(Received for publication, December 30, 1996, and in revised form, January 23, 1997)
From the Departments of Pathology and ¶ Medicine, Truncated forms of the NOTCH1 transmembrane
receptor engineered to resemble mutant forms of NOTCH1 found in certain
cases of human T cell leukemia/lymphoma (T-ALL) efficiently induce
T-ALL when expressed in the bone marrow of mice. Unlike full-sized
NOTCH1, two such truncated forms of the protein either lacking a major portion of the extracellular domain ( NOTCH1 (also referred to as TAN-1), a
homolog of Drosophila NOTCH, was first identified at the
breakpoint of a recurrent (7;9)(q34;q34.3) chromosomal translocation
associated with a subset of human T cell acute lymphoblastic
leukemia/lymphoma (T-ALL)1 (1-3). RNA
transcribed from the normal NOTCH1 gene is found in most
cells but is present at highest levels in developing thymus and brain
(2, 4). The product of this gene is a 2555 amino acid type I
transmembrane receptor protein of about 350 kDa (p350) containing a
series of structural motifs originally described in other polypeptides
(Fig. 1), including 36 epidermal growth factor-like
repeats and three lin-12-like repeats in the extracellular domain and
six ankyrin-like repeats in the intracellular domain. All chromosomal
breakpoints in human T-ALLs occur within a single intron dividing the
coding sequence for epidermal growth factor repeat 34 and result in
overexpression of novel mRNAs containing sequences from the 3
The oncogenicity of truncated NOTCH1 has been confirmed by inserting
cDNAs encoding portions of the NOTCH1 gene into murine bone marrow progenitor cells which were then transplanted into syngeneic recipients (6). In these experiments, NOTCH1 missing the
entire extracellular domain except for the leader peptide and 61 amino
acids immediately external to the transmembrane region ( Removal of extracellular amino acid sequences from NOTCH1 and
NOTCH-related proteins has effects on differentiation similar to those
observed when cells expressing full-length versions of these proteins
are exposed to ligand (7-11), findings consistent with the
interpretation that truncation leads to constitutive activation of the
intracellular domain. Signaling by NOTCH1 appears to be mediated, at
least in part, by the transcription factor RBP-J When overexpressed, fragments of NOTCH1 representing the intracellular
domain can associate with RBP-J Production, purification, and characterization
of polyclonal rabbit antibodies against two portions of the
intracellular domain of NOTCH1 (25), T3 (amino acids 1763-1877) and TC
(amino acids 2277-2470), and an amino-terminal portion of RBP-J Cell culture reagents were obtained from Life
Technologies. All cell lines were cultured at 37 °C under 5%
CO2. SUP-T1, a human T lymphoblastic cell line containing
two copies of a balanced (7;9)(q34;q34.3) chromosomal translocation and
no normal chromosome 9 (27) was grown in RPMI 1640 supplemented with
10% fetal calf serum. T6E and I22 cells, derived from murine tumors
induced by cDNAs encoding The cDNA constructs
NOTCH1 (codons 1-2555), Expression and
purification of GST-NOTCH1 ankyrin repeat (5) (codons 1872-2123) and
GST-RBP-J NOTCH1 cDNAs in pSP72 (Promega)
and RBP-J Two-hybrid analysis was performed
as described by Fields and Song (31). A 1.1-kilobase NcoI
fragment encoding the NOTCH1 ankyrin repeats and immediate flanking
sequences (codons 1858-2206) was subcloned into the NcoI
site of the plasmid pAS2, while a NcoI-BamHI
fragment containing the complete cDNA for RBP-J For retroviral transduction,
NOTCH1 cDNAs were subcloned into pBABEpuro (32) and packaged by
transient transfection of Bosc23 cells (6). After addition of Polybrene
(4 µg/ml), the resultant supernatants containing retrovirus were used
to infect NIH 3T3 cells. For transient expression, cDNAs were
subcloned into pcDNA3 (Invitrogen) and transfected into cultured
cells by calcium phosphate precipitation (28).
