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Protein Degradation: Four E3s For

The Notch Pathway

Review

Eric C. Lai

545 Life Sciences Addition, University of California,

Department of Molecular and Cell Biology, Berkeley, California 94720-3200, USA.

The Notch pathway is a conserved signal transduction cascade which is essential for pattern formation and the proper execution of a wide array of cell-fate decisions. As only modest differences in Notch pathway activity suffice to determine dramatic differences in cellular behav-ior, this pathway is tightly regulated by a variety of molecular mechanisms. Several recent studies now highlight the importance of the ubiquitination pathway in the control of Notch pathway activity. This review will summarize recent advances in understanding the function of four E3 ubiquitin ligases that regulate levels of the Notch receptor and other components of this pathway. These include Suppressor of deltex/Itch and Sel-10, which both regulate Notch, Neuralized, which regulates the Notch ligand Delta, and LNX, which regulates the Notch antagonist Numb.

Introduction

The ubiquitination pathway regulates protein fate through covalent attachment of ubiquitin, a 76-amino acid peptide. This process is initiated when ubiquitin is transferred from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2). E3 ubiquitin ligases confer target specificity and associate with both E2 and substrate to catalyze the transfer of ubiquitin to the substrate or to the end of a polyubiq-uitin chain. Two of the most common uses of this pathway include polyubiquitination of soluble teins, which targets them for degradation by the pro-teasome, and monoubiquitination of transmembrane proteins, which signals their internalization by the endocytic pathway and subsequent degradation by the lysosome [1,2].

Two structurally distinct types of E3 are known, defined by possession of either a HECT domain or a RING finger. RING finger E3s are further subdivided into two classes, single peptide E3s, in which both substrate recognition and RING domains reside in the same protein, and multiprotein SCF E3 complexes, in which these activities are found in different proteins. Remarkably, four proteins representing each class of E3 have recently been shown to regulate the Notch (N) pathway.

The N pathway is a conserved signal transduction cascade of fundamental importance to the develop-ment of all metazoan organisms studied to date, from worms to humans. Both extrinsic and intrinsic mecha-nisms regulate activity of the transmembrane N recep-tor. For example, extracellular interactions with its transmembrane ligand Delta (Dl) allow neighboring cells to activate N. This results in the release of a pro-teolytic fragment of the N intracellular domain (NIC), which subsequently functions as a nuclear coactivator for a CSL transcription factor. Numb is an example of an intrinsic regulator; this intracellular protein autonomously antagonizes N activity and is typically distributed asymmetrically between cells whose fate is selected by N signaling. The N pathway is perhaps best known for mediating lateral inhibition, whereby the adoption of a particular cell fate is restricted within a group of equipotent cells, although it functions in some other types of developmental process as well.

Early evidence for the involvement of ubiquitination in the N pathway came from a study of conditional mutations in Drosophila proteasome subunits [3]. Although complete loss of proteasome function is lethal, a temporally restricted, genetic reduction in proteasome activity was found to result in specific cell-fate transformations during development of exte-rior mechanosensory organs. These defects, including shaft-to-socket and neuron-to-sheath transforma-tions, phenocopy excesses in N signaling, suggesting that the N pathway is negatively regulated by the pro-teasome. Moreover, the stability of NICwas increased in cells with compromised proteasomes, raising the possibility that N itself is a target of the proteasome. This hypothesis has been borne out by a group of recent papers that identify two types of ubiquitin ligase involved in degradation of N.

Notch gets Scratched by Itch

The Drosophila mutant Suppressor of deltex (Su(dx)) was originally identified as a suppressor of the wing vein thickening caused by deltex (dx), another muta-tion that displays extensive genetic interacmuta-tions with the N pathway [4,5]. In a seemingly unrelated research front, a mouse mutant (itchy) was noticed to have a persistent itching behavior and a variety of underlying immunological defects, including inflammation of the lung and stomach and hyperplasia of lymphoid and hematopoietic cells [6]. Curiously, these different mutations turn out to affect homologous proteins, which contain a C2 domain, four WW motifs, and a carboxy-terminal HECT-type E3 ubiquitin ligase module [7,8] (Figure 1).

Work from the Baron lab [9] demonstrated that

Su(dx) has genetic properties of a negative regulator

of N signaling. Loss of Su(dx) was found to suppress

N and Dl haploinsufficient phenotypes, and to enhance

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enhance N and Dl mutations. Interestingly, an anti-morphic allele was correlated with a small deletion in the HECT domain, and may encode a protein that can recognize its substrate but is unable to mediate its ubiquitination.

