From the Department of Neurobiology, Pharmacology, and Physiology, The University of Chicago, Chicago, Illinois 60637
Received for publication, December 30, 2002, and in revised form, January 16, 2003
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ABSTRACT |
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The evolutionary conserved Notch signaling
pathway is involved in cell fate specification and mediated by
molecular interactions between the Notch receptors and the Notch
ligands, Delta, Serrate, and Jagged. In this report, we demonstrate
that like Notch, Delta1 and Jagged2 are subject to presenilin
(PS)-dependent, intramembranous " Mutations in genes encoding presenilins (PS1 and
PS2)1 cosegregate with the
vast majority of pedigrees with early-onset familial Alzheimer's
disease (FAD) (1). Multiple lines of evidence indicate that PS
expression is essential for intramembranous " The Notch signaling pathway is an evolutionarily conserved signal
pathway for local cell-cell communication between neighboring cells
involved in cell fate determination (11). The Notch receptors undergo
proteolytic processing in the trans-Golgi network by a furin-like
convertase in the ectodomain (12), resulting in a mature heterodimeric
receptor that accumulates on the cell surface (13). Ligand binding
triggers sequential proteolytic processing within the extracellular
juxtamembrane region by a member of the ADAM (a disintegrin and
metalloprotease domain) family, termed TACE (tumor necrosis factor
(TNF) Several Notch ligands have been identified in vertebrates and
invertebrates, including Delta, Serrate, and Jagged, transmembrane proteins that share several structural features including a DSL (Delta,
Serrate, Lag-2) domain, required for Notch binding, and multiple
epidermal growth factor-like repeats in respective extracellular domains (18, 19). Like Notch, Delta is a substrate for proteolysis by a
metalloprotease of the ADAM family, termed Kuzbanian (20), resulting in
shedding of the ectodomain segment. The precise role of
Kuzbanian-dependent proteolytic processing of Delta is not fully understood, but recent studies suggest that this event
down-regulates Delta-mediated Notch signaling (21). In this regard,
ectodomain shedding of Jagged has not been described to date.
In this report, we demonstrate that like Notch, Delta1 and Jagged2, are
subject to presenilin (PS)-dependent, Cell Culture and Inhibitor Treatment--
Mouse neuroblastoma
N2a cells stably expressing mouse Delta1 and NIH 3T3 cells stably
expressing human Jagged2 with a COOH-terminal Myc-epitope tag,
were maintained in 200 µg/ml G418 (Invitrogen) and 2.5 µg/ml
puromycin (Clontech), respectively. Transfections--
Mouse N2a cells constitutively expressing
Myc-epitope Swedish APP695 (23) were cotransfected
with 10 µg of PS1 cDNAs and 100 ng of pIREShyg
(Clontech) and selected with 400 µg/ml
hygromycin. Hygromycin-resistant colonies were further screened with
400 µg/ml of zeocin to generate a stable "pool" of ~100-200 colonies.
Antibodies and Western Blot Analysis--
Cells were lysed in
immunoprecipitation buffer containing detergents and protease
inhibitors as described (24). Solubilized proteins were fractionated by
electrophoresis on SDS-polyacrylamide gels and electrophoretically
transferred to polyvinylidene difluoride membranes (Bio-Rad). Membranes
were blocked and then probed with primary antibodies and horseradish
peroxidase-coupled secondary antibodies (Pierce). Myc-tagged
Delta1 and Jagged2 derivatives were detected using monoclonal
Myc specific antibody, 9E10. Polyclonal antibody,
PS1NT (25), was used to detect full-length PS1 and PS1
NH2-terminal fragment. Cell Surface Biotinylation--
Cells were grown to near
confluence in a 10-cm dish and subjected to cell surface biotinylation
with 0.5 mg/ml sulfosuccinimodobiotin (Sulfo-NHS-SS-biotin,
Pierce) essentially as described previously (24). Cells were then lysed
in immunoprecipitation buffer, and biotinylated proteins were captured
with streptavidin-agarose beads (Pierce).
