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Bray SJ.. Notch signaling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678-689

October 2006
Nature Reviews Molecular Cell Biology 7(9):678-89

DOI:10.1038/nrm2009

Source
PubMed

Authors:

University of Cambridge

A small number of signalling pathways are used iteratively to regulate cell fates, cell proliferation and cell death in development. Notch is the receptor in one such pathway, and is unusual in that most of its ligands are also transmembrane proteins; therefore signalling is restricted to neighbouring cells. Although the intracellular transduction of the Notch signal is remarkably simple, with no secondary messengers, this pathway functions in an enormous diversity of developmental processes and its dysfunction is implicated in many cancers.

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Notch signalling has a simple framework that is highly

conserved throughout the animal kingdom

1–3

(FIG. 1).

Both the Notch receptor and its ligands,

Delta and Serrate

(known as Jagged in mammals), are transmembrane

proteins with large extracellular domains that consist

primarily of epidermal growth factor (EGF)-like repeats

(BOX 1). Ligand binding promotes two proteolytic cleavage

events in the Notch receptor

(FIG. 1). The first cleavage

is catalysed by

ADAM-family metalloproteases, whereas the

second is mediated by

γ-secretase, an enzyme complex

that contains presenilin, nicastrin,

PEN2 and APH1

(REFS 4–6). The second cleavage releases the Notch intra-

cellular domain (Nicd), which then translocates to the

nucleus and cooperates with the DNA-binding protein

CSL (named after

CBF1, Su(H) and LAG-1) and its co-

activator

Mastermind (Mam) to promote transcription

(FIG. 1; BOX 1). The precise numbers of Notch paralogues

differ between species

(TABLE 1) — for example, there are

four Notch receptors in mammals (

Notch1–4), two in

Caenorhabditis elegans (

LIN-12 and GLP-1) and one

in Drosophila melanogaster (

Notch) — but the basic para-

digm is common throughout

1–3

The Notch pathway functions during diverse

developmental and physiological processes, which can

broadly be subdivided into three categories

(BOX 2). The

first functions of Notch to be well characterized were

those affecting neurogenesis in flies and vertebrates

From these studies it became evident that Notch acts at

different stages of development even within one tissue.

For example, Notch first regulates the number of cells

that acquire neural potential (lateral inhibition;

BOX 2),

and subsequently it determines whether progeny will

adopt neural or glial fates

(lineage decisions; BOX 2).

Notch inhibits neural differentiation in many lineages,

but recently Notch was shown to promote neural fates in

mouse and human embryonic stem cells,

underscoring

the importance of the cellular context in the determi-

nation of the outcome of signalling. Iterative activation

of Notch has now been detected in multiple lineages.

A recent example is the midgut progenitor cells in both

mammals and D. melanogaster, in which Notch main-

tains proliferating progenitor cells and regulates binary

cell-fate decisions in the stem cell progeny

9–12

. In addition

to the ever-increasing examples of biologically impor-

tant roles for the Notch pathway during development,

Notch activity also emerges as a contributory factor to

many cancers

. For example, mutations in Notch1 were

detected in more than 50% of T-cell acute lymphoblastic

leukaemias

The basic core Notch-transduction pathway is the

same in most Notch-dependent processes. However,

the mechanisms that regulate the pathway are different.

Although it is still unclear how the interaction between

a ligand and the Notch extracellular domain (ECD)

results in the activation of the receptor, many factors

are emerging that influence whether or not a produc-

tive ligand–receptor interaction occurs, including the

precise location of the receptor in the cell. This review

aims to summarize our current understanding of the

mechanisms that function on the core Notch pathway

(CSL-independent signalling and crosstalk with other

pathways are beyond the scope of this article). One

challenging question is, what determines in which cells

the ligands and the receptor are active? Often, dramatic

differences in signalling and signal reception between

cells do not correlate with obvious differences in the

expression levels of the ligands or the receptor. It has

recently become evident that post-translational modifi-

cations and trafficking of the Notch ligands and receptor

affect the activation of the pathway. Another important

Department of Physiology,

Development and

Neuroscience, University of

Cambridge, Downing Street,

Cambridge CB2 3DY, UK.

e-mail: sjb32@cam.ac.uk

doi:10.1038/nrm2009

ADAM-family

metalloproteases

Transmembrane disintegrins

and metalloproteases that

proteolytically cleave the

juxtamembrane region of

cellular transmembrane

proteins and detach their

extracellular regions — this

process is known as

ectodomain shedding.

γ-secretase complex

Presenilin, a multispan

membrane protein, is the

catalytic subunit, and the

transmembrane proteins

nicastrin and APH1 stabilize

the presenilin holoprotein.

PEN2, a two-pass

transmembrane protein,

induces endoproteolysis of

presenilin and maturation of

the γ-secretase complex.

Notch signalling: a simple pathway

becomes complex

Sarah J. Bray

Abstract | A small number of signalling pathways are used iteratively to regulate cell fates,

cell proliferation and cell death in development. Notch is the receptor in one such

pathway, and is unusual in that most of its ligands are also transmembrane proteins;

therefore signalling is restricted to neighbouring cells. Although the intracellular

transduction of the Notch signal is remarkably simple, with no secondary messengers, this

pathway functions in an enormous diversity of developmental processes and its

dysfunction is implicated in many cancers.

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Delta

Notch

ADAM10 or

TACE

(S2 cleavage)

γ-secretase

(S3 cleavage)

Nicd

CSL

Target genes

repressed

Target genes

active

Co-R

Mam

E3 ubiquitin ligase

An adaptor protein that links

ubiquitin-conjugating E2

enzymes with substrates and

contributes to the catalytic

transfer of ubiquitin onto the

substrate.

Epsin

A clathrin and phosphatidyli-

nositol-4,5-bisphosphate-

binding protein that contains

ubiquitin-interaction motifs. It

is thought to facilitate

endocytosis of ubiquitylated

cargo proteins.

Auxilin

A J-domain-containing protein

that is implicated in the

disassembly of clathrin from

clathrin-coated vesicles.

Recycling endosome

A compartment that sorts

transmembrane proteins that

are recycled to the plasma

membrane following

endocytosis.

Exocyst

A heteromeric protein complex

that is required for polarized

exocytosis of post-Golgi

secretory vesicles.

question is, what happens once Nicd enters the nucleus?

Both the duration of signalling and the identity of target

genes have impacts on the output of Notch activation,

so their regulation is of major importance. Together, the

different mechanisms give a perspective on how this

simple pathway can be manipulated, but they also show

that we are still just beginning to understand the full

complexities of Notch regulation.

Regulation of Notch-ligand activity

Expression of Notch ligands during development is quite

dynamic and contributes significantly to differential

activity of the pathway. In some developmental con-

texts, the ligand is produced by a distinct population of

cells (boundaries/inductive signalling;

BOX 2). However,

under many circumstances, differential ligand transcrip-

tion is not sufficient to explain why certain cells become

the signal-sending cells. Other post-transcriptional

mechanisms are clearly at work.

Ubiquitylation and ligand activity. The identification

of the

E3 ubiquitin ligases, Neuralized (Neur) and Mind

bomb (Mib), that interact directly with Notch ligands

and are required for ligand activation

(FIG. 2) was a strik-

ing and surprising recent discovery

14,15

. Loss of Neur in

D. melanogaster or Xenopus laevis and of Mib1 in zebrafish

results in neurogenic phenotypes

16–19

. In D. melanogaster,

mib1 mutants have a later defect of arrested appendage

(imaginal disc) development (possibly due to persistence

of maternal protein or redundancy with MIB2). The

MIB1-associated defects can be rescued by expression of

Neur, which indicates that these two proteins — although

they share few structural similarities apart from RING

domains — can perform the same function

20–22

. Much

of the difference between these two E3 families might be

attributed to their expression patterns and to their regula-

tion (see below), although it remains possible that they

preferentially interact with different Notch ligands.

