The Notch Ligands, Jagged and Delta, Are SequentiallyProcessed by �-Secretase and Presenilin/�-Secretase andRelease Signaling Fragments*

Received for publication, March 14, 2003, and in revised form, June 12, 2003Published, JBC Papers in Press, June 25, 2003, DOI 10.1074/jbc.M302659200

Matthew J. LaVoie and Dennis J. Selkoe‡

From the Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital,Boston, Massachusetts 02215

The cleavage of Notch by presenilin (PS)/�-secretase isa salient example of regulated intramembrane proteol-ysis, an unusual mechanism of signal transduction. Thiscleavage is preceded by the binding of protein ligands tothe Notch ectodomain, activating its shedding. We hy-pothesized that the Notch ligands, Delta and Jagged,themselves undergo PS-mediated regulated intramem-brane proteolysis. Here, we show that the ectodomain ofmammalian Jagged is cleaved by an A disintegrin andmetalloprotease (ADAM) 17-like activity in culturedcells and in vivo, similar to the known cleavage of Dro-sophila Delta by Kuzbanian. The ectodomain sheddingof ligand can be stimulated by Notch and yields mem-brane-tethered C-terminal fragments (CTFs) of Jaggedand Delta that accumulate in cells expressing a domi-nant-negative form of PS or treated with �-secretaseinhibitors. PS forms stable complexes with Delta andJagged and with their respective CTFs. PS/�-secretasethen mediates the cleavage of the latter to release theDelta and Jagged intracellular domains, a portion ofwhich can enter the nucleus. The ligand CTFs competewith an activated form of Notch for cleavage by �-secre-tase and can thus inhibit Notch signaling in vitro. Thesoluble Jagged intracellular domain can activate geneexpression via the transcription factor AP1, and thiseffect is counteracted by the co-expression of the�-secretase-cleaved product of Notch, Notch intracellu-lar domain. We conclude that Delta and Jagged undergoADAM-mediated ectodomain processing followed by PS-mediated intramembrane proteolysis to release signal-ing fragments. Thus, Notch and its cognate ligands areprocessed by the same molecular machinery and mayantagonistically regulate each other’s signaling.

Converging lines of evidence from several disciplines haverecently identified regulated intramembrane proteolysis (RIP)1

as an unusual and hitherto unrecognized mechanism of signaltransduction (1). A salient example of RIP is provided by thesequential processing of certain type 1 membrane glycoproteinsby A disintegrin and metalloprotease (ADAM) and then bypresenilin/�-secretase (2). Presenilin (PS) was first identifiedas a polytopic membrane protein bearing mutations that causethe most aggressive form of familial Alzheimer’s disease (3).Subsequently, PS was shown to be a facilitator of signaling byNotch receptors during cell fate determination in Caenorhab-ditis elegans (4), Drosophila (5, 6), and mammals (7). There isnow compelling evidence that PS represents the active sitecomponent of �-secretase, a multiprotein complex that affectsthe intramembranous cleavages of Notch, the amyloid �-pro-tein precursor (APP), Erb-B4, E-cadherin, and several othertype 1 membrane proteins (8–16).

The requirement for PS in Notch signal transduction ap-pears to be explained by the finding that a PS-mediated cleav-age within the single transmembrane domain of Notch releasesits intracellular domain (NICD) to the nucleus, where it regu-lates transcription of target genes (17, 18). Based on the findingthat APP, like Notch, is sequentially cleaved by an ADAMfamily protease and PS/�-secretase, the APP intracellular do-main (AICD) was recently shown to reach the nucleus (19, 20),where it can regulate transcription of target genes (21, 22).Thus, emerging data suggest that presenilins, which are ubiq-uitously expressed in metazoans, serve as crucial switches inthe signaling of a variety of single pass transmembranereceptors.

The initiation of Notch signaling at the plasma membrane isbelieved to require the binding of an extracellular ligand (e.g.Delta or Jagged) to the Notch ectodomain, triggering the shed-ding of that domain by an ADAM protease (e.g. ADAM 10/kuzand/or ADAM 17/TACE) (23–25) and the subsequent PS-medi-ated intramembrane cleavage of the retained C-terminal frag-ment (CTF). Release of the large ectodomains of Notch, APP,and other PS substrates may remove steric hindrance on PS/�-secretase and allow intramembrane cleavage to occur (26).Based on these findings, we postulated that numerous type 1single transmembrane proteins that undergo ADAM-mediatedectodomain shedding are substrates of PS-mediated RIP. Be-cause the Notch ligand, Delta, undergoes cleavage by Kuzba-nian (related to mammalian ADAM 10/kuz) in flies (27), wehypothesized that another Notch ligand, Jagged, would un-dergo a similar ADAM-mediated ectodomain shedding to gen-erate a suitable PS substrate in the RIP mechanism. Here, wereport several lines of evidence that Delta and Jagged are

* This work was supported by National Institutes of Health (NIH)postdoctoral training award AG00222 (to M. J. L.) and NIH GrantsAG15379 and AG06173 (to D. J. S.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Harvard Institutesof Medicine, 77 Ave. Louis Pasteur, Boston, MA 02215. Tel.: 617-525-5200; Fax: 617-525-5252; E-mail: DSelkoe@rics.bwh.harvard.edu.

1 The abbreviations used are: RIP, regulated intramembrane prote-olysis; PS, presenilin; APP, amyloid �-protein precursor; NICD, Notchintracellular domain; AICD, APP intracellular domain; CTF, C-termi-nal fragment; JICD, Jagged intracellular domain; aa, amino acids;CHO, Chinese hamster ovary; HEK, human embryonic kidney; DMEM,Dulbecco’s modified Eagle’s medium; ADAM, A disintegrin and metal-loprotease; IP, immunoprecipitation; E13, embryonic day 13; FL, full-

length; ICD, intracellular domain; SEAP, secreted alkaline phospha-tase; NLS, nuclear localization signal; TACE, tumor necrosis factor�-converting enzyme.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 36, Issue of September 5, pp. 34427–34437, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 34427

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

indeed cleaved by ADAM family proteases to generate freeectodomains and CTF fragments and that this ectodomainshedding can be stimulated in a Notch-dependent manner.Further, we show that following ectodomain shedding, theDelta and Jagged CTFs undergo intramembrane cleavages byPS/�-secretase and can thus compete with NEXT, the analo-gous CTF of Notch, for �-secretase cleavage, resulting in de-creased Notch signaling. We then show that the Jagged intra-cellular domain (JICD) can stimulate AP1-dependent geneexpression and that this effect of JICD is inhibited by theNotch-derived NICD. Taken together, these findings demon-strate that both Notch and its ligands are processed by thesame molecular machinery and suggest that the regulatedintramembrane proteolysis of both receptor and ligand mayplay important, potentially competitive roles in cell signaling.

EXPERIMENTAL PROCEDURES

Plasmids—The expression plasmids for FL Jagged (pBOS-SN3T)and FL Delta (pBOS-rDelta1HA) of rat origin were generous gifts of G.Weinmaster. The CBF reporter construct, JH23, was a gift of S. D.Hayward, and the N�E and NICD constructs were gifts of R. Kopan.The J�E construct was generated by a series of PCRs. Starting with twoindependent PCRs, one insert was amplified that encoded the first 34amino acids of rat Jagged (aa 1–28 contain the signal peptide) such thatthe 3�-end of the oligonucleotide would anneal with the 5�-end of asecond PCR product encoding the predicted Jagged CTF (aa 1057–1220)followed by a C-terminal HA tag and stop codon. Aliquots of these twoPCR products were annealed and amplified in a third PCR to generatethe full-length J�E cDNA insert. This PCR product was then purifiedand TA-cloned into the pEF6/V5-His vector (Invitrogen), colonies werescreened for orientation, and sequence was verified. The Jagged JICDand JICD�NLS plasmids were cloned by PCR amplification of theappropriate cDNA regions from the full-length plasmid encoding aa1087–1220 and aa 1103–1220, respectively, cloned into the above men-tioned vector, and then screened and sequence-verified as well.

Cell Culture, Transfection, and �-Secretase Inhibitor Treatment—Chinese hamster ovary (CHO) cells, African green monkey kidney cells(COS7), and human embryonic kidney (HEK) cells were cultured inDulbecco’s modified essential medium (DMEM) plus 10% fetal bovineserum, penicillin, and streptomycin. The 12-19 and 2A-2 CHO cell lineswere cultured as previously described (28). For transient transfections,10 �g of plasmid DNA per 10-cm dish were incubated with Lipo-fectAMINE 2000 (Invitrogen) and aliquoted onto cells cultured in min-imal volumes of Opti-MEM (Invitrogen) medium. 4–6 h after transfec-tion, cells were washed, returned to their appropriate growth media,and maintained for an additional 24–30 h prior to collection. For ex-periments involving �-secretase and �-secretase inhibitor treatment,cell culture media containing premixed aliquots of inhibitor or theMe2SO vehicle alone were placed on cells 4 h after transfection. Cellswere then maintained for an additional 12–16 h in the presence ofvehicle or inhibitor and harvested.

