Regulation of Notch Signaling Through Intracellular Transport

CHAPTER FOUR

Regulation of Notch Signaling Through Intracellular Transport Sean D. Conner Department of Genetics, Cell Biology, and Development, University of Minnesota, Twin Cities, Minneapolis, MN, United States of America E-mail address: [email protected]

Contents 1. Introduction 2. Notch Internalization 2.1 Endocytic Activation Model 2.2 Internalization-Independent Notch Signaling 2.3 Ubiquitination and Notch Endocytosis 3. Endosome Entry 4. Notch Recycling 4.1 Numb Inhibits Notch Recycling in Drosophila 4.2 Differential Regulation of Notch by Numb 4.3 Receptor Recycling Promotes Notch Activity 5. Notch Degradation Within Lysosomes 5.1 ESCRT-Dependent Notch Degradation 5.2 ESCRT-Associated Factors Facilitate Notch Transport 5.3 ESCRT Mutations Mistarget Notch and Elevate Signaling 5.4 E3 Ubiquitin Ligase Activity Controls Notch Degradation 6. Deltex: Critical Determinant in Endosomal Transport Decisions 6.1 Deltex Promotes Signaling in the Absence of Ligand 6.2 Mammalian DTX1 Downregulations Notch Activity 7. Concluding Remarks Acknowledgments References

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Abstract The highly conserved Notch-signaling pathway performs a central role in cell differentiation, survival, and proliferation. A major mechanism by which cells modulate signaling is by controlling the intracellular transport itinerary of Notch. Indeed, Notch removal from the cell surface and its targeting to the lysosome for degradation is one way in which Notch activity is downregulated since it limits receptor exposure to

International Review of Cell and Molecular Biology, Volume 323 ISSN 1937-6448 http://dx.doi.org/10.1016/bs.ircmb.2015.12.002

© 2016 Elsevier Inc. All rights reserved.

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ligand. In contrast, Notch-signaling capacity is maintained through repeated rounds of receptor recycling and redelivery of Notch to the cell surface from endosomal stores. This review discusses the molecular mechanisms by which Notch transit through the endosome is controlled and how various intracellular sorting decisions are thought to impact signaling activity.

1. INTRODUCTION The canonical Notch-signaling pathway is central to processes ranging from cell-fate specification to cell viability (Andersson et al., 2011; ArtavanisTsakonas and Muskavitch, 2010). Notch is a type I integral membrane cell surface receptor that forms a heterodimer after being cleaved by furin during its transit through the biosynthetic pathway (Logeat et al., 1998). Signaling is initiated when Notch binds one of several ligands belonging to the Delta, Serrate, and Lag-2 (DSL) family of integral membrane proteins, which are expressed on the surface of neighboring cells (D’Souza et al., 2008, 2010; Kopan and Ilagan, 2009). After ligand binding, the Notch extracellular region undergoes a conformational change (Gordon et al., 2007), which experimental evidence suggests results from a pulling force generated by Notch-bound ligand internalization into the signaling cell (Fig. 1, Musse et al., 2012; MelotyKapella et al., 2012; Gordon et al., 2015). The resulting conformational

[(Figure_1)TD$IG] Signaling cell

Ligand pulling by endocytosis drives Notch conformational change

DSL Notch

ADAM

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Endocytosisindependent signaling γ-Secretase

Endocytosis

Recycling

Notch conformational change exposes ADAM cleavage site

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Early/sorting endosome γ-Secretase

Lysosome

NICD

Nucleus

Figure 1 Model illustrating Notch activation by ligand followed by endocytosisdependent (A) or endocytosis-independent (B) γ secretase-mediated Notch cleavage and subsequent NICD release from the membrane.

