A Novel Proteolytic Cleavage Involved in Notch Signaling

Molecular Cell, Vol. 5, 207–216, February, 2000, Copyright 2000 by Cell Press

A Novel Proteolytic Cleavage Involved in Notch Signaling: The Role of the Disintegrin-Metalloprotease TACE Christel Brou,* Fre´de´rique Logeat,* Neetu Gupta,* Christine Bessia,* Odile LeBail,* John R. Doedens,§ Ana Cumano,† Pascal Roux,‡ Roy A. Black,§ and Alain Israe¨l*k * Unite´ de Biologie Mole´culaire de l’Expression Ge´nique URA 1773 CNRS † Unite ´ de De´veloppement des Lymphocytes URA 1961 CNRS ‡ Physicochimie des Macromole ´ cules Biologiques URA 1773 CNRS Institut Pasteur 25 rue du Dr Roux 75724 Paris Cedex 15 France § Department of Protein Chemistry Immunex Corporation 51 University Street Seattle, Washington 98101

Summary The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a ␥-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the ␥-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNF␣-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE⫺/⫺ bone marrow–derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway. Introduction The Notch gene, first identified in Drosophila melanogaster, encodes a 300 kDa single-pass transmembrane receptor. Its extracellular domain contains 36 EGF-like repeats, some of which are responsible for interacting with the ligands. During its maturation in the secretory pathway, the mammalian Notch1 receptor is constitutively cleaved by a convertase of the furin family (at the S1 site), generating an extracellular (p200) a and transmembrane (p120) fragment, which remain associated. Only the heterodimeric molecules are present at the cell surface (Blaumueller et al., 1997; Logeat et al., 1998). k To whom correspondence should be addressed (e-mail: aisrael@ pasteur.fr).

The ligands (Delta and Serrate in Drosophila, Delta and Jagged in mammals) are expressed at the surface of adjacent cells, and their binding to Notch results in receptor activation. It has been shown that following activation, Notch is cleaved intracellularly (between Gly-1743 and Val-1744 in murine Notch1, hereafter named site S3) by a constitutive but still unknown protease (Kidd et al., 1998; Lecourtois and Schweisguth, 1998; Schroeter et al., 1998; Struhl and Adachi, 1998). This activity seems to be very similar if not identical to the ␥-secretase activity known to generate the A␤ peptide involved in Alzheimer’s disease from the ␤-APP precursor. The activity of presenilin 1 (PS1) is required for both Notch and ␤-APP cleavages (De Strooper et al., 1999; Song et al., 1999; Struhl and Greenwald, 1999; Ye et al., 1999). The Notch cleavage liberates the intracellular part of the protein, which migrates into the nucleus where it associates with the DNA-binding factor CSL (CBF-1, Suppressor of hairless, Lag-1; or RBP) to reconstitute an effective transcription factor (Jarriault et al., 1995; Tamura et al., 1995; Aster et al., 1997). This complex binds to regulatory sequences of the E(Spl) genes (Hes 1, Hes 5 in mammals) and upregulates expression of their encoded bHLH transcription factors (Jarriault et al., 1995, 1998; Kuroda et al., 1999; Ohtsuka et al., 1999), which in turn affect the regulation of downstream target genes (Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995; Eastman et al., 1997). A constitutively active mutant of Notch, N⌬E, which contains the intracellular portion of the receptor, the transmembrane domain, and 20 amino acids of the extracellular region, has been shown to be cleaved by the intracellular protease in a constitutive manner at the S3 site (Kopan et al., 1996; Lecourtois and Schweisguth, 1998; Struhl and Adachi, 1998). This fact explains the constitutive transcriptional activity of this mutant, although it is originally membrane anchored. On the other hand, the LNG construct (Kopan et al., 1994, 1996), which includes 277 amino acids of the extracellular domain, is neither constitutively active nor constitutively cleaved, although it does not contain any EGF repeat. Therefore, the conformation of the molecule is likely to be important for the accessibility of the substrate to the S3 protease. Ligand binding could result in a conformational change or/and an additional event could take place after ligand binding to permit cleavage of Notch by the S3 protease. The kuzbanian (kuz) gene that encodes a metalloprotease of the ADAM (a disintegrin and metalloprotease domain) family has been genetically linked to the Notch signaling pathway (Rooke et al., 1996; Pan and Rubin, 1997; Sotillos et al., 1997). The kuz gene interacts in a dosage-sensitive manner with the Notch gene in Drosophila and operates genetically upstream of Notch (Pan and Rubin, 1997). The human Kuz ortholog (ADAM 10) has been suggested to play a critical role in the regulation of sympathoadrenal cell fate (Yavari et al., 1998). Furthermore, an ADAM 10–deficient mouse dies earlier in development than the Notch1 knockout mouse (D. J.

