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INTRODUCTION |
The Notch receptor pathway is a highly conserved signal
transduction mechanism that is critical for cell fate decisions during tissue patterning and morphogenesis in a number of organ systems in
metazoans (1, 2). Signals through the Notch receptor are initiated by
interaction with one of two known single-pass transmembrane ligands,
Delta or Serrate (mammalian Jagged) (3-5), presented on an adjacent
cell. Delta is cleaved to release its extracellular domain by a
mechanism involving Kuzbanian
(Kuz),1 an ADAM
metalloprotease (6) required for appropriate Notch signaling in several
contexts (7-10). Mishra-Gorur et al. (11) recently
elucidated that Delta is cleaved at Ala581 and
Ala593 to give soluble but inactive extracellular products,
Dlec581 and Dlec593. Nonetheless, controversy continues about the
activity or inactivity of extracellular forms of Notch ligands (6,
11-13), and thus the functional role of Delta proteolysis in
propagating Notch signals is unclear.
There is growing evidence for a functional role of the intracellular
domain of Notch ligands. In Drosophila and mammalian systems, truncated forms of Delta and Serrate lacking the intracellular sequences are ineffective activators of Notch, indicating the intracellular domain of these ligands is critical for appropriate Notch
signals (14-16). Even more intriguing, Jagged1 has an intrinsic signaling activity that relies upon a C-terminal PDZ-ligand domain (17). Thus, the role of proteolytic processing in modulating the
activity of Delta, Serrate and Jagged has become an increasingly important question.
We now show that Drosophila Delta undergoes three
proteolytic cleavages in the region of the juxtamembrane and
transmembrane domains. Only one of these cleavages, corresponding to
Ala581, is dependent on Kuz. The two additional cleavages
correspond to the previously described cleavage at Ala593
and a novel unidentified site within or close to the transmembrane domain. Processing of Delta is up-regulated when exposed to Notch expressing cells and is similarly induced by APMA, a well documented activator of metalloproteases. Finally, we show that a truncated intracellular isoform of Delta shows prominent nuclear localization. Altogether, these data demonstrate Notch-induced Delta proteolysis and
implicate a nuclear function for Delta and therefore support a model of
bi-directional signaling in the Notch pathway.
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EXPERIMENTAL PROCEDURES |
Cloning of Delta Expression Constructs--
The cDNA for
Drosophila Delta (gift from Spyros Artavanis-Tsakonas,
Harvard Medical School) was cloned into the pIZV5His vector (Invitrogen) inframe with the C-terminal V5 epitope tag and
polyhistidine sequence to create the pIZDlV5His plasmid. Delta
intracellular domain expression constructs were generated by first
creating a hemagglutinin (HA) epitope with a start codon by annealing
the oligonucleotides:
5'phos-AGCTTATGTACCCCTACGACGTGCCCGACTACGCCGAGCT and
5'phos-CGGCGTAGTCGGGCACGTCGTAGGGGTACATA and cloning into the HindIII/SacI site of the pIZV5His vector to
generate pIZ5'HAV5His. The Delta intracellular domain (amino acids
620-833, which includes the natural stop codon) was PCR amplified with
primers 5'-TGGAATTCTAAGCGCAAGCGTAAGCGTGC and
5'-CCGCTCGAGTTACATATGCGGAGTGCCGCAG and cloned into the
EcoRI/XhoI site in pIZ5'HAV5His to create an
N-terminal HA epitope inframe with the Delta intracellular domain (pIZDlicHA).
Cell Culture--
Drosophila S2 and Kc167 cells were
routinely cultured at 25-27 °C in Sang's M3 medium (JRH
Biosciences, Lenexa, KS) with 10% fetal bovine serum (FBS) and
bactopeptone (2.5 g/liter) and
yeastolate (1 g/liter) (1× BPYE) supplement (Difco).
Spodoptera frugiperda (SF9) cells were cultured in Graces
medium (JRH Biosciences) with 10% FBS and 0.5× BPYE. The stable S2
cell lines, Dl-S2 and N-S2, which express Drosophila Delta
or Drosophila Notch under the metallothionien promoter are
described previously (18). Protein expression in these cells was
routinely induced with 0.7 mM CuSO4 addition to the medium for 16 h prior to assay.