Various
NOTCH1 cDNAs cloned into pcDNA3 were used in co-transfections
of 293 cells along with pSG5-RBP-J Unless otherwise noted, all incubations
described below were performed at 4 °C with mixing. To prepare
immunoprecipitates with polyclonal rabbit antibodies, cells were washed
twice with ice-cold Hank's buffered saline and then lysed in ice-cold
25 mM Hepes, pH 7.5, containing 1% Nonidet P-40, 70 mM KCl, 20 mM sodium fluoride, 2 mM
sodium orthovanadate, 2 mM sodium molybdate, 0.2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (lysis buffer). After 30 min on ice, insoluble material was removed by centrifugation at 4 °C
and 14,000 × g for 15 min. Supernatants were divided
into two aliquots which were incubated with 3 µl of rabbit serum or
10 µl of affinity purified rabbit antibody for 60 min on ice. Ten
µl of protein A-Sepharose beads (Pharmacia) were then added and the
incubation allowed to proceed for an additional 60 min at 4 °C with
mixing. To prepare immunoprecipitates with anti-myc
monoclonal antibody 9E10, 2 µl of ascites was pre-bound to 10 µl of
protein A-Sepharose in 200 µl of lysis buffer by mixing for 2 h
at 4 °C. Cell lysates were precleared by incubation with 10 µl of
protein A-Sepharose for 30 min and then allowed to mix with 9E10
antibody bound to beads for 2 h. Beads were washed four times with
lysis buffer and bound polypeptides eluted by boiling in SDS-PAGE
buffer. Immunoprecipitated polypeptides were then separated by SDS-PAGE
and analyzed by Western blotting.
Cells grown on slides were washed and
fixed with 3% paraformaldehyde in phosphate-buffered saline for 15 min
at room temperature. For immunoperoxidase staining of NIH 3T3 cells,
cells were permeabilized with To assess the interaction of RBP-J Human T cells contain two major RBP-J
In converse experiments, RBP-J ICN
precipitated with RBP-J
As expected, ICNW, a polypeptide consisting of the entire intracellular
domain of NOTCH1 and differing from ICN by only nine amino acids at the
amino terminus (Fig. 3A), bound RBP-J
Yeast two-hybrid analysis of interaction between the NOTCHI ankyrin
repeats (ARI) and RBP-J +++, ~90% of colonies intensely blue at 30 min; Program in Virology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
E) or consisting only of the
intracellular domain (ICN) were found to activate transcription in
cultured cells, presumably through RBP-J
response elements within
DNA. Both truncated forms also bound to the transcription factor
RBP-J
in extracts prepared from human and murine T-ALL cell lines.
Transcriptional activation required the presence of a weak
RBP-J
-binding site within the NOTCH1 ankyrin repeat region of the
intracellular domain. Unexpectedly, a second, stronger RBP-J
-binding site, which lies within the intracellular domain close
to the transmembrane region and significantly augments
association with RBP-J
, was not needed for oncogenesis or for
transcriptional activation. While ICN appeared primarily in the
nucleus,
E localized to cytoplasmic and nuclear membranes,
suggesting that intranuclear localization is not essential for
oncogenesis or transcriptional activation. In support of this
interpretation, mutation of putative nuclear localization sequences
decreased nuclear localization and increased transcriptional activation
by membrane-bound
E. Transcriptional activation by this mutant form
of membrane-bound
E was approximately equivalent to that produced by
intranuclear ICN. These data are most consistent with NOTCH1
oncogenesis and transcriptional activation being independent of
association with RBP-J
at promoter sites.
end
of NOTCH1 (5). These transcripts encode a series of
truncated NOTCH1 polypeptides of ~100-125 kDa lacking the epidermal
growth factor-like and lin-12-like repeats (5).
Fig. 1.