Rat Embryo Preparation and Cell Fractionation—E13 rat (Sprague-Dawley, Taconic Farms) embryos were harvested on ice, diced in 10 mM

HEPES, 1 mM EDTA, 0.25 M sucrose, pH 7.4, run through 10 strokes ofa Dounce homogenizer, and passed four times through a 27-gaugeneedle. The homogenate was then spun at 3000 � g to pellet unbrokencells, debris, and nuclei, and the supernatant was then spun at100,000 � g for 1 h to pellet microsomal membranes. This pellet waseither resuspended in 150 mM sodium citrate, pH 6.4, for the in vitrogeneration of JICD or else lysed in 1% Nonidet P-40 for the detection ofendogenous full-length and CTF Jagged. For cell culture experimentsand cell fractionation, cells were scraped from their dishes in phos-phate-buffered saline plus 20 mM EDTA, pelleted by centrifugation,swollen for 10 min in 1 ml of a hypotonic lysis buffer (10 mM NaCl, 3 mM

MgCl2, 1 mM EDTA, and 10 mM Tris, pH 7.4) containing a spectrum ofprotease inhibitors, and then mechanically disrupted by Dounce homo-genation and several passages through a 27-gauge needle. Nuclei werepelleted at 800 � g for 10 min. The supernatant was centrifuged at100,000 � g for 1 h. Supernatant from this high speed spin constitutedthe cytosolic fraction. The pelleted microsomes were solubilized in 1%Nonidet P-40, constituting the membrane fraction. In parallel, thenuclear pellet was washed three times in 0.1% Nonidet P-40 and pel-leted at 375 � g for 5 min. The pellet was then solubilized in 1 ml of 1%SDS (such that cytosolic and nuclear fractions were prepared in equalvolumes) and then sonicated to shear genomic DNA prior to analysis by

SDS-PAGE electrophoresis. All preparations were kept at 4 °C through-out the subcellular fractionation.

Immunoprecipitation and Western Blotting—Presenilin co-immuno-precipitations were preformed with antiserum X81 to the N terminus ofPS1 at a dilution of 1:200 in the presence of protein A-agarose (RocheApplied Science) and incubated at 4 °C for 16 h, as described (29).Immunoprecipitates were washed once in with 0.5 M NaCl STEN bufferfor 20 min and then in 0.125 M STEN for an additional 20 min prior toelution in 100 mM glycine, pH 2.5, as described (30). Samples were thenloaded onto precast Tris-glycine SDS-PAGE gels (Invitrogen), trans-ferred to polyvinylidene difluoride membrane, and probed with anti-HAmonoclonal antibodies 12CA5 or 3F10 (Roche Applied Science), JaggedC-terminal antibodies (sc-8303 and sc-6011; Santa Cruz Biotechnology,Inc., Santa Cruz, CA), Delta C-terminal antibody (sc-12531; Santa CruzBiotechnology), or Notch1 (8G10; Calbiochem). For the detection of theJagged ectodomain in conditioned media, cells were transfected withFL-Jagged or in the absence of plasmid and grown for 16 h. The cellswere then conditioned for 6 h and concentrated 25� with a Microconcentrifuge filter (Mr cut-off � 50,000; Centricon) and blotted with sc-12531, as described above. For preabsorption experiments, sc-6011 wasincubated for 16 h with the immunizing peptide at 25 mg/�l antiserumprior to Western blotting. For Western blotting of FL APP and the APPCTFs C83 and C99, membranes were probed with 13G8 (gift of ElanPharmaceuticals). For PS1 detection, membranes were probed witheither Ab14 (to PS NTF; gift of Dr. S. Gandy) or 13A11 (to PS CTF; giftof Elan Pharmaceuticals).

For densitometric analyses, films were scanned and analyzed usingAlphaEase version 5.5 (Alpha Innotech). In the case of FL Delta quan-tification, images were captured from exposures lighter than presentedin Fig. 1A to ensure that densities were within the linear range of thephotographic film.

Notch Reporter Assay—COS cells were plated into poly-D-lysine-coated 24-well plates (Becton Dickinson) at least 24 h prior to transfec-tion. Cells were transfected with the TK-Luc vector (20 ng/well; Pro-mega) to normalize for transfection efficiency as well as with the JH23CBF-Luc reporter vector (75 ng/well). Minimum concentrations of N�EcDNA required for CBF-Luc expression were determined, and thisconcentration of N�E cDNA was held constant for all experiments (150ng/well). In some cases, the J�E expression vector was co-transfected aswell. 24–30 h after transfection (see above) cells were lysed in passivelysis buffer (Promega) and assayed using the Dual Luciferase Assay kit(Promega) on a Wallach 1420 multilabel counter. All CBF-Luc meas-urements were normalized against the Renilla luciferase to control fortransfection efficiency.

Alkaline Phosphatase Reporter Assay for Transcriptional Signal-ing—For screening experiments, COS cells were plated onto poly-D-lysine-coated 96-well dishes (Becton Dickinson) and grown to �90%confluence. Cells were transfected with 200 ng of various reporterDNAs (Mercury Pathway Profiling System; Clontech) along with 200 ngof either pJICD or pJICD�NLS, and cells were maintained in 500 ml ofserum-free DMEM. Media were collected after 48 h of growth, and a�25-�l aliquot was measured for alkaline phosphatase activity as pre-viously described (31). Experiments were performed in triplicate orquadruplicate, and we conducted a total of at least three independentexperiments. For follow-up experiments in 24- or 6-well dishes, COS,HEK, and CHO cells were cultured as described above and transfectedwith proportional amounts of reagents. Cells were cultured for 16 h, andtransfection media were replaced with 500 �l (24-well) or 1 ml (6-well)of serum-free DMEM. Following 48 h of conditioning, the media wereremoved and spun at 300 � g for 5 min to remove cellular debris andfloating cells, and then a 25-�l (24-well) or 10-�l (6-well) aliquot wasassayed for alkaline phosphatase activity as above. Experiments wererepeated 2–4 times per condition.

Statistics—Data were analyzed by either two-way Student’s t test(Fig. 1a) or one-way analysis of variance followed by Fisher’s LSD orTukey’s post hoc analysis for pairwise comparisons (see Fig. 7).

RESULTS

Ectodomain Shedding of Full-length Jagged and Delta—Theintramembranous cleavage of type 1 integral membrane pro-teins by PS/�-secretase is preceded by an initial endoproteoly-sis just outside of the membrane that results in the shedding ofthe large ectodomain. For example, ADAM-mediated cleavageof the ectodomain of the Delta/Jagged receptor, Notch, occursprior to the PS/�-secretase-mediated release of the NICD (23–25). We hypothesized that the mammalian Notch ligands,

Regulated Delta and Jagged Proteolysis and Signaling34428

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

Jagged and Delta, also undergo these �- and �-secretase scis-sions. To assess whether full-length (FL) Jagged and Deltaundergo ectodomain shedding to yield appropriate PS/�-secre-tase substrates, COS cells were transiently transfected withplasmids encoding either FL Jagged (Fig. 1a, left panel) or FLDelta (Fig. 1a, middle panel) with C-terminal HA tags, and celllysates were analyzed by Western blotting. Both Jagged andDelta were expressed at high levels, and single C-terminalimmunoreactive fragments were consistently identified in celllysates and bicarbonate-washed membrane fractions at theappropriate molecular weights predicted for the respectivemembrane-associated CTFs. These data suggest that theectodomains of both Jagged and Delta are shed to generatesuitable candidate substrates of PS/�-secretase (i.e. CTFJagged and CTF Delta). Previous work in Drosophila demon-strated the Kuzbanian-dependent cleavage of Delta (27); how-ever, there is no prior report of such �-secretase-type cleavage(or any other proteolysis) of Drosophila Serrate or its mamma-lian homolog, Jagged. We next performed experiments in whichCOS cells were transiently transfected to express Delta andco-cultured for 4 h with mock-transfected cells or COS cellstransiently transfected to express Notch. Following this briefco-culture with Notch-expressing cells, densitometric analyses

of the ratio of CTF-Delta to FL-Delta revealed an �51% in-crease (n � 5, p � 0.05) in the levels of CTF-Delta (Fig. 1a, rightpanels). A more dramatic effect was observed following co-transfection with of Delta and Notch in the same cell; however,it is likely that this represents both increased ectodomain shed-ding of Delta and competition between CTF-Delta and theanalogous NEXT fragment of Notch for �-secretase (see below).