Luciferase Reporter Assay--
To generate a construct encoding
the Delta1-Gal4VP16 fusion protein, the primer pairs,
5'-CCATCGATTTAAGAAGCTACTGTCTTCTATC-3' and
5'-CCATCGATCACCGTCCTCGTCAATTCC-3', were incubated with pMst-GV-APP (26)
in a PCR. The PCR product was inserted between the Delta1 COOH terminus
and Myc sequences. 0.4 µg of the resulting
Delta1-Gal4VP16-Myc construct was cotransfected the Gal4
reporter plasmid (pG5E1B-luci) (26), and 50 ng of a control plasmid
encoding Rellina luciferase into HEK293 cells. Cells were harvested
48 h after transfection, and luciferase activities were determined
using dual-luciferase reporter assay system (Promega) following
manufacture's instructions. Values shown are the averages from
triplicate experiments for each condition.
The family of Notch receptors and Notch ligands are type I
integral membrane proteins. It is now well established that Notch and
the Notch ligand, Delta1, are substrates for processing by metalloproteases of the TACE/ADAM family (14, 15, 20), resulting in
shedding of respective ectodomains. In the case of Notch, TACE cleavage
generates a membrane-tethered derivative, termed S2/NEXT, that is the
substrate for intramembranous proteolysis by a
presenilin-dependent -secretase"
processing, resulting in the production of soluble intracellular
derivatives. Moreover, and paralleling the observation that expression
of familial Alzheimer's disease-linked mutant PS1 compromises
production of Notch S3/NICD, we show that the PS-dependent
production of Delta1 cytoplasmic derivatives are also reduced in cells
expressing mutant PS1. These studies led us to conclude that a similar
molecular apparatus is responsible for intramembranous processing of
Notch and it's ligands. To assess the potential role of the
cytoplasmic derivative on nuclear transcriptional events, we expressed
a Delta1-Gal4VP16 chimera and demonstrated marked transcriptional
stimulation of a luciferase-based reporter. Our findings offer the
proposal that Delta1 and Jagged2 play dual roles as activators of Notch
receptor signaling and as receptors that mediate nuclear signaling
events via
-secretase-generated cytoplasmic domains.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase" processing of a number of type I membrane proteins, including the
-amyloid precursor protein (APP) (2, 3), the developmental signaling
receptor, Notch1 (4, 5), the receptor tyrosine kinase, ErbB4 (6), cell
adhesion molecules, N- and E-cadherins (7), the low density
lipoprotein receptor-related protein, LRP (8), the cell surface
adhesion protein, CD44 (9), and the synaptic adhesion protein,
nectin-1
(10).
-converting enzyme) (14, 15), and subsequent intramembranous
cleavage of this membrane-tethered derivative, termed S2/NEXT, by a
PS-dependent
-secretase activity (4). The resulting
soluble cytoplasmic domain, termed S3/NICD (Notch intracellular
domain), translocates to the nucleus and interacts with the DNA-binding
proteins CSL, resulting in transcriptional activation of target
genes (16, 17).
-secretase processing, resulting in the production of soluble intracellular derivatives. We show that a plasma membrane-resident ~40 kDa
carboxyl-terminal fragment (CTF) that is presumably generated by a
Kuzbanian-like activity serves as substrate for
-secretase,
resulting in the liberation of a cytosolic, ~38-kDa CTF. We
demonstrate that expression of a Delta1-Gal4VP16 chimera is capable of
activating transcription of a luciferase reporter and that nuclear
transactivation is abrogated by a highly potent and selective
-secretase inhibitor. In parallel, we demonstrate that a ~27-kDa
Jagged2 CTF is also a substrate for
-secretase. Our findings suggest
that Delta1 and Jagged2 may play dual roles as activators of Notch
receptor signaling and as receptors that mediate nuclear signaling
events via
-secretase-generated cytoplasmic domains. Finally, we
report that expression of FAD-linked PS1 variants lead to compromised
intramembranous cleavage of Delta1. These observations mimic earlier
studies showing reduced cleavage at the Notch S3 site and the APP
"
" site within respective transmembrane domains in cells
expressing FAD-linked mutant PS1. Thus, we argue that a similar
molecular apparatus is responsible for intramembranous cleavage of
Notch and it's ligand, Delta1.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Secretase inhibitor treatments were for 16 h with 2 µM of
L-685,458 (22).