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change in Notch exposes an ADAM family metalloprotease cleavage site (Brou et al., 2000; van Tetering et al., 2009). This leads to proteolytic release of the ectodomain, leaving a membrane-tethered Notch extracellular truncation fragment (NEXT). NEXT then serves as a substrate for γ secretasemediated intramembrane cleavage (De Strooper et al., 1999). This liberates the Notch intracellular domain (NICD, a transcription factor) into the cytoplasm, which can then target to the nucleus and coordinate gene expression (Kitagawa et al., 2001). In general, Notch-signaling capacity is influenced by receptor exposure to ligand at the surface of signal-receiving cells. As a consequence, Notch transit to and from the cell surface is tightly controlled by a host of factors. This review focuses on the mechanisms that coordinate Notch entry into cells through the endocytic pathway, the sorting decisions that occur following delivery to endosomes, and the role of ubiquitination in controlling Notch activity. Although not covered in this review, the mechanisms governing ligand transport in signal-sending cells are equally critical to controlling the Notch pathway. Thus, the reader is directed to several excellent reviews on the topic (D’Souza et al., 2010; Musse et al., 2012; Weinmaster and Fischer, 2011).

2. NOTCH INTERNALIZATION Notch is constitutively internalized (McGill et al., 2009; Sakata et al., 2004; Wilkin et al., 2004). Thus, endocytosis modulates signaling capacity by controlling the extent of Notch exposure to ligand at the cell surface. While there is general agreement in the field that Notch removal from the plasma membrane limits signaling, the role of endocytosis following Notch activation by ligand remains hotly debated. To account for myriad, and sometimes contradictory findings arising from studies to resolve how Notch transport impacts signaling, two contrasting, but not mutually exclusive, models have been proposed (Fig. 1). In the first model, ligand-activated Notch (NEXT) must be internalized and delivered to the acidic endosomes for γ secretase-mediated cleavage. In the second model, γ secretase cleaves NEXT at the plasma membrane and thus, signaling occurs independent of Notch endocytosis.

2.1 Endocytic Activation Model The idea that NEXT must be internalized and delivered to endosomes before γ secretase-dependent cleavage was formulated based on two genetic

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studies in Drosophila where mutations in core components of the endocytic machinery lead to defects in Notch endocytosis and signaling. For example, follicle cells of the developing ovary are unable to activate the Notch-signaling pathway when dominant-negative forms of dynamin are expressed (Vaccari et al., 2008). Similarly, Notch internalization and signaling are impaired in cells expressing a mutant form of clathrin (Windler and Bilder, 2010). Curiously, signaling was unaffected in cells lacking AP-2, an adaptor complex that links clathrin to endocytic cargo (Brodsky, 2012; Robinson, 2004), despite the fact that Notch uptake was robustly inhibited. These observations, combined with the firmly established roles for dynamin, clathrin, and AP-2 in receptor-mediated endocytosis (McMahon and Boucrot, 2011), were interpreted to indicate that Notch activity is differently regulated by receptor uptake through two functionally distinct endocytic routes: (1) an AP-2-dependent pathway that downregulates Notch by removing the receptor from the cell surface in the absence of ligand, and (2) an AP-2-independent route that requires clathrin and dynamin to deliver ligand-activated NEXT to γ secretase-containing endosomes to promote signaling (the Endocytic Activation model, Fig. 1A). In support of the Endocytic Activation model, in vitro biochemical studies demonstrated that γ secretase, isolated from rat liver, is optimally active at a pH <6.5 (Pasternak et al., 2003). This finding is consistent with the idea that NEXT would be efficiently processed by γ secretase following delivery to acidic endosomes—the pH progressively drops during transit through the endosome from ∼6.0 to 6.3 in the early/sorting endosome to ∼5.0–5.5 within the lysosome (Maxfield and McGraw, 2004; Yamashiro and Maxfield, 1987). Additional evidence suggesting that NEXT must be delivered to acidic endosomes derives from the observation that mutations which disrupt vacuolar ATPase activity, a pump critical for endosome acidification (Mindell, 2012), lead to Notch-signaling defects and receptor accumulation within the late endosome (Yan et al., 2009).

