From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267-0524
Received for publication, November 8, 2002, and in revised form, December 20, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
JAGGED1 is a member of the Delta/Serrate/Lag-2
(DSL) family of proteins that are cell-bound ligands for Notch
receptors. Initiation of Notch signaling occurs through a series of
proteolytic events upon the binding of Notch to a DSL protein presented
on neighboring cells. Whether DSL proteins themselves are capable of
initiating an intrinsic signaling mechanism within the cell they are
expressed is not known. Aberrant misexpression of JAGGED1 and DELTA1
has been documented in several human tumors; however, the mechanism by
which misexpression of JAGGED1 contributes to oncogenesis has not been
elucidated. We report that expression of human JAGGED1 transforms RKE
cells in culture, therefore providing a model system to elucidate the
function of DSL proteins. JAGGED1-mediated transformation occurs in a
dose-dependent manner and requires a PDZ-ligand at the C
terminus. Mutation of the PDZ-ligand did not affect the ability of
JAGGED1 to initiate Notch signaling in neighboring cells. However, the
PDZ-ligand is required for changes in the expression of JAGGED1 target
genes and transcriptional activation of luciferase reporter constructs.
Our data indicate the existence of a novel PDZ-dependent
signaling pathway intrinsic to JAGGED1. We propose a bi-directional
signaling model such that DSL proteins may have two distinct functions:
to initiate Notch signaling in a neighboring cell and to initiate a
PDZ-dependent signaling mechanism in the DSL-expressing
cell. Moreover, we conclude that this intrinsic signaling
mechanism of JAGGED1 may partly provide a link between aberrant
misexpression of JAGGED1 and tumorigenesis.
A developing paradigm in signal transduction is that of the
importance of cell-to-cell communication. Many fundamental regulatory decisions are mediated by signal transduction pathways that are initiated by the engagement of a receptor-ligand pair through cell-to-cell contact. The Notch signal transduction pathway, which governs cell fate decisions, is regulated primarily through this sort
of mechanism (1-5). JAGGED1 is a member of the Delta/Serrate/Lag-2 (DSL)1 family of proteins
that are thought to be cell-bound ligands that regulate Notch signaling
(6-9). DSL genes encode Type I membrane-spanning proteins that have an
extracellular domain consisting of multiple highly conserved EGF-like
motifs (16 copies in human JAGGED1) and a conserved DSL domain that is
rich in cysteine residues (Fig. 2A). The DSL family is
classified into either the Delta-like or serrate/Jagged subgroups. The
defining motif of serrate/Jagged DSL proteins is the presence of a
cysteine-rich region (CR) between the EGF-like repeats and
transmembrane domain. The intracellular domains of DSL proteins vary in
length and are not conserved in primary amino acid sequence. For
example, there is no sequence conservation between the 125 residues in
the intracellular domain of JAGGED1 and the 154 residues in the
intracellular domain of human DELTA1. Furthermore, the intracellular
domains of DSL proteins do not share any significant similarities to
other known proteins. However, deletions of the intracellular domains
of serrate and delta result in similar mutant phenotypes in
Drosophila, indicating that there is some function
associated with this portion of the molecules (10-12). DSL proteins
expressed on the surface of the signal-transmitting cell are thought to
function by activating Notch in a neighboring signal-receiving cell
(Notch-expressing cell). However, an important question remains to be
addressed: Does the signal-transmitting cell itself receive a signal
that can be transmitted through the expressed DSL protein?
Interestingly, the C terminus of JAGGED1 encodes a putative PDZ
(PSD-95/Dlg/Zo-1)-ligand (13). Therefore, a potential role for JAGGED1
in novel PDZ-dependent signaling mechanisms exists.
However, biological or biochemical evidence for this type of intrinsic
JAGGED1 signaling has not been observed. Although it is not clear if
signaling events occur in both the DSL-expressing and Notch-expressing
cells upon receptor-ligand binding, bi-directional signaling mechanisms
such as the Eph/Ephrin pathway have been documented (14).
There are four mammalian Notch genes (Notch1-4) encoding
membrane-spanning receptors that are activated through interaction with
DSL proteins across cell boundaries. The binding of DSL and Notch
proteins is thought to result in a conformational change that renders
Notch susceptible to proteolytic processing mediated by
metalloproteases such as TNF Our laboratory has demonstrated that expression of activated NOTCH
proteins (Nic) in RKE cells results in neoplastic
transformation. Although there is evidence that JAGGED1 gene
expression is altered in several human tumors, such as cervical and
colon carcinomas, there is no evidence for a causal role in oncogenesis
(29). Here, we report that expression of JAGGED1 results in cellular
transformation of RKE cells in a dose-dependent manner.
Both the extracellular and intracellular domains are required for this
activity since expression of either a soluble form of the extracellular
domain or a membrane-tethered intracellular domain fails to transform cells. Furthermore, JAGGED1-mediated transformation requires an intact
C terminus that constitutes a PDZ-ligand. Our data indicate that
cellular transformation by JAGGED1 expression is likely due to a
PDZ-dependent signaling mechanism intrinsic to JAGGED1,
providing evidence of a causal role for misexpression of JAGGED1 in
oncogenesis and for a bi-directional mode of signaling in the Notch/DSL pathway.
Plasmids--
pcDNA expression vectors encoding human
JAGGED1, DELTA1, and NOTCH1 were
kindly provided by S. Artavanis-Tsakonas (29-31). MFG-LacZ and
SV
The following expression plasmids were generated:
pcDNA-AF6
Jagged1 promoter reporter constructs were used in
luciferase assays. For pJ1pro
Glutathione S-transferase (GST) fusion protein expression
constructs were generated by ligation of the pGEX-4T3 vector (Amersham Biosciences) to coding sequences for the intracellular domain of
JAGGED1 (GST-Jic, aa 1094-1218) or for the deletion mutant
that lacks the PDZ-ligand (GST-Jic Cell Culture and Retroviral Infections--
RKE and RK3E cells
have been previously described (31, 37). All cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin,
and 2 mM glutamine (DMEMc). All cell culture reagents were
purchased from Invitrogen.
In order to establish clonal lines, RKE cells were transfected with
plasmid DNA (10 µg) as indicated in the text using either Lipofectamine (10 µg, Invitrogen) according to manufacturer's protocols or a modified BES-calcium phosphate method. Cells were seeded
in media containing 400 µg/ml G418 (Invitrogen) to select for
expression of drug resistance markers.
To analyze transformation efficiency by focus formation, 1 × 103 to 1 × 105 RKE-derived cells
expressing JAGGED1, Jex, Jtmic, or
J1
The retroviral vector MFG-LacZ was packaged into retroviral particles
by cotransfection of 293T cells with 5 µg of MFG-LacZ and 5 µg of
SV Analysis of Protein Expression--
Crude membrane fractions
were prepared by hypotonic lysis at 4 °C in hypotonic lysis buffer
(25 mM Hepes pH 7.5, 10 mM KCl, 0.5 mM dithiothreitol) supplemented with protease inhibitors (2 mM pefabloc, 5 µg/ml leupeptin, and 2 µg/ml aprotinin
(Roche Molecular Biochemicals)). Following dounce homogenization with a
B-type pestle (Kontes Glass Company), nuclei were pelleted at
1,600 × g for 10 min at 4 °C. Cell debris were
removed from the crude cytoplasmic fraction (supernatant) by
centrifugation at 13,000 rpm for 10 min at 4 °C. Membrane fractions
were isolated by ultracentrifugation at 100,000 × g
for 30 min at 4 °C. Membrane pellets were washed with 1 ml of
hypotonic lysis buffer and solubilized in 200 µl of Nonidet P-40
lysis buffer (150 mM NaCl, 50 mM Hepes pH 7.4, 1.5 mM EDTA, 0.5 mM dithiothreitol, 10%
glycerol, 1% Nonidet P-40, and protease inhibitors). Whole cell
lysates were obtained by lysing cells in 1 ml of Nonidet P-40 lysis
buffer. Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C.