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Pan, personal communication cited in Nye, 1997). Injection of a truncated dominant-negative derivative of Kuz (KuzDN) into Xenopus embryos results in overproduction of primary neurons (Pan and Rubin, 1997), as is the case for injection of a dominant-negative form of Delta (Chitnis et al., 1995). In a two-cell coculture assay, Dl1induced transactivation of HES1 is inhibited by KuzDN, suggesting that Kuz activity is needed for transcriptional response (Jarriault et al., 1998). On the other hand, neither the constitutive cleavage of N⌬E (Schroeter et al., 1998) nor its transcriptional effect on HES1 (Jarriault et al., 1998) are affected by coexpression of a dominantnegative Kuz, indicating that Kuz acts probably upstream of the S3 processing step. It has been recently proposed that Kuz could actually be responsible for Delta maturation, giving rise to a soluble form of the ligand (Qi et al., 1999). We have proposed (Logeat et al., 1998) that the Kuz metalloprotease might be responsible for an extracellular cleavage of the receptor, occuring after binding of the ligand (site S2 hereafter). This would give rise to a substrate that is readily recognized by the protease responsible for S3 processing. This substrate should have a structure close to that of the constitutively active but membrane-anchored form N⌬E. We show here that a processing event does indeed take place in the extracellular part of the receptor, preceding the proteolytic release of the intracellular domain occuring at the S3 site. Furthermore, the purification of this activity using an in vitro assay shows that it can be attributed to the metalloprotease TACE (TNF␣-converting enzyme, also known as ADAM 17). We finally show that PMA-induced differentiation of TACE⫹/⫹ bone marrow–derived monocytic precursor cells (which is accompanied by the induction of the ligand Jagged1) is inhibited in a ␥-secretase-dependent manner, while PMA treatment of TACE⫺/⫺ cells results in normal differentiation (although PMA induces Jagged1 expression in both cell types). These results support a role for TACE in Notch activation, at least in certain cell types. Results It has been previously reported, using transient transfection of N⌬E in 293T cells followed by pulse-chase analysis (Schroeter et al., 1998), that a processing product resulting from cleavage between amino acids Gly-1743 and Val-1744 of Notch1 (site S3) becomes visible after 30 min chase. This cleavage is sensitive to the protease inhibitor MG132 or to MW167, a recently described inhibitor of ␥-secretase (De Strooper et al., 1999). In order to visualize possible intermediate processing products, we designed a chimeric substrate of lower molecular weight (called ⌬E-GALVP16; Figure 1A) containing the Notch peptide signal, followed by Notch amino acids 1703 to 1809 (this molecule has the same N terminus as N⌬E), fused to the transactivator GALVP16 (Tora et al., 1989). Using this construct, it was possible to detect the product generated by S3 cleavage (hereafter designated P3, to specify the result of processing at the S3 site; Figure 1B, compare lane 2 to 1). Interestingly, an intermediate migrating product could also be observed,

Figure 1. Pulse-Chase Analysis of ⌬E-GALVP16 Constructs (A) Schematic map of the ⌬E-GALVP16 substrate used in (B). SP, signal peptide; TM domain, transmembrane domain; VSV, VSV epitope tag. The S2 and S3 sites are indicated, and the amino acid coordinates are from murine Notch1. (B) 293T cells were transfected with plasmids encoding ⌬EGALVP16 wild type (lanes 1–3), carrying the AV→VH mutation at the S2 site (lanes 4–6), or carrying the GCGV→LLFF mutation (R. Kopan, personal communication), at the S3 site (lanes 7–9). After 24 hr, cells were pulsed with [35S]Met for 15 min, then directly extracted (lanes 1, 4, and 7) or chased for 3 hr (lanes 2, 3, 5, 6, 8, and 9). MG132 (50 ␮M, lanes 3, 6, and 9) was applied continuously 1 hr before and during the pulse chase. Cells extracts were immunoprecipitated with anti-VSV antibody (P5D4). Immunoprecipitated proteins were eluted and analyzed on a 8% SDS-PAGE. ⌬E-GALVP16, P2, and P3 respectively indicate the uncut substrate and the P2 and P3 processing products.

which accumulated in the presence of MG132 (Figure 1B, lanes 2 and 3, band P2) or MW167 (data not shown). We conclude that this new product results from processing by another protease at a site that we named S2, located upstream of the S3 site. Moreover, when the S3 site was mutated in such a way that it could no longer be processed (Figure 1B, lanes 7–9, Mut S3), the P2 product accumulated in the absence of MG132, suggesting that in the wild-type context, at least part of the P3 product is derived from P2 (see Discussion). In order to identify the enzymatic activity responsible for processing at the S2 site, we then used as a substrate for in vitro experiments the ⌬E-GALVP16 construct (in Figures 4 and 6) or a substrate that starts at the furine cleavage site (amino acid 1654 in the Notch1 sequence, FuGALVP16 in Figure 2A [Logeat et al., 1998]). We obtained identical results with both substrates in all the in vitro experiments. These substrates were synthetized in a coupled in vitro transcription/translation system in the presence of [35S]Met. Following incubation with a membrane fraction derived from HeLa cells, a processing product was detected after SDS-PAGE and autoradiography (Figure 2B, compare lane 2 and lane 1). The detected proteolytic activity was sensitive to DTT (Figure 2B, lane 3), to EDTA, and to another metalloprotease inhibitor (1,10 o-phenantroline; data not shown). The same effect was observed on a substrate that does not contain GAL-VP16 (data not shown); on the other hand, GAL-VP16 alone was not processed in vitro (data not shown), confirming that this effect requires the Notch sequence. After increasing the scale of the reaction and using [3H]Leu instead of [35S]Met, we purified the band

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Figure 2. In Vitro Cleavage at the S2 Site (A) Schematic map of the FuGALVP16 substrate used in (B). The sequence of the region surrounding sites S2 and S3 is indicated. The transmembrane domain is boxed. (B) The substrates FuGALVP16, either wild type (lanes 1–3) or mutated at the S2 site (lanes 4–6), containing a Notch fragment (amino acids 1655–1809) in frame with a VSV epitope and the chimeric protein GALVP16, were in vitro transcribed and translated in the presence of [35S]Met. An aliquot was incubated without (lanes 1 and 4) or with (lanes 2, 3, 5, and 6) HeLa cells membranes. DTT was added to 1 mM when indicated (lanes 3 and 6). The products were analyzed on an 8% SDS-PAGE. UC and C represent the substrate and its P2 processing product, respectively.

