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Notch signaling ata glance

Kazuya Hori*, Anindya Sen* andSpyros Artavanis-Tsakonas`

Department of Cell Biology, Harvard Medical School,240 Longwood Avenue, LHRRB-418, Boston,MA 02115, USA

*These authors contributed equally to this work`Author for correspondence

(artavanis@hms.harvard.edu)

Journal of Cell Science 126, 2135–2140

� 2013. Published by The Company of Biologists Ltd

doi: 10.1242/jcs.127308

SummaryCell–cell interactions define a quintessential

aspect of multicellular development.

Metazoan morphogenesis depends on a

handful of fundamental, conserved cellular

interaction mechanisms, one of which is

defined by the Notch signaling pathway.

Signals transmitted through the Notch

surface receptor have a unique

developmental role: Notch signaling links

the fate of one cell with that of a cellular

neighbor through physical interactions

between the Notch receptor and the

membrane-bound ligands that are expressed

in an apposing cell. The developmental

outcome of Notch signals is strictly

dependent on the cellular context and can

influence differentiation, proliferation and

apoptotic cell fates. The Notch pathway is

conserved across species (Artavanis-

Tsakonas et al., 1999; Bray, 2006; Kopan

and Ilagan, 2009). In humans, Notch

malfunction has been associated with a

diverse range of diseases linked to changes

in cell fate and cell proliferation including

cancer (Louvi and Artavanis-Tsakonas,

2012). In this Cell Science at a Glance

article and the accompanying poster we

summarize the molecular biology of Notch

signaling, its role in development and its

relevance to disease.

IntroductionThe functional Notch receptor is expressed on

the cell surface as a processed heterodimer

resulting from a Furin-dependent cleavage

(S1 cleavage) in the Notch extracellular

domain (NECD), which occurs during

trafficking through the Golgi complex

(Logeat et al., 1998) (see poster). The

NECD undergoes O-linked glycosylation

during Notch synthesis and secretion, which

is crucial for proper folding of the Notch

receptor and the interaction with its ligand

DSL (Delta, Serrate, Lag-2) (Rana and

Haltiwanger, 2011). The Notch receptor on

the signal-receiving cell binds directly to

ligands located on the apposing signal-

sending cell (Bray, 2006; Kopan and Ilagan,

2009). Receptor–ligand engagement triggers

a second NECD cleavage (S2 cleavage) by

a metalloproteinase ADAM (known as

Kuzbanian in Drosophila melanogaster),

which in turn facilitates a further crucial

signaling cleavage within the Notch

transmembrane domain by a c-secretase

complex that contains Presenilin (S3

cleavage) (Struhl and Greenwald, 1999;

Brou et al., 2000); this then releases the

Notch intracellular domain (NICD) from the

membrane. The NECD is trans-endocytosed

into the signal-sending cell together with its

(See poster insert)

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ligand (Gordon et al., 2008). DSL ligands canalso be cleaved by a metalloproteinaseADAM, which in turn downregulates ligand

activity (Zolkiewska, 2008).

Trans-interactions between ligand andreceptor that are expressed in apposingcells define activating events, whereas cis-

interactions – between the receptor and aligand that is expressed in the same cell –are inhibitory in nature (Sprinzak et al.,2010; del Alamo et al., 2011). The

molecular biology of cis-interactions isstill being elucidated, but the interplaybetween cis- and trans-interactions is a

crucial factor that distinguishes thesignal-receiving cell from the signal-sending cell, an important decision for

development and disease. In addition tocis–trans interactions, this crucial fate-determination step is also regulated by

the ratio of ligand-competent receptors toreceptor-competent ligands on the cellsurface.

The released NICD translocates directlyto the nucleus, where it forms a

transcriptional complex with the DNA-binding protein CSL (CBF1, Suppressor ofHairless, Lag1), Mastermind (Mam) and

transcriptional co-activators to drive theexpression of Notch target genes (Bray,2006; Kopan and Ilagan, 2009). In the

absence of NICD, CSL forms complexeswith a variety of co-repressors to suppressthe transcription of Notch target genes(Bray, 2006; Kopan and Ilagan, 2009) (see

poster).