In normal cells, the extensive trafficking of Notch lig-

ands through the cell is evident from intracellular puncta

that are detected in different tissues and animals. This traf-

ficking is compromised in the absence of Neur or Mib, as

ligands accumulate at the cell surface but are inactive

18,21

This surprising observation indicates that regulation of

ligand activity by Neur and Mib is intimately associated

with endocytosis

(FIG. 2) and it requires the ubiquitin-

binding protein

Epsin

23–25

and probably the J-domain-

containing protein

auxilin (which can disassemble

clathrin coats)

Different models have been proposed to explain the

link between ubiquitylation, endocytosis and ligand

activity

14,15,23

. For example, ligand endocytosis could

generate a ‘pulling force’ on a bound receptor that causes

a conformational change in the juxtamembrane region

Another possibility is that ubiquitylation promotes ligand

clustering. Indeed, Notch activation is more effective

if soluble ligands are clustered through fusion to an

Fc moiety or through immobilization on plastic

28,29

A third possibility is that ubiquitylation permits traffick-

ing into an endocytic compartment, which enables ligand

modification or results in re-insertion of the ligand into

specific membrane domains. Two observations support

this model. Segregation of RAB11, a component of the

recycling endosome, influences signalling in the D. mela-

nogaster sensory organ precursors (SOP). Furthermore,

mutations in an

exocyst component, SEC15, compromise

SOP Notch signalling

30,31

Paradoxically, some functional ligands in C. elegans

are secreted (for example

DSL-1; REF. 32) and so

would presumably not be ubiquitylated. However, the

ubiquitin-binding protein Epsin is also required for

Notch (LIN-12) signalling activity in this animal,

implying that mechanisms of ligand activation are

conserved

. Whatever the mechanism for ligand activa-

tion, regulation of E3 ligases is potentially one signifi-

cant strategy for controlling the activity of the Notch

pathway, as exemplified by the

Bearded-related family

of small inhibitory polypeptides

34,35

(BOX 3). Therefore,

elucidating the mechanism of ligand activation is of

prime importance.

Ligand localization. The localization of ligands within

the cell is important for effective signalling and might

be influenced by other proteins. For example, Echinoid,

an immunoglobulin C2-type cell-adhesion molecule,

colocalizes with Notch and Delta at

adherens junctions in

D. melanogaster.

Genetic interactions indicate that Echinoid functions

as a positive regulator to promote Notch signalling

Echinoid colocalizes with Delta in endocytic vesicles,

and Echinoid overexpression depletes Delta from

the membrane. Therefore, it is possible that Echinoid

Figure 1 | The core Notch pathway. Binding of the Delta ligand (green) on one cell to

the Notch receptor (purple) on another cell results in two proteolytic cleavages of the

receptor. The ADAM10 or TACE (TNF-α-converting enzyme; also known as ADAM17)

metalloprotease (yellow) catalyses the S2 cleavage, generating a substrate for S3

cleavage by the γ-secretase complex (brown). This proteolytic processing mediates

release of the Notch intracellular domain (Nicd), which enters the nucleus and interacts

with the DNA-binding CSL (CBF1, Su(H) and LAG-1) protein (orange). The co-activator

Mastermind (Mam; green) and other transcription factors (see also

FIG. 4) are recruited to

the CSL complex, whereas co-repressors (Co-R; blue and grey) are released.

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RHR-N

CSL

BTD

RHR-C

Mam

Nicd

CSL–Nicd–Mam

complex

DNA

Notch

Delta/Dll1/Dll5 Jagged 1/2

RAM

Ank

LINEGF repeats

EGF

repeatsDSL EGF repeats CRDSL

PEST

Bearded-related proteins

Short polypeptides that were

identified in insects and

contain an N-terminal

amphipathic helix and 2 or 3

conserved motifs.

Adherens junction

A cell–cell junction that

mediates adhesion through

cadherins and regulates and/or

links to the actin cytoskeleton.

PDZ-binding motif

A motif at the C terminus of a

protein that is recognized by a

PDZ-domain-containing

protein. PDZ-domains are

conserved 80–90-residue

domains that fold into

β-sandwich

and are found in

many scaffold and signalling

proteins.

promotes endocytic activation of Delta. Alternatively,

Echinoid-mediated adhesion could favour Notch–Delta

interactions. Consistent with this notion, it has been

shown that altered cytoarchitecture of cells can affect

their signalling potential

. Furthermore, the intracellular

domains of some Notch ligands contain protein–protein

interaction motifs (for example,

PDZ-binding motifs)

that can bind to intracellular scaffolding proteins

38–40

Deletion of the cytoplasmic PDZ-binding motif in Delta

had minimal effects on most functions of Notch signal-

ling

. Nevertheless, some subtle neuronal defects were

observed

. So, sequences within the ligand intracellular

domain might modulate activity, affect localization or

mediate an independent reverse-signalling activity.

Ligand processing and soluble ligands. Structurally, the

ligands share many characteristics with Notch itself and

are prone to similar modifications (see below) includ-

ing proteolytic processing

41,42

. However, the purpose

of ligand cleavage remains unclear. One suggestion is

that proteolytic processing of the ligand contributes to

ligand downregulation

. For example, loss of the metallo-

protease Kuzbanian-like, which has been shown to cleave

Delta, results in ectopic Notch signalling in certain loca-

tions

. Another suggestion is that cleaved or secreted

ligands antagonize Notch signalling, because, under

most circumstances, soluble ligand fragments inhibit

receptor signalling

28,43,45

. However, several members of

the Notch-ligand family in C. elegans have no transmem-

brane domains, but their expression can rescue worms

that lack more conventional ligands, indicating that the

secreted ligands retain signalling potential

. It is also

possible that cleavage of transmembrane ligands could

transmit an intracellular signal through activities that are

associated with the ligand’s intracellular domains — that

is, reverse signalling. Further investigations are needed

to identify all of the functional consequences of ligand

proteolysis on Notch signalling in vivo.

Tuning of Notch-receptor activation

Notch receptors have broad expression patterns in many

tissues, but analyses of where cleavage occurs or where

target genes are expressed reveal a limited profile of activ-

ation. Furthermore the ability to respond to a specific

ligand is spatially regulated in the chick inner ear or the

D. melanogaster wing

46,47

. These observations indicate

that the activity of the receptor must also be regulated

through post-transcriptional mechanisms

(FIG. 3).

Role of glycosylation. Notch proteins have a large ECD

that consists of multiple EGF-like repeats, which are sites

for glycosylation

. The enzyme O-fucosyl transferase

(O-Fut) adds the first fucose and is essential for the gen-

eration of a functional receptor

49–51

. Depletion of O-Fut

in D. melanogaster and mice results in phenotypes that

resemble those associated with lack-of-Notch signalling

Box 1 | The Notch-pathway players

DSL ligands

Notch ligands (a) are transmembrane proteins that are

characterized by an N-terminal DSL (Delta, Serrate and

LAG-2) domain that is essential for interactions with the

Notch receptor. The extracellular domains of the ligands

contain varying numbers of epidermal growth factor (EGF)-

repeats. The ligands are subdivided into two classes, Delta

or Delta-like (Dll) and Serrate (Jagged in mammals),

depending on the presence or absence of a cysteine rich

(CR) domain.

Notch receptors

The mature Notch receptor (b) is produced through a furin

cleavage during biosynthesis (see

FIG. 3). Notch

extracellular domains contain 29–36 EGF repeats, 3

cysteine rich LIN repeats and a region that links to the

transmembrane and intracellular fragment. This linker

region is important in preventing premature activation of

the receptor and is altered in 26% of activating mutations

that are associated with T-cell acute lymphoblastic

leukaemia

. EGF-repeats 11 and 12 (orange) are essential

for ligand binding. The intracellular portion consists of a RAM domain, six ankyrin (Ank) repeats and a C-terminal PEST

domain. It also contains nuclear localization signals. Individual types of Notch receptor have additional protein–protein

interaction motifs.

Nuclear effectors

The key transducer of the Notch-signalling pathway is a DNA-binding protein, CSL (CBF1, Su(H) and LAG-1) (c). CSL is

similar to the Rel family of transcription factors. However, CSL differs from Rel in the insertion of a central modified

β-trefoil domain (BTD) between the two Rel-homology regions (RHR-N, RHR-C)

. DNA contacts are predominantly made

through the RHR-N and BTD domains. The BTD domain contains a hydrophobic pocket that is thought to mediate the

interaction with the Notch intracellular domain (Nicd). To activate transcription, the co-activator Mastermind (Mam) is

required. Mam proteins from different species share little sequence homology apart from an N-terminal region that forms

an extended α-helical domain that contacts the RHR-N and RHR-C domains of CSL and the Ank domain of Nicd in a

trimeric complex

94,95

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Fringe

A Golgi-resident glycosyl-

transferase that was first

identified in D. melanogaster

and has three homologues in

mammals: Lunatic Fringe,

Radical Fringe and Manic

Fringe.

(for example, presenilin mutants). Not only is the

enzymatic activity important, O-Fut also functions as a

chaperone to promote the folding and transport of Notch

from the endoplasmic reticulum to the cell membrane

O-Fut that has lost the capability to glycosylate, because

of mutations in the active site, retains the capability to

chaperone Notch proteins to the cell-surface. This find-

ing shows that the chaperone function of O-Fut is not a

secondary effect of glycosylation.