Schematic representation of full-sized and
oncogenic forms of NOTCH1. The scale above the diagram
of full-length NOTCH1 is in codons/amino acid residues. E is encoded
by a cDNA consisting of codons 1-22 fused to codons 1673-2555 and
is predicted to result in the synthesis of a mature polypeptide with an
amino terminus lying 61 amino acids external to the transmembrane
domain. ICN is encoded by a cDNA consisting of the first two codons
of NOTCH1 fused to codon 1770, which lies 13 amino acids internal to
the transmembrane domain. Structural motifs and important landmarks within these polypeptides are labeled as follows: L, leader
peptide; EGFR, epidermal growth factor-like repeats;
LNR, Lin-12-like repeats; CC, conserved cysteine
residues Cys1685 and Cys1692, which lie 49 and
42 amino acids external to the transmembrane domain; TM,
transmembrane domain; tryptophan residue Trp1767;
N1 and N2, nuclear localization signal sequences;
AR, ankyrin-like repeats; O, opa sequence;
P, PEST sequence.
[View Larger Version of this Image (19K GIF file)]
E), or
deleted for all protein sequence up to amino acid 14 of the
intracellular domain (ICN) (shown schematically in Fig. 1) induced
T-ALL in ~50% of the recipient animals. In contrast, normal,
full-length NOTCH1 produced no tumors in similar transplant experiments. The tumors induced by
E contained an abundant
~120-kDa NOTCH1 that localized to perinuclear and cytoplasmic
membranes, while tumors induced by ICN contained an abundant
~100-115 kDa NOTCH1 within the nucleus. Therefore, different
amino-terminal deletions created cytoplasmic and nuclear forms of
truncated NOTCH1 that appeared to be equally oncogenic.
(12-15), which is
homologous to the proteins Su(H) in the Drosophila and lag-1
in Caenorhabditis elegans. Evidence for this functional link
between the proteins derives from genetic studies which have placed
Drosophila NOTCH and its nematode counterparts, lin-12 and
glp-1 (16), upstream of Su(H) (17-19) and lag-1 (20, 21),
respectively. Furthermore, the murine HES1 promoter
activated by NOTCH1 contains an RBP-J
-binding site (22), suggesting
that RBP-J
is also downstream of activated NOTCH1.
bound to DNA (22), and when tethered
to a promoter by covalent linkage to a DNA-binding protein, NOTCH1 has
transactivating effects (5, 23), indicating that NOTCH1 might
transactivate through physical interaction with RBP-J
bound to
promoter elements. If this mechanism of transactivation were involved
in NOTCH1 transformation, it might be expected that subcellular
localization would influence oncogenicity. However,
E transforms as
well as ICN despite its cytoplasmic localization. Moreover, based on
studies performed in transiently overexpressing cells, NOTCH1
association with RBP-J
has been reported to require a tryptophan
residue (Trp1767) lying 10 amino acids internal to the
transmembrane domain that is absent from ICN (24). These discrepancies
raised the possibility that NOTCH1 oncogenicity might not involve
RBP-J
or nuclear localization. To investigate these possibilities,
we have evaluated the physical and functional interaction of RBP-J
and NOTCH1 in a variety of cell types, including NOTCH1-induced T-ALL
cell lines.
Antibodies
(26), have been previously described.
E and ICN (6), respectively, were
grown in RPMI supplemented with 20% fetal calf serum and interleukin-2 (4 units/ml). Human embryonic kidney 293 cells and two cell lines derived from 293 cells, 293T and Bosc23 (28), were all grown in
Dulbecco's modified Eagle medium supplemented with 10% fetal calf
serum.
E (codons 1-22 fused to codons
1673-2555),
E
AR (a subclone of
E with a deletion removing
codons 1858-2206), ICN (codons 1770-2555), and ICN
N1
N2 (a
subclone of ICN with two deletions removing codons 1771-1857 (
N1)
and codons 2097-2204 (
N2)) have been previously described (5, 6).