Endocytosis and endosomal processingat different stages of Notch traffickinghave been shown to have important and

complex roles in regulating the activity ofNotch signaling (Fortini, 2009; Yamamotoet al., 2010). Notably, endocytic trafficking

has not only been implicated in ligand-dependent signaling (Coumailleau et al.,2009), but it has also been shown that

within endocytic compartments thereceptor can, under certain circumstances,be activated in a ligand-independentfashion. The physiological significance of

such phenomena remains to be elucidated(Vaccari et al., 2008; Wilkin et al., 2008;Vaccari et al., 2009; Hori et al., 2011).

Structures of Notch and its ligandsNotch structure

Notch receptors are multidomain proteins,and have been conserved frominvertebrates to humans. The poster panel

‘Structure of the Notch receptor andligands’ shows representations of theDrosophila Notch receptor and the four

mammalian Notch receptors (NOTCH 1, 2,

3 and 4). The NECD consists of 29 to36 epidermal growth factor (EGF)-likerepeats, which are post-translationally

modified by a variety of glycans andhave been implicated in Notch function(Rana and Haltiwanger, 2011); mostnotably, the EGF-like repeats 11–12 have

been shown to be necessary and sufficientfor receptor-ligand interactions (Rebayet al., 1991). The NECD is followed by

the negative regulatory region (NRR),which is composed of the three cysteine-rich LNR Notch repeats and the

heterodimerization domain. The NRR hasbeen reported to prevent the access ofmetalloproteinases to the S2 cleavage site

of Notch in the absence of ligand (Bray,2006; Kopan and Ilagan, 2009). The NICDconsist of a RAM domain, ankyrin (ANK)repeats flanked by two nuclear localization

signals (NLS), a transcriptional activationdomain (TAD) and a C-terminal Pro GluSer Thr (PEST) domain. The RAM and

ANK domains are essential for interactingwith CSL in the nucleus.

Notch ligand structure

The DSL ligands of the Notch receptorshave been also conserved throughoutevolution (D’Souza et al., 2008).

Drosophila Notch has two DSL ligands,Delta and Serrate, whereas there are fivemammalian ligands, three of which belongto the Delta-like family (DLL1, DLL3 and

DLL4) and two belong to the Jagged familyof Serrate homologs, Jagged 1 and 2 [alsoknown as JAG1 and JAG2, respectively

(see poster)]. DSL ligands aretransmembrane proteins with anextracellular domain that contains a

characteristic number of EGF-like repeatsand a cysteine-rich N-terminal DSLdomain. The DSL domain is a conserved

motif that is found in all DSL ligands and isrequired for their interaction with Notch.Serrate, Jagged 1 and Jagged 2 contain anadditional cysteine-rich domain. In contrast

to the canonical DSL ligands, non-canonical ligands lack the DSL domainand comprise a group of structurally diverse

proteins, which includes integral andglycosylphosphatidylinositol (GPI)-linkedmembrane proteins, and are presumed to

modulate Notch receptor activity (D’Souzaet al., 2010).

Glycosylation of NotchThe EGF repeats of Notch are subjectedto three types of O-linked modification:O-glucosylation, O-fucosylation and

O-GlcNAc addition (Rana and Haltiwanger,2011). These post-translational modifications

regulate Notch activity during its synthesisand secretion (Kopan and Ilagan, 2009). AnO-fucosyltransferase, which is encoded by O-

fut1 (POFUT1 in mammal), adds O-fucose to

several Notch EGF-like repeats that harborthe consensus C2-x-x-x-x-(S/T)-C3 motif(Okajima and Irvine, 2002). It has also been

shown that O-FUT1 functions as a chaperoneto promote the folding and/or export of Notchto the plasma membrane (Okajima et al.,

