Intracellular Cleavage of Notch Leads to a Heterodimeric Receptor on The

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Cell, Vol. 90, 281–291, July 25, 1997, Copyright 1997 by Cell Press Intracellular Cleavage of Notch Leads to a Heterodimeric Receptor onthePlasmaMembrane

Christine M. Blaumueller, Huilin Qi, controlling cell fate decisions (reviewed in Blaumueller Panayiotis Zagouras, and and Artavanis-Tsakonas, 1997). Spyros Artavanis-Tsakonas* Ligands, cytoplasmic effectors, and nuclear elements Howard Hughes Medical Institute of Notch signaling have been identified in Drosophila, Department of Cell Biology and Biology and vertebrate counterparts have also been cloned Boyer Center for Molecular Medicine (reviewed in Artavanis-Tsakonas et al., 1995). While pro- Yale University tein interactions among the various elements have New Haven, Connecticut 06536-0812 been documented, the biochemical nature of Notch signaling remains elusive. Expression of truncated forms of Notch reveals that Notch proteins without transmembrane and extracellular domains are translocated to the Summary nucleus both in transgenic flies and in transfected mammalian or Drosophila cells (Fortini et al., 1993; Lieber et Previous models for signal transduction via the Notch al., 1993; Ahmad et al., 1995; Zagouras et al., 1995). pathway have depicted the full-length Notch receptor Sequence comparisons between mammalian and Dro- expressed at the cell surface. We present evidence sophila Notch molecules, along with deletion analysis, demonstrating that the Notch receptor on the plasma have found two nuclear localization sequences that re- membrane is cleaved. This cleavage is an evolution- side on either side of the Ankyrin repeats (Stifani et al., arily conserved, general property of Notch and occurs 1992; Lieber et al., 1993; Kopan et al., 1994). These in the trans-Golgi network as the receptor traffics to- findings prompted the speculation that Notch may be ward the plasma membrane. Although full-length directly participating in nuclear events by means of a Notch is detectable in the cell, it does not reach the proteolytic cleavage and subsequent translocation of surface. Cleavage results in a C-terminal fragment, the intracellular fragment into the nucleus. However, TM N , that appears to be cleaved N-terminal to the trans- conclusive functional evidence for such a hypothesis EC membrane domain, and an N-terminal fragment, N , remains elusive (Artavanis-Tsakonas et al., 1995). that contains most of the extracellular region. We pro- In characterizing the human Notch2 gene, which is vide evidence that these fragments are tethered to- highly homologous to the other known Notch receptors, gether on the plasma membrane by a link that is sensi- we find that the majority of the protein in the cell is tive to reducing conditions, forming a heterodimeric cleaved. Cell fractionation and biotinylation analysis receptor. identify a predominantly cleaved Notch2 fragment on the cell surface that carries a small portion of the extra- Introduction cellular sequences, the transmembrane region, and the intracellular domain. This cleavage profile is not peculiar The Drosophila melanogaster Notch gene encodes an to Notch2 but seems to be a general feature of the Notch kDa transmembrane protein that acts as a recep- receptors. We demonstrate that Notch is proteolytically 300ف tor in a cell–cell signaling mechanism controlling cell cleaved in the trans-Golgi network before it reaches the fate decisions throughout development (reviewed in Ar- plasma membrane. We also present evidence indicating tavanis-Tsakonas et al., 1995). Closely related homologs that the extracellular domain of Notch is tethered to the of Drosophila Notch have been isolated from a number cell via a DTT-sensitive link with the truncated Notch of vertebrate species, including humans, with multiple fragment found on the cell surface, forming a heterodi- paralogs representing the single Drosophila gene in ver- meric receptor. On the basis of the experimental evi- tebrate genomes. We have previously reportedthe isola- dence we have gathered, we propose that the active, tion of cDNA clones encoding the C terminus of a human ligand-accessible form of the receptor is the heterodim- Notch paralog, originally termed hN (Stifani et al., 1992). eric form, whereas full-length Notch reflects newly syn- We have isolated and sequenced the entire gene and thesized, intracellular and, hence, inactive molecules. designated this protein human Notch2 because of its close relationship to the Notch2 proteins found in other Results species (Weinmaster et al., 1992). The hallmark Notch2 structures are common to all the Notch-related proteins, Characterization of the Human Notch2 Gene including, in the extracellular domain, a stretch of 34 to The full-length cDNA encoding the human Notch2 pro- 36 tandem epidermal growth factor-like (EGF) repeats tein is 7.8 kb in length, and the predicted protein product and three Lin-12/Notch (LN) repeats, and, in the intracel- is 2471 amino acids long. This protein has all of the lular domain, six Ankyrin repeats and a PEST-containing expected domains of Notch family proteins and is 92% region. Like Drosophila Notch and the related Caeno- identical to the rat Notch2 amino acid sequence overall. rhabditis elegans genes lin-12 and glp-1 (Sternberg, An alignment of human Notch2 with human Notch1, 1993; Greenwald, 1994), the vertebrate Notch homologs Xenopus Notch (Xotch), and Drosophila Notch is shown play a role in a variety of developmental processes by in Figure 1. In all of the proteins shown, 36 EGF repeats are present, and each is more closely related to the correspond- *To whom correspondence should be addressed. ing EGF repeat in the other Notch homologs than to Cell 282