It is possible that O-Fut might contribute to the spatial

regulation of Notch activity, as its expression pattern

is not uniform

49–51

. Furthermore, a Notch mutation

that introduces a fucosylation site into EGF-repeat 14

results in ectopic receptor activity in a subset of neural

cells in D. melanogaster, which indicates that differences

in primary fucosylation could contribute to differential

activity of the receptor in different cell types

After the addition of the first fucose, the carbohydrate

chains can subsequently be extended by other glycosyl

transferases, such as those of the

Fringe family

. Multiple

EGF repeats in Notch have the potential to be modified

and, therefore, a large repertoire of differentially modi-

fied receptors could be generated. It has been shown

that these glycosyl-modifications alter the capability

of ligands to activate Notch. For example, in dorsal cells of

the D. melanogaster wing,

Fringe potentiates activation

by Delta and renders Notch resistant to activation by

Serrate

Using soluble ligand or receptor fragments these

differences can be correlated with effects on binding

affinities in cell-culture assays

54,55

. For example, Serrate

binds with higher affinity to Notch fragments that have

been fucosylated and with lower affinity to fragments

that have been further modified by Fringe

. Mutation

Table 1 | Notch-pathway components and auxiliary factors in different species

Component type Drosophila melanogaster Vertebrates and mammals Caenorhabditis

elegans

Receptor Notch Notch1–4 LIN-12, GLP-1

Ligand Delta, Serrate Delta1–4/A–D, Serrate,

Jagged1–2

APX-1, LAG-2, ARG-1,

DSL-1

CSL DNA-binding protein Su(H) CBF1/RBPkJ LAG-1

Co-activator Mastermind Mastermind1–3 LAG-3

Co-repressor Hairless, SMRTR* SMRT

γ-secretase complex

Presenilin, nicastrin,

APH1, PEN2

Presenilin1–2, nicastrin,

APH1, PEN2

SEL-12/presenilin,

APH-2/nicastrin,

APH-1, PEN-2

Glycosyl transferase Fringe Lunatic Fringe, Radical Fringe,

Manic Fringe

Metalloprotease,

receptor cleavage

Kuzbanian,

Tace CG7908*

ADAM10, TACE/ADAM17 SUP-17/Kuzbanian,

ADM-4/TACE

Metalloprotease,

receptor cleavage

Kuzbanian-like

Ring finger E3

(ligand regulation)

Mind bomb 1 Mind bomb 1–2

Ring finger E3

(ligand regulation)

Neuralized Neuralized1–2 F10D7.5*

Ring finger E3

(receptor regulation)

Deltex Deltex

HECT domain E3

(receptor regulation)

Su(dx), NEDD4 Itch, NEDD4* WWP-1

F-box E3 (nuclear) Archipelago* FBW7/SEL10 SEL-10

Numb, cytoplasmic Notch

inhibitor

Numb Numb, Numb-like

Numb-associated kinase Numb-associated kinase AP2-associated kinase SEL-5

4-pass transmembrane

protein, positive regulator

Sanpodo

Immunoglobulin C2-type

cell-adhesion molecule

Echinoid IGMC-1*

bHLH repessors, target

genes

E(spl)bHLH HES/ESR/HEY REF-1

Neuralized E3 inhibitors Bearded, Tom, M4

*Protein homologues that have not yet been tested for roles in Notch signalling. Where no protein is listed, relatives have not been

identified. AP2, adaptor protein-2; CSL, CBF1, Su(H) and LAG-1; E3, ubiquitin ligase; bHLH, basic-helix–loop–helix.

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Ommatidium

Stem cell lineage

SOP lineage

Boundary

Niche

Support/

stromal cell

Proneural cluster

Equivalent cells Distinct cell fates

of a glycosylation site in EGF-repeat 12, a crucial repeat

for ligand binding, allows activation of Notch by Serrate

even in the presence of Fringe, which indicates that this

is a key site for modification

. The results are therefore

most simply reconciled with a model in which Fringe

glycosylation affects binding affinities between ligands

and specific EGF-repeats. Under some conditions,

Jagged still binds to Notch in the presence of Fringe,

although the receptor is not activated, raising the pos-

sibility that the stability and/or the duration of inter-

actions are important

. Furthermore, Lunatic Fringe,

a mammalian homologue of Fringe, potentiates Delta

binding in in vitro studies and promotes Notch activity

at the somite clefts, but behaves as a Notch inhibitor

within the Delta-driven oscillatory somite clock in

some species

59,60

. These observations indicate that gly-

cosylation patterns might do more than producing an

all-or-none effect on different ligands.

Proteolytic cleavage of Notch. The discovery that

Notch activation entails proteolytic cleavage (S3 cleav-

age) that is mediated by γ-secretase was an important

breakthrough

4–6

(FIG. 1). However, this finding raised

some perplexing questions, such as where in the cell

does S3 cleavage occur and what renders Notch into a

substrate?

Truncation of the Notch ECD stimulates S3 cleavage,

and it seems that the efficiency of cleavage correlates

with the length of ECD

. This explains the constitu-

tive activity that is observed in the human TAN1 (also

known as Notch1) and INT3 (also known as Notch4)

oncoproteins for which chromosomal rearrangements

have truncated the ECD

3,6

. In vivo, the S3 cleavage occurs

in response to a prior (S2) cleavage that is mediated by

ADAM metalloproteases within the ECD, and is elicited

by productive ligand binding

6,62–64

. Two metalloproteases

have been implicated in the S2 cleavage,

ADAM10

(also known as Kuzbanian; Kuz) and tumour-necrosis

factor-α (TNFα)-converting enzyme (TACE; also

known as

ADAM17), and evidence indicates that these

have partially redundant roles

63–65

. The S2 protease cleav-

age remains an important aspect for investigation, par-

ticularly because studies of metalloproteases reveal the

potential for regulation by external factors, membrane

environment and intracellular signalling pathways

There is also potential for regulation of γ-secretase.

Ubiquitylation on a juxtamembrane lysine residue was

necessary for S3 cleavage in mammalian cell assays, indi-

cating that cleavage occurred after Notch endocytosis

Another recent study proposed that the transmembrane

protein Crumbs, a regulator of epithelial polarity, medi-

ates a negative feedback on γ-secretase activity

. Further

studies are needed to determine the importance and

diversity of γ-secretase regulation in vivo. However, pre-

senilin is already being investigated as an important target

for drug interventions — presenilin-mediated cleavage of

amyloid precursor protein is associated with the accumu-

lation of plaques in

Alzheimer’s disease. Drugs are now

being tested in clinical trials for selected types of cancer

in which mutations in Notch contribute to the pathology,

including T-cell acute lymphoblastic leukaemias

Box 2 | Different modes of Notch action

Lateral inhibition

Notch signalling amplifies small or weak differences within roughly equivalent

populations of cells. The diagrams (

a) represent Notch signalling (black arrows) in

ommatidia (upper) and neural preclusters (lower) that resolves equivalent (purple)

cells into distinct fates (blue and pink; cells with the highest Notch activity are

coloured pink). A confocal image shows Notch activity (E(spl) mδ0.5 expression; pink)

in developing ommatidia of a fly eye (the green staining marks cell membranes and

the thin peripheral cells demarcate each ommatidium). At early stages, mδ0.5

expression is sometimes detected in both posterior photoreceptors (see double

arrow) before signalling is refined, and then it is only detected in one (R4) cell (see

single arrows).

Lineage decisions

Notch signalling between two daughter cells is dependent on asymmetrical

inheritance of Notch regulators (for example, Numb). Diagrams (

b) illustrate

segregation of regulators (green) in progeny from a hypothetical stem cell lineage

(upper) and the Drosophila melanogaster sensory organ precursors (SOP) lineage

(lower). Thin black arrows indicate the direction of Notch signalling, pink cells acquire

highest Notch activation. The confocal image shows Numb distribution (green, by

Partner of Numb (PON)-GFP) in SOP lineages (nuclei are pink). Confocal bottom row

left; A Numb crescent is evident prior to division. Confocal bottom row right; Numb is

present in one of two daughter cells. Image courtesy F. Wirtz-Peitz and J. Knoblich,

both at the University of Vienna, Austria.

Boundaries/inductive

Notch signalling occurs between two populations of cells and can establish an

organizer and/or segregate the two groups. Diagrams (

c) of signalling at a boundary

(upper) or between stromal and progenitor cells (lower). Black arrows indicate the

direction of Notch signalling, pink cells have Notch activation. The confocal image is

of the fly wing primordium, in which Notch activity, as measured by Wg expression

(pink), is detected at the boundary of Serrate-expressing cells (green).