2005; Sasamura et al., 2007). The b1,3-N-acetylglucosaminyltransferase Fringe (thethree mammalian paralogs are known asLunatic, Radical and Manic fringe) catalyzes

the addition of N-acetylglucosamine(GlcNAc) to the primary O-fucose (Ranaand Haltiwanger, 2011). These modifications

by Fringe alter the responsiveness of theNotch receptor to DSL ligands in a context-specific manner (Panin et al., 1997). Further

elongation of the sugar modification with agalactose and sialic acid has been documentedfor mammalian Notch, but not in Drosophila

(Rana and Haltiwanger, 2011). Rumi, anendoplasmatic reticulum protein, adds O-glucose to serine at the consensus sequenceC1-x-S-x-P-C2 (Acar et al., 2008; Rana et al.,

2011), and the O-glucose can be elongated bythe addition of xylose (Rana and Haltiwanger,2011).

Regulation of Notch signaling bymembrane traffickingNotch trafficking

Membrane trafficking has been shown tohave an important and indeed complexfunction in the activation and regulation of

Notch signaling (Yamamoto et al., 2010;Baron, 2012). The first evidence came fromgenetic studies on Drosophila shibire,

which encodes the GTPase Dynamin, akey regulator of endocytosis (Seugnetet al., 1997). Classical genetic analysis

demonstrated a requirement of Dynaminin both the signal-sending and the signal-receiving cell (Seugnet et al., 1997). Furtherstudies, mostly in flies, identified several

factors that modulate the membranetrafficking of Notch and its ligands.Notably, such membrane trafficking events

have not only been associated with ligand-dependent activation of Notch but alsowith its ligand-independent activation, a

phenomenon that is still not wellunderstood. In either case, the activation isdependent on the S3 cleavage by c-

secretase (Struhl and Greenwald, 1999;Wilkin et al., 2008; Coumailleau et al.,2009).

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Several proteins have been involved inthe early steps of endocytosis – including

Dynamin (as mentioned above), Numb, acytoplasmic protein and Sanpodo, amultipass transmembrane protein that islocalized to the cell membrane – and are

required in the signal-receiving cell forNotch activation (Fortini, 2009). Inasymmetric cell divisions, which are

crucial for the differentiation of sensoryorgan precursors (SOPs), the interplaybetween Numb and Sandopo guides the

asymmetric endocytic trafficking of theNotch receptor, which determines cell fate(Berdnik et al., 2002; Hutterer andKnoblich, 2005; Babaoglan et al., 2009).

In this process, elegant work demonstratedthat the receptor is activated in asubpopulation of Rab5-positive early

endosomes, which are marked by SARA,an adaptor protein (Coumailleau et al.,2009). The SARA-containing endosomes

comprise Notch and Delta, and alsopossess c-secretase activity that cangenerate the NICD and hence activate the

receptor (Coumailleau et al., 2009) (seeposter).

Upon endocytosis, internalized Notchcan recycle back to the plasma membrane

or can be sorted to multivesicular bodies(MVBs) or late endosomes (Yamamotoet al., 2010; Baron, 2012). Sorting of

Notch into MVBs or late endosomes isregulated by the ESCRT (endosomalsorting complex required for transport)

complexes, which consist of ESCRT-0and Hrs, ESCRT-I, -II, -III, and Vps4(Henne et al., 2011). Loss of ESCRTfunction can result in the ectopic

activation of Notch signaling in a ligand-independent manner (Vaccari et al., 2008;Vaccari et al., 2009). Lethal (2) giant discs

(lgd), which encodes a C2-containingphospholipid-binding protein, interactswith the ESCRT-III complex and

regulates ligand-independent activation ofNotch (Childress et al., 2006; Gallagherand Knoblich, 2006; Jaekel and Klein,

2006; Troost et al., 2012).