Protein concentrations were determined using the bicinchoninic acid
assay (Pierce).
Equal amounts of each lysate (20-25 µg) were separated in
SDS-polyacrylamide (8 or 14%) gels, followed by transfer to either nitrocellulose (Schleicher & Schuell) or polyvinylidene difluoride (Millipore) membranes. Western blot analysis was performed by immunoblotting with the indicated antibodies (TS1.15h for Jagged1, Ref.
29, 9E10 for Myc, Ref. 38, Na+/K+ ATPase
(KETYY) (kindly provided by Dr. Jerry Lingrel)). Proteins were
visualized using the appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories) followed by enhanced chemiluminescence (ECL, Amersham Biosciences).
RT-PCR Analysis--
Total RNA was extracted from confluent
cultures using Trizol Reagent (Invitrogen) according to the
manufacturer's specifications. cDNA was synthesized from total RNA
(2 µg) using M-MLV Reverse Transcriptase (Promega) with oligo(dT)
primers. PCR reactions were performed in a 50-µl mixture containing
0.5 µM of each gene-specific primer, 1.5 mM
MgCl2, 200 µM of each dNTP (Roche Molecular
Biochemicals), 1× PCR buffer (Hybaid), 0.2 units of DNA polymerase
(Hybaid), and 0.1 µCi of [
Gene-specific primers were designed to specifically amplify endogenous
rat mRNA. RT-PCR primer sequences: GST Pulldown Assays--
Whole cell lysates were prepared from
transiently transfected 293T cells expressing either AF6myc or
AF6 Luciferase Reporter Gene Assays--
The 8× CBF1 luciferase
reporter plasmid was kindly provided by P. D. Ling and used to analyze
ligand-induced Notch activation in coculture luciferase experiments
(39). HeLa cells were seeded in six-well plates and transfected with
8× CBF1Luc (600 ng), CMV-
For the Jagged1 promoter luciferase assays, HeLa cells were
transfected with the luciferase reporter plasmid (0.5 ng) as indicated in the text, CMV-
HeLa cells were transfected using Lipofectamine (8 µg) in a total
volume of 2 ml of OptiMem. Lysates were prepared in 1X Passive Lysis
Buffer (Promega). Luciferase and Expression of JAGGED1 in RKE Cells Results in Cellular
Transformation--
There is now significant evidence that
constitutively active forms of all four Notch receptors can contribute
to oncogenesis (31, 40-44). We previously reported that expression of
NOTCHic (Nic) results in neoplastic
transformation of E1A-immortalized rat kidney epithelial cells (RKE);
in contrast, overexpression of wild-type NOTCH1 failed to transform
these cells (31). The current model for Notch signaling proposes that
activation of Notch occurs through ligand-induced proteolysis via
cell-to-cell contact. We reasoned that wild-type NOTCH1 does not
transform RKE cells due to a lack of ligand-induced activation, and
therefore, we attempted to establish a coculture focus assay to test
for ligand-induced transformation activity in the context of
cell-to-cell contact. Although cell-to-cell contact failed to induce
the transformation of NOTCH1-expressing cells, we observed that the
JAGGED1-expressing cells displayed a transformed phenotype (data not
shown and Fig. 1).
To confirm our initial observation that JAGGED1 expression resulted in
transformation, several clonal cell lines expressing JAGGED1 were
generated (Fig. 1). The JAGGED1 expression vector was transfected into
RKE cells, and colonies were obtained by selecting for expression of
the linked drug resistance marker. Colonies either displayed a
transformed morphology, as indicated by a multilayered growth of cells,
or they grew as a flat monolayer, which is consistent with a
non-transformed phenotype (data not shown). Cell lines were established
from six colonies that appeared morphologically transformed (J1, J3,
J12, J4, J7, and J8) and three colonies that appeared to have a
flat non-transformed morphology. To determine if the
JAGGED1-expressing clonal cell lines were transformed, J1 and J12
clonal cell lines were maintained in culture for 3 weeks. J1 and J12
cells did not exhibit contact inhibition and produced a dense
multi-layered mat of cells compared with the control plate of parental
RKE cells, which formed a uniform monolayer (Fig. 1A). The
intensity of methylene blue staining was greater on the J1 plate
compared with J12 plate, indicating that J1 cells grew to a greater
cell density. Since a dense layer of cells was formed by
JAGGED1-expressing cells, it was difficult to assess the differences in
transformation efficiency among the clonal cell lines. To determine the
degree of transformation efficiency, 1 × 104 clonal
cells were plated in the presence of excess parental RKE cells. Five of
the six clonal cell lines that initially displayed a transformed
morphology formed foci (Fig. 1B). However, the number and
size of the foci were different among the clonal cell lines, indicating
that there was a variation in transformation efficiency. J1 and J3 cell
lines produced a greater number of foci compared with J12 and J4 cell
lines, whereas parental RKE cells and drug resistant clones displaying
a non-transformed morphology did not form foci (Fig. 1B and
data not shown, respectively).
Crude membranes extracted from the clonal cell lines were analyzed for
JAGGED1 expression (Fig. 1C). J1 and J3 cells, which displayed the highest transformation efficiency, expressed the highest
level of JAGGED1. In contrast, expression of JAGGED1 was lowest in the
J4 clone, which produced the fewest foci, whereas J12 cells displayed
an intermediate level of JAGGED1 expression and transformation
efficiency, indicating that there is a link between expression and
transformation efficiency (Fig. 1C). No JAGGED1 expression
was observed in either the J7 clonal cell line that did not form foci
or in the drug resistant clones that displayed a non-transformed
morphology (Fig. 1 and data not shown). Na+/K+
ATPase expression in the crude membrane fractions is shown as a
normalization control for these samples (Fig. 1C). To
confirm that the JAGGED1-expressing cell was the transformed cell type in the coculture, JAGGED1-expressing cells were infected with MFG-LacZ
retrovirus to mark these cells with Deletion of Either the Extracellular or Intracellular Domain of
JAGGED1 Results in the Loss of Transforming Activity--
To determine
the functional domains in JAGGED1 that are necessary and/or sufficient
for transformation, we generated deletion mutants of JAGGED1 that
separate the extracellular and intracellular domains (Fig.
2A). While the extracellular
domain is thought to function by binding Notch to initiate signaling,
the intracellular domain has not been associated with any known
function. The extracellular domain of Jagged1 was constructed as either
a membrane-tethered or soluble protein. The membrane-bound form of the
extracellular domain was transfected into RKE cells, and isolated
drug-resistant cells did not form foci in transformation assays.
However, we were not able to detect expression of the membrane-tethered
protein using an antibody against the C-terminal Myc tag, but we did
detect protein expression in transiently transfected 293T cells,
indicating that the Myc tag must have been cleaved in the clonal RKE
cells (data not shown). Clonal cell lines that expressed the soluble form of the extracellular domain (Jex) and a
membrane-tethered form of the intracellular domain (Jtmic)
were generated and tested for transformation. While focus formation of
J12 cells was readily visible, there were no foci on plates containing
either Jex or Jtmic clonal cell lines (Fig.