Assays for Cleavage of Delta--
Both stable Dl-S2 and
transiently transfected S2, Kc167, and SF9 cells with pIZDlV5 were
analyzed. After transient transfection using Cellfectin (Invitrogen)
and 16-20 h of recovery, assays were carried out in serum-free M3
medium/BPYE to better resolve the Dlec product in the medium.
APMA (5 mM in H20 or Me2SO
(Sigma)) was added to the medium at various concentrations. Cells were incubated 4 h, and the medium and cells were harvested separately. Cells were lysed with 50 mM Tris, 1% IGEPAL CA-630
(Sigma), 150 mM NaCl containing the protease inhibitors
EDTA (5 mM), phenylmethylsulfonyl fluoride (2 mM), aprotinin, leupeptin, and pepstatin (5 µg/ml each).
Samples were run on SDS-PAGE and Western blotted by standard procedures
with the anti-Delta 9B antibody (6) (gift from Spyros Artavanis-Tsakonas, Harvard Medical School) and anti-V5 antibody (Invitrogen).
Inhibitory RNA--
Double-stranded RNA (RNAi) treatment of S2
or Kc167 cells was done essentially as described (19)
(dixonlab.biochem.med.umich.edu/protocols/RNAiExperiment.html). A PCR-amplified segment created using T7 primers with the Kuz sequences
(5'-ATGTCATCAAAATGTGCTTTCAAC and 5'-GTGACTGTTGTTGCTGAGGATTGT) was used as a template for RNA synthesis. RNAi was added to cells at concentrations up to 6 µg/well of a 12-well culture plate and incubated 3 days prior to expression of Delta by transient transfection of pIZDlV5. Cell lysates and media were prepared and analyzed by
Western blotting as described above.
Notch-induced Cleavage of Delta--
pIZDlV5-transfected Kc167
or S2 cells were incubated with S2 or N-S2 cells in M3/BPYE without FBS
for various time points prior to harvest and preparation of cell
lysates for Western blot analysis.
Immunohistochemistry--
DlV5 and DlicHA expressing S2 and
Kc167 cells were grown on poly-L-lysine-coated glass
coverslips. Cells were fixed with paraformaldehyde and stained in
phosphate-buffered saline with 1% normal goat serum and 0.1% Triton
X-100. The anti-V5 (1/500 dilution, Invitrogen) and anti-HA (1/1000
dilution, Babco, Berkeley, CA) antibodies were used and detected with
fluorescein isothiocyanate-conjugated anti-mouse secondary antibody
(1/250 Jackson Immunologicals, West Grove, PA). Nuclei were stained
with 4',6-diamidino-2-phenylindole. Images were captured using
Leitz Orthoplan2 fluorescent microscope and Spot Insight QE digital
camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Images
were processed in Adobe Photoshop (Adobe, San Jose, CA).
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RESULTS |
APMA-induced Cleavage of Delta--
Two isoforms of Dlec are
generated by endogenous proteolytic activity in Drosophila
S2 cells raising the possibility that more than one enzyme acts upon
the Delta protein (11). We have therefore explored the potential role
of additional sheddase activity in Delta processing using the
organomercurial compound APMA, which is an effective activator of
cysteine-switch metalloproteases used in numerous experimental systems
(20, 21). Incubation of Delta-expressing cells in increasing
concentrations of APMA results in a reduction of full-length Delta
(Dlfl) and a corresponding increase in the amount of Dlec released in
the medium (Fig. 1A). Interestingly, two Dlec isoforms are seen that migrate identically with
the previously described Dlec581 and Dlec593 isoforms (not shown) (11).
We have assigned these products DlecP1 and DlecP2, respectively (see
Fig. 1A). This APMA-induced proteolysis clearly indicates
that there are latent pools of endogenous protease(s) in S2 cells, most
likely metalloproteases, that are able to act on Delta.

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Fig. 1.
Delta proteolysis with APMA.