Figure 1. Notch Sequence Comparisons The Drosophila Notch (DrosN), Xenopus Notch/Xotch (XenN), human Notch1 (humN1), and human Notch2 (humN2) protein sequences are aligned, with names indicated to the left and numbering to the right (Wharton et al., 1985; Coffman et al., 1990; Ellisen et al., 1991; Stifani et al., 1992). Major Notch protein motifs are enclosed in boxes. Starting from the N terminus, the boxed regions indicate: EGF repeats, Lin-12/ Notch (LN) repeats, transmembrane domain (TM), Ankyrin repeats, and PEST-containing region. Also indicated are the putative CcN motif components (Stifani et al., 1992) nuclear localization signal (NLS, BNTS), and putative CKII and cdc2 phosphorylation sites. The calculated signal cleavage site is indicated with an arrow. Intracellular Cleavage of Notch 283

Figure 2. Intracellular Notch Epitopes Are Carried by a 110 kDa Fragment Western blot analysis of human cell lines, human tissues, Drosophila cell lines, rat and Drosophila embryos. The cell source of each lysate is indicated above the lanes. Notch2 expression was monitored with antibody bhN6D and Notch1 with antibody bTAN 20. Both recognize intracellular epitopes of the protein (Zagouras et al., 1995). (A) and (B) show Notch2 expression. (C) and (D) show Notch1 expression. (E) shows the expression of Drosophila Notch in embryos, Drosophila KC cultured cells, which endogenously express Notch, and Drosophila S2 cells, which do not express endogenously Notch but have been stably transfected with a Notch expression vector. The antibody used (9C6) recognizes an intracellular epitope (Fehon et al., 1990). In all panels, the 110 kDa major breakdown product (NTM) and the position of the full- length Notch protein are indicated. Molecular weight markers are shown on the left of each panel. neighboring EGF repeats within the same protein. The processing pattern is not confined to cell lines. The overall identity for the EGF repeat region between the predominant polypeptide species recognized by the human Notch paralogs is 59%, while the identity levels same antibody in rat embryo extracts and in a variety between the Drosophila and human proteins in this re- of human tissue extracts is also the 110 kDa Notch gion are slightly lower (51% for human Notch1 and 52% breakdown product (Figure 2A, lane 3, and Figure 2B). for human Notch2). While the overall amino acid conser- We note that this Western blot analysis reveals differ- vation across the EGF repeat domain is low, the conser- ences in the relative ratio of the full-length protein and vation of individual EGF repeats from one protein to the NTM derivative among the examined tissues. another is variable (M. Baron and S. A.-T., unpublished data). Certain repeats, including numbers 11 and 12, Cleavage Is a General Property which are capable of ligand binding (Rebay et al., 1991), of the Notch Receptor are more highly conserved than others. The overall con- We examined whether the characteristic cleavage pat- servation of the LN repeats is similar to that for the tern of the human Notch2 paralog is peculiar only to EGF repeats, having 54% identity between the human this molecule or whether it reflects a general pattern for homologs and slightly lower values between Drosophila the Notch receptor family. Western blot analysis, using Notch and either human Notch1 or human Notch2 (49% an antibody raised against the human Notch1 paralog and 44%, respectively). (antibody bTAN20), demonstrates that this protein dis- In Notch2 the conservation of the intracellular domain plays a processing pattern that is similar to that of is high. All of the known structural hallmarks of the Notch Notch2 (Figures 2C and 2D). These results are compati- proteins are maintained, including the Ankyrin repeats, ble with earlier analyses involving Notch1. The existence the PEST-containing region, and the basic stretch of of a prominent approximately 120 kDa fragment was amino acids that can function as nuclear localization previously demonstrated in extracts of two different signals and target truncated forms of the protein into human cell lines that express the Notch1 paralog (Aster the nucleus (Stifani et al., 1992; Lieber et al., 1993). et al.,1994). When a Notch1 expression plasmid is transfected into a baby hamster kidney cell line (BHK cells), The Notch2 Protein Is Cleaved the major Notch peptide detected in these cells by West- Antibodies raised specifically against the human Notch2 ern blot analysis is a 110 kDa species (data not shown protein were used to study its expression in cultured and Zagouras et al., 1995). cells (antibody bhN6D). Western blotting of Notch2 pro- In order to determine whether the processing pattern tein from the human SJ-NB5 neuroblastoma cell line seen for Notch1 and Notch2 is specific to mammalian revealed the presence of an approximately 110 kDa Notch proteins, we performed Western blotting of Dro- (henceforth NTM) polypeptide in addition to the full-length sophila cell lysates, using an antibody raised against 300 kDa protein (Figure 2A, lane 1). This lower molecular intracellular epitopes of Drosophila Notch (Figure 2E) weight polypeptide is the predominant species recog- (Fehon et al., 1990). In an embryonic extract, in addition nized by the antibody used in this experiment. A similar to the clearly detectable full-length protein, several processing pattern is seen in HaCat cells (Figure 2A, smaller Notch polypeptides, including an approximately lane 2), a human keratinocyte cell line. The observed NTM band, are visible. In the KC cell line, which expresses Cell 284