2.2 Internalization-Independent Notch Signaling The Endocytic Activation model provides a framework with which to explain how Notch is internalized and why internalization is critical for signaling. However, additional lines of evidence support an alternative model where internalization of activated Notch and its delivery to endosomelocalized γ secretase is not essential for signaling. For example, Shaye and Greenwald (2002) identified a short region within the LIN-12/Notch cytoplasmic tail that is essential for robust receptor endocytosis and

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downregulation of the signaling pathway in Caenorhabditis elegans (Shaye and Greenwald, 2002). Importantly, they also discovered that an internalizationdefective form of LIN-12/Notch efficiently rescues the hallmark nonvuval defect that arises in animals genetically null for lin-12. This latter finding demonstrates that LIN-12/Notch endocytosis is not a prerequisite for signaling in C. elegans. Given that Notch signaling in worms, like that in flies and mammals, is γ secretase dependent (Francis et al., 2002), this finding provides in vivo evidence against the idea that Notch internalization is absolutely essential for signaling. Unfortunately, this work remains largely unnoticed in the literature where the relationship between Notch and endocytic transport is discussed (Baron, 2012; Fortini and Bilder, 2009; Le Bras et al., 2011; Moretti and Brou, 2013; Pratt et al., 2010; Sala et al., 2012; Tien et al., 2009; Yamamoto et al., 2010). If the model where Notch signaling occurs independent of endocytosis is correct (Shaye and Greenwald, 2002), γ secretase must be present and active at the plasma membrane without requiring an acidic environment to cleave ligand-activated Notch. Indeed, evidence from several independent research groups reveal that γ secretase is active over a broad physiologic pH range (5.5–8.4) with optimal activity between pH 6.8 and 7.5 (Lee et al., 2002; McLendon et al., 2000; Zhang et al., 2001). Moreover, in vitro studies reveal that γ secretase is not only present on the plasma membrane in mammalian CHO cells, but is proteolytically active and capable of cleaving Notch substrates (Chyung et al., 2005). Numerous studies also reveal that γ secretase cleaves NEXT at the plasma membrane in live cells. For example, γ secretase-dependent Notch signaling is unaffected in Drosophila melanogastor when endocytosis is acutely perturbed in cells expressing a dominant-negative mutant form of dynamin (Struhl and Adachi, 2000). Similarly, γ secretase-mediated signaling is unimpaired or even elevated in HEK293 or HeLa tissue culture cells when endocytosis is disrupted by dominant-negative K44A dynamin overexpression (Kaether et al., 2006; Sorensen and Conner, 2010; Tagami et al., 2008) or by siRNA-mediated depletion of clathrin or AP2 (Sorensen and Conner, 2010). Moreover, when γ secretase activity is impaired in cells expressing mutant forms of nicastrin or presenilin in D.melanogastor, NEXT accumulates at the cell surface (Guo et al., 1999; Lopez-Schier and St Johnston, 2002), similar to that observed in HeLa cells when γ secretase is inhibited with the drug Compound E (Sorensen and Conner, 2010). Additionally, Notch signaling is disrupted in HEK293 cells treated with a membrane-impermeable γ

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secretase inhibitor, MRL631, while γ secretase-mediated processing of amyloid precursor protein within the endosome remains unperturbed (Tarassishin et al., 2004). Finally, studies by Tagami et al. (2008) demonstrated that γ secretase-dependent Notch cleavage at the plasma membrane predominantly yields the stable, and well-established V1744-NICD cleavage product (Schroeter et al., 1998). In contrast, γ secretase cleavage within endosome-enriched fractions resulted in NICD products capped by the amino terminal serine or leucine, which was found to be less stable due to proteome-mediated degradation (Tagami et al., 2008). Collectively, these broad range of observations from a variety of systems using multiple experimental strategies lend strong support to the conclusion that endocytosis is not essential for ligand-dependent Notch signaling. Instead, they support a model where endocytosis downregulates signaling capacity by reducing the potential for Notch interaction with ligand at the cell surface. Given this, how does one reconcile the elegant genetic evidence demonstrating that Notch signaling requires clathrin and dynamin (Vaccari et al., 2008; Windler and Bilder, 2010)? In addition to roles in endocytosis, dynamin and clathrin function broadly along the membrane transport system to coordinate receptor traffic through the cell (Brodsky et al., 2001; Brodsky, 2012; Praefcke and McMahon, 2004). For example, dynamin family members facilitate transport to and from the Golgi apparatus (Jones et al., 1998; Lauvrak et al., 2004), as well as promoting receptor recycling from the endosome (van Dam and Stoorvogel, 2002). Likewise, clathrin is critical for promoting cargo transport to and from the Golgi and between endosomal compartments (Bonifacino and Rojas, 2006; Burgess et al., 2011; Sachse et al., 2002; Stahlschmidt et al., 2014), including recycling to the plasma membrane following endocytosis (Zhao and Keen, 2008). Thus, the Notch-signaling defect observed when clathrin or dynamin activity is disrupted (Vaccari et al., 2008; Windler and Bilder, 2010) may not result from a selective disruption in receptor internalization. Instead, defects in recycling or in the biosynthetic pathway might lead to insufficient Notch at the cell surface and lead to impaired signaling. Consistent with this idea, Notch accumulates in the trans Golgi when clathrin is depleted by siRNA treatment in HeLa cells (Sorensen and Conner, 2010).