Characterization of the Ectodomain Shedding of Jagged—Inflies, Kuzbanian is responsible for the ectodomain shedding ofboth Delta and Notch (25, 27). However, in mammals, it ap-pears that ADAM 17/TACE mediates the S2 cleavage of Notch(23, 24). Mammals share a single Kuzbanian/ADAM 10 gene,whereas these are distinct but highly related genes in the fly.Therefore, we surmised that the initial cleavage of Jagged wasprobably mediated by either ADAM 17/TACE or ADAM 10/kuz.By preferentially inhibiting either ADAM 10 or ADAM 17, wecharacterized the protease responsible for cleaving the Jaggedectodomain and generating its CTF. CHO cells were trans-fected to express FL Jagged and treated for 24 h with either theADAM 17 inhibitor, batimistat (10 �M), or the ADAM 10 inhib-itor, TIMP-1 (5–10 nM) (Fig. 1b, top panel). Batimistat virtuallyprevented Jagged CTF formation, whereas TIMP-1 had verylittle effect. At doses previously shown to inhibit the ADAM

FIG. 1. ADAM-mediated proteolysis of Jagged and Delta ectodomain generates membrane-associated CTFs. A, left panel, COS cellswere transiently transfected in the absence of cDNA (mock) or with HA-tagged constructs for FL Jagged (left panel) or FL Delta (middle panel),and whole cell lysates were electrophoresed and blotted with antibody 12CA5 to the C-terminal HA tags. Right panel, COS cells were transientlytransfected with Delta and co-cultured for 4 h with cells either mock-transfected or transfected to express rat Notch-1. Whole cell lysates were thenprobed with 12CA5 to the C-terminal tag present on the exogenous Delta or with 8G10 (Notch). Densitometric analysis was performed on the ratioof FL Delta to CTF Delta from multiple experiments. *, statistically significant (p � 0.05; n � 5). B, CHO cells were transiently transfected withvector alone or FL Jagged. 16 h after transfection, the cells were treated for 24 h with vehicle (Me2SO), the ADAM 17/TACE inhibitor batimistat(20 �M), the ADAM 10/kuz inhibitor TIMP-1 (5 nM) (top panel), or the ADAM 17/TACE inhibitor TAPI-1 (bottom panel). Lysates were blotted forFL Jagged and its CTF with 12CA5. C, CHO cells were transfected to express FL Jagged. After 18 h, the cells were treated for 1 h with vehiclealone or phorbol 12-myristate 13-acetate (10 �M) in the presence of the �-secretase inhibitor Compound E (3 nM). Cell lysates were probed for FLJagged and its CTF and, as a positive control, for APP CTF. D, CHO cells were transiently transfected in duplicate with vector alone or FL Jagged.After 16 h, cells were washed and conditioned for 6 h in serum-free DMEM, and the conditioned media (CM) were concentrated and blotted witha Jagged ectodomain-specific antibody (sc-11376). Whole cell lysate (L) was run in an adjacent lane on the same blot as a control for the specificityof the ectodomain-specific antibody and migration of FL Jagged.

Regulated Delta and Jagged Proteolysis and Signaling 34429

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

17-mediated (i.e. �-secretase) cleavage of APP (32), TAPI-1 (10�M) also blocked the generation of Jagged CTF (Fig. 1b, bottompanel). Furthermore, in the presence of a �-secretase inhibitor,the addition of the ADAM 17/TACE inhibitor, batimistat, in-creased FL Jagged levels while decreasing Jagged CTF,whereas TIMP-1 had no effect on FL or CTF Jagged (data notshown). Consistent with a previous report of Kuzbanian-medi-ated cleavage of Delta in Drosophila (27), we found that the�-secretase processing of Delta was not affected by the ADAM17/TACE inhibitors batimistat or TAPI-1 but was decreased bythe ADAM 10/kuz inhibitor TIMP-1 (data not shown).

The �-secretase processing of APP consists of both regulatedand constitutive components. The regulated component isthought to be mediated by ADAM 17 and can be enhanced byphorbol ester treatment (33–35). To determine whether thecleavage of Jagged by ADAM 17 could likewise be regulated, weinvestigated the effect of the phorbol ester, phorbol 12-myris-tate 13-acetate, on endogenous APP and exogenous JaggedCTF production. A 1-h treatment with phorbol 12-myristate13-acetate (10 �M) increased the production of both the APPCTF (C83), and the Jagged CTF (Fig. 1c). Furthermore, wedetected a soluble form of Jagged ectodomain in the condi-tioned medium of Jagged-transfected cells that was absent incontrol medium (Fig. 1d). We observed greater ectodomainprocessing of Delta and Jagged in CHO cells than in COS cells,and we speculated that the ectodomain shedding of Delta andJagged that generated membrane-associated CTFs could havebeen due to endogenous Notch expression. To determinewhether Notch was present endogenously in these cells, weperformed IP-Western blotting using two different rodent-spe-cific antibodies to the Notch ectodomain, one for IP and thesecond for detection. We found two immunopositive bands inCHO cell lysates (presumably FL and furin-processed Notch)that co-migrated with transfected rat Notch (data not shown).This result is reminiscent of previous work in HEK cells, inwhich endogenous ligand was expressed at sufficient levels toinduce exogenous Notch activation (23).

Detection of Endogenous Full-length and CTF Jagged Mole-cules in Vivo—Whereas the ectodomain shedding of DrosophilaDelta has been demonstrated in vivo (27), this has not beenconfirmed in mammals. Furthermore, there is no report de-scribing any proteolysis of the Serrate/Jagged family of Notchligands. We therefore searched for evidence of processing of theJagged ectodomain in the developing rat embryo. Jagged mes-sage levels in rat embryo rise during early development to peakat day E13 (36). We harvested whole rat embryos at this ageand prepared microsomal membranes. When solubilized mem-branes were probed with two different antibodies to the Cterminus of Jagged-1, high levels of both the full-length proteinand its CTF were readily detected (Fig. 2a). We believe this tobe the first demonstration of the existence of the Jagged CTF invivo. To test our hypothesis that this endogenous Jagged CTFis further cleaved by PS/�-secretase, we incubated E13 ratembryo membranes in a sodium citrate buffer for 4 h at 37 °Cin either vehicle alone (1% Me2SO) or one of three structurallyunrelated �-secretase inhibitors. Jagged CTF was proteolyticprocessed into a JICD fragment of the appropriate size, andthis was blocked by each of the three �-secretase inhibitors(Fig. 2b).

PS/�-Secretase-dependent Accumulation of Jagged and DeltaCTFs—Shortly after their biosynthesis, FL PS1 and PS2 areconverted by endoproteolysis into NTF/CTF heterodimers thatenter into stable multimeric complexes containing nicastrinand one or more additional membrane proteins (9, 37, 38).Overexpression of FL human PS1 in CHO cells leads to quan-titative replacement of the endogenous hamster PS het-

erodimers by exogenous human heterodimers (28). We previ-ously generated a CHO cell line stably co-expressing human FLAPP, wild type PS1, and wild type PS2 (28). This cell line(12–19) shows replacement of endogenous hamster NTF/CTFswith functional human NTF/CTFs (Fig. 3a, lane 1); the latterparticipate in active �-secretase complexes that mediate cleav-ages of C83 and C99 to generate the p3 and A� peptides,respectively. An analogous cell line (2A-2) stably expresseshuman FL APP plus human PS1 and PS2 each bearing muta-tions in one of the critical intramembrane aspartate residuesrequired for PS endoproteolysis and �-secretase activity (28). Inthese 2A-2 cells, endogenous PS is replaced by the aspartyl-mutant FL PS, and there are very few or no detectable PSheterodimers (Fig. 3a, lane 2). As a result, the 2A-2 cellsstrongly accumulate C83 and C99 (Fig. 3b, lane 2) and fail togenerate p3 and A� (not shown; see Kimberly et al. (28)).

We assumed that these dominant negative effects of theaspartate mutations would prevent the cleavage of other PSsubstrates. Indeed, the 2A-2 cells show defective Notch in-tramembrane cleavage and NICD signaling (39). To furthertest this assumption, we first ruled out potential differences inADAM-mediated ectodomain shedding between the 12-19 and2A-2 lines by quantifying the proteases that specifically medi-ate the APP, Notch, Delta, and Jagged ectodomain cleavages,ADAM 17/TACE and/or ADAM 10/kuz. The levels of both thezymogens and the mature enzymes were unaffected by the PS1and PS2 aspartate mutations (Fig. 3a). Therefore, accumula-tion of potential PS/�-secretase substrates in the 2A-2 cell line

FIG. 2. Both full-length and CTF Jagged are present in devel-oping rat embryonic tissue. A, microsomal membranes were pre-pared from whole rat embryos on day E13. Membrane lysates wereprobed with a rabbit polyclonal antiserum (lane 1) and an affinity-purified goat antiserum to the C terminus of Jagged (lane 2) or with theaffinity-purified goat antiserum preabsorbed with the immunizing pep-tide (lane 3). B, microsomal membranes were resuspended in sodiumcitrate buffer and incubated at either 4 °C (lane 1) or 37 °C for 4 h in thepresence of either 1% Me2SO alone (lane 2) or with 1 �M III-31C (lane3), 400 nM DAPT (lane 4), or 100 nM Compound E (lane 5).