The Liu lab [10] then performed complementary biochemical studies with Itch, the results of which suggested that its target may be N. Itch associates with the intracellular domain of N via its WW domains and promotes ubiquitination of NICthrough its HECT domain (Figure 1). Although the subcellular localiza-tion of Su(dx)/Itch has not been tested, its possession of a C2 domain — a phospholipid-binding motif — suggests that it may be targeted to the plasma mem-brane. Indeed, Itch can interact with and promote ubiquitination of a membrane-anchored form of N. Although we await in vivo confirmation of alterations in N metabolism in Su(dx) flies or itchy mice, these studies strongly suggest that Su(dx)/Itch directly regulates N.

It is worth noting that dx encodes a RING finger protein, and so its product is another candidate for being a ubiquitin ligase in the N pathway. Although the precise role of Dx in the pathway is not well under-stood, a truncated form of Dx lacking the RING finger can rescue the dx phenotype and induce some gain-of-function phenotypes [11]. Thus, it does not appear that this domain is absolutely required for Dx’s func-tion. Nevertheless, the presence of the RING finger as well as two copies of a WWE domain — a motif found in several other proteins involved in ubiquitination, including both RING and HECT domain proteins — suggests that Dx normally interacts in some way with the ubiquitination pathway [12].

Sel-10 Knocks NICOut

The sel-10 gene was first identified as a suppressor mutation of the egg-laying defect caused by a hypomorph of lin-12, which encodes one of two N receptors in the nematode Caenorhabditis elegans [13]. Sel-10 behaves as a negative regulator of lin-12 activity, as lowering sel-10 dosage increases lin-12 activity, while elevating sel-10 dosage decreases lin-12 activity. Sel-10 is a Cdc4-related protein containing an F box and seven WD40 repeats (Figure 1), and can be found in a physical complex with NIC [14]. By analogy with Cdc4p, it was suggested that Sel-10 uses its F-box to recruit an SCF complex that ubiquitinates NIC.

Evidence for this model has come recently from a trio of papers from the Kitajewski, Lendahl and Israel labs [15–17] which focus on the function of mam-malian Sel-10. These papers show that Sel-10 binds to NICvia its WD40 domains and mediates its ubiquitina-tion and subsequent rapid degradaubiquitina-tion (Figure 2). Conversely, Sel-10 lacking the F box (Sel-10∆∆FB) mimics the effect of proteasome inhibitors and can stabilize NIC. Wu et al. [15] further demonstrate that NICcan be ubiquitinated in vitro by an immunopurified SCF complex composed of Sel-10, Cul-1, Skp-1 and

the RING finger protein Hrt-1. Sel-10∆∆FB fails to recruit an SCF complex and does not ubiquitinate NIC, thus providing a molecular basis for its dominant-negative activity.

Regulation of N by Sel-10 differs in several ways from that by Su(dx)/Itch. First, regulation by Sel-10 requires the NICPEST domain, whereas regulation by Su(dx)/Itch does not. Second, these proteins are likely to regulate N in different cellular compartments, because Sel-10 interacts only with nuclear NIC, and not with membrane-anchored or cytoplasmic forms of N as Su(dx)/Itch is capable of doing. Finally, Sel-10 recognizes a specific hyperphosphorylated form of NIC[15,17]. Although NICis multiply phosphorylated, Gupta-Rossi et al. [17] show that at least one of these phosphorylation events occurs in the nucleus and is specifically required for recognition by Sel-10.

Importantly, these papers [15–17] explain a long-standing paradox concerning N localization: although N is an essential nuclear coactivator, repeated attempts to locate endogenous N in the nuclei of tissues that are actively using the pathway have failed. In fact, the nuclear presence of N is often inferred indirectly from its ability to activate target gene tran-scription. Practically speaking, it seems obvious that the activity of NICneeds to be tightly controlled once it is liberated from regulation by ligand. It now appears that this is achieved through the activity of Sel-10, which makes nuclear NICextremely labile.