of lower relative molecular mass and carried out Edman N-terminal sequencing. Counting of 3H in each fraction of the sequencing reaction led us to localize the processing site on the Notch sequence between amino acids Ala1710 and Val-1711, 13 amino acids upstream of the transmembrane domain (Figure 2A). We designed two mutants of this site (AV→ED and AV→VH) that were no longer processed in vitro (Figure 2B, lanes 4–6 for AV→ED, and data not shown). Pulse-chase analysis in cells transfected with the same mutated constructs showed no appearance of the P2 intermediate band, with or without MG132 (Figure 1B, lanes 4–6), confirming that the same processing event was most likely detected in vitro and in vivo. Interestingly, the MG132-sensitive processing at the S3 site could still occur to some extent in this context (Figure 1B, lane 5, and see Discussion). We then tried to demonstrate that the S2 cleavage event could take place in vivo, in the context of the full-length Notch molecule. Based on the genetic data involving Kuz, a membrane metalloprotease of the ADAM family, in the Notch pathway, as well as on the inhibition of the S2 cleavage by metalloprotease inhibitors (Figure 2B), we postulated that the enzyme responsible for the S2 cleavage could be a member of the ADAM family. The activity we identified is constitutive in the cells used, suggesting that it is probably the conformation of the substrate that is modified following ligand binding. Since activation of the Notch pathway by mixing cells carrying either the receptor or a ligand apparently does not mobilize enough Notch molecules to allow visualization of processed forms, although it results in CSL-

dependent transcriptional activation of a reporter gene (Jarriault et al., 1998), we reasoned that we would visualize the S2 cleavage event more easily under conditions where the substrate is present in an “open” configuration and where the enzyme activity can be induced in a controlled manner. This situation is represented by the C12D cells (Logeat et al., 1998). These cells constitutively express a furin convertase inhibitor (␣1PDX), with the result that Notch is mostly retained in the endoplasmic reticulum or Golgi compartment in a full-length form instead of being presented at the plasma membrane in a heterodimeric form. As it has been postulated that in the context of the heterodimeric molecule, which is normally present at the cell surface, the extracellular region is responsible for preventing activation of the receptor (Kopan et al., 1996; Artavanis-Tsakonas et al., 1999), we reasoned that a molecule that has not been matured by furin might represent an “open” substrate. In extracts from C12D cells, Notch is indeed present essentially as a full-length molecule and few p120 (the C-terminal furin-generated processing product) can be detected (Logeat et al., 1998). As maturation of the metalloproteases of the ADAM family most likely requires processing by a furin-like enzyme at a tetrabasic site that marks the limit of the prodomain (Wolfsberg et al., 1995; Amour et al., 1998; Loechel et al., 1998), we reasoned that these proteases would be inactive in C12D cells. It has been previously reported that the latent state of the precursors for ADAM proteases is due to the formation of an intramolecular complex between a Cys residue localized in the prodomain and the essential Zn atom in the catalytic domain (Van Wart and BirkedalHansen, 1990; Loechel et al., 1998). These latent forms can be activated by oxidants when they are in an unprocessed form that includes the prodomain (Van Wart and Birkedal-Hansen, 1990). We treated Jurkat and C12D cells for 3 hr with glucose oxidase, which is known to generate oxygen radicals (see Experimental Procedures), and analyzed extracts with antisera directed against the intracellular part of Notch1 or against metalloproteases of the ADAM family. We first observed that, according to our hypothesis, these metalloproteases are mostly under the proform in C12D cells, in contrast to Jurkat cells where the mature form is the most abundant. This is true both for Kuz (data not shown) and for another related member of the ADAM family, TACE (Figure 3A, compare lanes 1 and 2). We also confirmed our previous observations showing that in C12D cells Notch1 is mostly under the p300 precursor form, with almost no detectable p120, while in Jurkat cells p300 is almost entirely processed (Figure 3B, compare lanes 1–2 with 3–4). Glucose oxidase treatment of C12D cells (Figure 3B, lane 4) led to the appearance of a new Notch1-derived band (P3; see below) migrating faster than the furin-generated p120 fragment. Concomitantly, the amount of the p300 precursor decreased (compare lanes 3 and 4 in Figure 3B). Interestingly, in accordance with the hypothesis mentioned above, glucose oxidase treatment of Jurkat cells, where maturation of membrane metalloproteases and of Notch by the furin convertase would be expected to take place normally, did not result in the generation of the P3 band (Figure 3B, lane 2; see also Figure 3C, lanes 1–2). In order to analyze the 100–120 kDa region in more detail,

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Figure 3. The Second Processing Event Can Occur In Vivo (A) Total extracts were prepared from Jurkat (lane 1) or C12D cells (lanes 2 and 3) and analyzed by Western blotting using P1, an antibody recognizing the extracellular region of TACE (the results concerning pro-TACE were confirmed using an antiserum directed against the prodomain of TACE [data not shown]). When indicated, the cells were treated for 3 hr with glucose oxidase (GO, 0.02 U/ml) before extracts were prepared. The positions of the precursor form (proTACE) and the mature form (TACE) are indicated on the right. Ju, Jurkat. A molecular weight marker (kDa) is shown on the left. (B) The extracts were analyzed using an antiserum recognizing the intracellular region of the Notch1 molecule (Logeat et al., 1998). GO, glucose oxidase. The p300 Notch1 precursor, the p120 furin-generated product, and the P3 product are indicated on the right. (C) An analysis similar to (B) was performed, except that a longer gel with a higher concentration of acrylamide was used, to focus on the 100–120 kDa region. Jurkat (lanes 1–3) or C12D cells (lanes 4–9) were treated for 3 hr with glucose oxidase (0.02 U/ml), 1,10 o-phenanthroline (5 mM), and/or MG132 (50 ␮M) as indicated below the lanes. The P1 (p120), P2, and P3 products are indicated on the right. n.s. represents a nonspecific band. (D) Nuclear extracts from C12D cells, treated or not with glucose oxidase as indicated, were analyzed by Western blotting with the anti-Notch antibody (top) or an anti-CSL antibody as a control for extraction of nuclear proteins (bottom). The P3 processing product and CSL protein are indicated on the right. (E) Total RNA extracted from untreated or glucose oxidase treated Jurkat or C12D cells, as indicated, was analyzed by Northern blotting using a HES-1 probe (top) or an S26 probe for normalization. Quantification by Phosphorimaging and normalization with respect to S26 showed a reproducible 2-fold increase in HES-1 expression in glucose oxidase-treated C12D cells and no increase in Jurkat cells.