A, Dl-S2 cells were incubated in medium
containing the indicated amount of APMA. Full-length (Dlfl) and
extracellular (Dlec) Delta were detected in cell lysates and
medium, respectively, by Western blotting with monoclonal
antibody 9B directed to the extracellular domain of
Drosophila Delta. An increase in two Dlec products,
designated DlecP1 and DlecP2, is seen in the media with a corresponding
decrease in Dlfl in the cells with increasing APMA. B,
DlV5-transfected S2, Kc167, and SF9 cells were incubated with the
indicated concentration of APMA and cell lysates analyzed by Western
blotting with anti-V5 antibody. Three C-terminal domain products
(DlcdP1/P2/P3) are seen in the cell lysates of S2 and Kc167 cells with
increasing APMA. The DlcdP2 product is not generated in SF9 cells.
C, a schematic representation of the cleavage sites in
Drosophila Delta is shown based on Mishra et al.
(11) and the results in Fig. 1B.
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To clarify the nature of these proteolytic cleavages in Delta, we
examined the fate of the C-terminal portion of the protein using a
V5-epitope-tagged construct, DlV5. In addition, we examined Delta
processing in three distinct insect cell lines: Drosophila S2 and Kc167 lines and the Lepidopteran SF9 cell line. Treatment of
DlV5-expressing S2 cells with increasing concentrations of APMA showed
a progression of C-terminal cleavage products with three distinct bands
that migrate at ~30, 28, and 26 kDa (Fig. 1B). We have
designated these bands DlcdP1, DlcdP2, and DlcdP3, respectively (see
Fig. 1, B and C). In the absence of APMA, the predominant band seen in S2 cells is the DlcdP2 band indicating that it
is the major product of steady state levels of endogenous protease
activity in these cells. With 50 µM APMA DlcdP1 is the predominant band detected, whereas at 200 µM APMA the
predominant product is the DlcdP3 band (Fig. 1B, first
panel). By apparent molecular weight, the DlcdP1 and DlcdP2 bands
are predicted to correlate with the cleavages that generate DlecP1
(DlEC581) and DlecP2 (DlEC593), respectively (see Fig. 1C).
It follows that a third uncharacterized cleavage must occur with APMA
induction that gives rise to the DlcdP3 product. Furthermore, given
that the DlecP2/DlcdP2 products likely result from cleavage at Ala593 (i.e. DlecP2 co-migrates with Dlec593 (not shown)), which is
positioned at the N-terminal portion of the transmembrane domain, the
DlcdP3 band must arise from a cleavage at an intramembrane or
intracellular site (see Fig. 1C).
Delta processing also occurs in Kc167 and SF9 cells (Fig.
1B). In contrast to the S2 cells, the DlcdP1 band is seen as
the major product in the untreated Kc167 and SF9 cells. Furthermore, a
significantly greater amount of the DlcdP1 band, in proportion to the
full-length Delta, is seen in SF9 cells suggesting that the enzyme
activity required for this product is present at a much higher level
than in S2 or Kc167 cells. It is of note that the DlcdP2 is absent in
SF9 cells (Fig. 1B, third panel). Altogether, the
data confirm that Delta is a substrate for three distinct proteolytic
processing events and that the activity of the enzymes responsible for
these cleavages varies among different cell lines, both at a resting
state and upon APMA induction.
Kuzbanian-dependent Delta Processing--
To begin
identifying the enzymes responsible for these cleavages, we examined
which of the three Dlcd products arises from Kuz-dependent
cleavage using RNA interference (RNAi) to remove Kuz activity.
Reduction of Kuz in Kc167 (Fig. 2) or S2
(not shown) cells resulted in the disappearance of the DlcdP1 band,
while the DlcdP2 and DlcdP3 bands remain unaffected. The Dlec in the medium shows a shift to predominantly the DlecP2 with increasing KuzRNAi, confirming that the DlecP1 and DlcdP1 are the corresponding products of the same cleavage.

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Fig. 2.
Kuzbanian RNAi inhibition of Delta
Cleavage. Kc167 cells treated with Kuz RNAi at the concentrations
indicated were transfected to express DlV5. Delta processing was
determined by Western blot analysis of cell extracts with anti-V5
antibody (top panel) to detect C-terminal domain products
and of medium with monoclonal 9B (bottom panel) to
detect extracellular domain products.