2B). Western blot analysis revealed that Jex was
found in the media and that Jtmic was targeted to the
plasma membrane (Fig. 2C). Furthermore, the expression
levels of Jex and Jtmic deletion mutants were
greater than the expression of JAGGED1 in the transformed J12 cells
(Fig. 2D, top panel). In order to compare the
expression levels of Jex and JAGGED1 using two different
antibodies (anti-Myc and anti-Jagged1 antibodies, respectively), we
determined the avidities of these antibodies using a Myc-tagged
Jtmic (Jtmic Transformation by JAGGED1 Requires a C-terminal PDZ-Ligand--
A
putative binding site for PDZ-domain proteins was identified at the C
terminus of JAGGED1 through sequence comparison with a known
PDZ-ligand, EphB2 receptor-protein tyrosine kinase (Fig. 3A) (13). To determine if
these residues were required for transformation by JAGGED1, we
generated a mutant that has a deletion of the six C-terminal residues
that comprise the PDZ-ligand (J1
The presentation of JAGGED1 proteins to Notch results in the initiation
of Notch signaling in the neighboring cell. This interaction is thought
to be mediated by the extracellular domain of JAGGED1; however, it is
not known if the intracellular domain is necessary to cluster and
present JAGGED1 proteins. Whether or not the PDZ-ligand at the C
terminus of JAGGED1 is required for ligand presentation remains
unknown. In order to determine if the deletion mutation of the
PDZ-ligand did not effect ligand-induction of Notch proteins, a
coculture luciferase assay was used to measure ligand-induced Notch
activity as described by Wu et al. (45). Briefly, HeLa cells
were transiently cotransfected with a CSL-Luciferase reporter, and
expression plasmids for NOTCH1
To determine that the C terminus of JAGGED1 is capable of binding to
PDZ-domain proteins, GST pulldown assays were performed with GST fusion
proteins expressing either the intracellular domain of JAGGED1 with or
without the PDZ-ligand (GST-Jic and
GST-Jic Expression of JAGGED1 Results in Changes in Gene
Expression--
To determine the expression levels of Notch signaling
components, RNA was extracted from parental RKE and RKE-derived clonal cell lines and analyzed by RT-PCR. Primers were designed to
specifically analyze rat mRNA and not mRNA derived from the
ectopic human cDNA in the clonal cell lines. A basal level of
mRNA for Notch1, 2, and 3, Delta 1, Jagged1 and 2, and Radical
Fringe was detected in RKE cells (Fig.
4). We observed that Notch3 and Jagged1
were induced in J1 and Nic-expressing cells compared with
parental and NOTCH1-expressing (N) RKE cells, while mRNA levels for
Notch1, Notch2, Jagged2, and Radical Fringe were not effected (Fig.
4A).
In order to determine the importance of the PDZ-ligand in
regulation of gene transcription, RT-PCR analysis was performed using
total RNA extracted from parental RKE and the clonal cell lines J1 and
J1
To determine if the induction of Jagged1 mRNA in cells is a
consequence of transcriptional activation of the gene, we assayed the
ability of JAGGED1 to induce transcription of Jagged1
promoter reporter constructs. There was a 2-fold induction of the
reporter constructs containing an 8-kb genomic DNA fragment
(pJ1pro Cellular Transformation by Human JAGGED1--
Although aberrant
misexpression of DSL proteins has been documented in several tumor
types, a causal role in oncogenesis has not been demonstrated (29,
47-49). Here, we report that expression of JAGGED1 transforms RKE
cells in culture. These cells readily escape contact inhibition, but
fail to form colonies in soft agar or tumors in nude mice (data not
shown), indicating that these cells are not malignantly transformed.
This is in contrast to expression of activated alleles of NOTCH
proteins (Nic) in RKE cells, which results in complete
malignant transformation. We present evidence that cellular
transformation by JAGGED1 expression is due, at least in part, to a
PDZ-dependent signaling mechanism intrinsic to JAGGED1 and
may be independent of Notch activation.
The current model of Notch signaling proposes that DSL proteins serve
to activate Notch. Binding of DSL ligands to Notch across cell
boundaries induces proteolytic processing that results in the release
of Nic from the plasma membrane. Nic
subsequently translocates to the nucleus and regulates a specific set
of genes. RKE cells express a basal level of mRNA for Notch1, 2, and 3 (Fig. 4A). Since both Jagged1 and Notch1 proteins are expressed in JAGGED1-expressing cells, it is plausible that Notch signaling may be activated in an autocrine-like manner where Notch1 is
activated by interaction with JAGGED1 within the same cell. However, we
have been unable to demonstrate any differences in Notch processing,
localization, or signaling between parental and JAGGED1-expressing RKE
cells. One possible explanation for these results is that the presence
of Radical Fringe in RKE cells may render Notch insensitive to
JAGGED1-induction (Fig. 4). There is substantial evidence that Fringe
proteins have N-acetylglucosaminyltransferase activity that
decreases the affinity of Notch for serrate/Jagged proteins and
increases the affinity of Notch for Delta proteins (25-28,50).
Although it remains a possibility that the level of Notch activation is
sufficiently low so that changes at protein level are undetectable in
RKE cells, we do not think this is the case. Moreover, we present
evidence that JAGGED1 is able to mediate cellular transformation
through a PDZ-dependent signaling mechanism.
A PDZ-Ligand at the C-terminal End of JAGGED1 May Be Involved in an
Intrinsic Signaling Pathway Independent of Notch Activation--
An
emerging paradigm is the existence of bi-directional signaling, in
which cell-to-cell contact stimulates cellular responses from both the
signal-receiving cell and the signal-transmitting cell. Receptor-ligand
interactions result in receptor clustering and recruitment of
cytoplasmic proteins such as kinases and adaptor proteins that
ultimately transmit the signal (51). Although there is much more known
about the downstream events that occur upon activation of Notch within
the Notch-expressing cell, there is little known about the effects of
the receptor-ligand interaction within the DSL-expressing cell. Here,
we present a model indicating that Jagged1 can mediate a signal through
a PDZ-dependent mechanism. We have demonstrated that the
six C-terminal residues of Jagged1 that comprise a PDZ-ligand are
required for Jagged1-mediated transformation, as shown by a dramatic
reduction in focus formation by J1
In our initial analysis to determine which domain of JAGGED1 is
necessary and sufficient for transformation, we created deletion mutations to separate the two major domains. The extracellular domain
is thought to function by binding Notch to initiate signaling, and
therefore we generated a soluble form of the extracellular domain
(Jex). Overexpression of Jex did not transform
RKE cells even though Jex molecules were expressed at a
higher level compared with JAGGED1 in the J12 cell line (Fig. 2).
Soluble forms of Jagged1 have been reported to physically interact with
Notch1, 2, and 3 receptors, indicating that Jex molecules
should be capable of binding to endogenous Notch proteins in RKE cells
(24). Furthermore, soluble forms of Jagged1 have been reported to have
biological function (52-55). A soluble form of the extracellular
domain of JAGGED1 is not sufficient to transform RKE cells, even though
a large amount of Jex remains in the media, indicating that
the intracellular domain is required for some signaling event. However,
expression of a membrane-tethered intracellular domain of JAGGED1
(Jtmic) failed to transform cells even though it encodes
the PDZ-ligand. Why then does the intracellular piece not transform
cells? One simple explanation is that the extracellular domain is
required to localize or cluster the intracellular domain in order to
initiate downstream signaling, this model is consistent with other
receptor-mediated signals.
In order to determine if JAGGED1 could interact with PDZ-domain
proteins, GST pulldown assays were performed. More specifically, we
assessed if the PDZ-domain protein AF6 could bind the intracellular domain of JAGGED1, since AF6 was previously reported to interact with
six residues (RMEYIV) found at the C terminus of Jagged1 using a
directed yeast two-hybrid analysis (13). Furthermore, AF6 is the
mammalian homolog of Drosophila canoe which has been genetically linked to the Notch pathway (46). Our data demonstrates that the interaction between JAGGED1 and AF6 occurs in a
PDZ-dependent manner, since deletion of the PDZ-ligand of
JAGGED1 or of the PDZ-domain of AF6 abolished this interaction (Fig. 3,
E and F). Although AF6 may link JAGGED1 to
downstream signaling proteins such as members of the RAS superfamily,
the role of AF6 in JAGGED1-mediated transformation remains to be
elucidated (56).