Regulated Delta and Jagged Proteolysis and Signaling34430

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

should be attributable to the loss of function of this protease.2A-2 cells transiently transfected with FL Jagged or FL Deltashowed accumulation of the Jagged and Delta CTFs, comparedwith levels obtained in simultaneously transfected 12-19 cells(Fig. 3c). Both lines were then transfected with FL Jagged (Fig.3d) or FL Delta (not shown) and treated for 12 h with vehiclealone or 500 nM III-31-C, a well characterized, cell-permeantPS/�-secretase inhibitor (IC50 � �250 nM). As reported previ-ously (9), treatment with III-31-C substantially augmented thelevels of the APP CTFs, C83 and C99, although not as stronglyas the PS1 and PS2 aspartate mutations did (Fig. 3d, lowerpanel). In accord, we observed an inhibitor-mediated increasein the Jagged CTF in the 12-19 cells that was not as great asthe elevation caused by the aspartate mutations in the 2A-2cells (Fig. 3d, upper panel). III-31-C had a modest additionaleffect on the levels of APP C83/C99 and Jagged CTF in theaspartate mutant (2A-2) line. In addition, the level of JaggedCTF was increased by the addition (at 3 nM) of another �-secre-tase inhibitor, “Compound E” (Fig. 1c, lower panel), stronglysuggesting that the Jagged CTF is a substrate of PS/�-secre-tase following processing of Jagged by ADAM 17/TACE. Thus,both mutagenic and pharmacological manipulations lead to theaccumulation of the Jagged and Delta CTFs in a PS/�-secretase-dependent manner, in full agreement with the effects on APPprocessing observed in the same cells.

Complex Formation of PS with the CTFs of Jagged andDelta—We previously reported that small amounts of FL APP

and, in particular, C83 and C99, can be co-immunoprecipitatedwith PS (29, 30). Furthermore, the levels of such complexes ofPS with C83 and C99 are markedly enhanced in cells lacking�-secretase activity (29). We therefore investigated whetherantibodies to human PS1 could immunoprecipitate the FLJagged and Delta proteins and their CTFs. Relying on condi-tions previously used for the successful co-precipitation of PS1with FL APP as well as C83 and C99 and also PS1 with theNotch membrane-anchored CTF (i.e. N�E, an ectodomain-truncated form of Notch) (29, 30, 41, 42), FL Jagged and Deltaas well as their respective CTFs were efficiently co-precipitatedby the PS1 antibody X81 in both the 12-19 and 2A-2 celllysates, whereas the preimmune serum of X81 did not bringdown any form of the Notch ligands (Fig. 4a). In agreementwith our previous finding that co-immunoprecipitation of PSwith C83 and C99 is more efficient in PS aspartate mutantthan wild type cells (29), the co-precipitation of the Jagged CTFwith the PS1 antibody was also greater in the 2A-2 than 12-19cells (Fig. 4a). Analysis of the co-precipitation of PS1 with FLDelta and its CTF revealed similar results; FL Delta waseffectively co-precipitated from both 12-19 and 2A-2 cell mem-branes, and more Delta CTF was recovered from the latter cells(Fig. 4b). Similar experiments were performed after co-express-ing neprilysin, an integral membrane protein not predicted tobe a substrate of �-secretase, and there was no evidence of co-IPwith PS (data not shown).

The 12-19 and 2A-2 cells used in these experiments stably

FIG. 3. The CTFs of the Notch li-gands, Delta and Jagged, accumulatein a PS/�-secretase-dependent man-ner. A, CHO cells stably co-expressinghuman wild-type (12–19) or dominant-negative aspartyl mutant (2A-2) isoformsof both PS1 and PS2 were probed for anydifferences in proteases that serve as �-and �-secretases. Bicarbonate-washedmicrosomes were blotted for the PS1NTF/CTF heterodimers (�-secretase), andfor the immature and mature forms ofADAM 10 and ADAM 17 (�-secretases).The faint band immediately above the po-sition of the PS1 NTF in the 2A-2 cells isa background band, not PS1 NTF; notethat there is also no detectable PS1 CTFin these cells. B, the effect of stably over-expressing aspartyl mutant PS1 and PS2on the levels of APP CTFs (C83 and C99)was assessed by blotting with monoclonalantibody 13G8. *, a band corresponding toa probable APP CTF dimer. C, 12-19 and2A-2 cells were transiently transfected toexpress FL Jagged (A) or FL Delta (B).Cells were collected 36 h later, and ly-sates were blotted with 12CA5 (to the C-terminal HA tags). D, 12-19 and 2A-2cells were transiently transfected to ex-press FL Jagged and treated with vehiclealone or the �-secretase inhibitor, III-31-C, for 16 h. Lysates were collected, andthe levels of Jagged CTF were determinedby 12CA5 Western blotting (top panel).Levels of the APP ��secretase substratesC83 and C99 were determined by blottingthe same lysates with APP C-terminal an-tibody 13G8 (bottom panel).

Regulated Delta and Jagged Proteolysis and Signaling 34431

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

overexpress wild type or mutant PS1 and PS2, and FL Jaggedand Delta were transiently expressed. Therefore, we askedwhether X81 could co-precipitate the substrates at endogenouslevels of both presenilin and substrate. Mouse primary mixedcortical cultures were lysed under similar conditions in 1%Nonidet P-40, co-precipitated with X81 and probed for Delta. Asmall amount of FL Delta was detected that was absent in theX81 preimmune precipitate (Fig. 4c).

PS/�-Secretase-mediated Cleavage of the Jagged CTF andNuclear Translocation of Its Cytoplasmic Domain—The cleav-age of APP and Notch by PS/�-secretase releases their intra-cellular domains (AICD and NICD) into the cytoplasm. Bothfragments are believed to complex with other cytoplasmic pro-teins, traffic to the nucleus, and affect gene expression, al-though by different mechanisms. It appears that AICD re-quires a binding partner, the transcriptional co-activator Fe65,to gain entry into the nucleus. In contrast, NICD has twoconsensus NLS motifs C-terminal to the Notch transmembrane

domain that are thought to mediate its nuclear entry. BothJagged and Delta contain basic amino acid stretches withintheir respective intracellular domains (Fig. 5a), and these pu-tative NLS sequences are conserved among the principal Notchligands of flies, mice, and humans. AICD and NICD are highlylabile proteins that are rapidly degraded and thus very difficultto detect with standard biochemical techniques.

To increase the levels of NICD generated by PS/�-secretase,previous studies have used an ectodomain-truncated form ofNotch in which the signal peptide was placed adjacent to anN-terminally truncated Notch sequence. This construct (N�E)codes for a type 1 transmembrane protein with a very shortectodomain that undergoes intramembranous cleavage by PS/�-secretase in a constitutive, ligand-independent manner.Transient transfection with this construct yields a readily de-tectable NICD that translocates to the nucleus (17, 43). Wetherefore designed a similar plasmid encoding the Jagged sig-nal peptide immediately followed by the complete membrane-anchored CTF of Jagged, beginning at the predicted ADAMcleavage site and ending with a C-terminal HA tag (J�E; Fig.5b). Transfection with J�E resulted in the expression of amembrane-anchored protein of appropriate molecular weightin both COS and CHO cells (Fig. 6a). Furthermore, transfectionwith J�E resulted in JICD production only in cells with intact�-secretase activity (i.e. the COS and the 1219 CHO cells). The2A-2 CHO cells did not generate JICD (Fig. 6a). Treatmentwith the �-secretase inhibitor, Compound E (3 nM), virtually

FIG. 4. The Notch ligands, Jagged and Delta, co-immunopre-cipitate with PS. A, 12-19 and 2A-2 cells were transiently transfectedto express FL Jagged, and their lysates were precipitated with the PS1NTF-specific antiserum, X81, or the corresponding preimmune serum(pre). B, 12-19 and 2A-2 cells were transfected to express FL Delta andsubjected to X81 co-IP as in A. C, primary mouse cortical neurons werecultured for 14 days and then solubilized in 1% Nonidet P-40 lysisbuffer. Lysates were precipitated with X81 or its preimmune serum andprobed with the Delta CTF-specific antibody, sc-12531.

FIG. 5. Primary structures of some of the known �-secretasesubstrates. A, alignment of the transmembrane domains (boldfacetype, underlined) of human (H) APP, Notch, and ErbB4 as well as rat(R) Jagged and Delta. The putative NLS sequences are highlighted inblue. The identified cleavage/start sites of the NICD and AICD proteinsare shown in red. The start sites of our recombinant JICD (Val1087) andJICD�NLS (Ser1183) constructs are shown in green (Fig. 5b). B, sche-matics of the membrane anchored J�E protein as well as the solubleJICD and JICD�NLS proteins. Amino acids in red represent the puta-tive N-terminal residues of the predicted JICD fragment.

Regulated Delta and Jagged Proteolysis and Signaling34432

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

eliminated JICD production in COS cells transfected with J�E(Fig. 6b).

Moreover, the membrane-anchored J�E accumulated in thepresence of the inhibitor, as expected. For these experiments onthe processing of J�E, we constructed two additional expres-sion vectors (Fig. 5b). A recombinant JICD construct used theintramembranous V1087 as the initial amino acid for two rea-sons. First, both AICD and NICD are thought to be generatedvia cleavage immediately prior to an intramembranous valineresidue. Second, valine 1087 is closest to the analogous cleav-age site of the previously sequenced ICDs of APP and Notch(17, 44). For a JICD�NLS vector, we chose as the start site theSer1103 residue that occurs 3 amino acids downstream of theputative NLS (aa 1095–1100) (Fig. 5). The JICD endogenouslygenerated from J�E migrated close to, but slightly slower than,the recombinant JICD fragment we expressed as a positivecontrol (Fig. 6b) (see “Discussion”).