Ubiquitylation of Notch also has animportant function in the regulation ofNotch trafficking. A number of E3

ubiquitin ligases, including Suppressor ofdeltex [Su(dx)] (ITCH in mammals), andNedd4, Cbl, have been shown to target

Notch for degradation (Sakata et al., 2004;Wilkin et al., 2004; Jehn et al., 2002).Deltex (Dx) is a RING-finger E3 ubiquitin

ligase that regulates the late-endosomalactivation of Notch in a ligand-independent manner (Hori et al., 2004;

Hori et al., 2005). However, when Dxforms a complex with Kurtz (Krz), a

non-visual b-arrestin, it promotes thedegradation of Notch (Mukherjee et al.,2005). This degradation of Notch by Dxand Krz is suppressed in the mutant form

of Vps32, a component of the ESCRT-IIIcomplex (Hori et al., 2011). Thus, Dx canregulate Notch signaling in either a

positive or a negative manner, dependingon its interactions with other regulatoryfactors. Other factors that are involved

in trafficking or fusion between lateendosomes and lysosomes, such as theHOPS and AP3 complexes, are alsorequired for Dx-dependent activation of

Notch (Wilkin et al., 2008). Finally, theclathrin adaptor AP1 complex, whichlocalizes to the trans-Golgi network and

to recycling endosomes, has also beenshown to regulate the trafficking of Notchduring SOP (Benhra et al., 2011) and eye

(Kametaka et al., 2012) development inDrosophila.

It has become clear that the mechanisms

underlying Notch trafficking through theendosomal pathway are complex. This isreflected by the fact that genetic analysis ofthe various trafficking elements reveals

several distinct phenotypic characteristicsthat influence Notch signaling in a strictlycontext-dependent manner (Hori et al.,

2011).

DSL ligand trafficking

Endocytosis and endosomal traffickingof DSL ligands within the signal-sendingcell are also essential requirementsfor activation of the Notch receptor

(Yamamoto et al., 2010; Baron, 2012).Two RING-type E3 ligases, Neuralized(Neur) and Mindbomb (Mib), promote

ligand endocytosis by ubiquitylation (LeBorgne et al., 2005; Wang and Struhl,2005). Epsin, a ubiquitin-binding protein

that interacts with phosphoinositol lipidsand endocytic proteins, such as clathrinand AP-2, has been demonstrated toregulate ligand endocytosis (Wang and

Struhl, 2004). Rab11-positive recyclingendosomes have been shown to berequired for Delta activity during SOP

development in Drosophila (Emery et al.,2005). Recent studies show that ligandrecycling does not change the bond

strength of the ligand receptor, butappears to regulate the accumulation ofligands at the cell surface (Shergill et al.,

2012). Following interactions with Notchon the surface of adjacent cells, a secondendocytic event has been proposed that

generates a ‘pulling force’ to activateNotch proteolysis, subsequently triggering

trans-endocytosis of the NECD into thesignal-sending cell, thereby releasingNICD in the signal-receiving cell (Tien

et al., 2009; Musse et al., 2012).

Nuclear events in Notch signalingThe core components of the nuclear Notchcomplex include NICD, CSL and Mam(Bray, 2006; Kovall and Blacklow, 2010).

In the absence of NICD, CSL is associatedwith co-repressors, such as CtBP, Hairless,Groucho, SMRT, SHRP (also known as

Sav), MINT (also known as X11L) andSPEN (Schweisguth and Posakony, 1994;Zhou and Hayward, 2001; Barolo et al.,

2002; Oswald et al., 2005). These co-repressors recruit histone deacetylases(HDACs) and other cofactors, therebyrepressing the activation of Notch target

genes until the NICD is presented in thenucleus (Hsieh et al., 1999; Zhou et al.,2000; Nagel et al., 2005). Upon activation

of the Notch receptor, the released NICDmoves from the cytoplasm to the nucleus.Here, the RAM domain of NICD allows its

interaction with CSL, which facilitates thebinding of MAM at the interface betweenthe ANK domain of NICD and CSL(Choi et al., 2012). This complex

further recruits coactivators, such ashistone acetyltransferases (CBP/p300) andchromatin remodeling complexes, which

mediate the transcription of Notch targetgenes (Wallberg et al., 2002; Kadam andEmerson, 2003; Gause et al., 2006).