A Bi-directional Signaling Mechanism through DSL
Proteins--
Jagged2 has been reported to facilitate cell cycle
progression through sustained activation of Notch signaling, which
results in a modest increase in CDK2 kinase activity (57). Although Jagged2-expressing fibroblasts no longer exhibit contact inhibition upon confluency, these cells do not form foci or grow in soft agar. We
previously reported that there are increased levels of CDK2 kinase
activity upon induction of Notch activity (58). However, we are unable
to detect a difference in CDK2 kinase activity between RKE and J1 cell
lines, indicating that JAGGED1 may be signaling independently of Notch
activation (data not shown). Although the functional differences
between Jagged1 and Jagged2 proteins remain unclear, the presence of
the PDZ-ligand likely provides a functional difference between these
DSL proteins (Fig. 6A).
Jagged1 proteins have a highly evolutionarily conserved sequence (RMEYIV) that comprises a PDZ-ligand; however, there is no resemblance of the PDZ-ligand consensus sequence at the C-terminal end of Jagged2
(RYAGKE). Therefore, the DSL protein JAGGED1 has the potential to
mediate a PDZ-dependent signaling mechanism through
downstream PDZ-domain proteins. Furthermore, we have compared the
different classes of DSL proteins and found the consensus sequences for putative PDZ-ligands (-(T/S/Y)XV; X is any amino acid) at
the C termini of Delta1, 2, and 4 proteins (Fig. 6A). In
contrast, the C termini of Delta3 and Jagged2 do not resemble
PDZ-ligands. Therefore, the possibility exists that there are
bi-directional signals in the Notch pathway involving a PDZ mechanism
mediated by both Delta and Jagged proteins.
What are the factors that mediate the specificity in DSL/Notch
signaling? It is proposed that temporal and spatial expression of DSL
proteins provide some level of specificity in Notch signaling during
growth and development. Fringe proteins provide another level of
specificity between the two classes of DSL proteins. Fringe modifies
Notch such that there is a decreased affinity for Jagged proteins and a
higher affinity for Delta proteins (Fig. 6) (25-28). Therefore, upon
expression of Fringe, Delta proteins would still be able to initiate
Notch signaling, while Jagged proteins could not. Here, we have
provided evidence that a PDZ-ligand of JAGGED1 is required to mediate
transformation and changes in gene expression, indicating that DSL
proteins have an intrinsic signaling mechanism that can be activated in
the cells in which they are expressed. We propose a bi-directional
signaling model such that DSL proteins may have two distinct functions:
(1) to initiate Notch signaling in a neighboring cell and (2) to
initiate an intrinsic PDZ-dependent signaling mechanism
(Fig. 6B). For example, Jagged1 could signal in both
directions in the absence of Fringe, whereas the Delta signal would
always be bi-directional. In the case of Jagged2, signaling would only
be in the Notch direction and this signal could be attenuated by
Fringe. In contrast, Delta3 is insensitive to Fringe and would always
allow signaling in the Notch direction. This model provides exquisite
flexibility for signal specificity and accounts for the multiple
distinct DSL proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-converting enzyme (TACE) (15-17). Constitutive presenilin-dependent
-secretase activity is
thought to then mediate an additional proteolytic cleavage that results in the release of the intracellular domain of Notch (Nic)
from the plasma membrane (16, 18, 19). Nic subsequently
translocates to the nucleus and effects gene expression (5, 17, 19,
20). All four Notch receptors have been shown to undergo the
constitutive
-secretase proteolysis, indicating that the specificity
in signaling is not at this level of processing (21). Where the
specificity in DSL-Notch signaling lies is not well understood.
Although it is not known which DSL proteins can activate which Notch
molecules, the DSL proteins Jagged1, Jagged2, and Delta1 have been
reported to bind Notch2 and subsequently induce processing, indicating
that multiple DSL proteins can activate a specific Notch molecule (22).
Furthermore, soluble forms of the extracellular domain of Jagged1 can
physically interact with Notch1, Notch2, and Notch3 in binding assays
(23, 24). Taken together, these data indicate that Jagged1 can bind all
Notch receptors leading to activation of signaling in a similar manner. One potential mechanism that provides specificity in signaling is at
the level of ligand-receptor interactions. Fringe proteins are
O-fucose-
1, 3-N-acetylglucosaminyltransferases
that modify the extracellular domain of Notch, resulting in an increase
in affinity for Delta proteins and a decrease in affinity for Jagged proteins (25-28).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ecotropic retroviral plasmids were kindly provided
by R. C. Mulligan and D. R. Littman, respectively (32, 33). Human
AF6 expression vector was kindly provided by L. Van Aelst
(34). The pcDNA-Nic (Nic) and
pcDNA-MASTERMIND-LIKE 1(MAML1myc) vectors were
described elsewhere (35).
PDZ (deletion of aa 994-1012),
pcDNA-Jex (aa 1-1067), pcDNA-Jtmic
(deletion of aa 24-1067), pcDNA-Jtmic
myc
(Jtmic plus a C-terminal Myc tag),
pcDNA-J1
PL (aa 1-1212), pcDNA-J1myc
(addition of a C-terminal Myc tag) and CMV-Notch
2444 (aa
1-2444). Detailed description of cloning strategies and primer sequences are provided as Supplemental Information.
8000/
98, the cDNA
sequence for Jagged1 was used to search the high throughput genomic
sequence data base (HTGS) on NCBI. A PAC clone was found to encode the
entire genomic locus of Jagged1, including sequences up and downstream.
PAC RPCI5 was obtained from The Sanger Centre. According to the genomic
sequence, a SacI fragment encoded sequences 8-kb upstream of
+1- and 2-kb downstream of the +1. This fragment was cloned into
pBluescript (Stratagene). This clone was then digested using
NotI, which digests the fragment immediately 3' of the 3'
JagPro-xho primer (described below). This fragment was blunted using
Vent polymerase and dNTPs at 72 °C for 20 min. This product was then
digested using SacI and cloned into pGL3basic SacI/SmaI. A 3.4-kb fragment of the
Jagged1 promoter was isolated by PCR from HeLa genomic DNA
using primers 5' JagPro3.4 and 3' JagPro. Sequences of the
primers are 5'-ATTCACTGGTGGACTGGAGG-3' and 5'-AGGGAAGGAGGTAGGTCAGC-3',
respectively. The PCR product was then cloned into the TA TOPO 2.1 vector (Invitrogen) following the manufacturer's protocol.
pJ1pro
629/
98 was then obtained from this clone using
PCR primers 5' JagPro0.7sacI and 3' JagPro-xho. The sequences of these
primers are 5'-GCGCGAGCTCCAACGATCCCTTCCAAGTA-3' and
5'-GCGCCTCGAGCGTCCCGGCTCTAATATAC-3', respectively. The PCR product was
digested with restriction enzymes SacI and XhoI
(NEBL) and cloned into the luciferase vector pGL3-basic (Promega).
Promoter constructs pJ1pro
629/
321 and
pJ1pro
321/
98 were isolated as above using the PCR
primers 5' Jagpro0.7sacI and 3' JagPro-UTR
(5'-GCGCCTCGAGAAAAACCAGCCTAGCTCG-3') or 5' JagProUTR (5'-GCGCGAGCTCCGAGCTAGGCTGGTTTTT-3) and 3' JagPro- xho, respectively.
PL, aa 1094-1213).
pGEX-Ras was kindly provided by L. Quilliam (36).
PL were seeded with 1 × 106 of
parental RKE on 100-mm diameter plates. Cultures were maintained for 3 weeks, and DMEMc was replenished every 3-4 days. To enhance visualization of foci, cells were fixed in methanol and stained in 70%
isopropyl alcohol containing 0.5% methylene blue.
Eco plasmids using Lipofectamine (12 µg).