Figure 4. The 110 kDa (NTM) Fragment Is Expressed on the Cell Figure 3. The 110 kDa (NTM) Fragment Is Associated with Mem- Surface branes SJ-NB5 cells were treated with biotin (ϩBiotin) while control cells Subcellular fractionation of SJ-NB5 cells followed by SDS-PAGE and were not (ϪBiotin). Each sample was lysed and divided into three Western blot with a Notch2 antibody raised against an intracellular equal portions precipitated with immobilized streptavidin, anti- epitope (bhN6D). Whole cell lysate is shown on the left lane. This Notch2 antibody PGHN, or normal rabbit serum (respectively lanes lysate was centrifuged at 900 ϫ g and the pellet (0.9K) is in the 1 and 4, 2 and 5, 3 and 6). Samples were run on a 4%–20% SDS- second lane. This pellet was resuspended and analyzed on a su- PAGE gel and blotted with antibody bhN6D. Molecular weight mark- TM crose step gradient 0%, 40% and 50%. The pellet of the gradient, ers are shown on the left. N accumulates on the surface, while which contains the nuclei (NP), and the interphases are analyzed full-length Notch is not precipitated by streptavidin. as indicated in the last three lanes. The supernatant of the initial low spin was centrifuged at 40,000 ϫ g and the pellet was analyzed in the lane indicated as 40K. Finally the supernatant of the 40K spin only biotin-labeled proteins, (2) the anti-Notch2 antibody was centrifuged again at 100,000 ϫ g (lanes indicated as 100K) and PGHN, a polyclonal antibody that recognizes an intracel- the resulting pellet (P) and supernatant (S) were loaded on the gel. lular epitope and immunoprecipitates human Notch2 (Zagouras et al., 1995), and (3) normal rabbit serum Notch endogenously, NTM is clearly detectable. Finally, (NRbS). Western blotting of the precipitated products in an S2 cell line, which does not express endogenous was performed using the anti-Notch2 antibody, bhN6D. Notch but has been stably transfected with a Notch The results of this experiment are shown in Figure 4. The expression plasmid, NTM is also prominent. We conclude only Notch2-related surface protein that was detected is that the processing of the Notch receptor is a general the NTM breakdown product. Immobilized streptavidin property of the Notch proteins. precipitated only the NTM product in the biotin-labeled samples (lane 1) and no protein in the unlabeled samples NTM Is Associated with Membranes (lane 4). In contrast, anti-Notch2 antibody PGHN effi- We sought to determine the subcellular localization of ciently precipitated both the full-length and breakdown the Notch polypeptides by cell fractionation. SJ-NB5 Notch2 products in biotinylated (lane 2) and nonbiotinly- cells were fractionated as described in the Experimental ated samples (lane 5). As expected, the negative control, Procedures, and the resulting fractions were examined NRbS, does not precipitate either protein form (lanes 3 by Western blotting. Figure 3 shows a fractionation ex- and 6). periment in which the NTM Notch fragment is associated We conclude that the NTM fragment is a transmem- with membrane lanes. Each fraction was also tested for brane Notch polypeptide that resides on the plasma the presence of syntaxin, a plasma membrane protein membrane and must be the result of a cleavage at a expressed in the same cell line (Bennett et al., 1992). In site in the extracellular domain. The molecular weight order to ensure that such a fractionation pattern is not of 110 kDa is compatible with this fragment, which con- confined to the SJ-NB5 cell line, we fractionated HaCat sists of the intracellular domain attached to extracellular cells and Drosophila S2 cells that were stably trans- sequences cleaved at a site close to the region between fected with a Notch expression plasmid (data not shown) the LN repeats and the EGF repeats. and obtained similar results. Notch Is Cleaved in the Trans-Golgi Network The Notch Receptor Presented at the Cell before Reaching the Surface Surface Is Cleaved The experiments described above demonstrate that the The association of the NTM Notch fragment with the steady-state form of the Notch receptor found at the plasma membrane was further examined by biotin label- cell surface is a cleaved form. In an attempt to determine ing of live SJ-NB5 cells (Figure 4). Biotin labeling of the cellular compartment where Notch is cleaved, we surface proteins was performed by incubating live cells carried out pulse-labeling analyses in the presence of on ice in medium containing biotin (control cells were drugs that are known to interfere with cellular trafficking. treated with the same medium lacking biotin). The cells Figure 5A demonstrates that Brefeldin A, which blocks were subsequently lysed and divided into three equal transport between the cis- and trans-Golgi network (Pel- portions that were incubated with the following re- ham, 1991), effectively blocks the breakdown of full- agents: (1) immobilized streptavidin, which precipitates length Notch. In contrast, monensin or chloraquinone Intracellular Cleavage of Notch 285

Figure 5. Processing of Notch2 Is Blocked by Brefeldin A and at 19ЊC (A) Shows the results of a pulse-labeling experiment in SJ-NB5 cells in the presence or absence of Brefeldin A. [35S]-Methionine was allowed to incorporate for 20 min and then chased for 0, 15, 30, 45, 60, 90 min at 37ЊC. The cell lysates were immunoprecipitated by PGHN (a polyclonal antibody raised against intracellular Notch2 epitopes) (Zagouras et al., 1995), analyzed by SDS-PAGE, and followed by fluorography. (B) SJ-NB5 cells were labeled with [35S]-methionine for 20 min, chased either at 37ЊCor19ЊC for 0, 30, 60, 90 min, immunoprecipitated by PGHN, and analyzed by SDS-PAGE, followed by fluorography. Two fragments accumulate during the chase and coimmunoprecipitate with PGHN: a 180 kDa fragment (NEC) and a 110 kDa fragment (NTM).