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Notch-induced Cleavage of Delta--
APMA induction of Delta
cleavage indicates that Delta processing is susceptible to activation
of latent proteases and raises the possibility that Delta processing
may be modulated in the context of cell signaling events. We therefore
asked whether Delta processing could be induced through interaction
with its receptor, Notch. Incubation of DlV5-expressing S2 (Fig.
3) or Kc167 (not shown) cells with N-S2
cells caused a transient accumulation of the DlcdP2 band at 1-2 h and
resolved to the DlcdP3 product at 6 h (Fig. 3, +N-S2
lanes). A corresponding decrease in the full-length Dl was
observed. Obvious aggregates were formed in the DlV5/Notch cell
mixtures (Fig. 3, lower right panel), indicative of a
Delta-Notch binding event described previously (18). In contrast, when
DlV5 cells were incubated with control S2 cells only a slight increase in the DLcdP2 band was seen at 6 h (Fig. 3, control lanes), and no
significant cell aggregates were observed (Fig. 3, lower left panel). These data confirm that Notch expressing cells promote processing of Delta.

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Fig. 3.
Notch-dependent cleavage of
Delta. DlV5-expressing S2 cells were co-cultured with S2 cells
(+S2) or Notch S2 cells (+N-S2) for the times
indicated. Cell extracts were analyzed by Western blotting with anti-V5
antibody to detect C-terminal domain products. Bottom panels
are bright field images of the corresponding cultures at 1 h of
co-culture.
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Localization of Full-length and Intracellular Delta--
The
cleavages within or close to the membrane that give rise to DlcdP3
predict a cytoplasmic product that is untethered from the membrane (see
Fig. 1C). We therefore asked what the fate of the
intracellular domain of Delta is by examining its subcellular localization. Expression of the entire Delta intracellular domain in
either S2 (Fig. 4) or Kc167 (not shown)
cells showed prominent nuclear localization. In contrast, full-length
Delta was localized to the plasma membrane and intracellular
compartments (Fig. 4). These data indicate that the products that
result from Delta processing can potentially translocate to the
nucleus.

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Fig. 4.
Localization of full-length and intracellular
Delta. S2 cells were transfected with DlV5 (full-length) or DlicHA
(intracellular). The localization of the Delta proteins relative to the
nucleus (indicated by 4',6-diamidino-2-phenylindole
(DAPI) staining) was revealed by immunostaining with anti-V5
or anti-HA, respectively.
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DISCUSSION |
The question of how signals conveyed by the Notch receptor direct
distinct cellular fates in development remains complex; however,
several advances have been made at the molecular level. Perhaps most
provocative is the discovery that regulated proteolysis by ADAM
proteases and Presenilin
-secretases are essential activation steps
to generate an intracellular Notch product with the ability to localize
to and act in the nucleus to modulate gene transcription (reviewed in
Refs. 22 and 23). It is now clear that Delta undergoes a similar
pattern of proteolytic processing. We demonstrate that Kuzbanian is
required for processing to give the DlecP1/DlcdP1 products and that the
DlcdP2 and DlcdP3 products point to additional intramembranous/intracellular cleavages. Consistent with our
observations, a very recent report by Ikeuchi and Sisodia (24) shows
presenilin-dependent processing of human Delta1 and
Jagged2. Altogether, the data suggest a receptor-like function for the
Delta protein: 1) processing is induced by binding to another protein
(e.g. Notch), 2) DlcdP3 predicts an untethered cytoplasmic
product, and 3) the cytoplasmic domain of Delta localizes to the
nucleus. Our data support a model whereby Notch-Delta interaction
between neighboring cells results in bi-directional signaling.