Infections were performed with MFG-LacZ viral supernatant containing 8 µg/ml hexadimethrine bromide (polybrene, Sigma). Cells were
washed three times with 1× phosphate-buffered saline prior to seeding
non-infected parental RKE cells. Cells were fixed in 0.05%
glutaraldehyde (Sigma) and stained with X-gal according to standard
protocols to detect
-galactosidase activity in MFG-LacZ-infected cells.
-32P]dCTP. In order to
perform semiquantitative PCR, cycling parameters were determined by
amplifications of serial dilutions of cDNAs so that there was a
linear range of amplification for each set of primers. Amplified PCR
products were separated in Tris acetate/EDTA (TAE)-4% polyacrylamide
gels and exposed to x-ray film.
-actin:
5'-ggccaggatagagccaccaatccac-3' and
5'-cgatatcgctgcgctcgtcgtcgac-3'; Notch1:
5'-gcggcacgcctggccgtggaaggca-3' and 5'-cggcatgctcgtgggcgggctagag-3';
Notch2: 5'-tcgaggaggcagctcagacctgag-3' and
5'-catctgcaccagcatccaggaggcg-3'; Notch3: 5'-acactgggagttctctgt-3' and
5'-gtctgctggcatgggata-3'; Jagged1: 5'-gcactgtgagaacaacataaatgac-3' and
5'-gcacaattgtcctggtaataagagt-3'; Jagged2: 5'-caacaccaatgactgcaacc-3' and 5'-cctctcacgttctttcctgc-3'; Delta1:
5'-cccgatggaggctacacctgccatt-3' and 5'tgctcttctcttctcctacagagcc-3';
Radical Fringe: 5'-cagacgttcattttcaccga-3' and
5'-cgtgtagggtcctgtcgaat-3'.
PDZmyc in Nonidet P-40 lysis buffer. Lysates were
precleared for 1 h with GST bound-glutathione agarose beads
(Sigma). Lysates were split into three equal aliquots and incubated for
2 h with relevant GST fusion proteins as indicated in the text.
Proteins associated with GST fusions were detected using appropriate
antibodies by Western blot analysis. AF6 proteins were detected by 9E10 antibody.
-gal (200 ng; transfection efficiency
control plasmid encoding
-galactosidase), CMV-NOTCH1
2444 (800 ng), and pcDNA-MAML1myc
(400 ng). 293T cells were transfected with the following CMV-driven expression plasmids (5 µg): pcDNA3.1A (pcDNA), Epidermal Growth Factor Receptor (EGFR), JAGGED1 (J1), JAGGED1
PL
(J1
PL), and DELTA1 (Dl1) using Lipofectamine (10 µg).
The following day, 293T cells were trypsinized, resuspended in 4 ml of
DMEMc, and cocultured with HeLa cells (1 ml/well) for 12 h. Three
separate experiments were performed in triplicate.
-Gal (0.5 ng), and the following expression vectors
(1 µg): pBP283 (pBabe), pcDNA, J1, J1
PL,
J1myc, or Nic. Three separate experiments were
performed in duplicate.
-galactosidase activities were
measured according to standard protocols. Luciferase values were
corrected for transfection efficiency based on the
-galactosidase activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
[in a new window]
Fig. 1.
Ectopic expression of JAGGED1 elicits
cellular transformation of RKE cells. A, saturation
plates of JAGGED1-expressing (J1 and J12) and parental RKE cells.
Culture plates were maintained for 3 weeks and stained with methylene
blue to enhance visualization of cell density. B,
transformation by JAGGED1 is dose-dependent. Focus
formation by 1 × 104 clonal JAGGED1-expressing RKE cells (J1,
J3, J12, J4, and J7) cocultured with 1 × 106 parental RKE
cells (Ratio = 1:100). C, JAGGED1 protein expression in
crude membrane fractions of clonal RKE cell lines was detected with
anti-Jagged1/TS1 antibody. Expression of a
Na+/K+ ATPase is shown as a loading control.
Molecular mass markers in kilodaltons (kDa) are indicated to
the left. D, representative focus formed by
MFG-LacZ infected JAGGED1-expressing RKE cells. Cells were stained with
X-gal to distinguish blue JAGGED1-expressing RKE cells from unstained
RKE cells.
-galactosidase activity prior to
coculturing with parental RKE cells. Following focus formation, cells
expressing
-galactosidase were visualized by staining with X-gal.
The presence of blue foci confirmed that JAGGED1-expressing cells were
the transformed cells (Fig. 1D). Furthermore, unstained RKE
cells were excluded from the foci and located only in the surrounding
monolayer, indicating that only the JAGGED1-expressing cells were transformed.
myc), which contains both
epitopes. Western blot analysis of equal amounts of protein from
Jtmic
myc cell lysates showed that the signal for
Jtmic
myc was slightly more intense with the anti-Jagged1
Ab in comparison to the anti-Myc Ab indicating that these antibodies
have similar avidities and can be used for comparing expression levels
(Fig. 2D, bottom panel). Therefore, the
expression level of Jex is much greater than JAGGED1
expression in J12 cells (Fig. 2D, top panel). In
addition, high levels of soluble Jex protein were found to
be stable in the media for at least 3 days (data not shown). These
results indicate that transformation by JAGGED1 requires an intact
protein, suggesting that both the extracellular and intracellular
domains might have important functions.
View larger version (41K):
[in a new window]
Fig. 2.
Deletion of either the extracellular or
intracellular domain of JAGGED1 results in the loss of JAGGED1-mediated
transformation. A, schematic diagram of JAGGED1:
Delta-Serrate-Lag2 (DSL), EGF-like repeats (EGF),
cysteine-rich (CR), and transmembrane (TM)
domains are indicated. Jex: a soluble form of the
extracellular domain. Jtmic: a membrane-tethered
intracellular domain. A Myc epitope (m) was fused to the
C-terminal ends of the Jex and Jtmic myc
deletion mutants. B, focus formation by Jex,
Jtmic, and JAGGED1-expressing (J12) clonal cells (1 × 104) was tested in a coculture focus assay with 1 × 106 parental RKE cells (Ratio = 1:100). C,
Western blot analysis of Jex, Jtmic, and
JAGGED1-expressing (J12) clonal cell lines. Proteins were detected in
the appropriate subcellular fractions from the following samples: media
(0.05% v/v), membrane (mem, 20 µg of protein) and
cytoplasmic (cyto, 20 µg of protein) fractions.
D, comparison of Jex, Jtmic
myc,
and JAGGED1 protein expression. Whole cell lysates (25 µg of protein)
were analyzed for the appropriate proteins. Anti-Jagged1/TS1 antibody
was used to detect JAGGED1 in J12 cell lysates (top right)
and anti-Myc/9E10 antibody was used to detect Jex
(top left). To compare the avidities of anti-Jagged1 and
anti-Myc antibodies for their epitopes, lysate from clonal cell line
expressing a Myc-tagged Jtmic
myc, which contains both
epitopes, was immunoblotted with both antibodies (bottom
panel). Molecular mass markers are indicated to the
left (kDa).
PL). Deletion of the
putative PDZ-ligand resulted in loss of transformation activity,
indicated by the inability of J1
PL-expressing cells (cl.
4 and cl. 2) to form foci compared with the transformed J1 and J12 cell
lines, which displayed a matching level of expression, respectively
(Fig. 3, B and C). These results indicate
that JAGGED1 may mediate an intrinsic signaling mechanism involving
downstream PDZ proteins.
View larger version (20K):
[in a new window]
Fig. 3.
Deletion of a putative PDZ-ligand results in
the loss of Jagged1 transformation. A, a putative
PDZ-ligand is located at the C-terminal end of JAGGED1, encoding
aa1213-1218. The deletion mutant, J1 PL, lacks these
last six residues. B, focus formation by J1
PL
(cl. 2 and 4) and JAGGED1-expressing (J1 and J12) clonal cells (1 × 104) was tested in a coculture focus assay with 1 × 106 parental RKE cells (Ratio = 1:100).