does not affect processing (data not shown). Cleavage consistent with the hypothesis that it carries most of is also effectively blocked at 19ЊC, a characteristic fea- the Notch extracellular domain (data not shown). ture of processing events that occur in the trans-Golgi network (Figure 5B). Full-Length Notch Does Not Reach the Cell Surface The Cleaved Extracellular Domain of Notch Is Tethered The Western blot analyses revealing the existence of to the NTM Transmembrane Fragment the NTM Notch fragment (Figure 2) also show varying In the aforementioned pulse-labeling experiments (Fig- amounts of full-length Notch. We were interested in ex- ure 5), the accumulation of the NTM fragment is closely ploring the fate of the full-length molecule by testing its paralleled by the accumulation of a larger fragment that expression at the cell surface. is approximately 180 kDa in molecular mass. This larger SJ-NB5 cells were labeled with [35S]-methionine for fragment is coimmunoprecipited by the antibody PGHN, 10 min and then chased for varying periods. The live which recognizes an intracellular epitope of human cells were incubated with Biotin as described above Notch2. However, blotting of the same immunoprecipi- and subsequently lysed and immunoprecipitated with tate by Western blot, using antibody bhN6D, also raised PGHN. The immunoprecipitate was divided into two against an intracellular epitope, detects only the NTM equal portions, one of which was reprecipitated with fragment. immobilized streptavidin (Figure 6). The two sets of sam- A single cleavage of the Notch protein that produces ples were then examined by SDS-gel electrophoreses a 110 kDa fragment would also generate a second frag- followed by fluorography. Figure 6 shows that negligible ment of approximately 180 kDa. We therefore presume amounts of full-length Notch are detected on the surface that the NEC fragment, which accumulates with kinetics throughout the chase, while substantial amounts of full- indistinguishable from those of NTM, corresponds to the length molecules are precipitated by the Notch antibody cleaved extracellular domain of the Notch2 protein that (total cellular Notch). As the full-length, newly synthe- remains attached to the NTM polypeptide by an SDS- sized Notch decreases during the chase, the NTM frag- and/or DTT-sensitive linkage. Antibodies recognizing ment begins to accumulate in the streptavidin-precipi- extracellular epitopes are not available for Western blot tated reaction. NTM accumulation is paralleled by the analysis. However, the relatedness of these fragments appearance of the NEC fragment, consistent with the is also supported by the fact that the appearance of NEC contention that this fragment represents the extracellu- is not inhibited by monensin or chloraquinone (data not lar domain of Notch and is tethered to the NTM Notch shown) but is inhibited by Brefeldin A and a 19ЊC block polypeptide. We conclude that Notch protein reaches (Figure 5). Additional supporting evidence comes from the surface in a cleaved form, and that newly synthe- pulse-labeling experiments done with a cysteine rather sized full-length Notch is not found on the plasma mem- than a methionine label. The predicted cysteine content brane. of the 180 kDa fragment resulting from a cleavage approximately between the EGF and the LN repeats is 220, Notch Heterodimers Bind the Ligand Delta while that of the 110 kDa fragment is 23. Labeling with The biological significance of the heterodimeric Notch cysteine shows that the NEC band incorporates nearly form would be questionable if it could not bind ligands. an order of magnitude more label than the NTM band, Physical interaction between the extracellular domains Cell 286

Figure 7. Delta Binds to the Heterodimeric Form of Notch Identical amounts of cell lysates were precipitated with Delta antibodies from: (1) S2 cells expressing Notch2, (2) S2 cells expressing Delta, and (3) and (4) Notch- and Delta-expressing cells, respectively, after 1 and 2 hr of aggregation. In addition, a cell lysate of Notch-expressing cells that has not been incubated with Delta antobody is shown in lane 5. All lanes are visualized with Notch antibody 9C6, which recognizes intracellular epitopes. The 110 kDa Notch NTM fragment is immunoprecipitated by the Delta antibodies in the extracts from Notch/Delta cell aggregates.

Notch-EGF repeats have been implicated in protein in- Figure 6. Full-Length Notch Does Not Accumulate on the Surface teractions, and missense mutations in both Drosophila 35 SJ-NB5 cells were pulse labeled with [ S]-methionine for 10 min, and humans have been associated with mutant pheno- chased for 0, 15, 30, 45, 60, 90 and 120 min, and this was followed by the biotinylation of the surface proteins. The cell lysates were types (Hartley et al., 1987; Kelley et al., 1987; Rebay et immunoprecipitated with the polyclonal Notch2 antibody PGHN al., 1991; Joutel et al., 1996). Functional data regarding (Zagouras et al., 1995). Lanes corresponding to those lysates are the cysteine-rich LN repeats are lacking. Nevertheless, designated T and show all the antigens recognized by PGHN. At all Notch homologs, from flies to humans, share similar each time point, part of the PGHN immunoprecipitate was resus- LN-repeat stretches in the equivalent extracellular re- pended and then immunoprecipitated by streptavidin, which would gion of the receptor. Within the intracellular domain of correspond to the Notch antigens on the surface (S lanes). The immunoprecipitation products were analyzed by SDS-PAGE fol- the Notch proteins, all six of the Ankyrin repeats are lowed by fluorography. The accumulation of the NTM and NEC frag- highly conserved. These repeats have been shown to ments is evident, while full-length Notch is not detected on the play a crucial role in Notch signaling and have been surface. implicated in molecular interactions between Drosophila Notch and the Deltex protein, which behaves as a positive regulator of Notch activity (Matsuno et al., 1995), of Notch and Delta have been demonstrated with the and with the downstream effector Suppressor of hairless help of aggregation assays involving Delta- and Notch- (Fortini and Artavanis-Tsakonas, 1994; Matsuno et al., expressing cells. If the heterodimeric form interacts with 1995). Consistent with the high degree of conservation, Delta after aggregation, then the 110 kDa NTM fragment Notch2 Ankyrin repeats were found to interact both with should coimmunoprecipitate using Delta antibodies. We Drosophila as well as with human Deltex (K. Matsuno find that after aggregation, Delta antibodies are capable and S. A.-T., unpublished data). of efficiently immunoprecipitating the NTM fragment, The biochemical evidence we present shows that the demonstrating that the heterodimeric form can bind Notch receptor is cleaved in the trans-Golgi network Delta (Figure 7). As expected, if the aggregation is dis- before reaching the cell surface. Pulse-labeling experi- rupted by depleting calcium from the medium by EGTA ments in combination with the biotinylation data indicate (Fehon et al., 1990), Delta antibodies fail to precipitate that the full-length Notch molecule does not reach the efficiently NTM (data not shown). plasma membrane. The varying amounts of full-length Notch detected in different cell extracts presumably re- Discussion flect a newly synthesized, inactive receptor that has not yet reached the Golgi and is inaccessible to ligands. The strong structural conservation among both the Dro- Several lines of evidence indicate that this cleavage is sophila and vertebrate Notch gene products, and among a general property of cellular Notch. First, the same homologs of other components of the same pathway, cleavage pattern is seen in all human cell lines and imply that the molecular and biochemical mechanisms human tissues examined. Second, both Notch1 and involved in Notch signaling are conserved across spe- Notch2 are processed in the same way. Third, the cleav- cies boundaries. The question of what particular roles age product NTM is seen in both freshly prepared embry- are played by the assortment of paralogs within the onic rat tissues as well as Drosophila extracts. Notch superfamily, in combination with the various para- The subcellular fractionation and biotinylation studies logs of the other pathway components, remains unclear. demonstrate that NTM is associated with the plasma Expression pattern comparisons, structural similarities, membrane, indicating a cleavage in the extracellular re- and the available functional data for distinct paralogs gion of Notch. The epitope recognized by the antibodies suggest that these molecules possess different expres- used here was raised against the least conserved region sion profiles but similar biochemical and developmental of the intracellular domain mapping between the PEST properties. sequence and the Ankyrin repeats (Zagouras et al., We find that the human Notch2 protein is a highly 1995). Hence, NTM must include the intracellular Notch2 conserved member of the Notch protein family. Specific sequences mapping between this C-terminal epitope Intracellular Cleavage of Notch 287