The pattern of Delta processing is in accordance with a growing body of
evidence showing that juxtamembrane and intramembrane proteolysis is a
widely conserved mechanism for modulating activity in a variety of
signaling pathways (25, 26). The ErbB-4 receptor and the amyloid
precursor protein are remarkably similar to Notch in that they undergo
sequential ADAM and presenilin-dependent cleavages and
nuclear translocation of their intracellular domains for subsequent
modulation of gene transcription (27-30). Interestingly, the
Neuregulin1 ligand for ErbB receptors is also a substrate for ADAM
protease activity and undergoes intramembrane proteolysis resulting in
nuclear translocation of its intracellular domain and changes in gene
transcription (31, 32). Together with Notch/Delta, these data suggest
the existence of a common mechanism of proteolytic regulation for
bi-directional cell signaling in diverse signaling pathways.
A bi-directional Notch/Delta signaling model is consistent with the
prevailing feedback-regulation model whereby transcription of Notch and
Delta genes in neighboring cells is modulated to elaborate the
asymmetries that propagate cell fate decisions (33-36). While the
function of DlcdP3 awaits further characterization, several molecular
and genetic studies implicate a role for the intracellular domain of
Notch ligands. Truncated forms of Delta and Serrate, which lack the
intracellular domain, act as non-autonomous dominant-negative
inhibitors of Notch signaling in vivo (14, 15). Likewise, a
truncated form of the human Delta1 ligand shows significantly
diminished activity toward activating Notch in a cell-based luciferase
reporter assay (16). Furthermore, a recent study by Ascano et
al. (17) describes a novel intrinsic signaling activity of the
mammalian Jagged1 ligand that relies upon interaction of the Jagged1
intracellular domain with PDZ proteins. Importantly, these authors also
demonstrate that ectopic Jagged1 expression up-regulates Notch3 and
Jagged1 mRNA levels in cultured cells, providing further evidence
for transcriptional regulation in elaboration of Notch signals. Our
data clearly show that the cytoplasmic domain of Drosophila
Delta has the intrinsic property of localizing in the nucleus. We have
thus far been unable to detect a Notch-induced DlcdP3 product in the
nucleus by conventional immunostaining methods (not shown). However,
data from Ikeuchi and Sisodia (24) indicate a Delta-Gal4VP16 chimera
acts in the nucleus in a luciferase reporter based assay. Altogether,
these data highlight the potential signal-receiving capacity of Notch
ligands. However, questions of how proteolysis might contribute to this
activity remain the subject of further investigation.
Alternatively, a model proposed recently for the role of Kuz cleavage
of Delta (11) argues that cleavage and inactivation of Delta on the
Notch/Delta-expressing cell is required to make that cell
preferentially a signal receiving cell. In this regard, the additional
cleavages of Delta reported here may similarly contribute to Delta
inactivation. However, it is important to note that the cleavages
generating DlcdP2 and DlcdP3 occur independent of Kuz activity and
would therefore represent alternative pathways to down-regulate Delta
ligand activity. Furthermore, the fact that the DlcdP2 and DlcdP3
cleavages, but not the Kuz-dependent DlcdP1 cleavage, are
promoted by interaction with Notch suggests these novel cleavages are
potentially linked directly to a Notch signaling mechanism.
There is growing evidence that several classes of enzymes can mediate
proteolysis at and within the plasma membrane (for review, see Refs. 26
and 37). In addition to the ADAM metalloproteases, aspartyl and serine
proteases are known to act on transmembrane protein substrates (26,
37). Our observations indicate that all three cleavage events in Delta
are up-regulated in the presence of APMA. Consistent with its
properties as a metalloprotease activator, APMA induces the
Kuz-dependent cleavage to generate the DlecP1/DlcdP1 products. The enzyme activities responsible for the DlcdP2 and DlcdP3 products are yet to be identified. However, it is of note that DlcdP2 is generated in S2 and Kc167 cells but not in SF9 cells,
indicating that the enzyme required for this cleavage is absent in SF9
cells. The fact that SF9 cells lack endogenous
-secretase activity
(20) implicates presenilin as a candidate for cleavage that generates
DlcdP2. The role of presenilins and other proteases in Delta processing
warrants further investigation.
In conclusion, our results demonstrate Notch-induced processing of the
Delta ligand and nuclear localization of the Delta intracellular
domain. These events, which are analogous to the proteolytic activating
steps in Notch and a number of other cell surface signaling molecules,
point to a receptor function for Delta and thus support a model of
bi-directional signaling in the Notch-Delta pathway.