Additional subclones with mutations and small deletions in potential
nuclear localization signal sequences were made by ligating polymerase
chain reaction products with compatible restriction sites to the ICN
and
E constructs. The construct ICNW was created by ligating a
double stranded DNA linker containing nucleotides
15 to +6 (relative
to the NOTCH1 start codon) to the ICN construct after digestion with
the restriction enzymes Bsu36I and BamHI. A
cDNA construct encoding RBP-J
3 in the eukaryotic expression plasmid pSG5 has been previously described (15). To express RBP-J
3
with a myc epitope tag, the cDNA was excised from pSG5 as a BamHI-SfcI restriction fragment and
subcloned using a SfcI/NotI adaptor into a
derivative of pcDNA3 that encodes three iterated copies of a
myc epitope. The resultant fusion cDNA encodes RBP-J
3 with myc epitopes replacing the six most carboxyl-terminal
amino acids (RBP-J
3-myc).
3 (26) fusion proteins have been described. A cDNA
spanning the region encoding the NOTCH2 ankyrin repeats (codons 9-271
in the sequence of Stifani et al. (29)) was amplified by
polymerase chain reaction using a human umbilical vein encdothelial
cell cDNA library (30) as template. After subcloning into pGEX-2TK
(Pharmacia), GST-NOTCH2 ankyrin repeat fusion protein was expressed in
Escherichia coli and purified on glutathione-Sepharose beads
(Pharmacia) as described for the NOTCH1 ankyrin repeats (5).
3 cDNA in pSG5 were transcribed and translated in
vitro in the presence of [35S]methionine using
rabbit reticulocyte lysates (Promega).
[35S]Methionine-labeled polypeptides were stored at
80 °C. Binding of in vitro translated polypeptides to
GST fusion proteins was performed under conditions described by
Jarriault et al. (22). Briefly, 5 µl of labeling mixture
containing polypeptides translated in vitro were diluted
into 0.5 ml of ice-cold 25 mM Hepes, pH 7.5, containing
0.5% Nonidet P-40, 100 mM KCl, 5 mM
MgCl2, 5 mM dithiothreitol, 0.2 mM
EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride (binding buffer). This mixture was
precleared by incubation with 50 µl of GST-glutathione Sepharose
beads at 4 °C with mixing for 60 min. After removal of the beads by
brief centrifugation in a microcentrifuge, the remaining supernatant
was divided into two aliquots of 0.25 ml and incubated with either GST
fusion protein-glutathione Sepharose beads or a equivalent volume of
GST-glutathione Sepharose beads at 4 °C with mixing for 60 min.
Beads were washed twice with binding buffer, once with binding buffer
containing 200 mM KCl, and once more with binding buffer,
and then boiled in SDS-PAGE loading buffer. 35S-Labeled
polypeptides were separated by SDS-PAGE in 10% gels and visualized by
fluorography.
was subcloned
into the NcoI-BamHI site of the plasmid pACTII.
Yeast strain Y190 was transformed with pAS2-AR1 and selected on Trp
plates, and then retransformed with pACTII-RBP-J
3 and selected on
Trp-Leu-agar plates in the presence of 25 mM
3-aminotriazole. Resultant colonies were then stained for
-galactosidase activity using 5-bromo-4-chloro-3-indoyl
-D-galactoside as substrate.
3, a CAT reporter containing a
concatamerized RBP-J
-binding sequence derived from the DNA of the
Epstein-jBarr virus Cp1 promoter (33), and an internal control cDNA
encoding
-galactosidase expressed from a glucokinase housekeeping
promoter. Cell extracts were routinely prepared 48 h
post-transfection and analyzed for
-galactosidase and CAT activity
according to standard protocols (34). CAT activities were normalized
with respect to
-galactosidase activity and quantitated by
comparison to results of control transfections performed with pcDNA3 lacking a cDNA insert and the reporter construct.
20 °C methanol for 1 min and then
stained with rabbit anti-NOTCH1 as described previously (5). For
immunfluorescent localization, 293T cells were stained with
affinity-purified rabbit anti-NOTCH1 (4 µg/ml) followed by goat
anti-rabbit antibody linked to fluorescein isothiocyanate (1:1000) in
the presence of 0.1% Triton X-100, using the conditions described by
Kopan et al. (35). Confocal and DIC microscopy was performed
on Zeiss epifluorescence microscope equipped with a MRC600 confocal
unit (Bio-Rad). For each field imaged, both confocal fluorescence and
DIC images were collected using SOM software (Bio-Rad). Image files
were translated to TIFF format using NIH Image software and composite
figures were subsequently created using Adobe Photoshop 2.5.