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Degradation Degradation

Epsin

Notch ligands

Active ligandInactive ligand

Neur or Mib

Ubiquitin donor (E2)

RAB GTPases

Members of the Ras

superfamily of small GTPases,

RAB proteins regulate vesicle

budding, fusion and motility.

HRS/VPS27

A protein that contains

ubiquitin-interaction motifs

and is important for sorting

ubiquitylated endosomal

cargoes.

Syntaxin I

Integral membrane protein

with sequence similarity to

t-SNAREs that is involved in

vesicle docking and vesicle

fusion.

β-arrestin

Also known as non-visual

arrestin, it is a cytoplasmic

protein that promotes

endocytosis of G-protein-

coupled receptors and is

present in coated vesicles.

ESCRT

(Endosomal sorting complex

required for transport). Three

heteromeric protein

complexes, ESCRTI, ESCRTII

and ESCRTIII, function

sequentially in the sorting of

membrane proteins into the

multivesicular body.

Notch endocytosis and trafficking. Notch is a cell-

surface receptor, so its expected location is the plasma

membrane. However, a substantial amount of Notch is

targeted for degradation and a large fraction of Notch

is detected in the cytoplasm in compartments of the

endocytic pathway. Studies in D. melanogaster have

shown that Notch colocalizes with the small

RAB

GTPases

RAB5 and RAB7, which are both markers of

the endocytic pathway. Moreover, Notch accumulates in

intracellular structures when the endocytic progression is

perturbed

70,71

. Mutations in several endocytic components

in D. melanogaster (for example,

HRS/VPS27, Syntaxin I

and

β-arrestin) result in elevated Notch protein levels,

without affecting the activity of the Notch pathway

71–73

By contrast, other mutations that compromise sorting of

ubiquitylated membrane proteins (most notably muta-

tions in the

ESCRT components VPS25 and TSG101 (also

known as VPS23)), result in dramatic hyperplasia that is

due to overactivation of the Notch pathway

74–76

But why does a block in one step in trafficking have

such profound effects on activity, whereas a block in

a different step does not? This is a puzzling question,

especially because mutations in vps25 and tsg101/vps23

perturb a later step than those in syntaxin I and hrs. One

suggestion is that Notch colocalizes with ligands and/

or γ-secretase only in certain compartments. Another

possibility is that the physiological composition of cer-

tain endocytic compartments favours ectodomain shed-

ding, a step that mimics ligand activation

. Activity of

the trapped Notch seems to be dependent on presenilin

activity because elevated Notch activity in cells that were

treated with RNA interference against ESCRT compo-

nents is sensitive to a γ-secretase inhibitor

. However,

the contribution of ligands to the activation has not been

fully assessed, and further studies are needed to ascertain

why certain endocytic sorting mutants result in such

potent Notch activation and whether this activation is

relevant to signalling under normal circumstances.

The activity of

Numb, a well characterized Notch

inhibitor, also involves endocytosis. Numb is asym-

metrically segregated into one of two daughter cells in

several lineages, and a search for mutants giving numb-

related phenotypes identified α-adaptin, a component

of the adaptor protein-2

(AP2) complex that links cargoes

to clathrin coats of transport vesicles

. Numb interacts

with the ear domain of α-adaptin and with Notch, so

it could directly recruit Notch into endocytic vesicles.

Furthermore, mammalian Numb promotes Notch ubiq-

uitylation

. However, in D. melanogaster, Numb associates

with the 4-pass transmembrane protein Sanpodo, which

performs an unknown but essential role in Notch signal-

ling wherever Numb-mediated regulation is crucial

79,80

Plasma-membrane accumulation of Sanpodo is reduced

by Numb in circumstances in which no detectable change

in Notch accumulation is observed, which indicates that

Sanpodo could be a primary endocytic target.

A further link between Numb, Notch and endo cytosis

comes from the identification of Numb-associated kinase

(NAK)

, which is related to AP2-associated kinases and

to SEL-5, a suppressor of dominant lin-12 phenotypes in

C. elegans

. However, partial rescue of Numb phenotype

is observed with Numb proteins that lack the α-adaptin-

interaction domain, which is indicative of alternative

mechanisms of Numb-mediated antagonism

. Further

studies are required to determine more fully the mecha-

nism of Numb action and the role of Sanpodo in the

regulation of Notch activity.

Ubiquitylation and Notch trafficking. Entry into the

endosomal and multivesicular-body-sorting pathway

is thought to be intimately linked with ubiquitylation

of transmembrane proteins. Several E3 ligases that

target Notch have already been identified

. The Itch/

NEDD4/Su(dx) family of HECT domain E3 ligases

are predominantly negative regulators of signalling,

which indicates that by modifying Notch they target

it for degradation. Studies in mammalian cells mapped

the interaction domain of Itch to the RAM–ankyrin

repeat region of Nicd

, which correlates with a ‘down-

regulation targeting signal’ that has been identified

in C. elegans LIN-12

(REF. 86). The targeting signal

was subsequently shown to interact with ALX-1, the

homologue of Bro1 — a Saccharomyces cerevisiae pro-

tein that is involved in a late step in multivesicular-

body sorting — and WWP-1, the homologue of Itch/

NEDD4/Su(dx)

. However, there might be more than

one Notch motif associated with recognition by this

E3-ligase family, as a more C-terminal PPXY motif

affected the capability of NEDD4 to promote Notch

degradation in D. melanogaster

The subtle phenotypes caused by mutations in the

HECT E3 ligases indicate that they do not make a crucial

contribution to Notch signalling. However, whether this

is indeed the case will remain an open question until the

consequences of eliminating all of the members of this

family are analysed. Nevertheless, by modulating turn-

over, Itch/NEDD4/Su(dx) could regulate the amount of

Notch that is available to interact with ligands. We do not

as yet know the extent to which these enzymes can them-

Figure 2 | Ligand activation entails ubiquitylation. The E3 ubiquitin (Ub) ligases

Neuralized (Neur) and Mind bomb (Mib) interact directly with Notch ligands. Prior to

modification by Neur or Mib, ligands are inactive, and can be endocytosed and degraded.

Neur- or Mib-mediated ubiquitylation of Notch ligands is required for Epsin-mediated

endocytosis. Ligands (in the light orange area) are then competent to signal either

because endocytosis is directly associated with receptor activation or because it allows

entry into a specific compartment or membrane domain that renders ligands active.

They can also be targeted for degradation. E2, ubiquitin-conjugating enzyme.

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AP2 complex

A heterotetrameric trafficking

adaptor complex that

comprises two large, one

medium and one small subunit.

It interacts with clathrin and

also, through the appendage

domain of a large subunit,

binds to accessory proteins,

including Epsins.

Rel family

Also known as nuclear factor

(NF)-κB proteins, this family of

transcription factors contains a

conserved domain (the Rel-

homology domain (RHD)) that

is required for DNA binding

and dimerization. These

proteins are important in

defence against infectious

diseases and cellular stress.

selves be regulated, although it has been shown that Itch

is a substrate for the mitogen-activated protein kinase

(MAPK) JNK1 in mammalian cells

and that the EGF-

receptor pathway regulates turnover through ALX-1

and/or WWP-1 in C. elegans vulval precursors

A second E3 ligase that binds to Nicd, within the

ankyrin repeats, is the RING finger protein

Deltex

Increased expression of Deltex in the D. melanogaster

wing promotes Notch signalling, antagonizes the nega-

tive effects of Su(dx), and results in increased Notch

accumulation in endocytic vesicles

90,91

. In this tissue,

Deltex has also been found to interact with the β-arrestin

Kurz, which might therefore mediate the internalization

of the Notch–Deltex complex

. In agreement with this

notion, loss of kurz results in elevated membrane levels of

Notch, whereas Deltex overexpression has the opposite

effect

73,91

. Although kurz mutants produce phenotypes of

elevated Notch signalling in the wing, deltex mutants pro-

duce the converse, so the precise relationship is complex.

Intriguingly, in several mammalian cells, including

lymphoid cells and neurons, Deltex antagonizes Notch,

further complicating the picture

16,92

. Perhaps the precise

balance of different E3-ligase activities dictates the out-

come on Notch localization and activity. These ubiquitin

modifications could potentially influence the length

of time that the receptor is located on the surface, its

accessibility to ligands, or its capability to interact with

γ-secretase

It is evident that Notch is subject to different types

of post-transcriptional regulation. Glycosylation and

proteolytic processing steps have a crucial influence on

receptor activity, and are potentially important steps for

drug intervention. Ubiquitylation and endocytic traffick-

ing can modulate the amount of receptor that is available

for signalling and could therefore provide powerful

mechanisms to tune the activity of the pathway.