2.3 Ubiquitination and Notch Endocytosis Ubiquitination is thought to regulate Notch cell surface levels by promoting receptor endocytosis (Le Bras et al., 2011). Indeed, Nedd4 depletion in

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mammalian cell culture disrupts Notch internalization kinetics (Sorensen and Conner, 2010). In comparison, overexpression of a Nedd4 mutant disrupts Notch endocytosis in D. melanogastor S2 culture cells (Sakata et al., 2004). Moreover, coimmunoprecipitation studies show that Notch interacts with and is ubiquitinated following Nedd4 overexpression (Sakata et al., 2004). Similar to findings for Nedd4, Notch is also ubiquitinated following Deltex overexpression (Hori et al., 2011). Notch accumulates at the plasma membrane in cells lacking the E3 ubiquitin ligase in Drosophila (Yamada et al., 2011) or following DTX-1 depletion in mammalian cell culture (Zheng et al., 2013). However, evidence supporting a direct role for Deltex in Notch internalization is currently lacking. Given that Deltex facilitates Notch-sorting decisions at the late endosome (Hori et al., 2004, 2011; Wilkin et al., 2008), it is possible that Notch accumulation at the cell surface does not arise from a direct internalization defect, but instead results from an endosomal sorting defect that leads to increased Notch recycling (see Section 5.3). Taken together, these findings demonstrate that Nedd4 and Deltex modulate Notch cell surface levels. However, given that neither factor is absolutely essential for Notch signaling (Sakata et al., 2004; Yamada et al., 2011), it may be that Notch ubiquitination by these E3 ligases serves to fine tune Notch-signaling capacity depending on the environmental context. Despite a clear role for ubiquitination in regulating Notch transport, ubiquitination sites on Notch have not been definitively mapped. Initial mammalian cell culture studies pointed to a critical juxtamembrane lysine (K1749 in mouse) as a site for Notch monoubiquitination—modification of which was thought to trigger internalization and processing by γ secretase (Gupta-Rossi et al., 2004). However, subsequent experiments revealed that Notch cleavage by γ secretase at the plasma membrane is not blocked by the K1749 site mutation. Instead, the cleavage site for γ secretase is shifted, which produces a proteosome-sensitive product (Tagami et al., 2008). Thus, determining the precise ubiquitination sites will be critical to resolving how Notch activity is differentially regulated by each E3 ubiquitin ligase.

3. ENDOSOME ENTRY Following endocytosis, Notch is delivered to the early/sorting endosome—a compartment where receptors are sorted toward one of the several

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[(Figure_2)TD$IG] Notch

rab5 Avl

Numb AP-1

BBS1 BBS4 RME-8

Itch/ Su(dx)

UB

ESCR Ts 0 I II III

Deltex Early/sorting endosome UB

AP3 HOPS

UB

Lysosome γ-Secretase

Nucleus

UB

MVE LIS

Figure 2 Notch endosomal sorting decisions the lead to Notch degradation, recycling, or ligand-independent signaling (LIS) from the lysosome.