C, Western blot analysis of crude membrane fractions (25 µg of protein) from J1
PL (cl. 2 and 4), J1, and J12
clonal cell lines. D, ligand-induced Notch signaling was
assayed by coculture luciferase reporter assay. HeLa cells
cotransfected with the Notch1
2444 expres- sion vector and the CSL-luciferase reporter were stimulated upon
coculture with 293T cells expressing JAGGED1 (J1),
J1
PL, and DELTA1 (DL1) or the control vectors
pcDNA and EGFR. Relative luciferase values were corrected for
transfection efficiency of
-galactosidase. The y-axis
shows fold activation expressed as the ratio of luciferase activity
measured for each construct and the luciferase activity measured for
pcDNA. Three individual experiments were performed in triplicate.
E, the intracellular domain of JAGGED1 interacts with
PDZ-domain protein AF6. Lysates were made from transiently transfected
293T cells with AF6 or AF6
PDZ and incubated with the
following beads: GST, GST-Jic (Jic),
GST-Jic
PL (
PL), GST-RAS (RAS). AF6
proteins were detected by Western blotting against the Myc epitope with
9E10.
2444 and
MAML1myc. NOTCH1
2444 that lacks the PEST
domain (aa 2445-2555) was used in these assays to generate a more
stable form of Notch. We previously reported that co-expression of
MAML1 enhances Nic-induced transactivation of a CSL
reporter gene; therefore, MAML1 was added to this assay to increase
detection of ligand-induced Notch activity (35). 293T cells were
transiently transfected with pcDNA vector control, EGFR, JAGGED1
(J1), J1
PL, or DELTA1 (Dl1) and subsequently cocultured
with transfected HeLa cells for 12 h. The J1, Dl1, and
J1
PL proteins were able to induce Notch transactivation
by 2.0-fold over pcDNA, indicating that J1
PL
retained the ability to activate Notch signaling. As a control, we
demonstrated that 293T cells expressing EGFR do not activate Notch
signaling (Fig. 3D). Therefore, deletion of the six
C-terminal residues comprising the PDZ-ligand does not affect the
ability of JAGGED1 to initiate Notch signaling across cell boundaries. However, the PDZ-ligand is essential for transformation, indicating that there is a potential for JAGGED1 to initiate Notch signaling in a
neighboring cell and to mediate a PDZ-dependent signaling mechanism within the JAGGED1-expressing cell.
PL, respectively). The PDZ-domain protein AF6 was
previously reported to interact with the amino acids RMEYIV at the C
terminus of Jagged1 in a directed yeast two-hybrid analysis (13).
Moreover, AF6 is the mammalian homolog of Drosophila canoe,
which has been genetically linked to the Notch pathway (46). Therefore,
we tested the ability of the GST-JAGGED1 fusion proteins to bind AF6.
While GST-Jic bound to AF6, GST-Jic
PL did
not efficiently pulldown AF6, indicating that the interaction requires
the PDZ-ligand (Fig. 3E). To determine if the PDZ-domain of
AF6 was necessary for this interaction, we generated a mutant AF6
lacking this domain (AF6
PDZ). Although
GST-Jic did not pulldown AF6
PDZ, this mutant
retained its ability to bind GST-Ras to a similar extent as AF6,
indicating that the structural integrity of AF6 was not compromised in
general (Fig. 3F). Therefore, the interaction of JAGGED1 and
AF6 occurs in a PDZ-dependent manner, indicating that
JAGGED1 has the potential to signal through interacting PDZ-domain proteins.
-actin mRNA was amplified to serve as a
normalization control for these samples. To show that induction of
Notch3 and Jagged1 gene expression was not due to
clonal variation, we analyzed mRNA isolated from several JAGGED1-expressing RKE cell lines (J1, J8, J3, and J4) (Fig.
4B). The mRNA levels of Notch3 and Jagged1 were greater
in J1, J8, and J3 clonal lines, which expressed the highest levels of
JAGGED1. In contrast, there was only a minor induction of
Notch3 and Jagged1 gene expression in the J4
clonal cell line, which expressed the lowest level of JAGGED1 (Figs.
4B and 1C, respectively). Although Notch3 and Jagged1 gene expression is
up-regulated in Nic-expressing cells, there is no evidence
indicating that these genes are direct targets of Notch signaling. Our
data indicate that JAGGED1 may be initiating a signal to downstream
effectors that cause changes in gene expression.
View larger version (19K):
[in a new window]
Fig. 4.
Changes in gene expression in
Jagged1-expressing cell lines. A, RT-PCR analysis of
total RNA extracted from parental RKE (RKE), JAGGED1 (J1), NOTCH1 (N),
and Nic-expressing (Nic) clonal cells. The
level of -actin mRNA is shown for normalization. B,
induction of Notch3 and Jagged1 expression in
different JAGGED1-expressing clonal lines (J1, J8, J3, and J4) compared
with RKE cells. C, comparison of mRNA levels in RKE, J1,
and J1
PL cl. 4 cell lines by RT-PCR. Only J1 cells have
increased levels of Delta1 and Jagged1 mRNA, while Notch3 is
up-regulated in both J1 and J1
PL cl. 4 cell lines.
PL cl. 4 (Fig. 4C). mRNA levels for
both Jagged1 and Delta1 were up-regulated only in the J1 cells and not
in J1
PL cells. However, the level of Notch3
gene induction was similar in both J1 and J1
PL cl. 4 cell lines, indicating that the PDZ-ligand is required for induction of
Jagged1 and Delta1, but not required for
induction of Notch3. RT-PCR analysis of Radical Fringe is
shown for normalization. These data demonstrate the importance of the
PDZ-ligand of JAGGED1 for mediating induction of certain JAGGED1 target
genes. Moreover, this demonstrates that induction of gene
expression by JAGGED1 may involve distinct signaling pathways.
8000/
98) and a 531-bp genomic DNA fragment
(pJ1pro
629/
98) in cells expressing JAGGED1 compared
with the vector control, indicating that the JAGGED1 responsive element
was contained between the sequences
629 to
98-bp upstream of the
start codon (Fig. 5A). In
contrast, there was a lower activation by JAGGED1 of the reporter construct pJ1pro
629/
321 that lacks the
sequences between
321 and
98 bp (Fig. 5A). Moreover, JAGGED1 induced the reporter activity by 7-fold using a construct containing the sequences
321 to
98 (pJ1pro
321/
98),
indicating that these sequences contain the JAGGED1 responsive element.
To further demonstrate that the PDZ-ligand is required for
Jagged1 gene expression, we compared the abilities of
JAGGED1 (J1), JAGGED1
PL (J1
PL), and a
C-terminal Myc-tagged JAGGED1 (J1myc) to activate the
Jagged1 promoter reporter constructs,
pJ1pro
629/
98 and pJ1pro
321/
98. J1
mediated a 2-4-fold activation of these reporters, whereas J1
PL and J1myc did not activate the
Jagged1 promoter (Fig. 5B). Since the PDZ-ligand must be at the C terminus, the addition of a C-terminal Myc tag potentially blocks binding of PDZ-domain proteins to Jagged1, indicating that the PDZ-ligand serves to mediate a downstream signaling
pathway through PDZ-domain proteins. These data indicate that there is
a vital role for the PDZ-ligand of JAGGED1 to mediate changes in
Jagged1 gene expression. Since Nic-expressing
cells have an increased level of Jagged1 mRNA, we assayed the
ability of Nic to activate the Jagged1 promoter
reporter. However, Nic did not activate the
Jagged1 promoters pJ1pro
629/
98 and
pJ1pro
321/
98 or reporter constructs containing DNA
fragments up to 8-kb upstream of the start codon within the
Jagged1 promoter (pJ1pro
8000/
98), indicating
that the Jagged1 gene is not likely to be a direct target of
Notch signaling (Figs. 4A and 5C and data not
shown). Interestingly, there is no induction of Jagged1 mRNA upon a
12-hour hormone induction of RKE cells expressing
Notchic-ER.2
Therefore, the Jagged1 promoter is responsive to a
PDZ-dependent signaling mediated by JAGGED1 and not to
activated Notch proteins, indicating that JAGGED1 has an
intrinsic signaling mechanism that likely is Notch-independent.