NOTCH1 and RBP-J Are Physically Associated in T Cell
Lines
and oncogenic forms
of NOTCH1, immunoprecipitates were prepared from lysates of human SUP-T1 cells, which contain two copies of the t(7;9) and no normal chromosome 9, from lysates of T6E cells, which are derived from a T
cell tumor induced by a retrovirus containing the
E cDNA, and
from lysates of I22 cells, which are derived from a T cell tumor
induced by a retrovirus containing the ICN cDNA.
isoforms, J
1 and J
3,
created by alternative splicing of the 5
exon. Western blot analysis
of immunoprecipitates prepared from SUP-T1 cells (Fig. 2A) demonstrated that RBP-J
3 and trace
amounts of RBP-J
1 were co-precipitated by affinity-purified
antibodies against two different portions of the intracellular domain
of NOTCH1, while neither isoform was precipitated by control
affinity-purified antibody against GST. Since approximately equal
amounts of RBP-J
1 and RBP-J
3 were precipitated by anti-RBP-J
,
enrichment for RBP-J
3 in the NOTCH1 immunoprecipitates probably
reflects a greater association of RBP-J
3 with NOTCH1, as has been
previously noted with Epstein-Barr virus transcriptional regulators
EBNA2 and EBNA3C (36). RBP-J
antiserum precipitated only RBP-J
3
from T6E and I22 lysates, and RBP-J
3 co-precipitated with
anti-NOTCH1 antibody but not with anti-GST antibody. The extent of
RBP-J
association with NOTCH1 forms in SUP-T1, T6E, and I22 cells
was substantial, since NOTCH1 antibody precipitated much more than 1%
of the RBP-J
3 and approximately as much as with the RBP-J
antibody.
Fig. 2.
Co-precipitation of NOTCH1 polypeptides and
RBP-J from lysates of T cell lines expressing oncogenic forms of
NOTCH1. A, immunoprecipitation of RBP-J
with NOTCH1
antibody. Equal amounts of lysates from SUP-T1 (SUPT-1),
T6E, and I22 cells were incubated with antiserum against RBP-J
,
affinity-purified anti-GST antibody, or affinity-purified anti-NOTCH
antibody directed against the T3 or TC regions, followed by the
addition of protein A-Sepharose. Immunoprecipitated proteins were
eluted, subjected to SDS-PAGE in 8% gels, and transferred to
nitrocellulose. A composite Western blot is shown that was stained with
RBP-J
antiserum. Input represents 1% of total protein. The position
of bands for RBP-J
and immunoglobulin heavy chain (HC)
are indicated. B, immunoprecipitation of NOTCH1 with
antiserum to RBP-J
. Lysates were incubated with antiserum to
RBP-J
or preimmune serum and immunoprecipitates were collected and
analyzed as described under A, except that electrophoresis was performed in a 6% gel. A composite Western blot is shown that was
stained with affinity-purified NOTCH1 antibody against TC.
[View Larger Version of this Image (48K GIF file)]
-specific antibody immunoprecipitated
NOTCH1 (Fig. 2B). From SUP-T1 cell lysates, multiple t(7;9)-specific polypeptides of 100-125 kDa co-precipitated with RBP-J
-specific antibody. From T6E lysates, a 120-kDa NOTCH1
corresponding in size to
E co-precipitated with RBP-J
antibody,
as did smaller amounts of a 350-kDa cross-reactive polypeptide. The
350-kDa polypeptide was recognized by multiple NOTCH1 antibodies and is
identical in size to human p350 (not shown). Several NOTCH1
polypeptides from I22 lysates were also co-precipitated with RBP-J
antibody, with the largest being slightly smaller than
E, as
expected for ICN.
3
antibody even though it lacks the residue
Trp1767 previously shown to be important in NOTCH1
association with RBP-J
both in vitro and in transiently
transfected cells (24). To investigate an alternative basis for the
association of ICN with RBP-J
, various forms of NOTCH1 (shown
schematically in Fig. 3A) were expressed
in vitro and tested for binding to RBP-J
3-GST fusion
protein immobilized on glutathione-Sepharose beads.