-Tubulin was detected by
anti-
-Tubulin antibody (Sigma). Bound antibodies were visualized by
enhanced chemiluminescence (ECL) detection system (PerkinElmer Life Science).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase activity (4). This
cleavage event, termed S3 cleavage, occurs between amino acids 1743 and
1744 (16); the P1 valine residue is indispensable for S3 cleavage and
subsequent nuclear signaling activity (16). Intramembranous cleavage at
the S3 site results in the generation of a soluble, cytoplasmic
derivative of Notch, termed S3/NICD, that is a transcriptional
coactivator. Intrigued by the finding that Delta1 undergoes ectodomain
shedding, and the presence of valine residues at analogous positions
within the transmembrane domains of Delta1 and the Jagged2 (Fig.
1A), we asked whether these
Notch ligands may be substrates for
-secretase cleavage, as
well.
View larger version (42K):
[in a new window]
Fig. 1.
PS-dependent
-secretase cleavage of Notch ligands. A,
amino acid sequences of transmembrane domain and intracellular
juxtamembrane region in Notch1 and Notch ligands, Delta1 and Jagged2,
are presented. The box represents the predicted
transmembrane domain. Mouse Notch1 is cleaved in a
PS-dependent manner at S3 site (arrow) before
valine 1744 (16). Arrowheads point to valine residue(s)
within the Delta1 and Jagged2 transmembrane domains. B, N2a
cells stably expressing Myc-tagged mouse Delta1 were incubated
in the absence (lane 1) or the presence (lane 2)
of
-secretase inhibitor, L-685,459. Additionally, we transiently
transfected N2a-pooled cells stably expressing human wild-type
(WT) PS1 (lane 3) or PS1 D385A variant
(lane 4) with cDNA encoding Delta1-Myc. The
bands corresponding to ~117-kDa full-length, ~40-kDa COOH-terminal
fragment (CTF1), and ~38-kDa CTF2 of Delta1, are indicated
(upper panels). The bands corresponding to ~45-kDa
full-length PS1 and ~29-kDa PS1 NH2-terminal fragment are
also indicated (lower panel). Molecular markers (in
kilodaltons) are shown at right. C, NIH 3T3 cells
stably expressing Myc-tagged human Jagged2 were incubated in
the absence (lane 1) or the presence (lane 2) of
L-685,458. We also transfected N2a stable pools expressing PS1 WT
(lane 3) or PS1 D385A (lane 4) with cDNA
encoding Jagged2-Myc. The band corresponding to ~170-kDa
full-length Jagged2 and ~27-kDa CTF1, and ~25-kDa CTF2 of Jagged2
derivatives, are indicated with arrows.
We first examined stable N2a cells that constitutively express
Myc-tagged mouse Delta1 harboring a carboxyl-terminal,
Myc-epitope tag. Western blot analysis revealed the presence of
full-length ~117-kDa Delta1-Myc and a prominent ~40-kDa
Delta1 carboxyl-terminal fragment (D-CTF1) that presumably represents
the membrane-tethered fragment generated following metalloprotease
cleavage within the ectodomain (20, 21) (Fig. 1B, lane
1). In addition, we observed low levels of a ~38-kDa CTF
(D-CTF2) at steady state (Fig. 1B, lane 1).
Importantly, the ~38-kDa D-CTF2 derivative fails to accumulate in
cells treated with a highly potent and selective -secretase inhibitor, L-685,458 (Fig. 1B, lane 2), findings
strongly suggesting that this fragments is generated by
-secretase.
To further establish that production of D-CTF2 is
PS-dependent, we transiently expressed Delta1-Myc in
N2a cells that constitutively express either wild-type human PS1 or a
dominant negative human PS1 variant that harbors the D385A mutation
(24); intramembranous processing of Notch1 is abrogated in cells
expressing PS1 D385A (24). As we had observed in cells treated with the
-secretase inhibitor, D-CTF2 failed to accumulate in cells
expressing PS1 D385A (Fig. 2B,
lane 4). Collectively, these data strongly suggest that
Delta1 is a substrate of PS-dependent,
-secretase
cleavage.
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We then examined the processing of the Notch ligand, Jagged2, in an NIH
3T3 cell line that stably expresses human Jagged2 harboring a
carboxyl-terminal Myc-epitope tag. Western blot analysis revealed the presence of ~170-kDa full-length Jagged2 and an
~25-kDa Jagged2 carboxyl-terminal fragment (J-CTF2) (Fig.