and the transmembrane domain. In the absence of the 1993; Lieber et al., 1993; Jarriault et al., 1995). Truncated N-terminal sequence for NTM, it is not possible to deter- Notch proteins without transmembrane and extracellu- mine the cleavage site accurately. However, a likely pos- lar domains are nuclear and result in the constitutive sibility is that cleavage occurs between the EGF and the activation of the receptor, demonstrated by the up-regu- LN repeats, producing two fragments with a calculated lation of downstream genes (Jennings et al., 1994; Sun molecular mass of 112 kDa and 180 kDa. Notwithstand- and Artavanis-Tsakonas, 1996). However, high resolu- ing the possibility that these molecular masses can only tion phenotypic studies have shown that the gain-of- be approximate in view of expected posttranslational function phenotypes associated with the expression of modifications such as glycosylation (Johansen et al., such truncated forms may not depend on nuclear trans- 1989), they closely correspond to the size of NTM and location of Notch. Both membrane-bound and nuclear NEC. It is likely the C. elegans Notch-like receptors lin- truncated proteins produce indistinguishable pheno- 12 and glp-1 are cleaved in an analogous fashion, since types in the Drosophila eye (Fortini and Artavanis-Tsa- N-terminal and C-terminal fragments of glp-1 were konas, 1993). At present there are no compelling data found to copurify (Crittenden et al 1994). A deletion anal- to support the contention that nonengineered forms of ysis involving Notch1 expression constructs transfected the Notch receptor are ever found in Drosophila nuclei in cell lines by Aster et al. (1994) led them to suggest (Kidd et al., 1989; Fehon et al., 1991). Nuclear Notch1 that, under their experimental conditions, Notch1 may immunoreactivity has been detected in human cervical be cleaved between the LN repeats and the transmem- tissues and rat retina (Ahmad et al., 1995; Zagouras et brane domain.We note that in the extracts we examined, al., 1995), but the biochemical nature of these nuclear the main Notch1 Notch-processing product in SJ-NB5 antigens recognized by the existing antibodies remains and HaCat cells is, as in Notch2, approximately 110 kDa. unknown, and their functional significance, if any, is also In the rat embryos, however, the main cleavage product unclear. The present, as well as previous, immunoblot appears to be larger. The significance of such qualitative studies consistently detect processed Notch polypep- differences in the processing pattern, or the additional tides and, hence, it is possible that small yet functionally breakdown products we detect in our Western blots, critical amounts of nuclear Notch do exist (Kidd et al., remains to be determined. 1989; Fehon et al., 1990; Aster et al., 1994; Zagouras et The accumulated evidence strongly indicates that NEC al., 1995). In vitro biochemical studies involving Notch contains the cleaved extracellular sequences of Notch, pathway elements and/or fragments of Notch domains even though the lack of appropriate antibodies pre- have raised the possibility that Notch sequences can vented us from directly demonstrating this hypothesis. influence transcription (Hsieh and Hayward, 1995; Jarri- The kinetics of NEC accumulation and its inhibition profile ault et al., 1995; Kopan et al., 1996; our unpublished are identical to NTM. The molecular weights of the Notch data). Such in vitro approaches, even if they show that breakdown products, as argued above, are also com- Notch sequences are capable of interfering directly with patible with such a notion. Finally, the relative incorpora- transcription, fail to demonstrate the relevance of such tion of radioactive cysteine in the two fragments reflects an activity in vivo. Therefore the intriguing question of the approximately 10:1 ratio predicted by the amino acid nuclear Notch remains open. composition of two fragments produced by a cleavage Tethering of NTM to NEC is compatible with both the approximately between the EGF and LN repeats. In this assumed mode of action of Notch, which necessitates regard, it is noteworthy that extracellular Notch frag- interactions between the extracellular domains of the ments are present in the conditioned medium of Dro- Notch receptor and its ligands, and with the cell autono- sophila cell cultures that express Notch (Rebay, 1993; mous nature of Notch signaling (Stern and Tokunaga, I. Rebay, R. Fehon and S. A.-T., unpublished data). Im- 1968; Hoppe and Greenspan, 1990; Markopoulou and munocytochemical studies with Drosophila tissues do Artavanis-Tsakonas, 1990; Heitzler and Simpson, 1991). not reveal differences in the cellular distribution of the On the other hand, any model of Notch biochemical intracellular versus the extracellular domain of Notch (R. activity and cellular function must take into account Fehon and S. A.-T., unpublished data). that Notch is cleaved. Several questions raised by this The coprecipitation of the NEC fragment together with finding are worth pointing out. The possibility that NEC NTM, and the simultaneous appearance of the two frag- may be released from the surface, acting as an inhibitor ments on the plasma membrane indicate that NEC and of the pathway, must be further examined, especially in NTM are tethered to one another. Since the link is sensi- view of reports that have appeared in the literature over tive to reducing agents, we assume that the two frag- the years suggesting that Notch may have nonautono- ments are linked via disulfide bridges. Our inability to mous activities (Gehring, 1973; Technau et al., 1987; detect full-length Notch on the surface indicates that Baker and Schubiger, 1996). Such a scenario must take the cleaved form is the active form of the receptor. This into account that the expression of truncated forms of argument is supported by the Notch-like mutant pheno- Notch, approximately corresponding to the postulated types associated with loss-of-function mutations in kuz- structure of NTM, results in the constitutive activation of banian, a gene encoding a protease implicated in the the receptor (Ellisen et al., 1991; Jennings et al., 1994; cleavage of Notch (see accompanying paper, Pan and Kopan et al., 1994; Sun and Artavanis-Tsakonas, 1996). Rubin, 1997 [this issue of Cell]). The notion that alterations in the extracellular domain Expression studies of truncated Notch molecules may facilitate signaling events hasbeen proposed on the have prompted the speculation that Notch signaling may basis of studies involving the expression of engineered depend on cleavage of the intracellular domain of Notch constructs in cultured cells (Kopan et al., 1996). Irre- followed by its translocation to the nucleus (Fortini et al., spective of how well these studies reflect the in vivo Cell 288