Fig. 3.
Demonstration of two sites within NOTCH1 that
associate with RBP-J in vitro . A, schematic
representation of NOTCH constructs tested in vitro for
binding to RBP-J
3. The amino-terminal sequences of ICNW and ICN are
also shown. B and C, comparison of binding of
various forms of NOTCH1 to RBP-J
3-GST. In each binding assay, 10 µl of 35S-labeled in vitro translated NOTCH1
was precleared with 25 µl of GST glutathione beads and then allowed
to mix with 25 µl of GST glutathione-Sepharose beads or RBP-J
3-GST
glutathione-Sepharose beads for 2 h. Bound proteins were eluted
and analyzed by SDS-PAGE in a 10% gel followed by fluorography. In
B and C, the input lane represents 5% of the
total protein. B shows a fluorogram resulting from a 12 h exposure, whereas in C the exposure time was 48 h. D, comparison of binding of RBP-J
3 to NOTCH ankyrin
repeats. In vitro translated RBP-J
3 (10 µl) was mixed
with 2.5 µl of GST, GST-NOTCH1 ankyrin repeat (AR1), or GST-NOTCH2
ankyrin repeat (AR2) glutathione-Sepharose beads, each
having ~12.5 µg of bound fusion protein. Binding, elution, and
analysis of bound proteins was performed as described in B
and C. The input lane represents 1% of the total
protein.
[View Larger Version of this Image (36K GIF file)]
-GST more strongly
than ICN (Fig. 3B). However, when compared with the level of
binding observed with control GST beads, ICN also bound specifically to
RBP-J
-GST (Fig. 3C). Binding was enhanced by deletion of
regions flanking the ankyrin repeats (ICN
N1
N2 in Fig.
3C) and abolished by a deletion that removed the ankyrin repeats (ICN
AR in Fig. 3C). The isolated ankyrin repeats
of NOTCH1 and of a closely related human homolog, NOTCH2, also bound
in vitro translated RBP-J
in amounts above that observed
with the GST control (Fig. 3D), indicating the presence of a
weak RBP-J
-binding site within the ankyrin repeat region that has
been conserved during divergence of these two mammalian NOTCH
receptors. The ankyrin repeat region of NOTCH1 was also found to
interact strongly with RBP-J
in the yeast two-hybrid assay (Table
I), as ~90% of colonies co-transformed with pAS2-AR1
and pACTII-RBP-J
3 showed high level expression of a
-galactosidase reporter gene. In contrast, no
-galactosidase
expression was observed when a control cDNA encoding an unrelated
protein, yeast SNF1, was substituted for AR1 or RBP-J
3. Therefore,
the ankyrin repeat region of NOTCH1 contains a second binding site for
RBP-J
that could mediate stable association of ICN and RBP-J
in
I22 cells.
3
, absence of
staining at 24 h.
Cells
Plasmid
-Galactosidase
pAS2-SNFI
pACTII-RBP-J
3
pAS2-ARI
pACTII-SNFI
pAS2-ARI
pACTII-RBP-J
3
+++
To further
investigate the RBP-J binding sites of NOTCH1, immunoprecipitates
were prepared from lysates of 293 cells transiently expressing
myc epitope-tagged RBP-J
3 and various forms of NOTCH1. NOTCH1,
E, and ICNW all co-precipitated with anti-myc
antibody (Fig. 4A, lanes 1-3, respectively),
as did
E
AR (not shown), while ICN did not co-precipitate in
detectable amounts (Fig. 4A, lane 4). These data indicate
that the binding site containing Trp1767 is necessary and
sufficient for stable association in transiently expressing cells. In
contrast, stable association of RBP-J
through the ankyrin repeat
region is not detectable in transiently expressing 293 cells.