Kinases, such as glycogen synthasekinase 3b (GSK3b), granulocyte colonystimulating factor (G-CSF) and cyclin C/

cyclin-dependent kinase-8 (CDK8), havebeen shown to phosphorylate NICD, whichis thought to be important for the stabilityof NICD and hence its activity (Ingles-

Esteve et al., 2001; Espinosa et al., 2003;Fryer et al., 2004). The E3 ubiquitin ligaseSEL10 (also known as FBXW7) modifies

NICD and targets it for proteasomaldegradation (Gupta-Rossi et al., 2001;Tsunematsu et al., 2004).

The role of Notch in developmentNotch function during development

Notch signaling is remarkably pleiotropicand there is hardly a tissue that is not

affected by cell fate choices that areregulated by Notch signaling. Thedevelopmental consequence of either

down- or upregulating of Notch signalingis strictly context-specific, and the sameNotch signal might, for instance, in one

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context promote proliferation, whereas inanother result in apoptosis. Hence, the way

in which Notch signaling is integrated withother signaling pathways in the context ofa particular cellular physiology, dictateshow Notch activity affects cell fate. In

spite of this spatial and temporalcomplexity, lateral specification eventsare the hallmark of Notch-regulated

cell fate (Greenwald, 1998). Lateralspecification (a more general andaccurate term than ‘lateral inhibition’,

which is often used when referring toNotch) describes the mechanism by whichcells that are in the process of adoptingone particular cell fate influence the fate

of a cellular neighbor (see poster).Nevertheless, many lineages rely onNotch-mediated lateral inhibition, as is

the case of neuroblast differentiationwithin the neurogenic region of theDrosophila embryo, the classic example

of the developmental action of Notch. Inthis case, a cell that is about to becomea neuroblast inhibits its immediate

neighbors from adopting the same fate. Interms of morphogenesis, this has twoimportant implications: Notch helps inthe segregation of specific lineages from

a field of developmentally equivalent cells,and Notch signaling is also one of severalcrucial mechanisms that specifies borders

between cellular fields (Bray, 2006).

An analogous function of Notch hasbeen documented during the differentiation

of the peripheral nervous system inDrosophila, where the analysis of theSOP cell lineage has provided a usefulmodel for understanding the role of Notch

signaling in binary cell-fate decisions (seeposter). After SOP cell division, one of thedaughter cells becomes the signal-sending

cell, whereas the other cell becomesthe signal-receiving cell, leading toasymmetric activation of Notch signaling.

The progeny again uses the Notch pathwayin a second round of cell division toestablish different cell fates (socket, hair,

sheath and neuron) (Bray, 2006). Asalluded to above, Notch signaling canalso contribute to the establishmentof boundaries between distinct cell

populations to segregate the two groupsof cells (see poster).

Notch in stem cells

In general, activation of the Notchsignaling pathway is associated with early

cell lineages in development, makingNotch a very good marker for lineagetracing (Fre et al., 2011). Notch signaling

has been implicated in the regulation of an

increasing number of stem cells in manydifferent tissues, leading to it beingcharacterized as a ‘stem cell pathway’

(Liu et al., 2010). It is not possible toreview the spectrum of stem cell systems,in which Notch has been implicated, in thecontext of this article. However, we need to

emphasize that Notch activity is pervasiveand important for the differentiation,maintenance and proliferation of stem

cells in almost every system examined,including stem cell lineages, intestines,haematopoietic system, germline, various

epithelia, muscle, mesenchyme, nervoussystem, and others (Liu et al., 2010;Perdigoto and Bardin, 2013; Kageyama

et al., 2010; Conboy and Rando, 2002;Conboy et al., 2003; Carlson et al., 2008).

As an example, the adult Drosophila

midgut contains intestinal stem cells

(ISCs), which divide to self-renew and toproduce committed progenitor cells calledenteroblasts (Micchelli and Perrimon,

2006; Ohlstein and Spradling, 2006)(see poster). The enteroblasts furtherdifferentiate terminally into absorptiveenteroctye cells and secretory

enteroendocrine cells (Micchelli andPerrimon, 2006; Ohlstein and Spradling,2006). Notch signaling has been shown to

mediate asymmetric cell divisions of ISCsin the adult midgut in Drosophila. Delta ishighly expressed in ISCs (Ohlstein and

Spradling, 2007) and induces enteroblaststo differentiate to the enteroctye cell fate,whereas its presence at only low levels has

been proposed to promote the secretoryenteroendocrine cell fate (Ohlstein andSpradling, 2007). The fucosylation ofNotch has been implicated in the ISC

commitment (Perdigoto et al., 2011).