View larger version (18K):
[in a new window]
Fig. 5.
An intact PDZ-ligand is required for
activation of the Jagged1 promoter. A,
activation of the Jagged1 promoter constructs by
JAGGED1. Genomic DNA fragments of the Jagged1 promoter
were cloned into the pGL-3b luciferase vector:
pJ1pro 8000/
98, pJ1pro
629/
98,
pJ1pro
629/
321, and pJ1pro
321/
98.
Cotransfection of HeLa cells with producer DNA (pBABE or JAGGED1
(J1)) and Jagged1 promoter constructs. pGL-3p
luciferase vector served as a negative control. B and
C, deletion of the PDZ-ligand results in loss of
transactivation of the Jagged1 promoter by JAGGED1. Hela
cells were transiently transfected with the
pJ1pro
629/
98 (B) and
pJ1pro
321/
98 (C) Luciferase reporter
constructs and the following expression plasmids: pcDNA vector
control, J1, J1
PL, J1myc, and
Nic.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PL-expressing cell
lines compared with expression matched JAGGED1-expressing cell lines
(Fig. 3B). However, both J1 and J1
PL proteins
are capable of activating Notch signaling to a similar extent,
indicating that deletion of the PDZ-ligand does not interfere with
Notch activation across cell boundaries (Fig. 3D). RT-PCR analysis of gene expression in J1 and J1
PL cell lines
demonstrated that the PDZ-ligand is required for induction of
Jagged1 and Delta1, since only the J1 cell line
and not the J1
PL cell line had increased levels of
Jagged1 and Delta1 mRNA (Fig. 4C). Furthermore,
J1
PL did not activate the Jagged1 promoter
construct, indicating that the PDZ-ligand is required for downstream
signaling events that lead to changes in gene expression (Fig. 5,
B and C). Therefore, we provide evidence for the
existence of a novel signaling mechanism intrinsic to JAGGED1 that
involves a PDZ-dependent pathway.
View larger version (15K):
[in a new window]
Fig. 6.
A bi-directional signaling mechanism through
DSL proteins. A, schematic diagram of a DSL protein.
The six C-terminal amino acids are listed for DSL proteins
Jagged1/2 and Delta 1/2/3/4. There are consensus sequences for putative
PDZ-ligands at the C terminus of Jagged1 and Delta1/2/4. Fringe
proteins are able to inhibit only Jagged proteins, and not Delta
proteins. B, a bi-directional model of NOTCH/DSL signaling.
JAGGED1 initiates Notch signaling in a neighboring cell upon
cell-to-cell contact. Fringe proteins are shown here to inhibit the
JAGGED1-NOTCH1 interaction, and thus blocking Notch activation. JAGGED1
proteins also have the potential to mediate a PDZ-dependent
signaling mechanism within the JAGGED1-expressing cell itself.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the members of the Capobianco laboratory for support and technical assistance. We also thank David Robbins for insightful comments on our work. We are grateful to S. Artavanis-Tsakonas, D. R. Littman, R. C. Mulligan, L. Quilliam, and L. Van Aelst for kindly providing reagents used in this study.
![]() |
FOOTNOTES |
---|
* This work was funded in part by Grant RPG LBC-99465 from the American Cancer Society (to A. J. C.) and Grant ROI CA 83736 from the National Cancer Institute (to A. J. C.) and the NCI National Institutes of Health Training Grant 5T32 CA59268 (to J. M. A.). A. J. C. is a scholar of the Leukemia and Lymphoma Society (Award 1298-02).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.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary data.
To whom correspondence should be addressed: 231 Albert Sabin Way,
Dept. of Molecular Genetics, University of Cincinnati, College of
Medicine, Cincinnati, OH 45267-0524. Tel.: 513-558-3664; Fax: 513-558-8474; E-mail: tony.capobianco@uc.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M211427200
2 C. Ronchini and A. J. Capobianco, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DSL, Delta/Serrate/Lag-2;
EGF, epidermal growth factor;
CMV, cytomegalovirus;
aa, amino acids;
GST, glutathione
S-transferase;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
PDZ, PSD-95/Dlg/Zo-1.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Artavanis-Tsakonas, S.,
Rand, M. D.,
and Lake, R. J.
(1999)
Science
284,
770-776 |
2. | Miele, L., and Osborne, B. (1999) J. Cell. Physiol. 181, 393-409[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Milner, L. A.,
and Bigas, A.
(1999)
Blood
93,
2431-2448 |
4. | Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R., and Israel, A. (1995) Nature 377, 355-358[CrossRef][Medline] [Order article via Infotrieve] |
5. | Struhl, G., and Adachi, A. (1998) Cell 93, 649-660[Medline] [Order article via Infotrieve] |
6. | Fleming, R. J. (1998) Semin. Cell Dev. Biol. 9, 599-607[CrossRef][Medline] [Order article via Infotrieve] |
7. | Lissemore, J. L., and Starmer, W. T. (1999) Mol. Phylogenet. Evol. 11, 308-319[CrossRef][Medline] [Order article via Infotrieve] |
8. | Nye, J. S., and Kopan, R. (1995) Curr. Biol. 5, 966-969[Medline] [Order article via Infotrieve] |
9. | Lindsell, C. E., Shawber, C. J., Boulter, J., and Weinmaster, G. (1995) Cell 80, 909-917[Medline] [Order article via Infotrieve] |
10. |
Sun, X.,
and Artavanis-Tsakonas, S.
(1997)
Development
124,
3439-3448 |
11. |
Sun, X.,
and Artavanis-Tsakonas, S.
(1996)
Development
122,
2465-2474 |
12. |
Hukriede, N. A.,
Gu, Y.,
and Fleming, R. J.
(1997)
Development
124,
3427-3437 |
13. |
Hock, B.,
Bohme, B.,
Karn, T.,
Yamamoto, T.,
Kaibuchi, K.,
Holtrich, U.,
Holland, S.,
Pawson, T.,
Rubsamen-Waigmann, H.,
and Strebhardt, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9779-9784 |
14. | Schmucker, D., and Zipursky, S. L. (2001) Cell 105, 701-704[CrossRef][Medline] [Order article via Infotrieve] |
15. | 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, 207-216[Medline] [Order article via Infotrieve] |
16. | 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, 197-206[Medline] [Order article via Infotrieve] |
17. | Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998) Nature 393, 382-386[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Struhl, G.,
and Greenwald, I.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
229-234 |
19. |
Kopan, R.,
Schroeter, E. H.,
Weintraub, H.,
and Nye, J. S.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1683-1688 |
20. |
Kato, H.,
Taniguchi, Y.,
Kurooka, H.,
Minoguchi, S.,
Sakai, T.,
Nomura-Okazaki, S.,
Tamura, K.,
and Honjo, T.
(1997)
Development
124,
4133-4141 |
21. |
Saxena, M. T.,
Schroeter, E. H.,
Mumm, J. S.,
and Kopan, R.
(2001)
J. Biol. Chem.
276,
40268-40273 |
22. |
Shimizu, K.,
Chiba, S.,
Hosoya, N.,
Kumano, K.,
Saito, T.,
Kurokawa, M.,
Kanda, Y.,
Hamada, Y.,
and Hirai, H.
(2000)
Mol. Cell. Biol.