Co-precipitation of membrane-bound forms of NOTCH1 (full-sized NOTCH1
and E) in T cell lines and transiently expressing 293 cells
suggested that RBP-J
can associate with NOTCH1 in the cytoplasm. Alternatively, association might occur ex vivo after cell
lysis. To distinguish between these possibilities, immunoprecipitates were prepared from lysates of cells co-expressing NOTCH1 and RBP-J
3 (Fig. 4B, lane 2) and mixtures of cells expressing either
full-size NOTCH1 or RBP-J
3 (Fig. 4B, lane 1).
Co-precipitation of NOTCH1 and RBP-J
myc with myc antibody
was only observed in lysates prepared from co-expressing cells,
consistent with RBP-J
3·NOTCH1 complexes being formed in the
cytoplasm of intact cells.
The roles of the two
RBP-J-binding regions and of the nuclear localization signal
sequences in transcriptional activation were characterized using the
RBP-J
binding element from the Epstein-Barr virus Cp1 promoter (33).
Transient expression of
E and ICN in 293 cells activated
transcription 8-12-fold, whereas full-length NOTCH1 had no effect
(Fig. 5A). Although a plasmid encoding
RBP-J
3 was routinely included in these transfections, similar
results were obtained when this plasmid was excluded, probably because endogenous RBP-J
levels in 293 cells are sufficient for full activation.2 ICN was slightly more active
than
E in all experiments (p < 0.06).
E
AR,
deleted for the ankyrin repeats, had only 20% of the activity of
E,
consistent with a key role for the ankyrin repeat region in
transcriptional activation. In contrast, ICNW, ICN, and ICN
N1
stimulated transcription to a similar degree (Fig. 5B),
despite the absence of Trp1767 from ICN and ICN
N1.
Therefore, the strong RBP-J
-binding site around Trp1767
is not required for transcriptional or oncogenic activity.
Transcriptional Activation Is Not Dependent on Nuclear Localization
Activation of transcription using the RBP-J
binding element of Cp1 promoter by
E and ICN, but not by NOTCH1,
indicated that deletion of the extracellular domain resulted in
activation of transcription through RBP-J
. Since
E localizes
predominantly to cytoplasmic and nuclear membranes, intranuclear
localization may not be required for its effects. Alternatively, a
small amount of
E, but not NOTCH1, might be cleaved within the
intracellular domain, freeing it from membranes and enabling it to
up-regulate transcription through RBP-J
bound to DNA. If NOTCH1 must
first translocate to the nucleus to be active, mutation of the putative NOTCH1 nuclear localization sequences (NLSs) should inhibit
transactivation and ICN should be a more potent transactivator than
E. To test these predictions, the nuclear localization signal
sequences of NOTCH1 were mapped by mutational analysis in NIH 3T3 cells
(Fig. 6, A-E, summarized in Fig.
6F), and NLS mutations were then evaluated for effects on
transactivation by ICN and
E.
Previous work has indicated the existence of at least one sequence
amino-terminal and one sequence carboxyl-terminal of the ankyrin
repeats with NLS activity (5, 37). The N1 region amino-terminal to the
ankyrin repeats contains two potential NLSs, Lys1779-Lys-Lys-Arg-Arg1783 and
Lys1820-Lys-Phe-Arg-Phe-Glu1825, while the
region carboxyl-terminal to the ankyrin repeats contains two closely
spaced stretches of basic amino acids,
Lys2156-Lys-Val-Arg-Lys2160 and
Lys2177-Ala-Arg-Arg-Lys-Lys2182. When coupled
with a deletion termed N2.1 that removed amino acids 2158-2206,
mutation of the sequence
Lys1820-Lys-Phe-Arg-Phe-Glu1825 to
LEFRFE (termed MN1) led to a marked increase in cytoplasmic localization (Fig. 6C), with most cells showing
approximately equal cytoplasmic and nuclear staining. Amino acid
residues 1820-1825 (designated N1) are conserved among all members of
the vertebrate NOTCH receptor family (Table II). In
contrast, deletion of the second amino-terminal basic sequence,
Lys1779-Lys-Lys-Arg-Arg1783, had no additional
effect on localization, either in concert with
N2.1 alone or
together with MN1 and
N2.1 (not shown).