New News of Neur

The Drosophila gene neuralized (neur) was identified over two decades ago as one of six loci whose zygotic function is strongly required for diverting neurecto-dermal cells from the neural fate towards the epider-mal fate. These loci, known as the neurogenic genes, include what we now understand to constitute the Figure 1. Domain structure of E3s that regulate N signaling. Interestingly, all four proteins have analogous structures, although they are unrelated by sequence. Each consists of mul-tiple copies of a demonstrated or presumed protein–protein interaction domain involved in substrate recognition (blue) and a domain involved in E3 ubiquitin ligation (red). HECT and RING domains have intrinsic ubiquitin ligase activity, while the F-box recruits a multiprotein, RING-containing SCF E3 complex. The importance of the E3 domain is emphasized by the dominant-negative effect on N signaling caused by E3-deleted forms of these proteins. F box WD40 repeats RING Current Biology NHR domains Sel-10 C2 HECT Su(dx)/Itch Neur LNX RING PDZ domains

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core of the N pathway. Cloning of neur revealed a pioneer protein that contains two copies of a novel domain, the neuralized homology repeat (NHR), and a carboxy-teminal RING finger [18,19] (Figure 1). Cell autonomy of neur mutant clones indicated that its function is important in N signal-receiving cells, while its predominant localization to the plasma membrane suggested that it might regulate a membrane target [20–22]. Neur deleted for the RING finger (Neur∆∆RF) exhibits strong dominant-negative activity in multiple developmental settings when expressed ectopically, indicating that this domain is crucial for Neur activity [20,21].

A host of recent studies from the Boulianne, Rubin, Kintner and Delidakis labs [23–26] have now examined the molecular function of Neur, and demonstrated that the Neur RING finger functions as an E3 ubiquitin ligase in vitro. Interestingly, several lines of evidence indicate that Dl is a primary target of Neur E3 activity (Figure 2). In particular, loss-of-function and gain-of-function experiments demonstrate that Neur promotes endocytosis and degradation of Dl in a RING finger-dependent manner in vivo, that Neur promotes ubiquitination of Dl in vitro, and that Dl can be found in a physical complex with Neur and Neur∆∆RF [24–26]. The ultimate effect of Neur on Dl–N signaling remains controversial. Dl activates the N pathway non-autonomously in neighboring cells, but it can also autonomously inhibit the reception of the N signal (‘in

cis’). Neur has been proposed to antagonize the

cis-inhibitory activity of Dl in N signal-receiving cells,

thus biasing them toward activation of the N pathway. However, Neur has also been proposed to facilitate N signaling non-cell-autonomously. Further work is necessary to resolve these different interpretations. Numb Succumbs to LNX

Numb is a PTB domain protein which is asymmetri-cally localized in sibling cells whose fates are selected by N signaling; it can bind directly to the intracellular domain of N and antagonizes N activity [27,28]. In

Drosophila, asymmetric localization of Numb into one

of two daughter cells is accomplished via a multitein complex which segregates Numb (and other pro-teins) to one side of the cell cortex during mitosis, and thus to only one daughter following cell division [29]. As several components of this system have so far been found only in flies, other mechanisms might be involved in asymmetric localization of Numb activity in other organisms. A new report by McGlade and col-leagues [30] suggests an alternative mechanism involving regulated degradation of Numb, mediated by the RING domain protein ‘ligand of Numb-protein X’ (LNX p80).

LNX was originally identified as a Numb PTB domain-binding protein and contains an amino-terminal RING finger and four PDZ domains [31] (Figure 1). Nie

et al. [30] demonstrate that LNX is an E3 ubiquitin

ligase that mediates ubiquitination and degradation of Numb [30] (Figure 2). It was proposed that LNX may augment N signaling by lowering the level of Numb. In support of this, the ability of an activated N receptor to induce transcription of a reporter gene was found to be increased upon coexpression of LNX. Interest-ingly, the last three PDZ domains are not required for LNX-mediated ubiquitination and degradation of Numb; this truncated variant also displays increased affinity for Numb. Whether these other PDZ domains are involved in negatively regulating LNX–Numb interac-tions, in the recognition of other substrates, or in both, remains to be seen.

Although LNX genes have been found in mice and humans, it does not have apparent worm or fly orthologs, suggesting that its activity may be specific to vertebrates. Curiously, an alternatively spliced LNX isoform (p70) that lacks the RING finger is expressed specifically in the brain [31]. Although the function of this LNX isoform has not yet been examined, its structure suggests the tantalizing possibility that it is a naturally occuring dominant-negative variant. Accentuating the Negative: Future Directions The flood of recent studies bring to our attention multiple roles for the ubiquitination pathway in the regulation of the N pathway, but much remains to be answered. Some of the key questions about the currently identified E3s include the following. The mechanism of regulation of N by Su(dx)/Itch remains unclear, and it will be necessary to understand whether they regulate internalization of membrane bound N, somehow influence N cleavage, or act upon Figure 2. Proposed roles of ubiquitin ligases (red) in regulating

N pathway activity.