destinations (Fig. 2): (1) the receptor can be recycled to the plasma membrane, enabling additional opportunity for interaction with ligand; (2) the receptor can be directed toward an endosomal storage compartment for future use, or (3) the receptor can be sent to the lysosome (Hsu et al., 2012; Maxfield and McGraw, 2004). Thus, blocking Notch delivery to the early/sorting endosome should disrupt receptor transport and impair Notch signaling. Indeed, mutations in early endosome transport genes in Drosophila like rab5 or the syntaxin7 homolog, Avalanche(Avl), disrupt Notch delivery to early endosomes and results in receptor accumulation within cells (Lu and Bilder, 2005; Vaccari et al., 2008). This, in turn, limits Notchsignaling capacity. While these findings illustrate an important role for Notch transit through the endosome, our understanding of the mechanisms that direct Notch toward a recycling, storage, or degradative route remain incomplete.

4. NOTCH RECYCLING 4.1 Numb Inhibits Notch Recycling in Drosophila Notch is constitutively internalized and recycled (McGill et al., 2009). This likely maintains the capacity of cells to respond to ligand. Thus, downregulating

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the recycling pathway is a critical step in controlling Notch activity. Indeed, live cell imaging studies combined with antibody uptake experiments in Drosophila reveal a critical role for Numb in suppressing Notch recycling in asymmetrically dividing cells (Cotton et al., 2013; Couturier et al., 2013). However, Numb-dependent inhibition of Notch recycling does not result from a direct interaction between Numb and Notch. Instead, Numb is thought to prevent Notch redelivery to the cell surface indirectly by suppressing the recycling of Sanpodo, an integral membrane protein that directly binds to and cotrafficks with Notch and is critical for signaling (Couturier et al., 2012; O’Connor-Giles and Skeath, 2003; Upadhyay et al., 2013). Similarly, the heterotetrameric endosomal adaptor protein complex, AP-1, also negatively regulates Notch activity by inhibiting Sanpodo recycling from rab11-positive endosomes in flies (Benhra et al., 2011). However, the generality of these findings in relation to other species is currently unclear given that orthologs to Sanpodo in other species have yet to be identified.

4.2 Differential Regulation of Notch by Numb A conserved role for Numb as a negative regulator of Notch signaling is supported by the fact that Numb loss correlates with increased Notch signaling in 50% of primary human breast cancer cells (Pece et al., 2004). Additionally, Numb overexpression suppresses Notch activity in preventing myogenesis in cultured C3H10T1/2 cells (Beres et al., 2011). Although, this latter observation is not thought to result from changes in Numb-mediated Notch transport. Instead, Numb is thought promote the recruitment of Itch to Notch. Itch is an E3 ubiquitin ligase (see Section 5.4) whose activity promotes Notch ubiquitination and proteosomal degradation (McGill and McGlade, 2003). This is consistent with a general role for Numb in promoting Itch-mediated receptor degradation (Di Marcotullio et al., 2006). However, Numb activity in inhibiting receptor recycling also appears to be conserved, although its precise mode of action is unclear. For example, Numb overexpression in mammalian cell culture promotes Notch sorting through late endosomes while Numb depletion enhances receptor recycling (McGill et al., 2009). Likewise, NUM-1, the C. elegans Numb homolog, was shown to perform a general role in inhibiting endosomal recycling (Nilsson et al., 2008). Curiously, Num-1 loss-of-function enhances LIN12/Notch-signaling defects in animals treated with lin12 RNAi (Korcsmaros et al., 2011), suggesting that NUM-1 promotes Notch signaling in C. elegans. Similarly, genetic loss-of-function studies in mice indicate that

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Numb promotes Notch activity during neurogenesis (Petersen et al., 2002, 2004; Zhong et al., 2000; Zilian et al., 2001). Collectively, these studies demonstrate that Numb can act both as a positive and a negative regulator of Notch. Unfortunately, they also illustrate the difficulty in developing a simple mechanistic model that explains how Numb regulates the Notch pathway. Thus, whether or not Numb promotes or inhibits Notch signaling may depend on cellular context and the interplay with cell-type specific components.