PS 12-19 and 2A-2 cells were each transiently transfectedwith J�E, and both cell lines were fractionated to enrich for

nuclei (19, 45). A protein corresponding to the predicted size ofJICD was detected in the washed nuclear fraction of the 12–19cells but not in that of the aspartate mutant 2A-2 cells (Fig. 6c).Next, we transfected COS cells with plasmids expressing eitherJICD or JICD�NLS; the latter form lacks the basic amino acidstretches believed to represent an NLS (Fig. 5b). Following cellfractionation, the JICD fragment was markedly enriched in thenuclei of the JICD transfectants, whereas it was dramaticallyreduced in the nuclei and abundant in the cytosol of theJICD�NLS transfectants (Fig. 6c). Many type 1 proteins con-tains basic amino acid stretches adjacent to the cytosolic face ofthe plasma membrane that are often considered stop-transfersequences needed for proper orientation within the lipid bi-layer. However, in the case of Jagged/JICD, we found that thisstretch of basic amino acids is also necessary for the nuclearaccumulation of the �-secretase-derived JICD fragment (Fig.6c). However, future work will be required to determinewhether this putative NLS can also confer nuclear localizationto an irrelevant protein, confirming its function as a bona fideNLS.

Ligand Processing Interferes with Notch Signaling at Multi-ple Levels—During the establishment of lateral inhibition, boththe Notch receptor and its ligand are initially expressed in thesame cell. Because the ectodomain shedding of both Notch andits ligands can be stimulated through ligand-receptor interac-tions and both result in the generation of suitable �-secretasesubstrates, we sought to determine whether ligand CTF in acell would compete for �-secretase cleavage, reduce NICD pro-duction, and thus inhibit Notch signaling. First, we establishedthe minimum amount of N�E cDNA required to activate CBF-luciferase expression in a well characterized Notch reportersystem (46). Then we titrated increasing concentrations of J�EcDNA and quantified their effects on Notch signaling. In allexperiments, we co-transfected the TK-Luc vector that consti-tutively expresses a variant of luciferase to control for trans-fection efficiency. Treatment with a �-secretase inhibitor re-sulted in a marked reduction in HES activity, confirming the�-secretase-dependent nature of our Notch reporter system(data not shown). We found that co-transfection with J�E re-sulted in a linear dose-dependent inhibition of N�E signaling(Fig. 7a), whereas co-transfection of vector only or HA-dynamin(another soluble, HA-tagged protein) had no effect (Fig. 7a).Surprisingly, co-transfection of N�E and the soluble JICD frag-ment also caused a modest inhibition of Notch signaling, al-though not as great as the membrane-anchored form of Jaggeddid (Fig. 7a). To extend these results, we also investigated theeffects of an APP-derived �-secretase substrate (C99) on N�Esignaling. In accord with the findings for J�E, co-transfectionwith C99 resulted in a dose-dependent inhibition of N�E sig-naling, but in this case, AICD, the soluble �-secretase product,had no effect.

Next, we transiently transfected COS cells with secretedalkaline phosphatase (SEAP) reporter constructs that aredriven by various transcriptional enhancer elements, and thesecells were then co-transfected with either vector alone or JICDor JICD�NLS. Conditioned media were collected 48 h post-transfection and analyzed for SEAP activity. The pTAL nega-tive control vector contains only a TATA box upstream of theSEAP gene and serves as a control for background alkalinephosphatase activity, whereas the pSEAP positive control vec-tor contains an SV40 promoter upstream of the SEAP gene toallow for robust constitutive alkaline phosphatase secretion. Ofthe eight candidate enhancer elements we screened, only theAP1 element produced a clear and consistent increase in SEAPactivity in response to JICD co-expression (Fig. 7b). JICD�NLSproduced slightly less response than did intact JICD (Fig. 7b).

FIG. 6. PS/�-secretase dependent generation of JICD and thenuclear localization of recombinant JICD. A, COS cells andPS12–19 and 2A-2 CHO cells were transiently transfected with J�Eand homogenized in 1% SDS lysis buffer. B, COS cells were transfectedwith J�E and cultured with the �-secretase inhibitor, Compound E, orvehicle alone for 16 h. Sister COS cultures transfected with recombi-nant JICD were run as a sizing control in the far right lane. C, COS cellswere transfected with either JICD or JICD�NLS. Cytosol (C) and nuclei(N) were prepared in equal volumes, and aliquots were separated on a14% Tris-Glycine gel and probed with 3F10 (upper panel). *, a nonspe-cific nuclear protein detected with the 3F10 antibody, demonstratingequal protein loading. As a control, the same cytosolic and nuclearfractions of the JICD-transfected cells were probed for the proteinkinase CdK5 as a cytosolic marker and for the DNA-binding proteinhistone H1 as a nuclear marker (lower panels).

Regulated Delta and Jagged Proteolysis and Signaling 34433

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

Transfection with the AP1 reporter construct alone served asthe base line for additional quantitative experiments thatclearly confirmed the activation of AP1-mediated transcrip-tional activity by JICD in COS cells (Fig. 7c). Importantly,these results occurred independently of cell type, since both theincrease in AP1 activity by JICD and the lack of response withcAMP-response element and six other enhancer elements werealso observed in both CHO and HEK cells (data not shown).

A recent study demonstrated that NICD is capable of re-pressing AP1 activation, using a similar reporter system (48).Therefore, we examined whether NICD could have an antago-nistic effect on the AP1-mediated activation produced by JICD.Indeed, co-expression of NICD with either JICD or JICD�NLSalmost completely blocked the AP1 activation caused by eachJagged fragment alone (Fig. 7c). These data suggest that JICDcan selectively stimulate AP1 activation in a manner that is notentirely dependent upon nuclear entry and that is opposed bythe effects of NICD.

DISCUSSION

Here, we report the discovery that the Notch ligands, Jaggedand Delta, like Notch itself, undergo ectodomain processing byADAM family proteases and subsequent cleavage by PS/�-secretase to release potential signaling fragments. In view ofthe fact that four previously reported PS/�-secretase sub-strates, APP, Notch, ErbB4, and E-cadherin, each generateintracellular fragments that are implicated in signal transduc-tion in the nucleus, we asked whether the JICD is also liber-ated by PS/�-secretase and translocates to the nucleus. Ourresults suggest that the regulated intramembrane proteolysisof the Notch ligands has implications for Notch signaling thatpartly involve the function of the soluble JICD.

We assigned several criteria to a potential PS/�-secretasesubstrate, and the Notch ligands meet all of them. First, it waspreviously shown that the ectodomain of Drospohila Delta iscleaved by Kuzbanian, a protease related to mammalian

FIG. 7. The proteolytic fragments ofJagged interfere with Notch signal-ing. A, COS cells were co-transfectedwith the Notch CBF-Luc reporter JH23,the positive control TK-Luc, and N�E andJ�E expression constructs. 24–30 h post-transfection, cells were lysed and assayedfor both luciferase activities. Data areshown as CBF-dependent luciferase activ-ity corrected for transfection efficiency(TK-Luc) (mean � S.E., n � 3). Data werethen normalized to the CBF-luciferase ac-tivity of N�E alone. *, statistically signif-icant compared with N�E alone (p �0.01). B, COS cells were co-transfected toexpress either JICD or JICD�NLS withvarious secreted alkaline phosphatase re-porters driven by a TATA box only(pTAL), AP1 response element (pAP1),calcium response element (pCRE), heatshock response element (pHSE), glucocor-ticoid response element (pGRE), nuclearfactor of activated T-cells (pNFAT), NF�B(pNF�B), Myc (pMYC), and serum re-sponse element (pSRE). Cells were main-tained in serum-free DMEM for 48 h, andaliquots of conditioned media were as-sayed for alkaline phosphatase activity(mean � S.E., n � 3). *, statistically sig-nificant (p � 0.05). C, COS cells weretransiently transfected with vector alone,JICD, or JICD�NLS along with either thepTAL negative control reporter or thepAP1 reporter. Conditioned media wereassayed as above (mean � S.E., n � 3). *,denotes statistically significant frompAP1 alone (p � 0.05). Similar data wereobtained in CHO and HEK cells (data notshown). D, COS cells were co-transfectedwith vector alone or JICD or JICD�NLSplus the pAP1 reporter in the presence orabsence of an NICD expression plasmid.Conditioned medium was assayed asabove (mean � S.E., n � 6). *, statisticallysignificant difference from pAP1 alone(p � 0.05); **, statistically significant dif-ference from pAP1 plus JICD (p � 0.05).