heterodimer may be engaged in homotypic, or conceivably heterotypic, interactions. The analysis of the Notch receptor on nonreducing gels is consistent with this notion. In the absence of reducing agents, NEC and NTM are not detected. However, instead of detecting the full- length molecule, we detect higher molecular weight complexes of a yet-undetermined nature (data not shown). Since full-length Notch appears to reflect a ligand inaccessible, intracellular form of the protein, cleavage provides an important tool to regulate the Notch pathway. Such cleavage can effectively control the number of active surface receptors. Genetic analysis in Drosoph- ila has demonstrated that the animal is unusually sensitive to the number of wild-type copies of the Notch gene. In fact, Notch is one of a handful of genes in Drosophila that are both haploinsufficient as well as triploid mutant (Lindsley and Zimm, 1992). It will be important to estab- lish whether proteases other than Kuzbanian may also be involved in the cleavage of the Notch receptor, as well as to determine if the NEC/NTM cleavage site is functionally unique.

Experimental Procedures

Isolating and Sequencing Human Notch2 cDNAs A human fetal brain cDNA Zap II library (from 17–18 week embryo; Figure 8. A Model for the Trafficking of the Notch Receptor Stratagene, La Jolla, CA) was used in the screening for human Notch homologs. The Notch cDNA clones were originally obtained by using Full-length Notch is synthesized in the ER (N) and then cleaved in a probe encoding portions of the human Notch2 protein (hN2K and the trans-Golgi network (TGN) extracellular region, producing two hN5K) (Stifani et al., 1992). A probe used to screen for cDNAs span- fragments, NTM and NEC. Full-length Notch (N) reflects an inactive, ning 5Ј regions of the human Notch2 gene was generated from the presumably newly synthesized form of the receptor, which is not hN2K cDNA. Because we did not isolate cDNAs localized to the seen on the surface. NTM and NEC, produced by a cleavage in the extreme 5Ј terminus of the human Notch2 gene using this probe, extracellular domain, are tethered together on the surface via a DTT- we took advantage of the fortuitous isolation of a human Notch2 sensitive link, constituting the active form of the receptor that can cDNA (Adams et al., 1993) that extends further 5Ј, as determined interact with ligands (striped circle) and/or interact homotypically by sequence comparison to the rat Notch2 cDNA isolated by Wein- with another Notch receptor, or, conceivably, with other surface master et al. (1992). Although this human cDNA does not extend to molecules. the extreme 5Ј end of the human Notch2 coding region, it was used to generate a new probe that was closer to the 5Ј end of the gene. This probe was used to isolate the 5Ј-most cDNAs encoding human situation, together with the well-documented in vivo ac- Notch2. Sequencing was done using the Sequenase Kit (United tion of truncated forms of Notch, they do raise the possi- States Biochemical Corporation, Cleveland, OH). bility that a ligand-dependent degradation or cleavage of the extracellular domain may result in the activation Cell Culture of the receptor. However, it seems unlikely that signaling SJ-NB5 cells were grown at 37ЊC in an atmosphere of 5% C02 and would involve a simple ligand-dependent “shedding” of 95% air, in RPMI (GIBCO-BRL, Grand Island, NY) supplemented EC N . For instance, cell adhesion mediated by Notch/ with 10% fetal bovine serum (GIBCO-BRL, Grand Island, NY), 2 mM ligand interactions has been shown to trigger an endo- L-Glutamine (ICN Biomedicals, Inc., Costa Mesa, CA), 100 ␮g/ml cytic flow of Delta molecules in the Notch-expressing penicillin, and 100 ␮g/ml streptomycin (ICN Biomedicals, Inc., Costa cells, where it is eventually found in multivesicular bod- Mesa, CA). Cells were dissociated using PBS with 0.25% trypsin ies (Fehon et al., 1990; R. Fehon and S. A.-T., unpub- and 0.03% EDTA (J.T. Baker, Inc., Phillipsburg, NJ), and subcultured at ratios of 1:3 to 1:10. HaCat Cells (cultured human keratinocytes) lished data). Detailed expression studies of Delta ex- were a gift from Dr. Michael Reiss (Yale University). Aggregation pression in cells known to undergo Notch signaling are experiments and the maintenance of Drosophila S2 and KC cells consistent with the cell culture findings (Kooh et al., were described in Fehon et al. (1990). 1993). At this stage it seems that the simplest working hypothesis on Notch signaling should involve the heter- Antibodies EC TM odimeric (N /N ) surface Notch complex proposed The antibodies bhN6D and bTAN20 are monoclonal antibodies (Rat, here, rather than the action of any single cleaved frag- IgG) directed against the nonconserved intracellular epitopes of ment (see the proposed model in Figure 8). The negative human Notch2 and Notch1, respectively (Zagouras et al., 1995). On complementation displayed by the Abruptex mutation, Western blots they recognize specifically Notch1 and Notch2 but a group of gain-of-function mutants affecting amino are not useful for immunoprecipitations. In contrast, PGHN antibody, acids in the EGF homologous region of Notch, has been a polyclonal antibody (Rabbit, IgG) directed against intracellular epitopes of human Notch homolog hN2, can be used to immunopre- thought to reflect homotypic interactions between cipitate Notch2 (Zagouras et al., 1995). The Drosophila antibody Notch receptors (Foster, 1975; Xu et al., 1990). Therefore 9C6 is a monoclonal antibody that recognizes intracellular epitopes akin, for example, to the insulin receptor, the NEC/NTM (Fehon et al., 1990). Intracellular Cleavage of Notch 289