1C, lane 1). However, the ~25-kDa CTF2 was
eliminated in cells treated with the -secretase inhibitor,
L-685,458, and interestingly, a new ~27-kDa J-CTF1 accumulates under
these conditions (Fig. 1C, lane 2). These
findings suggest that J-CTF1 is constitutively processed by a
-secretase-like activity, to generate J-CTF2. To establish that
production of J-CTF2 is PS-dependent, we transiently
expressed Jagged2-Myc into N2a cells that express the PS1 D385A
mutation (24). As we had observed in cells treated with the
-secretase inhibitor, the production of J-CTF2 is eliminated, and
J-CTF1 now accumulates (Fig. 1C, lane 4). These
results indicate that Jagged2 is also a substrate of
PS-dependent
-secretase cleavage.
To identify the subcellular site(s) at which the D-CTF2 and D-CTF1
derivatives of Delta1-Myc accumulate, we treated N2a cells that
constitutively express Delta1-Myc with the membrane-impermeant, biotinylation reagent, sulfosuccinimidobiotin at 4 °C. Biotinylated, cell surface polypeptides were recovered from detergent-solubilized lysates using streptavidin-conjugated agarose, and captured proteins were subject to Western blot analysis with the Myc-specific,
9E10 antibody. In cells expressing Delta1-Myc, we observed
biotinylated full-length Delta1-Myc and ~40-kDa D-CTF1 (Fig.
2A, lane 5). However, the ~38-kDa D-CTF2 was
not recovered by immobilized streptavidin, despite the presence of the
fragment in detergent lysates (Fig. 2A, lane 2).
Hence, D-CTF2, like the Notch S3/NICD derivative is not present at the
cell surface, but presumably present in the cytosol. Moreover, D-CTF2
failed to accumulate in cells treated with the -secretase inhibitor,
as expected, and streptavidin only recovered both full-length and
~40-kDa D-CTF1 from detergent lysates of these inhibitor-treated
cells (Fig. 2A, lane 6). These observations
strongly suggest that the soluble, cytoplasmic domain of Delta1 is
generated following sequential cleavage of full-length Delta1 species
by the concerted action of Kuzbanian-like metalloprotease(s) and a
PS-dependent
-secretase.
It is now clear that the soluble intracellular domains of Notch and APP
that are generated by -secretase are translocated to the nucleus and
serve as transcriptional coactivators (16, 17, 26). To test the
possibility that D-CTF2 could be transported to nucleus, and exhibit
nuclear signaling activity, we generated cDNA encoding a
Delta1-Gal4VP16 fusion protein and transfected this construct into
human embryonic kidney 293 (HEK293) cells together with the reporter
plasmid, pG5E1B-luciferase (26). Compared with cells expressing the
pG5E1B vector, expression of Delta1-Gal4VP16 fusion protein stimulated
transcription by ~70-fold (Fig. 2B). Notably,
transactivation of the reporter plasmid by the Delta1-Gal4VP16 fusion
protein was markedly inhibited upon treatment of cells with the
-secretase inhibitor (Fig. 2B). These results suggest
that intracellular domain of Delta1 that is generated by
PS-dependent
-secretase cleavage can be transported into
nucleus, findings that raise the possibility that the cytosolic
derivative of Delta1 may play a role in nuclear signaling events.
Finally, and intrigued by the finding that cells expressing FAD-linked
PS1 mutants enhance production of pathogenic -amyloid 42 peptides
(1), but exhibit impaired processing within the transmembrane domains
of Notch and APP that liberate NICD and AICD, respectively (27-29), we
analyzed processing of Delta1-Myc in pools of N2a cells that
constitutively express human wild-type PS1 or the PS1
E9, M146L,
E280A, or C410Y FAD variants. Compared with N2a cells expressing
wild-type human PS1 (Fig. 3A,
lane 1), in which Delta1-Myc was processed to D-CTF1
and low levels of D-CTF2, as described above (Fig. 1B), the
production of the D-CTF2 fragment is reduced in most cell lines
expressing FAD-linked PS1 variants, albeit with only a modest effect in
the M146L cells (Fig. 3A, lanes 2-5, quantified
in Fig. 3B), this despite comparable levels of expression of
human PS1 (Fig. 3A, lower panel). Taken together
with the curious observation that expression of FAD-linked PS1 variants
leads to reduced processing at the Notch S3 and APP
sites, our
findings that production of the D-CTF2 derived from Delta1 is also
reduced by expression of mutant PS1 argues that a similar, if not
identical, molecular apparatus is involved in substrate recognition and
intramembranous processing of these functionally divergent membrane
proteins.
