both HECT-type ligases: see page 469) (Figure 22.8 and Table 22.1). Ubiquitylation conveys Notch into
Epsin is described as a ‘natively disordered’ polypeptide, meaning that apart from the
N-terminal region, it has few structural features.50 It contains three ubiquitin-interacting motifs (UIM) which, however, bind weakly to ubiquitin. Tight binding therefore requires several interactions, and this may explain why polyubiquitylated proteins are favoured.
It connects to PI(4,5)P2 and it carries several AP-2 binding motifs that bind-adaptin. Epsin also interacts with RhoGAP, suggesting that it has a role in actin dynamics, possibly explaining
why it is an important component of the endocytotic machinery (see Figure 15.15, page 471).46,51,52
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Signal Transduction
FIG 22.7 Endocytosis of the Delta ligand.
(a) Delta is ubiquitylated by the E3-ligase neur. The polyubiquitylation chain is recognized by the UIM of epsin which links Delta to the adaptor complex AP-2, leading to its endocytosis with the help of the coat protein clathrin. (b) Endocytosis may facilitate Notch signalling in a number of ways. It may be re-expressed at the membrane with important cofactors and in a clustered manner, which may enhance Notch binding and activation. It may also undergo further glycosylation (symbolized as a blue asterisk). (c) Endocytosis may facilitate unmasking of the S2-cleavage site by withdrawing the Lin-12 repeats. Finally, endocytosis may be needed for the effective recycling of ligand after removal of the extracellular Notch segment.
Itch is a gene present in mice that continually scratch their skin. This behaviour is due to aberrant inflammatory responses in several organs, including the skin.53
an early endosome, identified by the colocalization of the small GTPase Rab5, and, if no further modifications occur, this is followed by its destruction in the lysosomal compartment (recognized by association with Rab7).
Why Notch is stimulated by ubiquitylation and endocytosis is less clear. One possibility is based on the finding that the catalytic component of -secretase (presenilin) predominantly resides in the early and late endosomes, and this might facilitate cleavage of the S2 site18 (Figure 22.9). Another possibility is that a second round of ubiquitylation, in the early endosomal compartment, rescues
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Notch from lysosomal destruction, so opening the way for an alternative, non-canonical, signalling pathway. This second round of ubiquitylation occurs through Deltex (a RING-finger type E3 ligase), which binds the ankyrin repeat region.54 Deltex-mediated ubiquitylation directs Notch towards a recycling endosome, identified by the colocalization of the small GTPase Rab11. From here, Notch either returns to the membrane, possibly modified, and contributes to ligand-mediated signalling or it remains in the cell and signals, by an as yet poorly characterized pathway, in a ligand-independent manner.55,56 Note that the E3–ubiquitin ligase Cbl, which regulates EGF receptor uptake (see Figure 12.21, page 350) is also associated with Notch receptor uptake and could also play a role in its trafficking. The domain architectures of proteins that take part in these processes are shown in Figure 22.10.
FIG 22.8 Endocytosis of the Notch receptor.
Notch, polyubiquitylated by Itch (Su(Dx) in Drosophila) or NEDD4. The ubiquitins are recognized by Epsin and this connects Notch with the adaptor protein complex AP-2. Endocytosis occurs with the help of the coat protein clathrin.
Rab GTPases: These are members of the Ras superfamily of monomeric GTPases involved in intracellular vesicle transport. They
regulate the assembly, on the surface of emerging transport vesicles, of (1) coat proteins, necessary for cargo selection and vesicle budding, (2) microtubule binding proteins, necessary
for transport, and (3) SNARE and docking proteins, necessary for fusion of the released vesicle with other subcellular membrane compartments.
AP-2: A complex made up of four subunits (two large, - and 2-adaptin, and two small, 2-and2-adaptin), of which-adaptin has the role of cargo receptor. The
‘ear’ domain of -adaptin interacts with accessory proteins, such as Epsin or numb, and coat proteins, such as clathrin.
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