Subcellular Fractionation and Western Blotting cysteine-free DMEM medium for 1 hr. 100 ␮Ci 35S-translabeled Met- SJ-NB5 cells were grown to 80%–90% confluence in six T-75 tissue Cys (ICN) was added to each plate, pulsed at 37ЊC for 20 min, and culture flasks, scraped in TBS, washed once, and resuspended in chased for varying times and temperatures (see the figure legends). 1 ml cold buffer A (75 mM KCl, 10 mM imidazole [pH 7.2], 1 mM The chase began by adding 2ϫ volumes complete medium plus

EGTA, 2.5 mM MgCl2, 0.02% NaN3, 1 mM DTT, and 1 mM Pefabloc 100 ␮g/ml cold methionine and cysteine to the plates. SC [Boehringer Mannheim]). During the fractionation process, all For Brefeldin A samples, Brefeldin A was maintained at a final samples were kept on ice and resuspended in cold buffer A. Pellet concentration of 10 ␮g/ml in the starving medium as well as in both samples at all stages of fractionation were resuspended in their pulse and chase. Cells were washed with cold PBS and lysed in original volumes so that stoichiometric ratios of all samples would lysis buffer (Zagouras et al., 1995) containing 1 mM Pefabloc SC, be equivalent. 0.7 ␮g/ml pepstatin A, and 0.5 ␮g/ml leupeptin. Cell lysates were Cells were homogenized using Omini’s hand homogenizer with centrifuged at 14,000 rpm for 5 min. The supernatants were trans- microscopic monitoring of cell lysis throughout homogenization. A ferred to fresh tubes and precleared by incubating with 5 ␮l normal 50 ␮l aliquot was kept as fraction 1 (whole cell lysate). The lysate rabbit serum and 50 ␮l 10% protein A-sephrose CL-4B (Pharmacia was then centrifuged at low speed, (900 ϫ g), for 5 min at 4ЊC. The LKB) for 1 hr at 4ЊC. The beads were pelleted by centrifugation and resulting pellet was resuspended in buffer A, with a 50 ␮l aliquot of the supernatants were divided into two equal aliquots. One aliquot the suspension as fraction 2 (0.9K/P). The suspension was centri- was immunoprecipitated by incubating with rabbit polyclonal anti- fuged again. The pellet was washed once with buffer A. After a third human Notch2 antibody PGHN and 50 ␮l protein A-sephrose CL- centrifugation, the pellet was resuspended in 200 ␮l of buffer A, 4B for 2–3 hr or overnight at 4ЊC. The other aliquot was immunopre- mixed with 1.8 ml of 60% sucrose made in buffer A containing 5 cipitated by normal rabbit serum as control. The beads were washed mM MgCl2, and then transferred to Beckman’s SW 50.1 centrifuge three times in RIPA buffer B (150 mM NaCl, 1% NP40, 0.5% DOC, tube. The suspension was overlaid with 2 ml of 40% sucrose-buffer 0.1% SDS, 50 mM Tris [pH 7.5]), washed once in 50 mM Tris-Cl, A and then 2 ml of buffer A. The sample was centrifuged at 100,000 ϫ 150 mM NaCl (pH 7.5), resuspended in 50 ␮l SDS sample buffer, g for 1 hr at 4ЊC. Two banded fractions were collected separately, boiled, and subjected to a 3–15% gradient SDS-PAGE. The gel was and 50 ␮l aliquots were kept. The upper and lower fractions were fixed in 25% isopropanol and 10% acetic acid for 30 min, soaked termed 40/0 and40/50, respectively, and the nuclear pellet atbottom in Amplify (Amersham) for 15–30 min, dried, and fluorographed to was resuspended in buffer A and designated NP. X-ray film at Ϫ70ЊC prior to developing. The supernatant from the 900 ϫ g spin was centrifuged again at 40,000 ϫ g for 15 min at 4ЊC using a Sorval SS-34 fixed angle rotor. Pulse Chase and Biotinylation -conflu %80ف The pellet from this midspeed spin was resuspended in buffer A SJ-NB5 cells were grown on 100 mm petri dishes until and designated 40K/P. The supernatant from the midspeed spin ent and pulse chased as described above. The pulse-chase times was further centrifuged at 100,000 ϫ g for 1 hr at 4ЊC using a shown are in the figure legends. After pulse chase, the plates were Beckman 70 Ti fixed-angle rotor. The pellet was again resuspended put on ice, washed three times in cold PBS (containing 0.1 mM in buffer A and termed 100K/P. The supernatant was labeled 100K/S. CaCl2 and 1 mM MgCl2), and then incubated 30 min at 4ЊCin2ml