Differing nuclear landscapes

Following Notch activation, Nicd enters the nucleus and

directly regulates the expression of target genes. Among

the Notch targets, the best characterized are the bHLH

(basic-helix–loop–helix) genes of the E(spl)/HES class.

However, the response to Notch differs greatly between

cell types — for example, Notch promotes cell prolifer-

ation in some contexts and apoptosis in others

. The

capability to elicit different responses might partly arise

from crosstalk with other pathways. It also depends on

the enhancers that are responsive to Notch regulation

in a given cell.

CSL proteins: pivotal in the switch. CSL proteins are the

essential effectors of the Notch pathway

(FIG. 4). These

DNA-binding proteins have been highly conserved

throughout evolution (for example, there is 84% iden-

tity between human and D. melanogaster proteins). The

crystal structure of the DNA-binding domain revealed

a striking similarity with the

Rel family of transcription

factors

(BOX 1). Nicd forms a trimeric complex with

CSL and the co-activator Mam, which is essential for

Nicd-dependent transcription in vitro and in vivo

94–97

Another Nicd-interacting protein, SKIP (Ski-interacting

protein), a transcriptional coregulator and component of

spliceosomes, is also recruited to promoters at the same

time

98,99

. Mam in turn recruits the histone acetylase p300,

which promotes assembly of initiation and elongation

complexes

100

The assembly of the co-activator complex not only

promotes transcription, but also results in turnover of

Nicd. The rapidly changing levels of pathway activity

require that the nuclear effectors do not have a long

half-life. This is achieved by recruitment of factors

such as cyclin-dependent kinase-8 (

CDK8), which

phosphorylates Nicd, rendering it into a substrate for

the nuclear ubiquitin ligase SEL10

(REFS 98,101). In

mammalian cells, SEL10 preferentially interacts with

a phosphorylated form of Nicd and the expression of a

dominant negative SEL10 leads to increased expression

of Notch targets

102–104

. This interaction requires the

C-terminal PEST region, consistent with observations

that Notch with C-terminal truncations behave as gain-

of-function alleles, and in humans they contribute to

Box 3 | Making a difference

Several mechanisms are used in different Notch-dependent processes to regulate

ligand and receptor activities.

Lateral inhibition

The following mechanisms could contribute:

• Receptor turnover — destabilization of Notch in the cell that will become the signal-

sending cell (for example, through NEDD4-family E3 ligases (WWP-1, Itch, NEDD4,

Su(dx))

• Regulation of Neuralized (Neur) E3-ligase activity — Neur inhibitors, encoded by the

Bearded family, are expressed in the cells in which Notch is activated

34,35

• E(spl)/HES bHLH (basic-helix–loop–helix) and REF-1 repressors — target genes that

are upregulated in response to Notch activation encode repressor proteins that

inhibit cell-fate promoting genes

107,135

Together these three mechanisms amplify small differences in signalling activity

between cells. However, they do not explain how a difference arises in the first place.

Lineage decisions

The following regulators are asymmetrically segregated into one of two daughter cells

in the Drosophila melanogaster sensory organ precursors (SOP) lineage to regulate

Notch signalling.

• Numb — inhibits Notch receptor through a mechanism that involves endocytosis and

another transmembrane protein, Sanpodo

• Neur E3-ligase activity — Neur is asymmetrically segregated into the same daughter

cell as Numb, favouring ligand activation

136

• Ligand trafficking — the recycling endosome (marked by RAB11) is asymmetrically

segregated with Neur and favours ligand activity

. Mutations in an exocyst

component, SEC15, also perturb asymmetrical trafficking

Together these mechanisms result in different signalling capabilities in two daughter

cells. The asymmetrical segregation is dependent on the apical basal and planar cell-

polarity machinery.

Boundaries/inductive signalling

The following intrinsic differences between two populations of cells translate into

distinct signalling populations

46,47,130,137

• Restricted expression of ligands.

• Restricted expression of Fringe glycosyl transferases — results in modifications to

Notch that alter its capability to respond to ligands

47,137

• Feedback regulation of Fringe expression — can be positive (for example,

rhombomere boundaries

137

) or negative (for example, chick somites)

130

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Furin-like

convertase

(S1 cleavage)

Glycosylation

Fringe

Degradation

Multivesicular

body

O-Fut

Endoplasmic reticulum

Golgi

Itch/

NEDD4/

Sudx

Deltex

ESCRT complexes

Syntaxin/

avalanche

Notch

oncogenicity

. Destruction of Nicd would result in the

dissociation of Mam and other co-activators, but it is

unclear whether CSL proteins would also be affected

or whether they remain intact on the DNA. Simple

models predict that they remain on the DNA, and CSL

is detected, by chromatin immunoprecipitation assays,

at the HES1 promoter after Nicd has dissociated

In the absence of Notch activity, CSL proteins recruit

co-repressors. In D. melanogaster, the adaptor Hairless

tethers the more global repressors Groucho and CtBP,

which recruit histone deacetylases

105–107

. So far, no

homologue of Hairless has been identified and, in mam-

malian cells, CSL co-repressors include

SMRT (also

known as NcoR) and SHARP (also known as MINT/

SPEN)

108,109

, which in turn recruit CtBP or other global

co-repressors. Two other CSL-interacting proteins,

SKIP and CIR (CBF1-interacting co-repressor), are

also part of the repression complex

99,110

. Homologues

of these mammalian proteins exist in D. melanogaster

and have been linked with CSL or Notch signalling. For

example, the SMRT homologue SMRTER is implicated

in CSL-dependent repression of Delta expression in the

D. melanogaster eye and the SHARP homologue Spen

affects CSL protein levels in the embryo

111,112

. However,

the relationship between the different co-repressors is

still unclear; are they all recruited simultaneously or are

there different co-repressor complexes, potentially with

spatial and temporal differences?

In mice and D. melanogaster, the phenotypes that are

produced by depleting the single CSL are similar but not

identical to loss-of-Notch function. Initially, these dif-

ferences led to the speculation about CSL-independent

Notch signalling. Subsequently, it became clear that

many differences could be explained by derepression

of target genes, as these CSL mutations also affect

the repressive function of CSL

113–115

. It is notable that

derepression is modest and is only detected in a small

number of cells

113

, although it is sufficient to partially

rescue the wing primordium defects in D. melanogaster

presenilin mutants

116

. One example of an essential role

for CSL repression is in the SOP lineage, in which it is

required for cell-fate specification and later physiological

function

107,117,118

. The fact that more global derepression

is not observed in the absence of CSL indicates that Nicd

and Mam supply essential co-activator function and/or

that CSL-repressor complexes are only essential for a

small component of target-gene repression.

Epigenetic regulators. The precise mechanisms that

are involved in Notch-dependent transcription are not

yet known, although studies in mammalian cells have

revealed a number of recruited cofactors

98,119

. These

include the histone acetyl transferase GCN5

(REF. 120)

as well as the SWI/SNF chromatin-remodelling enzyme

Brahma (also known as BRM), which interacts directly

with CSL proteins in co-immunoprecipitation assays

119

Figure 3 | Processing and trafficking regulate Notch-receptor activity. Notch (purple) is produced in the

endoplasmic reticulum where it interacts with the O-fucosyl transferase (O-Fut; green) and is transported to the Golgi. In

the Golgi, it is processed by Furin-like convertase (grey, S1 cleavage) and glycosylated (shown as dark grey protrusion from

Notch) by O-Fut and other glycosyltransferases (for example, Fringe; red) before export to the cell surface. Notch that is

endocytosed from the cell surface can be recycled or degraded through the multivesicular-body pathway. Actions of the

ubiquitin ligases Deltex (purple) and Itch/NEDD4/Su(dx) (pink) regulate trafficking, although their precise roles are not yet

clear. Other proteins (syntaxin, ESCRT complexes) that affect trafficking are indicated, but their sites of action are

hypothetical and remain to be fully clarified. Ub, ubiquitin.

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SHARP/

MINT/

SPEN

Target genes

active

Mam

CtBP

Mam

SKIP

Gro

SMRT

HDACs

SIN3A

CIR

Hairless

Target genes

repressed

CSL

CDK8

SEL10

HAT complexes

p300

GCN5/PCAF

Chromatin remodelling

BRM

TRA1/TRRAP

Dom

UbUb

Proteasome

Nicd

CSL

Consistent with these biochemical interactions, the

defects caused by reduced Brahma function (using a

dominant negative) in D. melanogaster are enhanced

by mutations that reduce the activity of the Notch

pathway

121

. Similarly, mutations in TRA1 (also known

as TRAPP; involved in the recruitment of the chro-

matin modification complexes SAGA and TIP60) and

in Domino (a histone exchange protein in the TIP60

complex) enhance the phenotypes of some Notch and

mam alleles

122

The emerging picture is that Notch signalling

requires recruitment of histone acetylase complexes and

exchange of histone variants to activate transcription.