For simplicity, associated SCF complex and/or E2 enzymes are not shown. Su(dx)/Itch may ubiquitinate and regulate plasma membrane-associated N. Sel-10 recruits an SCF complex that ubiquitinates nuclear, phosphorylated NIC, thereby targeting it for degradation by the proteasome. Neur targets Dl for endo-cytosis and subsequent degradation. LNX ubiquitinates Numb, targeting it for degradation by the proteasome.

Dl N CSL Su(dx)/Itch P P Sel-10 Cytoplasm Nucleus Neur Current Biology Numb LNX

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remains to be identified. The consequences of Neur-mediated internalization of Dl on Dl–N signaling remain to be fully understood. Finally, it remains to be seen if regulation of Numb by LNX is truly required for the proper adoption of cell fates in vivo.

It is clear, however, that things are never as simple as they seem at first. For example, Sel-10 has also been suggested to negatively regulate the presenilin Sel-12, which is required for the generation of an active N receptor [32], and so may conceivably regu-late the N pathway at multiple levels. In addition, there is evidence that the E3 domains of these regulators also serve a negative autoregulatory function. This appears to be most clear for Sel-10 and Neur, whose E3 domains likely target themselves for degradation by the proteasome [16,24,25]. Finally, other novel mol-ecules are likely to be involved. For example, the small ankyrin repeat protein Nrarp has recently been shown to form a ternary complex with NICand CSL and to negatively regulate NIClevels in Xenopus [33]; its pos-sible connection to protein degradation remains to be determined. Additional surprises are undoubtedly in store, and I welcome the increasingly complicated picture of negative regulatory mechanisms for the N pathway that is surely to come.

Acknowledgments

Thanks to Martin Baron, Neetu Gupta-Rossi, Urban Lendahl, Gisele Deblandre, Jan Kitajewski, and C. Jane McGlade for useful discussions. E. C. L. is sup-ported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation, DRG 1632. References

1. Hicke, L. (2001). A new ticket for entry into budding vesi-cles-ubiquitin. Cell 106, 527–530.

2. Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 425–479.

3. Schweisguth, F. (1999). Dominant-negative mutation in the

ββ2 and ββ6 proteasome-subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage. Proc. Natl. Acad. Sci. U.S.A. 96, 11382–11386.

4. Morgan, T.H., Bridges, C.B., and Schultz, J. (1931). The constitution of the germinal material in relation to heredity. Year Book Carnegie Inst. 30, 408–415.

5. Xu, T., and Artavanis-Tsakonas, S. (1990). deltex, a locus interacting with the neurogenic genes, Notch, Delta and mastermind in Drosophila melanogaster. Genetics 126, 665–677.

6. Hustad, C.M., Perry, W.L., Siracusa, L., Rasberry, C., Cobb, L., Cattanach, B., Kovatch, R., Copeland, N., and Jenkins, N.A. (1995). Molecular genetic chracterization of six recessive viable alleles of the mouse agouti locus. Genetics 140, 255–265.

7. Perry, W.L., Hustad, C.M., Swing, D.A., O’Sullivan, T.N., Jenkins, N.A., and Copeland, N.G. (1998). The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in a18H mice. Nat. Genet. 18, 143–146.

8. Cornell, M., Evans, D., Mann, R., Fostier, M., Flasza, M., Monthatong, M., Artavanis-Tsakonas, S. and Baron, M. (1999). The Drosophila melanogaster Suppressor of deltex gene, a regulator of the Notch receptor signaling pathway, is an E3 Class Ubiquitin ligase. Genetics 152, 567–576.

melanogaster Suppressor of deltex gene: a regulator of Notch signaling. Genetics 150, 1477–1485.

10. Qiu, L., Joazeiro, C., Fang, N.H.W., Elly, C., Altman, Y., Fang, D., Hunter, T., and Liu, Y. (2000). Recognition and ubiquitination of Notch by Itch, a HECT-type E3 ubiquitin ligase. J. Biol. Chem. 275, 35734–35737.

11. Matsuno, K., Diederich, R., Go, M., Blaumueller, C., and S.A.-T. (1995). Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121, 2633–2644.

12. Aravind, L. (2001). The WWE domain: a common interac-tion module in protein ubiquitinainterac-tion and ADP ribosylainterac-tion. Trends Biochem. Sci. 26, 273–275.

13. Sundaram, M., and Greenwald, I. (1993). Suppressors of a 12 hypomorph define genes that interact with both lin-12 and glp-1 in Caenorhabditis elegans. Genetics 135, 765–783.

14. Hubbard, E., Wu, G., Kitajewski, J., and Greenwald, I. (1997). sel-10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins. Genes Dev. 11, 3182–3193.