4.3 Receptor Recycling Promotes Notch Activity While genetic and ex vivo cell culture based data indicate that Numb can inhibit Notch recycling (Cotton et al., 2013; Couturier et al., 2013; McGill et al., 2009), recent studies demonstrate a role for Bardet–Biedl syndrome (BBS) proteins in regulating Notch activity. BBS proteins form a stable complex (the BBSome) consisting of seven highly conserved proteins (BBS1, 2, 4, 5, 7, 8, 9) that form a coat on membranes to facilitate receptor delivery to and from cilia (Jin et al., 2010). Depletion of BBS1 or BBS4 in mammalian cell culture was recently found to redistribute Notch from the plasma membrane to the late endosome (Leitch et al., 2014). This result, combined with a reduction in receptor localization in rab11-positive recycling endosomes, led to the conclusion that BBS proteins promote Notch redelivery to the cell surface from recycling endosomes (Leitch et al., 2014). Similar to that observed following reduced expression of BBSome proteins, loss of the highly conserved endosome-associated recycling factor, RME-8, leads to a reduction in Notch at the plasma membrane, accumulation within rab4-positive endosomes, and a decrease in Notch-signaling capacity in Drosophila (Gomez-Lamarca et al., 2015). Taken together, these published studies indicate an important role for receptor recycling to maintain appropriate levels of Notch and maintain signaling capacity.

5. NOTCH DEGRADATION WITHIN LYSOSOMES 5.1 ESCRT-Dependent Notch Degradation The targeting of integral membrane proteins for degradation within lysosomes is thought to require the coordinated activity of the endosomal sorting complex required for transport (ESCRT) machinery. Originally identified in yeast, the ESCRT machinery consists of four protein complexes: ESCRT 0,

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I, II, III, and several accessory factors which act in series to capture and deliver ubiquitinated cargo into intralumenal vesicles of multivesicular endosomes (MVEs, Henne et al., 2011; Wegner et al., 2011). MVE fusion with lysosomes then deliver cargo to the proteolytic enzymes within the lysosome lumen (Fig. 2). Not surprisingly, mutations in proteins belonging to each of the previously mentioned ESCRT complexes impact Notch endosomal transport, but differentially alter signaling capacity. For example, genetic analysis in the Drosophila reveals that mutations in ESCRT 0 (eg, Hrs and STAM) lead to enlarged Notch-containing endosomes, although Notch signaling is unaffected (Childress et al., 2006; Jekely and Rorth, 2003; Thompson et al., 2005; Vaccari et al., 2008). In contrast, mutations in ESCRT I components like Tsg101/erupted or Vps28 promote Notch accumulation in Hrs-positive endosomes and upregulate Notch signaling (Moberg et al., 2005; Vaccari et al., 2009). Similarly, receptor transport defects and/or elevated Notch signaling are observed when either ESCRT II (Vps22/EAP30, Vps25p/EAP20, Thompson et al., 2005; Herz et al., 2009; Vaccari et al., 2009; Vaccari and Bilder, 2005) or ESCRT III (Vps2/ CHMP2, Vps20/CHMP6, Vps32/shrub/CHMP4, Vaccari et al., 2008, 2009; Thompson et al., 2005; Aoyama et al., 2013) components are mutated. Curiously, vps36 mutations increase Notch protein levels in enlarged endosomes, yet ectopic Notch signaling is not observed, contrasting mutations in other ESCRT II components (Herz et al., 2009).

5.2 ESCRT-Associated Factors Facilitate Notch Transport Several associated factors have been identified that work in concert with ESCRTs to facilitate Notch transport along the degradative pathway. For example, Vps31/Alix is thought promote ESCRT-dependent cargo sorting and incorporation into MVEs (Bissig and Gruenberg, 2014). RNAi silencing of vps31/Alix in C.elegans impairs Lin12/Notch degradation (Shaye and Greenwald, 2005). Lethal giant discs (Lgd) is putative phospholipid binding protein that directly binds the ESCRT III component, Shrub, to coordinate activity of the complex (Troost et al., 2012). Lgd mutations or overexpression leads to defects in Notch transport and ectopic signaling (Childress et al., 2006; Gallagher and Knoblich, 2006; Jaekel and Klein, 2006; Parsons et al., 2014). Finally, Lin12/Notch transport defects result in C. elegans mutants lacking sel-2 (de Souza et al., 2007), a homolog to members of the mammalian neurobeachin family that influence endosomal sorting decisions and directly bind Hrs (de Saint Basile et al., 2010).