In addition, BRE1, a homologue of the yeast histone 2B

ubiquitin ligase, is crucial for Notch function in vivo and

stimulates Notch-dependent transcription in a transient

transfection assay

123

. Sumoylation might also regulate

the activity of key nuclear components

124

. Together the

data show that Notch activity is highly sensitive to

chrom atin modifications and histone re-arrangements

that could contribute to target-gene specificity.

Furthermore, overexpression of two Polycomb group

epigenetic silencers enhances Notch-induced over-

proliferation and also causes hypermethylation of a

tumour suppressor gene

125

, indicating further mecha-

nisms that could constrain the accessibility of enhancers

and cooperate with Notch to confer different programmes

of gene expression.

Cooperation with tissue-specific activators. CSL sites

alone are often poor at mediating activation in vivo, indi-

cating that Nicd functions in combination with tissue-

specific factors

126

. The best-characterized examples are

the proneural bHLH proteins, which synergize with

Nicd in D. melanogaster; there is as yet no evidence for

them having a similar role in vertebrates

107,114,127

. During

neurogenesis, the proneural proteins are expressed in

groups of cells in which they promote neural develop-

ment. Activation of Notch in a subset of cells within each

group results in expression of target genes such as those

of the E(spl) family. Analysis of the regulatory sequences

from these genes revealed both CSL- and proneural-pro-

tein-binding sites

114

. Mutations in the proneural-protein-

binding sites eliminate activation of the target genes

107

Therefore, binding of tissue-specific activators contrib-

utes to robust target-gene expression, and can explain

the specificity of Notch responses in different cell types.

In some cases, the precise arrangement of binding sites

influences the cooperation between Notch and other

DNA-bound activators

128,129

. Also, during lateral inhi-

bition, and in the somite clock, the targets themselves

feedback to inhibit their own transcription, thereby

ensuring a transient burst of transcription in response to

Notch activation

130

. However, barely a handful of tissue-

specific activators that work in cooperation with Notch

have been identified, so the extent and diversity of such

factors is still unclear.

Making a difference

How are these different regulatory mechanisms deployed

in different Notch-dependent processes? Examples are

given in

BOX 3. Here I would like to highlight two aspects

of this deployment. First, the activity of the receptor and

its ligands can be controlled in various ways. Different

mechanisms might be harnessed to establish or amplify

differences between cells in specific contexts. Much

of this regulation affects the subcellular distribution of

the proteins, most particularly their trafficking in the

endocytic pathway and/or their post-translational modi-

fication, such as glycosylation, ubiquitylation and most

probably phosphorylation. Furthermore, several mecha-

nisms are frequently employed together to enhance the

efficiency of signalling. For example, there might be

positive regulation of the ligand and downregulation of

the receptor in the same cell, making for a more robust

mechanism

32,86,131

. Furthermore, these mechanisms can

be targeted in feedback loops, as is exemplified by the

polypeptide inhibitors of Neur

34,35

(BOX 3).

Second, if the pathway responds to dynamic fluctua-

tions in its environment and/or if it is used iteratively,

there must be mechanisms to ensure that the response

is short-lived. This is particularly important in pro-

cesses such as the somite clock, in which Notch activity

oscillates — autoinhibitory feedback of the E(spl)/HES

Notch targets and the regulation of Lunatic Fringe are

two mechanisms that contribute to this oscillatory Notch

activity

59,130

. In addition to negative-feedback mecha-

nisms, transient signalling is favoured by destruction

of Nicd after recruitment of transcription initiation

factors on target enhancers

98,101

. Furthermore, the

Figure 4 | Nuclear cycle of CSL. The Notch intracellular domain (Nicd, purple) forms a

trimeric complex with the DNA-binding protein CSL (CBF1, Su(H) and LAG-1; orange)

and the co-activator Mastermind (Mam, green). SKIP (Ski-interacting protein), a protein

that interacts with the ankyrin repeat domain of Nicd and with CSL is also present.

Histone acetyl transferases (HATs; p300 and/or PCAF/GCN5) and chromatin-

remodelling complexes (BRM, TRA1/TRRAP and Dom) are recruited and contribute to

activation and elongation of transcription at target genes. Kinases such as cyclin-

dependent kinase-8 (CDK8) and the SEL10 E3 ligase modify Nicd, making it a substrate

for proteosomal degradation. In the absence of Nicd, CSL is associated with

co-repressors, the precise composition of which might vary according to species and

cell type. Two putative co-repressor complexes are illustrated: a mammalian complex

containing SMRT, SHARP (also known as MINT and SPEN) and CtBP, and a Drosophila

melanogaster complex containing Hairless, CtBP and Groucho (Gro). Although not

depicted, SKIP might also be part of the repression complex. The co-repressors recruit

histone deacetylases (HDACs; such as HDAC1, HDAC3, HDAC4 and RPD3) and other

cofactors (SIN3A and CIR). Target genes are repressed until more Nicd is produced to

re-initiate the cycle. P, phosphate; Ub, ubiquitin.

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Somitogenesis

The development of somites,

the segmental blocks of

mesoderm that give rise to the

axial skeleton, muscles and

dermis.

proteins and mRNAs from target genes are, in many cases,

rapidly degraded. For example, mutations that increased

the half-life of Hes7 mRNA disrupted

somitogenesis,

which highlights the importance of rapid turnover

132

The short half-life of many target-gene mRNAs can be

explained by the fact that they are subject to regulation

by microRNAs (miRNAs), as seen with the E(spl)/HES

genes

133,134

. Mutations that affect miRNA target sites or

miRNAs themselves can result in phenotypes resembling

increased Notch activity, underscoring the importance

of this regulation.

An important area that has not been discussed in

this review because of space limitations is the crosstalk

between Notch and other signalling pathways. For exam-

ple, what are the mechanisms that control the regulators

of the Notch pathway? And, to what extent can the core

components of the Notch pathway be modified by or

interact directly with components from other pathways?

These additional levels of regulation are likely to con-

tribute to Notch-pathway activity in development and

disease.

Future Directions

What emerges from much of the recent literature is that

the precise location of the Notch ligand and the receptor

in the cell can have profound effects on signalling.

Currently it is very difficult to determine what the different

routes of trafficking are and how exactly they impact on

activity. Other obvious holes in our understanding are;

how does a ligand interaction promote the second cleavage

of Notch, the key first step towards ligand–receptor

interaction; and what ubiquitin-dependent step creates

a productive ligand? More sophisticated in vivo imaging

with methods to allow tracking of proteins in different

states (for example after a specific ubiquitylation) is

required to unravel these mechanisms. We also need to

know how these modifications impact on the length of

time Notch and its ligands are actually at the cell surface

and able to interact and how they impact on the stability

of Notch following activation. It is evident, therefore,

that methods for monitoring activity in real time are

needed to understand the dynamics of signalling in each

developmental process.

Understanding how and why different target genes are

activated according to cell type and time are another set

of important issues. Currently, the characterized direct

targets of Notch activity can be counted on a few fingers,

but even in those cases we have little knowledge about

what makes a gene a target in specific cells. Furthermore,

with the few identified targets, it is difficult to explain

the varied consequences of Notch activation. Genome-

wide studies are likely to increase the spectrum of targets

and allow the development of a systematic approach for

understanding the different responses.

1. Artavanis-Tsakonas, S., Rand, M. D. & Lake, R. J.

Notch signaling: cell fate control and signal integration

in development. Science 284, 770–776 (1999).

This is an excellent review of the field prior to 1999.

2. Schweisguth, F. Notch signaling activity. Curr. Biol. 14,

R129–R138 (2004).

3. Radtke, F. & Raj, K. The role of Notch in

tumorigenesis: oncogene or tumour suppressor?

Nature Rev. Cancer 3, 756–767 (2003).

A review that summarizes the links between Notch

and cancer.

4. Fortini, M. E. γ-secretase-mediated proteolysis in cell-

surface-receptor signalling. Nature Rev. Mol. Cell Biol.

3, 673–684 (2002).

5. Selkoe, D. & Kopan, R. Notch and presenilin:

regulated intramembrane proteolysis links

development and degeneration. Annu. Rev. Neurosci.

26, 565–597 (2003).

6. Mumm, J. S. & Kopan, R. Notch signaling: from the

outside in. Dev. Biol. 228, 151–165 (2000).

7. Louvi, A. & Artavanis-Tsakonas, S. Notch signalling in

vertebrate neural development. Nature Rev. Neurosci.