20,
6913-6922 |
23. |
Shimizu, K.,
Chiba, S.,
Kumano, K.,
Hosoya, N.,
Takahashi, T.,
Kanda, Y.,
Hamada, Y.,
Yazaki, Y.,
and Hirai, H.
(1999)
J. Biol. Chem.
274,
32961-32969 |
24. | Shimizu, K., Chiba, S., Saito, T., Kumano, K., and Hirai, H. (2000) Biochem. Biophys. Res. Commun. 276, 385-389[CrossRef][Medline] [Order article via Infotrieve] |
25. | Hicks, C., Johnston, S. H., diSibio, G., Collazo, A., Vogt, T. F., and Weinmaster, G. (2000) Nat. Cell Biol. 2, 515-520[CrossRef][Medline] [Order article via Infotrieve] |
26. | Bruckner, K., Perez, L., Clausen, H., and Cohen, S. (2000) Nature 406, 411-415[CrossRef][Medline] [Order article via Infotrieve] |
27. | Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S., and Vogt, T. F. (2000) Nature 406, 369-375[CrossRef][Medline] [Order article via Infotrieve] |
28. | Panin, V. M., Papayannopoulos, V., Wilson, R., and Irvine, K. D. (1997) Nature 387, 908-912[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Gray, G. E.,
Mann, R. S.,
Mitsiadis, E.,
Henrique, D.,
Carcangiu, M. L.,
Banks, A.,
Leiman, J.,
Ward, D.,
Ish-Horowitz, D.,
and Artavanis-Tsakonas, S.
(1999)
Am. J. Pathol.
154,
785-794 |
30. | Zagouras, P., Stifani, S., Blaumueller, C. M., Carcangiu, M. L., and Artavanis-Tsakonas, S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6414-6418[Abstract] |
31. | Capobianco, A. J., Zagouras, P., Blaumueller, C. M., Artavanis-Tsakonas, S., and Bishop, J. M. (1997) Mol. Cell. Biol. 17, 6265-6273[Abstract] |
32. | Tani, K., Lin, T., Hibino, H., Takahashi, K., Nakazaki, Y., Takahashi, S., Nagayama, H., Ozawa, K., Saitoh, I., Mulligan, R., et al.. (1995) Leukemia 9 Suppl. 1, S64-65[Medline] [Order article via Infotrieve] |
33. | Landau, N. R., and Littman, D. R. (1992) J. Virol. 66, 5110-5113[Abstract] |
34. |
Boettner, B.,
Govek, E. E.,
Cross, J.,
and Van Aelst, L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9064-9069 |
35. |
Jeffries, S.,
Robbins, D. J.,
and Capobianco, A. J.
(2002)
Mol. Cell. Biol.
22,
3927-3941 |
36. |
Rebhun, J. F.,
Chen, H.,
and Quilliam, L. A.
(2000)
J. Biol. Chem.
275,
13406-13410 |
37. | Ruppert, J. M., Vogelstein, B., and Kinzler, K. W. (1991) Mol. Cell. Biol. 11, 1724-1728[Medline] [Order article via Infotrieve] |
38. | Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Medline] [Order article via Infotrieve] |
39. |
Fuentes-Panana, E. M.,
and Ling, P. D.
(1998)
J. Virol.
72,
693-700 |
40. | Rohn, J. L., Lauring, A. S., Linenberger, M. L., and Overbaugh, J. (1996) J. Virol. 70, 8071-8080[Abstract] |
41. | Soriano, J. V., Uyttendaele, H., Kitajewski, J., and Montesano, R. (2000) Int. J. Cancer 86, 652-659[CrossRef][Medline] [Order article via Infotrieve] |
42. | Pear, W. S., Aster, J. C., Scott, M. L., Hasserjian, R. P., Soffer, B., Sklar, J., and Baltimore, D. (1996) J. Exp. Med. 183, 2283-2291[Abstract] |
43. | Gallahan, D., Kozak, C., and Callahan, R. (1987) J. Virol. 61, 218-220[Medline] [Order article via Infotrieve] |
44. |
Bellavia, D.,
Campese, A. F.,
Alesse, E.,
Vacca, A.,
Felli, M. P.,
Balestri, A.,
Stoppacciaro, A.,
Tiveron, C.,
Tatangelo, L.,
Giovarelli, M.,
Gaetano, C.,
Ruco, L.,
Hoffman, E. S.,
Hayday, A. C.,
Lendahl, U.,
Frati, L.,
Gulino, A.,
and Screpanti, I.
(2000)
EMBO J.
19,
3337-3348 |
45. |
Wu, G.,
Lyapina, S.,
Das, I.,
Li, J.,
Gurney, M.,
Pauley, A.,
Chui, I.,
Deshaies, R. J.,
and Kitajewski, J.
(2001)
Mol. Cell. Biol.
21,
7403-7415 |
46. | Miyamoto, H., Nihonmatsu, I., Kondo, S., Ueda, R., Togashi, S., Hirata, K., Ikegami, Y., and Yamamoto, D. (1995) Genes Dev. 9, 612-625[Abstract] |
47. | Gutgemann, A., Golob, M., Muller, S., Buettner, R., and Bosserhoff, A. K. (2001) Arch. Dermatol. Res. 293, 283-290[CrossRef][Medline] [Order article via Infotrieve] |
48. | Kettunen, E., Nissen, A. M., Ollikainen, T., Taavitsainen, M., Tapper, J., Mattson, K., Linnainmaa, K., Knuutila, S., and El-Rifai, W. (2001) Int. J. Cancer 91, 492-496[CrossRef][Medline] [Order article via Infotrieve] |
49. | Leethanakul, C., Patel, V., Gillespie, J., Pallente, M., Ensley, J. F., Koontongkaew, S., Liotta, L. A., Emmert-Buck, M., and Gutkind, J. S. (2000) Oncogene 19, 3220-3224[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Fleming, R. J.,
Gu, Y.,
and Hukriede, N. A.
(1997)
Development
124,
2973-2981 |
51. | Hunter, T. (2000) Cell 100, 113-127[Medline] [Order article via Infotrieve] |
52. |
Karanu, F. N.,
Murdoch, B.,
Gallacher, L.,
Wu, D. M.,
Koremoto, M.,
Sakano, S.,
and Bhatia, M.
(2000)
J. Exp. Med.
192,
1365-1372 |
53. | Wong, M. K., Prudovsky, I., Vary, C., Booth, C., Liaw, L., Mousa, S., Small, D., and Maciag, T. (2000) Biochem. Biophys. Res. Commun. 268, 853-859[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Small, D.,
Kovalenko, D.,
Kacer, D.,
Liaw, L.,
Landriscina, M.,
Di Serio, C.,
Prudovsky, I.,
and Maciag, T.
(2001)
J. Biol. Chem.
276,
32022-32030 |
55. |
Varnum-Finney, B.,
Purton, L. E., Yu, M.,
Brashem-Stein, C.,
Flowers, D.,
Staats, S.,
Moore, K. A.,
Le Roux, I.,
Mann, R.,
Gray, G.,
Artavanis-Tsakonas, S.,
and Bernstein, I. D.
(1998)
Blood
91,
4084-4091 |
56. |
Quilliam, L. A.,
Castro, A. F.,
Rogers-Graham, K. S.,
Martin, C. B.,
Der, C. J.,
and Bi, C.
(1999)
J. Biol. Chem.
274,
23850-23857 |
57. | Cereseto, A., and Tsai, S. (2000) J. Cell. Physiol. 185, 425-431[CrossRef][Medline] [Order article via Infotrieve] |
58. |
Ronchini, C.,
and Capobianco, A. J.
(2001)
Mol. Cell. Biol.
21,
5925-5934 |
59. |
Jeffries, S.,
and Capobianco, A. J.
(2000)
Mol. Cell. Biol.
20,
3928-3941 |