Regulated Delta and Jagged Proteolysis and Signaling34434

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

ADAM 10, to yield soluble ectodomain fragments (27), and wenow show that the mammalian Jagged protein is similarlyprocessed by ADAM 17/TACE, both in vitro and in vivo (Figs. 1and 2a). The ectodomain processing can be stimulated by ex-pressing Notch, and the cleavage of Jagged by ADAM 17 canalso be regulated by PKC activation (Fig. 1), similar to whathas been previously shown for the classic �-secretase substrate,APP. Second, the CTFs of Jagged and Delta accumulate in cellmembranes in a PS/�-secretase-dependent manner, as shownboth pharmacologically and by mutagenesis of the PS in-tramembrane aspartates (Fig. 3). Third, Jagged and Delta ho-loproteins and, in particular, their CTFs co-immunoprecipitatewith PS, and this precipitation is quantitatively similar to thatpreviously reported for the extensively characterized PS/�-secretase substrate, APP (Fig. 4). Fourth, ectodomain cleavageof the Notch ligands generates membrane-anchored fragmentsthat compete with Notch for PS/�-secretase cleavage and de-crease Notch signaling. Furthermore, the soluble JICD pos-sesses some inhibitory activity against Notch signaling as wellas a Notch-independent signaling capacity (Fig. 7).

Regulated Ectodomain Cleavage of Jagged—We found thatthe mammalian Jagged-1 protein is subject to an S2 (Notch)-like or �-secretase (APP)-like cleavage that is consistent withADAM 17/TACE-like activity. This was established via phar-macological inhibition and phorbol ester stimulation of ADAM17 cleavage, two methods previously shown to alter the ADAM17/TACE-mediated shedding of APP (33–35, 49). We demon-strated ectodomain processing of Jagged in vitro followingtransfection with Jagged expression constructs and observedsignificant levels of the Jagged CTF in vivo in the developingrat embryo. Furthermore, low levels of a novel Jagged ectodo-main fragment are secreted into the medium of Jagged-ex-pressing cells and are absent in control medium. This proteinmigrates on SDS-PAGE gels at a smaller apparent Mr thanwould be predicted for the entire Jagged ectodomain, suggest-ing that it may be subject to degradation in the conditionedmedium. It is also possible that, like Notch in fly cells, theectodomain is not secreted directly into the medium but isendocytosed by adjacent cells, which could explain the lowlevels of ectodomain we observed in medium. We find that theS2-like cleavage of rat Delta appears to be mediated by anADAM 10/kuz-like activity, consistent with the report thatDrosophila Delta is shed by Kuzbanian (27). Taken together,these data demonstrate that both families of mammalianNotch ligands undergo the same type of ectodomain processingas the Notch receptor itself. These initial findings supportedthe possibility that the Notch ligands could also be PS/�-secre-tase substrates.

Generation of the Soluble JICD by PS/�-Secretase—Recentevidence has demonstrated that the predominant ICDs gener-ated by the intramembrane cleavage of both APP and Notchbegin at a valine residue close to the cytosolic face of thetransmembrane domain (17, 44). Therefore, we chose an anal-ogous valine (Val1087) present within the Jagged transmem-brane domain as the start site of our recombinant JICD (Fig.5b). However, in both COS and CHO cell lines, we detected anendogenously generated JICD from the constitutively cleavedJ�E that migrated slightly slower than our recombinant JICD-like fragment. This result suggests that either the �-secretasecleavage site of Jagged is slightly N-terminal to the cleavagesite of both APP and Notch or that some post-translationalmodification is responsible for the difference in migration. In-terestingly, a portion of JICD generated from J�E remainedmembrane-associated (not shown), similar to what has beenfound after hypotonic cell lysis with portions of NICD (50) and

AICD.2 Future experiments will address the question ofwhether there are multiple �-secretase cleavage sites, as hasbeen shown for APP and Notch, and where the cleavage site(s)occur within the ligand transmembrane domains.

After the completion of this work, we became aware of veryrecently published data consistent with some of the observa-tions in the current report. A study conducted in insect cellsdemonstrated the Notch-induced ectodomain shedding of Dro-sophila Delta (51), consistent with our findings in mammaliansystems. In addition, pharmacological and genetic evidencewas described suggesting that mammalian Delta1 and Jagged2are processed by �-secretase (52). However, this report did notinclude a characterization of the �-secretase processing of ei-ther protein, which is a prerequisite for downstream �-secre-tase cleavage; nor was there information provided regardingprotein-protein interactions between these substrates and com-ponents of the �-secretase complex. The current data provideimportant insights into the protease activities that mediate the�-like and �-secretase cleavages of both rat Delta1 andJagged1. Further, we demonstrate both activities from embry-onic tissue without relying on substrate overexpression. Fi-nally, we provide data suggesting an AP1-mediated signalingrole for Jagged.

The Impact of Ligand Proteolysis on Notch Signaling—Al-though expression of Delta or Serrate is required for activationof Notch in vivo, there is a paradoxical observation that over-expression of Delta or Serrate does not result in Notch overac-tivation but rather reduces Notch signaling (55–64). In onesuch study, high expression of Delta or Serrate correlated witha decreased sensitivity to receiving a Notch signal (64). Impor-tantly, the cells that had ectopic overexpression of Delta orSerrate but could not receive a Notch signal were still able tostimulate Notch in adjacent cells, demonstrating that the li-gands were present on the cell surface and functioned in recep-tor stimulation. Therefore, it is unlikely that the ectopicallyexpressed ligand interfered directly with Notch; rather, someother interaction resulted in the inhibition of Notch signaling.Whereas this observation has been made in many ways in vivo,the mechanisms responsible for the Notch down-regulation arenot known. Our finding that the Notch ligands undergo regu-lated intramembrane proteolysis raises several possible mech-anisms by which ligand expression levels might modulateNotch signaling, as follows.

Several studies have examined the soluble Delta ectodomainfor Notch activating potential, and most concluded that it wasnot an efficient Notch activator (65, 66). Indeed, a recent reportconfirms that purified Delta ectodomain has no activity inseveral cell-based assays of Notch signaling, consistent withthe previous studies (67). These authors suggest that theectodomain cleavage of Delta serves to limit the amount ofligand available at the cell surface to activate Notch. In thisregard, our study makes the novel observation that the ectodo-main processing of Delta can be stimulated by interaction withNotch. Therefore, if Notch and Delta interact under nonidealconditions (e.g. Notch and/or the ligand are not prepared forendoproteolysis, which is necessary for NICD signaling), theinteraction could result in premature cleavage of the Deltaectodomain, thus reducing the pool of full-length Delta avail-able for subsequent Notch activation.

Such abortive �-secretase cleavage of ligand is not the onlyrole that ligand proteolysis could play in modulating Notchactivation. Another possibility is that the resultant Delta andJagged CTFs may compete with the Notch S2 cleavage product,NEXT, for �-secretase processing. Such substrate competition

2 W. T. Kimberly and D. J. Selkoe, unpublished data.

Regulated Delta and Jagged Proteolysis and Signaling 34435

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

would be particularly relevant early during lateral inhibition,when both Notch and its ligands are expressed in the same cell;competition for �-secretase would decrease the ability of astimulated cell to release NICD. Importantly, such physicalcompetition has already been established between Notch andanother �-secretase substrate, APP. In primary cultured neu-rons, endogenous Notch and APP compete for �-secretase, in-dicating that changes in the levels of one substrate affect theprocessing of another, even when both are expressed at endog-enous levels (39). These data further suggest that the proteasecan be saturated, which is not unexpected, given that PS/�-secretase levels are very tightly regulated by limiting cellularco-factors (68). However, the ability of multiple substrates tocompete with one another for �-secretase cleavage and its ef-fects on signaling remain to be confirmed in vivo. Further workwill be needed to demonstrate the effects of competition in adeveloping organism.

Additional evidence that NEXT and the Delta and JaggedCTFs can compete for �-secretase comes from an in vitro studyin which co-expression of Notch and Delta in the same cellresulted in a phenotype opposite of that observed followingco-culture of independent pools of Notch and Delta expressingcells (69). In these co-transfection experiments, the expressionof Delta resulted in a phenotype that was Notch-hypomorphic,suggesting that the expression of both Notch and its ligand inthe same cell attenuates Notch signaling, as has been sug-gested in vivo (64). We modeled this competition by investigat-ing the effects of J�E expression on Notch signaling from themembrane anchored N�E in a CBF-luciferase reporter system(46). We found that membrane-bound J�E inhibited CBF-lu-ciferase activity in a dose-dependent manner (Fig. 7a, left pan-el). Similarly, the APP �-secretase substrate, C99, also inhib-ited N�E-derived reporter activity in a dose-dependentmanner, whereas the soluble APP product, AICD, had no effect(Fig. 7a, right panel). Unexpectedly, the soluble JICD doespossess some Notch-inhibitory activity, although not as potentas J�E (Fig. 7a, left panel), whereas empty vector and anirrelevant soluble HA-tagged protein had no effect. This directeffect of JICD on Notch signaling was also observed when weused soluble NICD rather than N�E as the mediator of CBF-luciferase reporter activity (data not shown), supporting a di-rect interaction between the ICDs of Jagged and Notch asregards the regulation of the CBF complex. Our data are en-tirely consistent with 1) the observations of several groups thatligand overexpression inhibits Notch function in vivo (dis-cussed above) and 2) the finding that ligand and Notch co-expression blocks the Notch-induced inhibition of neurite out-growth in a cellular model of Notch function (69). These variousexperiments do not establish whether NICD and JICD caninteract physically or rather that JICD somehow inhibits theassociation of NICD with its transcriptional activating com-plex, a question that requires further study.