we modified the gel electrophoresis conditions. Figure 3C shows the results of such an analysis: glucose oxidase treatment of C12D cells actually induces the appearance of two new bands (P2 and P3 in Figure 3C, lanes 5 and 8). The fastest band observed in these lanes most likely corresponds to P3, the final processing product generated by ␥-secretase cleavage, as it can be found in the nucleus (Figure 3D, lane 2), and its appearance is blocked by MG132 (Figure 3C, lane 9) and by the ␥-secretase inhibitor MW167 (data not shown). Treatment with these drugs induces an accumulation of the intermediate band (Figure 3C, lane 9, band P2), which most likely corresponds to cleavage at the S2 site described above. Treatment with 1,10 o-phenanthroline (Figure 3C, lane 6) or with the more specific membrane metalloprotease inhibitor IC-3 (data not shown) blocks the appearance of P2 and P3, confirming that the S2 cleavage is due to a metalloprotease and that it takes place before S3. To confirm that glucose oxidase treatment indeed activates the Notch pathway, we analyzed the expression of the HES-1 gene: Figure 3E shows that this treatment results in an increased level of HES-1 mRNA in C12D, but not in Jurkat cells. These results strongly suggest that a proteolytic event due to a furin-matured metalloprotease is necessary for cleavage by the ␥-secretase-like enzyme to take place, confirming and extending the results obtained in the pulse chase analysis of Figure 1. One hypothesis, also based on the genetic data implicating Kuz (ADAM 10) in the Notch pathway, was that Kuz could account for the S2 activity. In order to determine if cleavage at the S2 site was

due to the Kuz metalloprotease, we established a purification procedure of the S2 activity from HeLa cell membranes. It was possible to separate chromatographically the fractions containing the S2 activity (Load and flow through of the RED-TSK column; Figure 4, bottom) from those containing the Kuz protein, detected by Western blotting (Load, 0.3 M, 0.5 M, and 1 M of the RED-TSK

Figure 4. The Metalloprotease Kuz and the S2 Processing Activity Can Be Chromatographically Separated A HeLa cells membrane preparation was fractionated on Q-Sepharose and RED-TSK columns, and the fractions were tested in parallel for the presence of the Kuz protein by Western blotting (upper panel) and for their capacity to cleave ⌬E-GALVP16 at the S2 site in vitro (lower panel). The two lanes for each fraction in the lower panel correspond to 1 and 10 ␮l of fraction per assay. The apparent molecular weight of Kuz is about 60 kDa, as previously described (Howard et al., 1996).

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Figure 5. TACE and the S2 Activity Coelute throughout the Purification Procedure S2 activity was purified from HeLa cells by successive fractionation onto Q-Sepharose (first three lanes), RED-TSK (three following lanes), Concanavalin A-Sepharose, RED-TSK, and finally onto a MonoQ column (last nine lanes) (see Experimental Procedures). The fractions were tested by Western blotting with anti-TACE antibody (M222) or by in vitro assay on ⌬E-GALVP16. The plus signs indicate that the fraction contains the S2 activity, the size being proportional to the activity. It should be noted that the peak of activity detected in vitro correlates with the highest concentration of TACE. The apparent molecular weight of TACE is about 80 kDa, as described (Black et al., 1997).

column; Figure 4, top). Therefore, it is very unlikely that the S2 activity can be attributed to Kuz, although we cannot completely rule out this possibility (see Discussion). The complete purification scheme of the S2 activity through several columns (see Experimental Procedures), the apparent molecular weight of the purified bands (60–85 kDa), the sensitivity to metalloprotease inhibitors, and the sequence of the recognition site led us to postulate that the S2 activity could be due to another ADAM protease, namely TACE. The most common cleavage site for this metalloprotease is indeed between Ala and Val, for example in pro-TNF␣ or proTGF␣ (Black et al., 1997; Moss et al., 1997; Peschon et al., 1998). As shown in Figure 5, there is a perfect correlation throughout all the purification procedure between the S2 activity, detected in vitro on ⌬E-GALVP16 (Figure 5, bottom), and the presence of TACE detected by Western blotting (Figure 5, top). Furthermore, the S2 activity-containing fractions (Figure 6A, lanes 2 and 3) were also capable of digesting pro-TNF␣ into TNF␣ in vitro (Figure 6C, lane 2, and data not shown for the other fractions). On the other hand, native TACE purified from CHO cells on the basis of its ability to mature pro-TNF␣ was also able to cleave ⌬E-GALVP16 (Figures 6A, lane 4, and 6C, lane 3). Finally, recombinant TACE purified from culture medium of cells secreting either the extracellular domain or the catalytic domain of this metalloprotease were as active on ⌬E-GALVP16 as on proTNF␣ (Figures 6A, lanes 5 and 6, and 6C, lanes 4–7). The recognition site for recombinant TACE is probably also AV on ⌬E-GALVP16, since the Mut S2 mutants are not processed by any of the active fractions (AV→ED in Figure 6B; data not shown for AV→VH). Therefore, it is likely that the S2 activity that we have purified is due to TACE.

Figure 6. TACE Accounts for the S2 Activity Fractions derived from the purification of the S2 activity (Concanavalin A–eluted material, lanes 2 in [A], [B], and [C], or MonoQ-eluting fraction in lanes 3 of [A] and [B]) or from culture supernatants of cells overexpressing recombinant forms of TACE (TACE cat only contains the catalytic domain of the enzyme: lanes 5 in [A] and [B], two differents doses in lanes 4 and 5 of [C]; TACE EC contains the extracellular domain of the enzyme except for the prodomain: lanes 6 in [A] and [B], lanes 6 and 7 in [C], increasing doses) were tested in vitro using different substrates. The reactions were performed in the same conditions for the three substrates: ⌬E-GALVP16 wild type in [A], the AV→ED mutant in [B], or pro-TNF␣ in [C]. UC and C represent the substrate and its processing product, respectively. Extracts derived from TACE⫹/⫹ (lanes 7 in [A] and [B]) or TACE⫺/⫺ fibroblasts (lanes 8 in [A] and [B]) were tested in parallel.