Proliferation and apoptosis

Notch signaling has been shown to direct

cells into proliferative or apoptotic states ina context-specific manner (Pallavi et al.,2012). Interestingly, Notch has both cell-

autonomous and non-cell-autonomouseffects on mitotic activity, which it caneither promote or suppress depending on

the cellular context. Although manyaspects of Notch signaling in proliferationand apoptosis remain poorly understood,its potential to link these events to

differentiation might be of particularrelevance to dysproliferative states,including cancer (Bray, 2006; Fiuza and

Arias, 2007; Bray and Bernard, 2010).

The developmental context of a cellappears to dictate how activation of Notch

affects the cell cycle. The underlying

basis appears complex, as, for instance,activation of Notch signaling can haveeither oncogenic or tumor-suppressive

effects in tumors of the same type. As isthe case in stem cell differentiation, thisapparent complexity is likely to be theresult of crosstalk between Notch signaling

and other signaling pathways (Hurlbutet al., 2007; Koch and Radtke, 2010;Louvi and Artavanis-Tsakonas, 2012;

Colombo et al., 2013).

The role of Notch signaling indiseaseGiven the profound and widespread rolesof Notch signaling across a range oftissues, it is perhaps no surprise that

abnormal Notch signaling is involved inseveral inherited diseases in humans,notably cancer. The link between Notchsignaling and disease has been reviewed

recently (Gridley, 2003; Fiuza and Arias,2007; Louvi and Artavanis-Tsakonas,2012; Penton et al., 2012); the tables

shown on the poster summarize theinherited syndromes that are associatedwith abnormal Notch signaling and

emphasize the highly pleiotropic natureof Notch, as these syndromes present abroad range of clinical symptoms. Notch

has been increasingly appreciated to have amajor direct and indirect role in cancer.Both solid tumors and leukemias havebeen associated with Notch where it has

been shown, depending on the context, toact both as an oncogene and a tumorsuppressor.

PerspectivesSince the discovery of the Notch locus inDrosophila almost 100 years ago, great

progress has been made in elucidating thecore mechanism of the canonical Notchsignaling pathway. Recent genome-scale

studies reveal an extraordinarily complexnetwork of genes that can affect Notchactivity in Drosophila (Guruharsha et al.,2012), and this cohort of genes is

complemented and extended by studiesin other organisms. This highlyinterconnected network contrasts with any

conventional view of the Notch signalingpathway as a simple linear sequence ofevents. Although we now have an

unprecedented insight into the way inwhich such a fundamental signalingmechanism is controlled by the genome,

we are faced with serious challenges inanalyzing the underlying molecularmechanisms of Notch signal control.

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However, systems-level approaches shouldshed light on the complex molecular

circuitry that governs this pathway andprovide insights into the mechanisms thatare relevant to Notch-related pathologies.

Acknowledgements

A short, schematic summary as this one,

involving a pathway with so many different

developmental actions is inevitably

incomplete and hence also somewhat

biased, in spite of our best efforts. We

apologize to all those colleagues whose

important studies could not be covered due

to space limitations. We would like to thank

Robert A. Obar for critically reading the

manuscript.

Funding

This work was supported by the National

Institutes of Health [grant numbers

NS26084 and CA98402 to S.A.-T.]; a

JSPS Postdoctoral Fellowship for Research

Abroad (to K.H.); and a Postdoctoral

Fellowship from the FSMA (to A.S.).

Deposited in PMC for release after 12

months.

A high-resolution version of the poster is available for

downloading in the online version of this article at

jcs.biologists.org. Individual poster panels are available

as JPEG files at http://jcs.biologists.org/lookup/suppl/

doi:10.1242/jcs.127308/-/DC1

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