7, 93–102 (2006).

8. Lowell, S., Benchoua, A., Heavey, B. & Smith, A. G.

Notch promotes neural lineage entry by pluripotent

embryonic stem cells. PLoS Biol. 4, e121 (2006).

9. Fre, S. et al. Notch signals control the fate of immature

progenitor cells in the intestine. Nature 435,

964–968 (2005).

10. van Es, J. H. et al. Notch/γ-secretase inhibition turns

proliferative cells in intestinal crypts and adenomas

into goblet cells. Nature 435, 959–963 (2005).

11. Ohlstein, B. & Spradling, A. The adult Drosophila

posterior midgut is maintained by pluripotent stem

cells. Nature 439, 470–474 (2006).

12. Micchelli, C. A. & Perrimon, N. Evidence that stem

cells reside in the adult Drosophila midgut epithelium.

Nature

439, 475–479 (2006).

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human T cell acute lymphoblastic leukemia. Science

306, 269–271 (2004).

The analysis shows that 50% of patients with acute

lymphoblastic leukaemia have activating mutations in

the Notch1 locus. These map to two specific regions

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to the membrane in the ECD impose constraints that

prevent premature activation.

14. Chitnis, A. Why is Delta endocytosis required for

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886–894 (2006).

15. Le Borgne, R., Bardin, A. & Schweisguth, F. The roles

of receptor and ligand endocytosis in regulating Notch

signaling. Development 132, 1751–1762 (2005).

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essential for efficient activation of Notch signaling by

Delta. Dev. Cell 4, 67–82 (2003).

One of several papers reporting the importance of

E3 ubiquitin ligases in regulating Notch ligands (see

references 17–23). This is the first to identify Mind

bomb. It also demonstrates the importance of

ubiquitylation in promoting ligand activity in the

signal-sending cell.

17. Lai, E. C., Deblandre, G. A., Kintner, C. & Rubin, G. M.

Drosophila neuralized is a ubiquitin ligase that

promotes the internalization and degradation of Delta.

Dev. Cell 1, 783–794 (2001).

18. Pavlopoulos, E. et al. neuralized encodes a peripheral

membrane protein involved in Delta signaling and

endocytosis. Dev. Cell 1, 807–816 (2001).

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neuralized is a ubiquitin ligase that interacts with

XDelta1 and regulates Notch signaling. Dev. Cell 1,

795–806 (2001).

20. Pitsouli, C. & Delidakis, C. The interplay between DSL

proteins and ubiquitin ligases in Notch signaling.

Development 132, 4041–4050 (2005).

21. Le Borgne, R., Remaud, S., Hamel, S. & Schweisguth,

F. Two distinct E3 ubiquitin ligases have

complementary functions in the regulation of Delta

and Serrate signaling in Drosophila. PLoS Biol. 3, e96

(2005).

22. Lai, E. C., Roegiers, F., Qin, X., Jan, Y. N. & Rubin, G. M.

The ubiquitin ligase Drosophila Mind bomb promotes

Notch signaling by regulating the localization and

activity of Serrate and Delta. Development 132,

2319–2332 (2005).

23. Wang, W. & Struhl, G. Distinct roles for Mind bomb,

Neuralized and Epsin in mediating DSL endocytosis

and signaling in Drosophila. Development 132,

2883–2894 (2005).

24. Wang, W. & Struhl, G. Drosophila Epsin mediates a

select endocytic pathway that DSL ligands must enter

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Acknowledgements

My apologies to all those colleagues whose important contri-

butions have not been acknowledged due to the space con-

straints of this Review. My thanks to A. Krejci, M. Glittenberg,

A. Djiane and the reviewers for helpful comments, and to

F. Wirtz-Peitz and J. Knoblich (Institute of Molecular

Biotechnology, Vienna, Austria) for the beautiful image of

SOPs in BOX 2. Work on Notch signalling in my laboratory is

currently supported by grants from the Medical Research

Council, the Wellcome Trust and the Association for

International Cancer Research.

Competing interests statement

The author declares no competing financial interests.

DATABASES

The following terms in this article are linked online to:

UniProtKB: http://ca.expasy.org/sprot

ADAM10 | ADAM17 | CBF1 | CDK8 | Delta | Deltex | DSL-1 |

Fringe | GLP-1 | Itch | LAG-1 | LIN-12 | Mastermind | NEDD4 |

Su(dx) | Su(H) | PEN2

Flybase: http://www.flybase.org

mib1

OMIN: http://www.ncbi.nlm.nih.gov/entrez/query.

fcgi?db=OMIM

Alzheimer’s disease

FURTHER INFORMATION

Sarah Bray’s homepage:

http://www.pdn.cam.ac.uk/staff/bray_s

Access to this links box is available online.

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Feb 2025
CELL BIOL TOXICOL

Methamphetamine (Meth) is a potent central nervous system stimulant with high addictive potential and neurotoxic effects. Chronic use results in significant damage in various brain functions, including cognition, memory, and sensory perception. Olfactory dysfunction is a notable yet often overlooked consequence of Meth abuse, and its underlying mechanisms are not fully understood. This study investigates the mechanisms of Meth-induced olfactory impairment through a thorough examination of olfactory bulb (OB) neurogenesis. We found that chronic Meth abuse impaired olfactory function in mice by not only reducing the self-renewal of subventricular zone (SVZ) neural stem cells (NSCs) but also altering their differentiation potential, leading their differentiation into astrocytes at the expense of neurons. Mechanistically, Meth inhibits autophagosome-lysosome fusion by downregulating Syntaxin 17 (Stx17), which reduces autophagic flux. In NSCs, autophagy tightly regulates Notch1 levels, and impaired autophagic degradation of Notch1 leads to its abnormal activation. This alters NSCs fate determination, ultimately affecting OB neurogenesis. Our study reveals that Meth impairs olfactory function through autophagic dysfunction and aberrant Notch1 signaling. Understanding these mechanisms not only provides new insights into Meth-induced olfactory dysfunction but also offers potential targets for developing therapies to alleviate Meth-induced neurotoxicity and sensory damage in the future.

Synergistic effects of mutation and glycosylation on disease progression

Article

Full-text available

Feb 2025

Glycosylation, a post-translational modification, plays a crucial role in proper localization and function of proteins. It is regulated by multiple glycosyltransferases and can be influenced by various factors. Inherited missense mutations in glycosylated proteins such as NOTCH3, Low-density lipoprotein receptor (LDLR), and Amyloid precursor protein (APP) could affect their glycosylation states, leading to cerebral small vessel disease, hypercholesterolemia, and Alzheimer’s disease, respectively. Additionally, physiological states and aging-related conditions can affect the expression levels of glycosyltransferases. However, the interplay between mutations in glycosylated proteins and changes in their glycosylation levels remains poorly understood. This mini-review summarizes the effects of glycosylation on transmembrane proteins with pathogenic mutations, including NOTCH3, LDLR, and APP. We highlight the synergistic contributions of missense amino acids in the mutant proteins and alterations in their glycosylation states to their molecular pathogenesis.

Mutational constraint analysis workflow for overlapping short open reading frames and genomic neighbors

Article

Full-text available

Mar 2025
BMC GENOMICS

Understanding the dark genome is a priority task following the complete sequencing of the human genome. Short open reading frames (sORFs) are a group of largely unexplored elements of the dark genome with the potential for being translated into microproteins. The definitive number of coding and regulatory sORFs is not known, however they could account for up to 1–2% of the human genome. This corresponds to an order of magnitude in the range of canonical coding genes. For a few sORFs a clinical relevance has already been demonstrated, but for the majority of potential sORFs the biological function remains unclear. A major limitation in predicting their disease relevance using large-scale genomic data is the fact that no population-level constraint metrics for genetic variants in sORFs are yet available. To overcome this, we used the recently released gnomAD 4.0 dataset and analyzed the constraint of a consensus set of sORFs and their genomic neighbors. We demonstrate that sORFs are mostly embedded into a moderately constrained genomic context, but within the gencode dataset we identified a subset of highly constrained sORFs comparable to highly constrained canonical genes.