Furthermore, TACE is probably the only activity capable of processing ⌬E-GALVP16 in our conditions, since crude extracts obtained from fibroblasts derived from TACE⫺/⫺ mice (Peschon et al., 1998) did not exhibit any S2 activity, although control extracts from their wildtype counterpart did (Figure 6A, lanes 7–8). The mature form of Kuz could be detected in both TACE⫹/⫹ or TACE⫺/⫺ fibroblasts (data not shown). We then tried to demonstrate that TACE plays a role in the activation of the Notch pathway under physiological conditions. For this, we used bone marrow–derived monocytic precursor cells (DRM) obtained as follows: DRM cells derived from TACE⫺/⫺ bone marrow cultures (Peschon et al., 1998) were infected with a retrovirus expressing the murine TACE cDNA, or as a control with a retrovirus expressing the bacterial lacZ gene. One clone was isolated from each cell population and referred to as DRM-5 (expressing TACE) or DRM-lacZ (not expressing TACE), respectively. We actually obtained the same results with the pools of TACE⫹/⫹ or TACE⫺/⫺ cells, as well as with multiple individual clones derived from these populations (data not shown). Similar types of myeloid precursor cells can be induced to differentiate into macrophages by PMA, and activation of the Notch pathway in such cells has been

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Figure 7. Inhibition of PMA-Induced Differentiation of DRM Cells Is Dependent on the Presence of TACE (A) TACE-reconstituted bone marrow–derived monocytic precursor cells (DRM-5) (obtained as described in Experimental Procedures by infecting TACE⫺/⫺ cells with a retrovirus expressing murine TACE) were treated with 100 ng/ml PMA for 0 (panel 1), 24 (panels 2 and 4), or 48 hr (panels 3 and 5) in the absence (panels 2 and 3) or presence (panels 4 and 5) of 50 ␮M MW167. (B) TACE⫺/⫺ DRM cells were treated with 100 ng/ml PMA for 0 (panel 1), 24 (panel 2), or 48 hr (panels 3 and 4) in the absence (panels 2 and 3) or presence (panel 4) of 50 ␮M MW167. The cells were then fixed and stained with May-Gru¨nwaldGiemsa as described in the Experimental Procedures. Red arrows indicate blast precursor cells (small cells with a dark nucleus and almost no visible cytoplasm), while green arrows indicate differentiated cells (larger cells, pale pink, nucleus poorly visible). (C) Western analysis of Jagged1 in TACE-reconstituted (TACE⫹/⫹) or TACE⫺/⫺ DRM cells treated or not with PMA for 24 or 48 hr, as indicated.

shown to inhibit these differentiation events (Li et al., 1998; Carlesso et al., 1999). DRM cells express reasonable levels of endogenous Notch1 (data not shown). PMA is a known activator of NF-␬B, and it has been shown recently that the expression of the Notch ligand Jagged1 is under the control of NF-␬B (Bash et al., 1999). We therefore treated the DRM cells with PMA and measured the extent of differentiation at 24 hr and 48 hr. In parallel, we measured the expression of Jagged1. Figure 7C shows that PMA induces the expression of Jagged1 in both DRM-5 and DRM-lacZ cells. In DRMlacZ cells treated with PMA for 48 hr, a large proportion of cells acquired the morphology of macrophages (Figure 7B, panel 3); this differentiation was already clearly visible at 24 hr (Figure 7B, panel 2). Interestingly, PMA did not induce differentiation of DRM-5 cells after 48 hr (Figure 7A, panel 3). One possible explanation for this result is that activation of the Notch pathway elicited by the PMA-induced expression of Jagged1 results in inhibition of differentiation of DRM-5, but not of DRMlacZ cells, where Notch activation would not occur. To confirm this hypothesis, we treated both cell types with the ␥-secretase inhibitor MW167 (which inhibits the S3 cleavage and therefore the activation of the pathway)

together with PMA and indeed observed an increased differentiation (Figure 7A, panels 4 and 5) of DRM-5, similar to what is seen in DRM-lacZ cells. No effect of MW167 on PMA-induced differentiation of DRM-lacZ cells could be observed (Figure 7B, panel 4). MW167 alone did not induce differentiation of DRM-5 cells (data not shown). These results suggest that TACE indeed plays a role in Notch activation.

Discussion Our data demonstrate that a proteolytic event due to a membrane metalloprotease (TACE being a likely candidate, see below) can take place in the extracellular region of the Notch receptor (at the S2 site located between amino acids 1710 and 1711 of murine Notch1, 13 amino acids upstream of the transmembrane domain), preceding the proteolytic release of the intracellular domain. We propose that ligand binding to the Notch heterodimeric receptor somehow unmasks the extracellular metalloprotease recognition site. In the accompanying paper, Mumm and colleagues show that such a processing does indeed occur in vivo at the exact same