15. Wu, G., Lyapina, S., Das, I., Li, J., Gurney, M., Pauley, A., Chui, I., Deshaies, R., and Kitajewski, J. (2001). Sel-10 is an inhibitor of Notch signaling that targets Notch for ubiqui-tin-mediated protein degradation. Mol. Cell Biol. 21, 7403–7415.

16. Oberg, C.J.L., Pauley, A., Wolf, E., Gurney, M., and Lendahl, U. (2001). The Notch intracellular domain is ubiq-uitinated and negatively regulated by the mammalian Sel-10 homolog. J. Biol. Chem. 276, 35847–35853.

17. Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover, A., and Israel, A. (2001). Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor. J. Biol. Chem. 276, 34371–34378.

18. Price, B.D., Chang, Z., Smith, R., Bockheim, S., and Laughon, A. (1993). The Drosophila neuralized gene encodes a C3HC4 zinc finger. EMBO J. 12, 2411–2418. 19. Boulianne, G.L., de la Concha, A., Campos-Ortega, J.A.,

Jan, L.Y., and Jan, Y.N. (1991). The Drosophila neurogenic gene neuralized encodes a novel protein and is expressed in precursors of larval and adult neurons. EMBO J. 10, 2975–2983.

20. Lai, E.C., and Rubin, G.M. (2001). neuralized functions cell-autonomously to regulate a subset of Notch-dependent processes during adult Drosophila development. Dev. Biol. 231, 217–233.

21. Lai, E.C., and Rubin, G.M. (2001). neuralized is essential for a subset of Notch pathway-dependent cell fate decisions during Drosophila eye development. Proc. Natl. Acad. Sci. U.S.A. 98, 5637–5342.

22. Yeh, E., Zhou, L., Rudzik, N., and Boulianne, G.L. (2000). Neu-ralized functions cell autonomously to regulate Drosophila sense organ development. EMBO J. 19, 4827–4837. 23. Yeh, E., Dermer, M., Commisso, C., Zhou, L., McGlade, C.,

and Boulianne, G.L. (2001). Neuralized functions as an E3 ubiquitin ligase during Drosophila development. Curr. Biol. 11, 1675–1679.

24. Lai, E.C., Deblandre, G., Kintner, C., and Rubin, G.M. (2001). Drosophila Neuralized is a ubiquitin ligase that pro-motes the internalization and degradation of Delta. Dev. Cell 1, 783–794.

25. Deblandre, G., Lai, E.C., and Kintner, C. (2001). Xenopus Neuralized is a ubiquitin ligase that interacts with X-Delta1 and regulates Notch signaling. Dev. Cell 1, 795–806. 26. Pavlopoulos, E., Pitsouli, C., Klueg, K., Muskavitch, M.A.T.,

Moschonas, N., and Delidakis, C. (2001). neuralized encodes a peripheral membrane protein involved in Delta signaling and endocytosis. Dev. Cell 1, 807–816.

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27. Rhyu, M.S., Jan, L.Y., and Jan, Y.N. (1994). Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491.

28. Wang, S., Younger-Shepherd, S., Jan, L.Y., and Jan, Y.N. (1997). Only a subset of the binary cell fate decisions medi-ated by Numb/Notch signaling in Drosophila sensory organ lineage requires Suppressor of Hairless. Develop-ment 124, 4435–4446.

29. Lu, B., Rothenberg, M., Jan, L.Y., and Jan, Y.N. (1998). Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetrical localization in Drosophila neural and muscle progenitors. Cell 95, 225–235. 30. Nie, J., McGill, M., Dermer, M., Dho, S., Wolting, C., and

McGlade, C.J. (2002). LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin dependent degradation. EMBO J. 21, 93–102. 31. Dho, S., Jacob, S., Wolting, C., French, M., Rohrschneider, L., and McGlade, C.J. (1998). The mammalian numb phos-photyrosine-binding domain. J. Biol. Chem. 273

32. Wu, G., Hubbard, E., Kitajewski, J., and Greenwald, I. (1998). Evidence for functional and physical association between Caenorhabditis elegans SEL-10, a Cdc4p-related protein, and SEL-12 presenilin. Proc. Natl. Acad. Sci. U.S.A. 95, 15787–15791.

33. Lamar, E., Deblandre, G., Wettstein, D., Gawantka, V., Pollet, N., Niehrs, C., and Kintner, C. (2001). Nrarp is a novel intracellular component of the Notch signaling pathway. Genes Dev. 15, 1885–1899.

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