All samples were resuspended in 10ϫ sample buffer, boiled and biotinylation buffer (10 mM triethanolamine [pH 9.0], 2 mM CaCl2, subjected to 4%–20% SDS-PAGE, transferred to nitrocellulose, and 150 mM NaCl) containing 1 mg/ml NHS-SS-Biotin (Pierce) (freshly Western blotted as described in Stifani et al. (1992). For Western diluted from a 200 mg/ml DMSO stock stored at Ϫ20ЊC) with very blotting, a culture supernatant of anti-human Notch2 antibody gentle shaking, and subsequently incubated in PBS-CMG buffer bhN6D, which recognizes the intracellular domain of human Notch2, (0.1 mM CaCl2, 1 mM MgCl2, 100 mM Glycine) for another 30 min was used at a dilution of 1:10. to quench unreacted biotin. Postincubation plates were washed twice in PBS-CM buffer to wash away the quenched biotin. Finally, Biotinylation the cells were lysed and immunoprecipitated by PGHN as previously described. After the final wash, the beads were divided into two %80ف The cells were grown in 10 cm plastic tissue culture plates to confluence. Six plates were used per sample (ϩ biotin, or Ϫ biotin). aliquots. One aliquot was boiled in SDS sample buffer; the second Cells were washed four times with cold PBS-CMG (PBS, 0.1 M aliquot was incubated in 100 ␮l elution buffer (1% SDS, 50 mM Tris- ml of fresh, Cl, and 150 mM NaCl [pH 7.5]) at 80ЊC for 10 min, then 900 ␮lof 1.7 .(8.0ف CaCl2, 1.0 mM MgCl2, 1.0% glucose, pH cold PBS-CMG Ϯ Sulfo-NHS-biotin were added to each plate, then lysis buffer was added to the eluted protein. After centrifugation, incubated at 4ЊC for 15 min with shaking. This solution was replaced the supernatant was transferred to a fresh tube containing 50 ␮lof with cold RPMI without serum to absorb excess biotin, and cells packed streptoavidin beads (Pierce) and incubated at 4ЊC for 2–3 were pipetted off of plates in this medium and incubated at 4ЊC for hr. The beads were washed and boiled in SDS sample buffer as 15 min. Cells were washed three times in cold PBS-CMG solution described above. The samples were analyzed by 3–15% SDS-PAGE and were then lysed in 1.2 ml lysis buffer per sample (as described electrophoresis, fixed, dried, and fluorographed. in Zagouras et al., 1995). After addition of SDS to 0.2%, the samples ␮l each) for precipita- Acknowledgments 400ف) were divided into three equal portions tion: immobilized streptavidin (20 ␮l; Immunopure Immobilized Streptavidin, Pierce, Rockford, IL), anti-human Notch2 antibody We thank Laurent Caron, Robert Mann, Irene Moore, Karen Purcell, PGHN (2 ␮l; as described in Zagouras et al., 1995), or normal rabbit Stewart Frankel Lakshmi Bangalore, Esther Verheyen, Ping Xia, and serum (NRbS, 2 ␮l) as a negative control. Samples were incubated Deborah Eastman for their help and criticisms. We also thank Mi- overnight at 4ЊC. Staphylococcus aureus (Sigma Chemical Co., St. chele Solimena and Pietro DeCamilli for their criticisms, comments, Louis, MO) was added to PGHN and NRbS samples at 80 ␮l per and suggestions. Special thanks to Mark Mooseker for his intellec- sample, and incubations were continued at 4ЊC for 30 min. All sam- tual and practical support throughout this project. C. M. B. was ples were washed two times in 500 ␮l RIPA buffer A (10 mM Tris- supported by a Yale predoctoral fellowship and the Patrick and HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 1% Sodium Deoxycholate, Catherine Weldon Donaghue Medical Research Foundation; P. Z. 150 mM NaCl) plus antipain (2.5 ␮g/ml; Sigma Chemical Co., St. was supported by the Howard Hughes Medical Institute. S. A.-T. is Louis, MO), aprotinin (2.5 ␮g/ml; Sigma Chemical Co., St. Louis, supported by the Howard Hughes Medical Institute and by the Na- MO), leupeptin (2 ␮M; Sigma Chemical Co., St. Louis, MO), pepstatin tional Institutes of Health grant NS26084. (2.5 ␮g/ml; Sigma Chemical Co., St. Louis, MO), and phenylmethyl- sulfonyl fluoride (1 mM mg/ml; Sigma Chemical Co., St. Louis, MO). Received May 6, 1997; revised June 24, 1997. Samples were resuspended in 2ϫ sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis. References

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