Apparent Signaling Capacity of the C-Terminal Domain ofJagged—Several of the previously identified �-secretase prod-ucts have been shown to traffic to the nucleus and directlyaffect gene expression through association with a transcrip-tional activating complex. We found that JICD can translocateto the nucleus as well, and therefore we asked whether aJICD-Gal4 fusion protein would drive expression of a Gal4-luciferase vector. While an AICD-Gal4 control construct droveluciferase activity as reported (21), JICD-Gal4 was inactive(data not shown). Therefore, we examined the ability of JICD toalter gene expression through a candidate-based screen of sev-eral known enhancer elements. The results showed that JICDconsistently stimulated AP1-mediated reporter expression inintact cells, whereas several other broad spectrum enhancer

elements, including calcium response element, serum responseelement, and heat shock element, were completely unaffected(Fig. 7). The selective effect of the soluble JICD protein on AP1activation was observed in three different cell types (COS,CHO, and HEK), in all of which cAMP-response element andseveral other enhancers were not JICD-responsive. It appearsthat the activation of AP1 by JICD may not absolutely requirenuclear entry, because a JICD mutant that lacked the putativeNLS still increased AP1 reporter activity. These data are par-ticularly interesting given the emerging evidence that Notchfunction extends beyond the ability of NICD to affect geneexpression through an association with suppressor of hairless(e.g. see Refs. 70–72). An emerging principle from these in vivoand in vitro studies is that Notch has an inhibitory effect on theAP1 modulator, Jun n-terminal kinase. Indeed, a recent reportdirectly demonstrates that NICD is a repressor of AP1-drivengene expression in mammalian cells (48). These findings, alongwith other reports, suggest that some aspects of Notch signal-ing in vivo involve AP1 proteins (73, 74). Consistent with thiswork, we have now found that AP1 stimulation by JICD isabrogated by NICD. Our data support the conclusion thatJagged, too, has a role in signaling via the AP1 system.

Consistent with this activation of AP1 by cytoplasmic JICD,the C terminus of human Jagged1 has been shown to contain aPDZ-ligand domain that is capable of binding the putative Raseffector, AF6 (75). AF6 is homologous to the Drosophila canoe,a protein also involved in Notch signaling (76). It has recentlybeen shown that overexpression of Jagged1 transforms RKEcells and that the integrity of the PDZ-ligand motif is requiredfor this effect (47). Furthermore, these investigators found thatJagged expression resulted in increases in Jagged1, Delta1,and Notch3 mRNA levels but not those of Notch1, -2, or -4,demonstrating a specific effect on gene expression. These datawere also confirmed by a luciferase reporter construct driven bythe Jagged1 promoter. Further, the authors found that Delta-1,-2, and -4 share this same PDZ-ligand motif with Jagged1,whereas Jagged2 and Delta3 do not, suggesting that this dif-ference may be important to the heterogeneity of Notch ligandeffects in various tissues. How the binding of this motif to AF6plays a role in cellular transformation by Jagged1 or in the AP1activation by JICD remains to be seen. In our experiments, westudied the rat homolog of Jagged1, which has 100% conserva-tion of this C-terminal PDZ-ligand motif. The fact that we havenow demonstrated that Jagged1 undergoes intramembranouscleavage to release a soluble ICD that reaches the nucleusmakes these recent observations of Jagged’s potential for sig-naling even more compelling. The functional effects of JaggedRIP and signaling on both Notch-dependent and Notch-inde-pendent signaling await further elucidation.

Clinical Implications of the Multiple Substrates of PS/�-Secretase—Because presenilin is emerging as a ubiquitous andhighly conserved molecular switch for signaling by certain type1 transmembrane receptors, it may be difficult to therapeuti-cally target individual substrates of �-secretase (e.g. APP).Growing evidence has implicated the Notch signaling pathwayin adults in both hematopoiesis and disease. Loss-of-functionmutations within human Jagged-1 are associated with Alagillesyndrome, and aberrant Notch/DSL signaling is thought to beinvolved in some forms of myeloid leukemia (53, 54, 77, 78).Therefore, a better understanding of the biochemical process-ing and downstream targets of the Notch-DSL signaling cas-cade as well as the roles of both ligand and receptor in cellgrowth and differentiation in health and disease could providenovel protein targets for combating abnormal cell proliferationor degeneration. Nevertheless, it remains likely that efforts toselectively block the �-secretase cleavage of a specific substrate

Regulated Delta and Jagged Proteolysis and Signaling34436

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

(e.g. APP) without inducing side effects from another mayprove difficult.

Acknowledgments—We thank Beth Ostaszewski for superlative tech-nical expertise and assistance and W. T. Kimberly and D. M. Walsh foradvice and helpful discussion. The PS/�-secretase inhibitors III-31-C,DAPT, and Compound E were kind gifts of M. Wolfe. We are indebtedto G. Weinmaster for providing the rat full-length Jagged and full-length Delta expression plasmids and thank S. D. Hayward for theCBF-Luc reporter construct and R. Kopan for the N�E expressionvector.

REFERENCES

1. Brown, M. S., Ye, J., Rawson, R. B., and Goldstein, J. L. (2000) Cell 100,391–398

2. Selkoe, D. J. (2001) Physiol. Rev. 81, 742–7613. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda,

M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin, J.-F.,Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L.,Chumakov, I., Pollen, D. A., Roses, A. D., Fraser, P. E., Rommens, J. M., andSt. George-Hyslop, P. H. (1995) Nature 375, 754–760

4. Levitan, D., and Greenwald, I. (1995) Nature 377, 351–3545. Struhl, G., and Greenwald, I. (1999) Nature 398, 522–5256. Ye, Y., Lukinova, N., and Fortini, M. E. (1999) Nature 398, 525–5297. De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm,

J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A., andKopan, R. (1999) Nature 398, 518–522

8. Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. s., Moore, C. L.,Tsai, J.-Y., Rahmati, T., Xia, W., Selkoe, D. J., and Wolfe, M. S. (2000) Nat.Cell Biol. 2, 428–434

9. Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Ye, W., Diehl, T. S., Selkoe,D. J., and Wolfe, M. S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2720–2725

10. Herreman, A., Serneels, L., Annaert, W., Collen, D., Schoonjans, L., and DeStrooper, B. (2000) Nat. Cell Biol. 2, 461–462

11. Lee, H. J., Jung, K. M., Huang, Y. Z., Bennett, L. B., Lee, J. S., Mei, L., andKim, T. W. (2002) J. Biol. Chem. 277, 6318–6323

12. Li, Y.-M., Xu, M., Lai, M.-T., Huang, Q., Castro, J. L., DiMuzio-Mower, J.,Harrison, T., Lellis, C., Nadin, A., Neduvelli, J. G., Register, R. B., Sardana,M. K., Shearman, M. S., Smith, A. L., Shi, X.-P., Yin, K.-C., Shafer, J. A.,and Gardell, S. J. (2000) Nature 405, 689–694

13. Ni, C. Y., Murphy, M. P., Golde, T. E., and Carpenter, G. (2001) Science 294,2179–2181

14. Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., andSelkoe, D. J. (1999) Nature 398, 513–517

15. Zhang, Z., Nadeau, P., Song, W., Donoviel, D., Yuan, M., Bernstein, A., andYankner, B. A. (2000) Nat. Cell Biol. 2, 463–465

16. Marambaud, P., Shioi, J., Serban, G., Georgakopoulos, A., Sarner, S., Nagy, V.,Baki, L., Wen, P., Efthimiopoulos, S., Shao, Z., Wisniewski, T., and Robakis,N. K. (2002) EMBO J. 21, 1948–1956

17. Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998) Nature 393, 382–38618. Huppert, S. S., Le, A., Schroeter, E. H., Mumm, J. S., Saxena, M. T., Milner,

L. A., and Kopan, R. (2000) Nature 405, 966–97019. Kimberly, W. T., Zheng, J. B., Guenette, S. Y., and Selkoe, D. J. (2001) J. Biol.