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5.3 ESCRT Mutations Mistarget Notch and Elevate Signaling As mentioned earlier, when the ESCRT machinery is mutated in Drosophila, elevated Notch signaling is observed. It has long been appreciated that defects in ESCRT activity can trigger mistargeting of biosynthetic and endocytic cargo to the recycling pathway or to the lysosome limiting membrane. For example, endocytosed epidermal growth-factor receptor is rapidly recycled back to the plasma membrane in mammalian cells expressing mutated Tsg101 (Babst et al., 2000). Thus, the ectopic Notch signaling observed in Drosophila lacking functional Tsg101/Erupted (Moberg et al., 2005) likely arises from receptor recycling and increased Notch exposure to ligand at the cell surface. In contrast, ectopic, ligand-independent Notch activation occurs when either vps25 or lgd is mutated (Childress et al., 2006; Gallagher and Knoblich, 2006; Jaekel and Klein, 2006; Vaccari and Bilder, 2005). These findings suggest that when early steps in the ESCRT pathway are disrupted (eg, Tsg101 mutations), Notch can be shunted to the recycling pathway and promote ligand-dependent signaling from the plasma membrane. In comparison, when later steps in the pathway are impaired (eg, vps25 or lgd mutations), Notch cannot be recycled. Instead, Notch remains on the endosome limiting membrane because it cannot be targeted into MVEs. From this location ligand-independent signaling can be initiated following endosome fusion with the lysosome (Fig. 2, Schneider et al., 2013).

5.4 E3 Ubiquitin Ligase Activity Controls Notch Degradation The suppressor of deltex (Su(dx)) encodes a HECT domain E3 ubiquitin ligase that negatively regulates Notch signaling in Drosophila (Cornell et al., 1999; Fostier et al., 1998; Mazaleyrat et al., 2003). Biochemical studies reveal that Itch, the mammalian Su(dx) homolog, directly ubiquitinates Notch (Qiu et al., 2000), while genetic studies reveal a critical role for Su (dx) in directing Notch postendocytic sorting toward late endosomes (Wilkin et al., 2004). Notch degradation is impaired in fibroblasts lacking Itch, which reinforces the conclusion that the E3 ligase directs Notch for lysosomal degradation (Chastagner et al., 2008). Consistently, RNAi silencing of wwp-1, the Su(dx) homolog in C. elegans, disrupts Lin12/ Notch downregulation and enhances receptor stability (Shaye and Greenwald, 2005). Moreover, Notch abundance also increases in mice lacking Itch (Matesic et al., 2006). Collectively, these findings support a model where Su(dx)/Itch/WWP-4 ubiquitinates Notch to direct its degradation within lysosomes.