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site following ligand binding. Processing by this metalloprotease would give rise to an accessible substrate for the ␥-secretase-like protease (S3 site), permitting the release of the transcriptionally active intracellular part of Notch. Since furin, the activity described here and in the accompanying paper (Mumm et al., 2000 [this issue of Molecular Cell]), as well as ␥-secretase have been shown to exhibit activity on appropriate substrates in the absence of an external stimulus, we conclude that constitutive proteases are responsible for transmitting the ligand-induced Notch signal. The pulse-chase experiments presented in Figure 1 demonstrate that a processing event occurs upstream of the ␥-secretase-like, MG132-sensitive cleavage. The P2 product generated by this cleavage accumulates when cells are treated with MG132 or MW167, an inhibitor of ␥-secretase, or when the S3 site is mutated such as ␥-secretase cleavage is inhibited. In the context of N⌬E, the ␥-secretase-like processing can take place independently of the upstream processing step, as shown with the ⌬E-GALVP16 Mut S2 construct. This is because the extracellular part of N⌬E is short enough to allow constitutive cleavage at both the S2 and S3 sites. Other deletion mutants of Notch, like the LNG construct, which includes 277 amino acids in the extracellular domain (Kopan et al., 1994, 1996), are neither constitutively active nor processed. It could be that the S2 site is masked by the heterodimerization that follows furin maturation during the transport of newly synthetized molecules to the membrane, even though the extracellular fragment is shorter than in the full-length receptor. This would imply that any construct whose N terminus is at or downstream of the furin site should be constitutively active: we have recently confirmed this prediction for a construct that starts at the furin site (data not shown). A similar conclusion has been reached by others (Artavanis-Tsakonas et al., 1999). Interestingly, full-length Notch itself can be a substrate for TACE or another member of the ADAM family in cells where maturation by furin and consequently heterodimerization and presentation at the membrane are blocked. This is the case in C12D cells (Logeat et al., 1998), which express ␣1PDX, a furin convertase inhibitor. In these cells, TACE is in an inactive precursor conformation since furin or a related convertase have been proposed to be responsible for the release of the prodomain in this family of metalloproteases (Wolfsberg et al., 1995; Loechel et al., 1998). When the latent metalloprotease is activated by oxidants, a processed nuclear form of Notch corresponding to the P3 product generated by the ␥-secretase-like activity can be visualized. To our knowledge, this is the first demonstration of nuclear Notch derived from the endogenous receptor. Treatment with MW167 or MG132 results in the disappearance of the P3 and in the accumulation of the P2 product, and treatment with metalloprotease inhibitors such as 1,10 o-phenanthroline or IC-3 reduces the amount of both P2 and P3 products, suggesting that in the context of the endogenous Notch molecule, cleavage at the S3 site requires prior cleavage at the S2 site. The same treatment applied to control Jurkat cells does not induce appearance of the P2 and P3 processing products, confirming that the mature form of Notch normally present at the cell surface is not sensitive to the action of the

ADAM proteases (even though the enzyme is active) and that an additional event, in the circumstances, ligand binding, is probably needed to unmask the S2 site. Purification of the enzyme responsible for Notch1 processing at the S2 site in vitro led to the identification of TACE (ADAM 17). The TACE cleavage site in the Notch1 sequence is located between Ala-1710 and Val-1711, 13 amino acids upstream of the transmembrane domain. This site is perfectly conserved in murine or human Notch1 and in Xenopus Notch. In murine Notch2 and Notch3 and Drosophila Notch, similar sites can be found (SV in murine Notch2 and Drosophila Notch at the same position upstream of the transmembrane domain, AV 24 amino acids upstream of the transmembrane domain in murine Notch3), but their sensitivity to TACE or to another ADAM protease have not been tested so far. On the other hand, it is difficult to identify a similar site in Lin12 or Glp1, because of the weak general conservation. However, a database search indicates that a TACE ortholog exists in C. elegans. Thus, it is possible that TACE could recognize a degenerated site in C. elegans Notch homologs; alternatively, other mechanisms may exist to lead to a conformational change after ligand binding. Interestingly, some constitutively active mutants of C. elegans Lin12 carry mutations in the region surrounding the putative S2 site (Greenwald and Seydoux, 1990): we are currently testing whether these mutations result in unmasking of this site in the absence of ligand, therefore allowing constitutive processing at the S2 and S3 sites. The AV cleavage site in Notch matches perfectly known TACE preferred recognition sites such as those found in pro-TNF␣ or pro-TGF␣ (Black et al., 1997; Moss et al., 1997; Peschon et al., 1998). Furthermore, the purification procedure led to the identification of only one activity capable of processing ⌬E-GALVP16 in vitro. In addition, this activity was not present in extracts from TACE⫺/⫺ cells. However, we cannot completely rule out the possibility that another enzyme could also be involved in the in vivo activity (see below). Another argument in favor of TACE being involved in the Notch signaling pathway comes from experiments using bone marrow–derived monocytic precursor cells (DRM) derived from TACE⫺/⫺ bone marrow cultures, reconstituted or not with the murine TACE cDNA. These cells express Notch, and when they are treated with PMA, the Notch ligand Jagged1 is induced, and in the case of the TACE-reconstituted cells, differentiation is inhibited. PMA treatment for 48 hr induces differentiation of most TACE⫺/⫺ cells into macrophages, whereas TACE-reconstituted cells proliferate as blast cells. Treatment with PMA and MW167, an inhibitor of the ␥-secretase (which prevents the S3 cleavage required for the final step of Notch activation), led to the differentiation of TACE-reconstituted cells, while it had no effect on PMA-induced differentiation of TACE⫺/⫺ cells. These results support the idea that activation of the Notch pathway by the ligand Jagged1 inhibits differentiation of these precursor myeloid cells and suggest that TACE is likely to be required for this activity. Another possibility would be that the lack of processing of a membrane protein different from Notch might be responsible for accelerated differentiation in TACE⫺/⫺ cells. One candidate for such a protein is the membrane-anchored form

Molecular Cell 214

of TNF, one of the known substrates of TACE. To test this hypothesis, we added soluble TNF to TACE⫺/⫺ DRM cells but could not observe any difference in the kinetics of differentiation (data not shown). Together with the sensitivity of the differentiation-inhibiting activity to the MW167 ␥-secretase inhibitor, these results support a role for TACE in the Notch signaling pathway in these myeloid precursors. Experiments are in progress to determine whether activation of the Notch pathway inhibits or just delays differentiation of the TACE-reconstituted cells. Genetic evidences in Drosophila and C. elegans have shown that Kuzbanian (ADAM 10) is also involved in the Notch signaling pathway (Rooke et al., 1996; Pan and Rubin, 1997; Sotillos et al., 1997). During the purification of the Notch-processing activity, Kuz eluted in fractions devoid of in vitro activity. In the absence of a bona fide substrate for Kuz, it is hard to evaluate whether this protease is active in the fractions where we detect it; however, the purification procedure we followed, which is similar to the one used by others, as well as the presence of mature Kuz in the extracts strongly suggests that it is active and cannot cleave Notch, at least under our in vitro conditions. We cannot completely rule out the possibility that Kuz could cleave Notch in certain tissues or that other members of the ADAM family could do so. However, it is shown in the accompanying paper (Mumm et al., 2000) that the S2 cleavage does still take place in Kuz⫺/⫺ cells. In addition, it has been recently proposed that Delta, one of the Notch ligands, could be processed, and Kuz has been proposed to account for this activity (Qi et al., 1999). Therefore, the genetic data could be explained by a role of Kuz in processing of the Notch ligands, if this processing is somehow required for signaling. Further experiments are required to clarify this point. TACE⫺/⫺ mice, which carry a deletion of the catalytic domain of the enzyme, die between embryonic day 17.5 and the first day after birth and do not exhibit a strong neurogenic phenotype (Peschon et al., 1998). This is in partial contradiction with a prominent role for TACE in the Notch signaling pathway, since Notch1⫺/⫺ mice die before 11.5 days of gestation. It is possible that because of a functional redundancy, Kuz or a related protease could in this case partially account for the S2 activity. Indeed, TACE and Kuz are the closest members of the ADAM family (29% amino acid identity). An interesting analogy is the ␤-APP protein, involved in familial forms of Alzheimer’s disease (for a review, see Selkoe, 1998); this protein undergoes several alternative proteolytic steps, and one of the possible combinations involves cleavage inside the transmembrane region by ␥-secretase, which also cleaves Notch at the S3 site and at an upstream membrane proximal site by an activity that has been attributed to either Kuz or TACE (Buxbaum et al., 1998; Lammich et al., 1999). However, it must be noted that even though TACE might not be the unique protease involved, the experiments describing an inhibition of differentiation of the DRM cells suggest that this enzyme is likely to play a prominent role in the activation of the Notch pathway, at least in certain cell types.