Chem. 276, 40288–4029220. Cupers, P., Orlans, I., Craessaerts, K., Annaert, W., and De Strooper, B. (2001)

J. Neurochem. 78, 1168–117821. Cao, X., and Sudhof, T. C. (2001) Science 293, 115–12022. Gao, Y., and Pimplikar, S. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,

14979–1498423. Mumm, J. S., Schroeter, E. H., Saxena, M. T., Griesemer, A., Tian, X., Pan,

D. J., Ray, W. J., and Kopan, R. (2000) Mol Cell 5, 197–20624. Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J. R., Cumano,

A., Roux, P., Black, R. A., and Israel, A. (2000) Mol. Cell 5, 207–21625. Lieber, T., Kidd, S., and Young, M. W. (2002) Genes Dev. 16, 209–22126. Struhl, G., and Adachi, A. (2000) Mol. Cell 6, 625–63627. Qi, H., Rand, M. D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and

Artavanis-Tsakonas, S. (1999) Science 283, 91–9428. Kimberly, W. T., Xia, W., Rahmati, R., Wolfe, M. S., and Selkoe, D. J. (2000)

J. Biol. Chem. 275, 3173–317829. Xia, W., Ray, W. J., Ostaszewski, B. L., Rahmati, T., Kimberly, W. T., Wolfe,

M. S., Zhange, J., Goate, A. M., and Selkoe, D. J. (2000) Proc. Natl. Acad.Sci. U. S. A. 97, 9299–9304

30. Xia, W., Zhang, J., Perez, R., Koo, E. H., and Selkoe, D. J. (1997) Proc. Natl.Acad. Sci. U. S. A. 94, 8208–8213

31. Flanagan, J., Cheng, H., Feldheim, D., Hattori, M., Lu, Q., and Vanderhae-ghen, P. (2000) Methods Enzymol. 327, 19–35

32. Slack, B. E., Ma, L. K., and Seah, C. C. (2001) Biochem. J. 357, 787–79433. Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J.,

Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) J. Biol.Chem. 273, 27765–27767

34. Jolly-Tornetta, C., and Wolf, B. A. (2000) Biochemistry 39, 15282–1529035. Skovronsky, D. M., Zhang, B., Kung, M. P., Kung, H. F., Trojanowski, J. Q.,

and Lee, V. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7609–761436. Lindsell, C. E., Boulter, J., diSibio, G., Gossler, A., and Weinmaster, G. (1996)

Mol. Cell Neurosci. 8, 14–27

37. Capell, A., Grunberg, J., Pesold, B., Diehlmann, A., Citron, M., Nixon, R.,Beyreuther, K., Selkoe, D. J., and Haass, C. (1998) J. Biol. Chem. 273,3205–3211

38. Yu, G., Nishimura, M., Arawaka, S., Levitan, D., Zhang, L., Tandon, A., Song,Y. Q., Rogaeva, E., Chen, F., Kawarai, T., Supala, A., Levesque, L., Yu, H.,Yang, D. S., Holmes, E., Milman, P., Liang, Y., Zhang, D. M., Xu, D. H.,Sato, C., Rogaev, E., Smith, M., Janus, C., Zhang, Y., Aebersold, R., Farrer,L. S., Sorbi, S., Bruni, A., Fraser, P., and St. George-Hyslop, P. (2000)Nature 407, 48–54

39. Berezovska, O., Jack, C., Deng, A., Gastineau, N., Rebeck, G. W., and Hyman,B. T. (2001) J. Biol. Chem. 276, 30018–30023

40. Li, Y. M., Lai, M. T., Xu, M., Huang, Q., DiMuzio-Mower, J., Sardana, M. K.,Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Proc. Natl.Acad. Sci. U. S. A. 97, 6138–6143

41. Ray, W. J., Yao, M., Nowotny, P., Mumm, J., Zhang, W., Wu, J. Y., Kopan, R.,and Goate, A. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3263–3268

42. Ray, W. J., Yao, M., Mumm, J., Schroeter, E. H., Saftig, P., Wolfe, M., Selkoe,D. J., Kopan, R., and Goate, A. M. (1999) J. Biol. Chem. 274, 36801–36807

43. Kopan, R., Schroeter, E. H., Weintraub, H., and Nye, J. S. (1996) Proc. Natl.Acad. Sci. U. S. A. 93, 1683–1688

44. Sastre, M., Steiner, H., Fuchs, K., Capell, A., Multhaup, G., Condron, M. M.,Teplow, D. B., and Haass, C. (2001) EMBO Rep. 2, 835–841

45. Thomas, J. E., Smith, M., Rubinfeld, B., Gutowski, M., Beckmann, R. P., andPolakis, P. (1996) J. Biol. Chem. 271, 28630–28635

46. Hsieh, J. J., Henkel, T., Salmon, P., Robey, E., Peterson, M. G., and Hayward,S. D. (1996) Mol. Cell Biol. 16, 952–959

47. Ascano, J. M., Beverly, L. J., and Capobianco, A. J. (2003) J. Biol. Chem. 278,8771–8779

48. Chu, J., Jeffries, S., Norton, J. E., Capobianco, A. J., and Bresnick, E. H. (2002)J. Biol. Chem. 277, 7587–7597

49. Hooper, N. M., Trew, A. J., Parkin, E. T., and Turner, A. J. (2000) Adv. Exp.Med. Biol. 477, 379–390

50. Kidd, S., Lieber, T., and Young, M. W. (1998) Genes Dev. 12, 3728–374051. Bland, C. E., Kimberly, P., and Rand, M. D. (2003) J. Biol. Chem. 278,

13607–1361052. Ikeuchi, T., and Sisodia, S. S. (2003) J. Biol. Chem. 278, 7751–775453. Joutel, A., and Tournier-Lasserve, E. (1998) Semin. Cell Dev. Biol. 9, 619–62554. Tohda, S., and Nara, N. (2001) Leuk. Lymphoma 42, 467–47255. Thomas, U., Jonsson, F., Speicher, S. A., and Knust, E. (1995) Genetics 139,

203–21356. Speicher, S. A., Thomas, U., Hinz, U., and Knust, E. (1994) Development 120,

535–54457. Kim, J., Irvine, K. D., and Carroll, S. B. (1995) Cell 82, 795–80258. Couso, J. P., Knust, E., and Martinez Arias, A. (1995) Curr. Biol. 5, 1437–144859. Diaz-Benjumea, F. J., and Cohen, S. M. (1995) Development 121, 4215–422560. de Celis, J. F., de Celis, J., Ligoxygakis, P., Preiss, A., Delidakis, C., and Bray,

S. (1996) Development 122, 2719–272861. de Celis, J. F., Garcia-Bellido, A., and Bray, S. J. (1996) Development 122,

359–36962. Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L. Y., and Jan, Y. N. (1996)

Genes Dev. 10, 421–43463. Jonsson, F., and Knust, E. (1996) Dev. Genes Evol. 206, 91–10164. Micchelli, C. A., Rulifson, E. J., and Blair, S. S. (1997) Development 124,

1485–149565. Shimizu, K., Chiba, S., Saito, T., Takahashi, T., Kumano, K., Hamada, Y., and

Hirai, H. (2002) EMBO J. 21, 294–30266. Varnum-Finney, B., Wu, L., Yu, M., Brashem-Stein, C., Staats, S., Flowers, D.,

Griffin, J. D., and Bernstein, I. D. (2000) J. Cell Sci. 113, 4313–431867. Mishra-Gorur, K., Rand, M. D., Perez-Villamil, B., and Artavanis-Tsakonas, S.

(2002) J. Cell Biol. 159, 313–32468. Thinakaran, G., Borchelt, D. R., Lee, M. K., Slunt, H. H., Spitzer, L., Kim, G.,

Ratovitsky, T., Davenport, F., Nordstedt, C., Seeger, M., Hardy, J., Levey,A. I., Gandy, S. E., Jenkins, N. A., Copeland, N. G., Price, D. L., and Sisodia,S. S. (1996) Neuron 17, 181–190

69. Franklin, J. L., Berechid, B. E., Cutting, F. B., Presente, A., Chambers, C. B.,Foltz, D. R., Ferreira, A., and Nye, J. S. (1999) Curr. Biol. 9, 1448–1457

70. Zecchini, V., Brennan, K., and Martinez-Arias, A. (1999) Curr. Biol. 9,460–469

71. Ordentlich, P., Lin, A., Shen, C. P., Blaumueller, C., Matsuno, K., Artavanis-Tsakonas, S., and Kadesch, T. (1998) Mol. Cell Biol. 18, 2230–2239

72. Yamamoto, N., Yamamoto, S., Inagaki, F., Kawaichi, M., Fukamizu, A., Kishi,N., Matsuno, K., Nakamura, K., Weinmaster, G., Okano, H., and Nakafuku,M. (2001) J. Biol. Chem. 276, 45031–45040

73. Fanto, M., Weber, U., Strutt, D. I., and Mlodzik, M. (2000) Curr. Biol. 10,979–988

74. Weber, U., Paricio, N., and Mlodzik, M. (2000) Development 127, 3619–362975. Liu, K. Y., Timmons, S., Lin, Y. Z., and Hawiger, J. (1996) Proc. Natl. Acad.

Sci. U. S. A. 93, 11819–1182476. Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K.,

Ikegami, Y., and Yamamoto, D. (1995) Genes Dev. 9, 612–62577. Ellisen, L. W., Bird, J., West, D. C., Soreng, A. L., Reynolds, T. C., Smith, S. D.,

and Sklar, J. (1991) Cell 66, 649–66178. Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P.,

Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E., Maciazek, J.,Vayssiere, C., Cruaud, C., Cabanis, E. A., Ruchoux, M. M., Weissenbach, J.,Bach, J. F., Bousser, M. G., and Tournier-Lasserve, E. (1996) Nature 383,707–710

Regulated Delta and Jagged Proteolysis and Signaling 34437

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

Matthew J. LaVoie and Dennis J. Selkoe-Secretase and Release Signaling Fragmentsγand Presenilin/

-SecretaseαThe Notch Ligands, Jagged and Delta, Are Sequentially Processed by

doi: 10.1074/jbc.M302659200 originally published online June 25, 20032003, 278:34427-34437.J. Biol. Chem. 

  10.1074/jbc.M302659200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/278/36/34427.full.html#ref-list-1

This article cites 78 references, 43 of which can be accessed free at

by guest on Novem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from