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DISCUSSION |
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A wealth of evidence has emerged to support a role for PS in
intramembranous -secretase processing of a host of type I membrane proteins. Intrigued by the similarity in the amino acid sequence of the
transmembrane domains of Notch and it's ligands, Delta and Jagged, we
hypothesized that these ligands might also serve as substrates for
-secretase. In the present report, we confirm our prediction and
offer several insights relevant to the molecular apparatus responsible
for intramembranous processing of Delta1 and Jagged2 and the potential
functional significance of this processing event.
First, we demonstrate that the production of cytosolic derivatives of
Delta1 and Jagged2, termed DICD and JICD, respectively, are inhibited
either by a highly potent, and selective transition-state isostere of
-secretase activity or by expression of the dominant negative D385A
PS1 mutant. Thus, the Notch ligands, Delta1 and Jagged2, are novel
substrates of PS-dependent
-secretase processing.
Second, and in view of earlier demonstrations that FAD-linked PS1
mutations impair cleavage at the Notch S3 and APP sites that lead
to production of S3/NICD and AICD (27-29), respectively, we assessed
the effects of FAD-linked PS1 variants on
-secretase processing of
Delta1. We show that the generation of DICD is impaired in most of cell
lines stably expressing four independent FAD-linked PS1 mutants. Most
interestingly, the relative levels of reduction in DICD parallel the
reported effects on S3/NICD production (29); expression of the C410Y
variant has the most pronounced effect, while the M146L variant has
only a modest effect on
-secretase processing. Thus, we argue that
the molecular apparatus involved in production of NICD, AICD, and DICD
are similar, if not one in the same.
Third, and in view of earlier conclusions that the intracellular
domains of Notch (S3/NICD) and APP (AICD) are transcriptional coactivators (16, 17, 24), we hypothesized that -secretase-generated DICD could be translocated to the nucleus and activate transcription of
a reporter gene. Our present results support this prediction. Despite
the strengths of these observations, the factors responsible for
translocating DICD into the nucleus are not known. For the APP
derivative, AICD, a cytosolic adaptor protein, Fe65, promotes nuclear
translocation (24). For Notch1, three putative nuclear localization
signals (NLS) are present within the ICD, and these may serve as
recognition motifs by members of the importin/karyopherin
and
receptors involved in nuclear import. Similarly, the Delta1 intracellular domain contains two putative NLSs,
PDRKRPE at amino acids 686-692, and
RKRP at amino acids 688-691, while the Jagged2
intracellular domain contains putative NLSs RKRR at amino
acids 1107-1110, and KRRK at 1108-1111. Hence, it
is conceivable that DICD and JICD may be imported via the classical
importin pathway, but further mutagenesis studies of the putative NLSs
will be required to validate this hypothesis.
In any event, our results offer the suggestion that
PS-dependent -secretase processing of Delta1 or Jagged2
and production of DICD and JICD may play roles in activating nuclear
transcriptional events. While the significance of these findings in
relation to cell-intrinsic versus cell-extrinsic aspects of
Notch signaling remains to be determined, the proposal that the Notch
ligands, Delta1 and Jagged2, may perform roles both as ligands and
receptors that directly participate in transcriptional activation
warrants further investigation.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. J. S. Nye and J. Sklar for providing cell lines expressing Delta1 and Jagged2, respectively.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AG021494 (to S. S. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of research fellowships from Uehara Memorial Foundation.
§ To whom correspondence should be addressed: Center for Molecular Neurobiology, The University of Chicago, Abbott 510, 947 East 58th St., Chicago, IL 60637. Tel.: 773-834-9186; Fax: 773-834-5311; E-mail: ssisodia@drugs.bsd.uchicago.edu.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.C200711200
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ABBREVIATIONS |
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The abbreviations used are:
PS, presenilin;
FAD, familial Alzheimer's disease;
APP, -amyloid precursor protein;
ADAM, a disintegrin and metalloprotease domain;
NEXT, Notch
extracellular truncation;
NICD, Notch intracellular domain;
CTF, COOH-terminal fragment;
DICD, Delta1 intracellular domain;
JICD, Jagged intracellular domain;
AICD, APP intracellular domain;
NLS, nuclear localization signal.
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