Single-Chain Nanobody Inhibition of Notch and Avidity Enhancement Utilizing the β-Pore-Forming Toxin Aerolysin

Article

Mar 2025
ACS CHEM BIOL

Modes of Notch signalling in development and disease

Article

Mar 2025
NAT REV MOL CELL BIO

Notch2 Signaling Drives Cardiac Hypertrophy by Suppressing Purine Nucleotide Metabolism

Article

Full-text available

Mar 2025

Gain-of-function mutations of Notch2 cause the rare autosomal dominant disorder known as Hajdu–Cheney syndrome (HCS). Most patients with HCS develop congenital heart disease; however, the precise mechanisms remain elusive. Here, a murine model expressing the human Notch2 intracellular domain (hN2ICD) in cardiomyocytes (hN2ICD-TgCM) was generated and the mice spontaneously developed ventricular diastolic dysfunction with preserved ejection fraction and cardiac hypertrophy. Ectopic hN2ICD expression promoted cardiomyocyte hypertrophy by suppressing adenylosuccinate lyase (ADSL)-mediated adenosine 5′-monophosphate (AMP) generation, which further enhanced the activation of the mammalian target of rapamycin complex 1 pathway by reducing AMP-activated kinase activity. Hairy and enhancer of split 1 silencing abrogated hN2ICD-induced cardiomyocyte hypertrophy by increasing Adsl transcription. Importantly, pharmacological activation of AMP-activated kinase ameliorated cardiac hypertrophy and dysfunction in hN2ICD-TgCM mice. The frameshift mutation in Notch2 exon 34 (c.6426dupT), which causes early-onset HCS, induces AC16 human cardiomyocyte hypertrophy through suppressing ADSL-mediated AMP generation. Thus, targeting Notch2-mediated purine nucleotide metabolism may be an attractive therapeutic approach to heart failure treatment.

Notch dependent chromatin remodeling enables Gata3 binding and drives lineage specific CD8+ T cell function

Article

Feb 2025
IMMUNOL CELL BIOL

Activation of CD8 ⁺ T cells enable them to control virus infections and tumors. This process involves the differentiation of naïve CD8 ⁺ T cells into effector and memory states, driven by specific transcription factors (TFs). Previously, we have shown that Granzyme A (Gzma) induction in activated CD8 ⁺ T cells depends on Gata3 and the establishment of a permissive chromatin landscape at the Gzma locus. Interestingly, Gzma expression is independent of IL‐4 signaling, which typically upregulates Gata3 in CD4 ⁺ T cells, suggesting an alternative pathway for Gata3 induction. Here we demonstrate that Notch signals during CD8 ⁺ T cell activation promote Gzma expression. Inhibition of Notch signaling or loss of the Notch transactivator Rbp‐j leads to reduced Gzma expression, with transcriptionally repressive chromatin at the Gzma locus. The genome targets of Gata3 differ in effector CD8 ⁺ T cells activated with IL‐4 compared with those activated with Notch signals or isolated after IAV infection. This indicates that the signals received during CD8 ⁺ T cell activation can alter the chromatin landscape, affecting Gata3 function. Furthermore, Gata3 deficiency results in reduced IAV‐specific CD8 ⁺ T cell responses and decreased Gzma expression, although the Gzma locus maintains a permissive chromatin landscape. These findings suggest that Notch signals received by virus‐specific CD8 ⁺ T cells prepare the chromatin landscape for Gata3 binding to CD8 ⁺ lineage‐specific gene loci, promoting effective CD8 ⁺ T cell immunity.

Characterization of the Genomic Landscape in HPV-positive Cervical and Head and Neck Squamous Cell Carcinomas by Whole Genome Next Generation Sequencing

Article

Full-text available

Feb 2025

An Infant With Congenital Scalp, Nail, and Limb Anomalies

Article

Feb 2025
PEDIATR DERMATOL

Functional Interaction between SEL-10, an F-box Protein, and the Nuclear Form of Activated Notch1 Receptor

Article

Full-text available

Sep 2001

The Notch signaling pathway is essential in many cell fate decisions in invertebrates as well as in vertebrates. After ligand binding, a two-step proteolytic cleavage releases the intracellular part of the receptor which translocates to the nucleus and acts as a transcriptional activator. Although Notch-induced transcription of genes has been reported extensively, its endogenous nuclear form has been seldom visualized. We report that the nuclear intracellular domain of Notch1 is stabilized by proteasome inhibitors and is a substrate for polyubiquitination in vitro. SEL-10, an F-box protein of the Cdc4 family, was isolated in a genetic screen for Lin12/Notch-negative regulators in Caenorhabditis elegans. We isolated human and murine counterparts of SEL-10 and investigated the role of a dominant-negative form of this protein, deleted of the F-box, on Notch1 stability and activity. This molecule could stabilize intracellular Notch1 and enhance its transcriptional activity but had no effect on inactive membrane-anchored forms of the receptor. We then demonstrated that SEL-10 specifically interacts with nuclear forms of Notch1 and that this interaction requires a phosphorylation event. Taken together, these data suggest that SEL-10 is involved in shutting off Notch signaling by ubiquitin-proteasome-mediated degradation of the active transcriptional factor after a nuclear phosphorylation event.

Radtke F, Raj KThe role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 3: 756-767

Article

Full-text available

Oct 2003
NAT REV CANCER

Notch signalling participates in the development of multicellular organisms by maintaining the self-renewal potential of some tissues and inducing the differentiation of others. Involvement of Notch in cancer was first highlighted in human T-cell leukaemia, fuelling the notion that aberrant Notch signalling promotes tumorigenesis. However, there is mounting evidence that Notch signalling is not exclusively oncogenic. It can instead function as a tumour suppressor.

SEL-5, A Serine/Threonine Kinase That Facilitates lin-12 Activity in Caenorhabditis elegans

Article

Dec 1999
GENETICS

Ligands present on neighboring cells activate receptors of the LIN-12/Notch family by inducing a proteolytic cleavage event that releases the intracellular domain. Mutations that appear to eliminate sel-5 activity are able to suppress constitutive activity of lin-12(d) mutations that are point mutations in the extracellular domain of LIN-12, but cannot suppress lin-12(intra), the untethered intracellular domain. These results suggest that sel-5 acts prior to or during ligand-dependent release of the intracellular domain. In addition, sel-5 suppression of lin-12(d) mutations is tissue specific: loss of sel-5 activity can suppress defects in the anchor cell/ventral uterine precursor cell fate decision and a sex myoblast/coelomocyte decision, but cannot suppress defects in two different ventral hypodermal cell fate decisions in hermaphrodites and males. sel-5 encodes at least two proteins, from alternatively spliced mRNAs, that share an amino-terminal region and differ in the carboxy-terminal region. The amino-terminal region contains the hallmarks of a serine/threonine kinase domain, which is most similar to mammalian GAK1 and yeast Pak1p.

12. neuralized Encodes a peripheral membrane protein involved in delta signaling and endocytosis.

Article

Dec 2001
DEV CELL

Elias Pavlopoulos

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized (neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.

Endocytic control of epithelial polarity and proliferation in Drosophila

Article

Jan 2005

The role of Notch in tumorigenesis: oncogene or tumour suppressor?

Article

Jan 2003
NAT REV CANCER

Notch activity in neural cells triggered by a mutant allele with altered glycosylation

Article

Jul 2003

Yanxia Li

The receptor protein Notch is inactive in neural precursor cells despite neighboring cells expressing ligands. We investigated specification of the R8 neural photoreceptor cells that initiate differentiation of each Drosophila ommatidium. The ligand Delta was required in R8 cells themselves, consistent with a lateral inhibitor function for Delta. By contrast, Delta expressed in cells adjacent to R8 could not activate Notch in R8 cells. The split mutation of Notch was found to activate signaling in R8 precursor cells, blocking differentiation and leading to altered development and neural cell death. split did not affect other, inductive functions of Notch. The Ile578→Thr578 substitution responsible for the split mutation introduced a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. The O-fucose monosaccharide did not require extension by Fringe to confer the phenotype. Our results suggest functional differences between Notch in neural and non-neural cells. R8 precursor cells are protected from lateral inhibition by Delta. The protection is affected by modifications of a particular EGF repeat in the Notch extracellular domain. These results suggest that the pattern of neurogenesis is determined by blocking Notch signaling, as well as by activating Notch signaling.

Processing of the Notch Ligand Delta by the Metalloprotease Kuzbanian

Article

Jan 1999

Huilin Qi

Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.

A Novel Proteolytic Cleavage Involved in Notch Signaling

Article

Feb 2000
MOL CELL

The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a γ-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the γ-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFα-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE−/− bone marrow–derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway.

Glycosylation regulates Notch signaling

Article

Oct 2003

Intracellular post-translational modifications such as phosphorylation and ubiquitylation have been well studied for their roles in regulating diverse signalling pathways, but we are only just beginning to understand how differential glycosylation is used to regulate intercellular signalling. Recent studies make clear that extracellular post-translational modifications, in the form of glycosylation, are essential for the Notch signalling pathway, and that differences in the extent of glycosylation are a significant mechanism by which this pathway is regulated.

Bray SJ.. Notch signaling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7: 678-689

Abstract

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