Experimental Procedures Reagents The membrane metalloprotease inhibitor IC-3 was from Immunex (Seattle, WA). MW 167 was from Partners Neurology (Boston, MA). Plasmids ⌬E-GALVP16 construct was obtained by PCR using N⌬E, which has the methionine at position 1727 replaced with a valine (Kopan et al., 1996), as a template with the following primers: 5⬘-CGGGATCCC CACGGCTCCTGACGCCC-3⬘ and 5⬘-CTAGCTAGCCTCCAGGTCT TCGTCTCC-3⬘, and inserted into the BamHI and NheI sites of a pCDNA3-derived vector, which allowed the in vitro and in vivo expression of proteins fused in C terminal to the VSV epitope. It includes amino acids 1703 to 1809 of Notch1, plus the signal peptide. GAL-VP16 (Tora et al., 1989) was also amplified and cloned in frame downstream of the VSV epitope. This last plasmid was used as a backbone to clone FuGALVP16, where the Notch fragment corresponding to amino acids 1655 to 1809 was amplified using as a first primer 5⬘-CGGGATCCGAGCTGGACCCCATGGAC-3⬘ and the same second primer as above. The Mut S2 constructs were obtained by site-directed mutagenesis by using the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Each mutant was sequenced to confirm the mutation. The oligonucleotides used were 5⬘-AATATTCCTTACAAGATTGAGGAAGACAAGAGTGAGCCGGTG G AG-3⬘ to generate the AV→ED mutant and 5⬘-ATTCCTTACAAGAT TGAGGTGCACAAGAGTGAGCCGGTGGAG-3⬘ to generate the AV→VH mutant. The same results were obtained in vitro and in vivo with both constructs. The GCGV→LLFF mutant of N⌬E was a kind gift of E. H. Schroeter and R. Kopan (St. Louis, MO). Transfection and Pulse Chase 293T cells and wild-type or TACE⫺/⫺ mouse fibroblasts (Peschon et al., 1998) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. Transfections and pulse-chase experiments were performed as described in Kopan et al. (1996), except that no cycloheximide was added during the chase. Cells were lysed in RIPA buffer (50 mM Tris [pH 7.9], 150 mM NaCl, 1% Nonidet P40, 0.1% SDS, 0.5% Deoxycholate) supplemented with protease inhibitors, and proteins were immunoprecipitated with the anti-VSV P5D4 monoclonal antibody for 1 hr at 4⬚C. Protein G-Agarose was added and incubated at 4⬚C for an additional hour, washed three times with RIPA buffer, and boiled for 5 min in SDS sample buffer. The samples were analyzed by SDS-PAGE. When mentioned, MG132 (50 ␮M) was added 1 hr before the pulse and during the chase period. Culture of DRM Cells DRM cells were obtained from Dexter cultures derived from bone marrow, and the myeloid fraction was immortalized by infection with a ras-myc encoding retrovirus (J. Peschon and R. B., unpublished data). DRM-5 and DRM-lacZ were obtained by infecting DRM cells derived from TACE⫺/⫺ bone marrow cultures with recombinant retrovirus expressing murine TACE cDNA or the lacZ gene, respectively. After infection, cells were cloned and selected for lacZ expression by staining and for TACE activity by measuring shed TNF in the culture medium following LPS stimulation. The cells were grown in medium supplemented with 10% FCS, 100 ␮M ␤-mercaptoethanol, and 20 ng/ml GM-CSF. When indicated, the cells were treated with 50 ␮M MW167 and either left untreated or treated with 100 ng/ml PMA. After 24 or 48 hr, the cells were cytospinned, fixed, and stained with May-Gru¨nwald Giemsa. Cell Treatment, Extract Preparation, and Western Blotting Jurkat and C12D cells (Logeat et al., 1998) were grown in RPMI supplemented with 10% fetal calf serum. Cells were treated for 3 hr with 0.02 U/ml of glucose oxydase with or without 5 mM 1,10 o-phenanthroline or 50 ␮M MG132 (generation of oxygen radicals requires glucose plus glucose oxidase, but glucose is already present in the culture medium [Farber et al., 1984]). Cells were then washed with cold PBS and lysed in TNT buffer (50 mM Tris [pH 8], 200 mM Nacl, 0.5% NP40, and 1 mM Pefabloc Sc [Boehringer Mannheim, Mannheim, Germany]). After 20 min on ice, cell lysates