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6. DELTEX: CRITICAL DETERMINANT IN ENDOSOMAL TRANSPORT DECISIONS 6.1 Deltex Promotes Signaling in the Absence of Ligand Deltex (Dx), a RING domain containing E3 ubiquitin ligase, is thought to positively regulate Notch activity in Drosophila. This is supported by two basic lines of evidence: (1) hypomorphic deltex mutations suppress Notch gain-of-function phenotypes (Xu and Artavanis-Tsakonas, 1990) and (2) Dx overexpression promotes ligand-independent Notch gain-of-function phenotypes (Hori et al., 2011). Mechanistically, Dx is thought to ubiquitinate Notch at the plasma membrane and promote receptor endocytosis (Hori et al., 2004; Matsuno et al., 1995; Yamada et al., 2011). Receptor ubiquitination is then thought to mark Notch for delivery to the lysosome-limiting membrane via an AP3/HOPs-dependent pathway (Wilkin et al., 2008). Once delivered, ligand-independent signaling can be initiated by γ secretase-mediated cleavage (Hori et al., 2011; Wilkin et al., 2008). However, the ability of overexpressed Dx to promote ligand-independent signaling depends on the cellular context as it does not universally activate Notch. In comparison, dx null mutations in Drosophila demonstrate that endogenous Dx is not essential for Notch signaling in any developmental context (Fuwa et al., 2006). However, Dx loss stabilizes Notch and prevents receptor degradation within lysosomes (Yamada et al., 2011). Whereas when Dx is coexpressed with the nonvisual β-arrestin, Kurtz, Notch protein levels are reduced (Mukherjee et al., 2005). Thus, on the one hand, overexpression studies indicate that Dx promotes ligand-independent signaling from late endosomes and/or lysosomes (Hori et al., 2011; Wilkin et al., 2008). On the other hand, loss-of-function analyses reveal that Dx is essential for Notch for degradation within lysosomes (Yamada et al., 2011). Mechanistically reconciling these findings with regard to how Dx regulates Notch endosomal sorting decisions is challenging. However, future studies may help to resolve the complexities of Dx mode of action.

6.2 Mammalian DTX1 Downregulations Notch Activity In comparison to Drosophila, genetic experiments in mice indicate that mammalian deltex 1 (DTX1) negatively regulates Notch activity in mammals. For example, Notch signaling promotes T-cell differentiation at the expense of B cells (Radtke et al., 2013). However, enforced DTX1

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expression in mice enhances B-lymphocyte development and impairs T-cell differentiation (Izon et al., 2002; Yun and Bevan, 2003). These findings indicate that DTX1 does not promote signaling, but instead downregulates Notch activity in mammals. Consistently, T-cell development occurs normally in mice lacking DTX1 (Hsiao et al., 2009). A negative regulatory role for DTX1 in Notch signaling does not appear to be cell-type specific given that liver stem cells genetically null for DTX1 and DTX2 reveal increased levels of Notch signaling (Lehar and Bevan, 2006). Moreover, DTX1 depletion in mammalian cell culture not only elevates signaling, but enhances Notch cell surface levels (Zheng et al., 2013). This later finding reinforces the idea that mammalian DTX1, like that in Drosophila, controls Notch transport decisions within cells. However, the transport step/pathway in which DXT1 acts has yet to be defined.

7. CONCLUDING REMARKS Our progress in understanding how receptor transit through the endosome impinges on Notch activity has been rapid over the last few years. While many of the core components that activate and downregulate the Notch pathway are known, the mechanisms that fine tune Notch-signaling capacity remain to be discovered. A combination of genetic, ex vivo, and biochemical approaches point to multiple levels of regulation where ubiquitination is critical to controlling Notch-sorting decisions. This presumably occurs by differential ubiquitination of Notch, although specific modification sites have yet to be determined. As mentioned earlier, ubiquitination controls Notch endocytosis (Sakata et al., 2004; Sorensen and Conner, 2010) and its subsequent targeting to either the lysosome lumen for degradation (Chastagner et al., 2008; Jehn et al., 2002; Wilkin et al., 2004; Yamada et al., 2011) or the lysosome limiting-membrane where ligand-independent signaling can be initiated (Hori et al., 2004, 2011; Wilkin et al., 2008). However, despite the well-documented role for E3 ubiquitin ligases in controlling the Notch transport and signaling, important questions remain. (1) Are there spatiotemporal mechanisms that control which E3 ligase activity predominates during Notch transit through the endosome? (2) Are the activities of each E3 ligase constitutive or regulated? (3) Which sites on Notch are modified and what types of modifications occur? (4) Which factors decode the modification status of Notch to direct receptorsorting decisions? Answers to these questions will undoubtably enrich our

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understanding of how receptor transit through the endosome is coordinated to regulate Notch activity.

ACKNOWLEDGMENTS I thank Wendy Gordon, Lihsia Chen, and Li Zheng for critical manuscript feedback and helpful discussions. Research in the Conner lab was supported by grants from the American Cancer Society (IRG-58-001-46-IRG46-01) and the National Institute of Health (R01 GM085029).

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