Involvement of TACE Protease in Notch Signaling 215

were centrifuged at 14,000 rpm for 20 min, and the supernatants were used for Western blot analysis. To prepare nuclear extracts, 109 cells were washed with PBS, resuspended in buffer B (10 mM Tris [pH 7.4], 15 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 5% glycerol, 1 mM DTT, 1 mM Pefabloc) containing 0.25 M sucrose, then broken with a Dounce homogenizer (pestle B; 100 strokes). After low-speed centrifugation, the nuclear pellet was resuspended in buffer B containing 2.3 M sucrose, loaded on a 1 ml sucrose cushion (2.3 M), and submitted to 1 hr centrifugation in a TL 100.2 at 4⬚C at 28,000 rpm. The nuclear pellet was washed in buffer B containing 0.25M sucrose, then nuclei were extracted in 0.4 M NaCl-containing buffer. Purification of the S2 Activity A membrane fraction was prepared from 1010 HeLa cells: pelleted cells were resuspended in three volumes of buffer A (50 mM Tris [pH 7.9], 10 mM NaCl), then allowed to swell for 30 min on ice, and lysed by 25 strokes of a Dounce homogenizer (type B pestle). The homogenate was centrifuged for 15 min at low speed, and the supernatant was subjected to a second centrifugation at 48,000 rpm for 1 hr in a 60Ti rotor. The resulting pellet was resuspended in 1 vol of buffer A containing 1% Nonidet P40, left on ice for 30 min, then centrifuged at 48,000 rpm in the same rotor for 1 hr. The resulting supernatant was decanted, and glycerol was added to 10%. This crude membrane preparation was fractionated on Q-Sepharose, the 0.2 M eluting fraction was diluted and loaded onto a RED-TSK column at 125 mM NaCl, then the material eluting at high ionic strength (0.3 M to 1 M) was passed over ConcanavalinA–Sepharose 4B. The S2 activity was eluted with buffer containing 50 mM Tris (pH 7.9), 50 mM NaCl, 0.2% Nonidet P40, and 250 mM methyl mannopyranoside, then loaded again onto a RED-TSK column, which was equilibrated with 50 mM NaCl. In these conditions, the S2 activity was detectable only in the flow through fraction. Finally, the S2-containing fraction was passed over a MonoQ column using a SMART system (Pharmacia, Uppsala, Sweden) and eluted in a gradient from 50 to 300 mM NaCl. Samples from each fraction were assayed for S2 activity, and the protein profile was analyzed by SDS-PAGE (silver staining). It revealed three major bands migrating between 60 and 85 kDa (data not shown). For the separation of Kuz from S2 activity presented in Figure 4, the membrane extract was loaded on Q-Sepharose, then the 0.15 M-eluting fraction was diluted twice and loaded on a RED-TSK column equilibrated in 75 mM NaCl. In Vitro Processing Assay and Sequencing The substrates were prepared by coupled in vitro transcription/ translation in rabbit reticulocyte lysate (Promega, Madison, WI) in the presence of [35S]methionine and [35S]cysteine (Promix Amersham, Pharmacia). For pro-TNF␣, the reaction was performed in a mix without methionine and cysteine, since labeling of the cysteine residues is necessary to detect the TNF␣. The substrate (0.25 ␮l) was incubated in buffer P (50 mM Tris [pH 7], 75 mM NaCl, 1 mM MgCl2, 10% Glycerol) with 10 ␮l of membrane extract or of each fraction in 20 ␮l final, at 30⬚C for 1 hr. The reaction was stopped by adding Laemmli buffer and analyzed on a 8.5% SDS-PAGE. The gel was fixed, dried, and revealed by overnight autoradiography. For sequencing of the processing product, [3H]Leu was used instead of 35S, and the reaction was scaled up. After transfer to a PVDF membrane, the processing product was excised and Edman sequencing was performed on an Applied Biosystems (PerkinElmer, Norwalk, CT) 473A sequencer: the radioactivity was counted in the fractions corresponding to each cycle and was found in fractions 10, 14, and 16, therefore localizing the processing site between Ala-1710 and Val-1711. Northern Blot Analysis Total RNA was isolated from cultured cells using TriZol (Life Technologies, GIBCO-BRL, Rockville, MD), and 25 ␮g were analyzed by Northern blot as described previously (Jarriault et al., 1998). The probe used to detect HES-1 expression was the full-length rat HES-1 cDNA (gift from Drs. R. Kageyama and S. Nakanishi). To normalize RNA amounts, we used as a probe the S26 cDNA, which encodes

a ribosomal protein expressed at a high and constant level in human adult tissues.

Antibodies M222 is a mouse monoclonal antibody directed against the extracellular domain of TACE. P1 is an affinity-purified rabbit polyclonal antibody directed against the extracellular region of TACE (for Western analysis using M222 and P1, nonreducing gel conditions were used). The anti-Kuz antibody was obtained by immunizing rabbits with a peptide corresponding to the 17 last amino acids of mammalian Kuz coupled to KLH. Anti-Notch IC antibody was described in Logeat et al. (1998). The anti-Jagged antiserum was from Santa Cruz Biotechnology (antiserum C-20 [Santa Cruz, CA]). The antiCSL antibody was obtained by immunizing rabbits with the peptide CKFGERPPPKRLTREA, coupled to KLH.

Acknowledgments We would like to thank Jacques Peschon and Pranitha Reddy (Immunex) for the TACE⫺/⫺ fibroblasts and DRM cells and for TACEreconstituted DRM cells, Jacques Peschon for useful advice on culturing DRM cells, Charles Rauch, Martin Wolfson, and Beverly Castner (Immunex) for preparing recombinant TACE, Paul Glynn (Leicester, UK) for Kuz antiserum and useful advice, Michael Wolfe (Boston, MA) for the synthesis of the MW167 ␥-secretase inhibitor, and R. Kopan (St. Louis, MO) and E. H. Schroeter for the kind gift of the GCGV→LLFF mutant of N⌬E. We also would like to thank Gilles Courtois for careful reading of the manuscript. N. G. is a recipient of a Biomed European Community grant. This work was supported in part by grants from ANRS, Association pour la recherche sur le cancer, Ligue Nationale contre le Cancer, and European Community to A. I.

Received August 4, 1999; revised January 4, 2000.

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