|
When coupled with MN1, mutation
Lys2156-Lys-Val-Arg-Lys2160 to KEFRK
(designated MN2a, Fig. 6D) or mutation of
Lys2177-Ala-Arg-Arg-Lys-Lys2182 to
KARRGT (designated MN2c, Fig. 6E) increased
cytoplasmic localization, indicating that these two basic sequences may
function as a bipartite NLS. This sequence, designated N2, is also
conserved (Table II). Neither mutation or deletion of both N1 and N2
completely abolished nuclear staining, consistent with previous reports
that the ankyrin repeat region has some intrinsic capacity for nuclear localization (37). No attempt was made to further reduce nuclear localization through ankyrin repeat mutations, since involvement of
this region in binding and activation of RBP-J would confound interpretation of "loss-of-function" mutations.
To produce a truncated NOTCH1 with limited (if any) capacity for
nuclear localization, MN1 and N2.1 were subcloned into
E to
create
EMN1
N2.1. Localization of various forms of NOTCH1 in
transiently expressing 293 cells was then determined by confocal microscopy (Fig. 7). Inactive, full-sized NOTCH1
localized to perinuclear regions and cytoplasmic vesicles (Fig.
7A). Cells expressing
EMN1
N2.1 showed staining of
nuclear membrane and variable perinuclear staining compatible with
endoplasmic reticulum and/or Golgi, with no detectable nuclear staining
(Fig. 7B). In contrast, ICNW produced exclusively nuclear
staining (Fig. 7C).
The effect of NLS mutations on transcriptional activation was then
determined in transient transfection studies using 293 cells (Fig.
8A). ICNMN1N2.1 had similar activity to
ICN, whereas
EMN1
N2.1 unexpectedly had consistently increased
activity relative to
E (p < 0.05). This increase in
activity was probably not due to diminished nuclear localization
per se, because a derivative of
EMN1
N2.1 with a
functional SV40 NLS sequence inserted into the
N2.1 site maintained
levels of transactivation similar to
EMN1
N2.1 (not shown). To
compare the potency of nuclear and membrane-bound versions of truncated
NOTCH1, transactivation by ICNW and
EMN1
N2.1 was measured over a
range of input plasmid (Fig. 8B). Both constructs had
similar dose-response curves, with a trend toward slightly higher
stimulation with ICNW at low input levels of plasmid. However,
EMN1
N2.1 (Fig. 8C, lane 1) also produced less protein
than ICNW at low levels of input plasmid, (Fig. 8C, lane 2),
suggesting that minor differences in transactivation could result from
differences in protein level rather than differences in potency. These
data indicate that both intranuclear and extranuclear forms of
truncated NOTCH1 transactivate the Cp1 promoter, suggesting that
transactivation may not require association of NOTCH1 and RBP-J
at
promoter sites.
Recurrent, specific chromosomal translocations found in tumors
frequently exert their oncogenic effects by increasing some normal
activity associated with the proteins encoded by genes located at the
translocation breakpoints. It seems likely that this theme will extend
to the role of truncated NOTCH1 in T cell neoplasia since normal
NOTCH1 is highly expressed in thymocytes; oncogenic forms of
NOTCH1 exhibit "gain-of-function" activity in various assays of
transcription and differentiation; and these gain-of-function forms are
particularly oncogenic in T cell progenitors. However, the precise
mechanism by which truncated NOTCH1 contributes to tumor development is
not clear and may be complex. Besides interacting physically and
functionally with RBP-J, NOTCH1 also interacts with components of
the NF-
B signaling pathway (5, 38) and some NOTCH phenotypes in the
fly appear to be independent of Su(H) (18). The data presented here
indicate that the oncogenic capacity of truncated NOTCH1 may be
mediated at least in part by RBP-J
. In this regard, it is relevant
that four Epstein-Barr viral proteins required for B cell
transformation, EBNA2 (39), EBNA3A, EBNA3B, and EBNA3C (26), bind to
and alter the activity of RBP-J
(14, 15, 26, 40-47).
Extensive binding of these transforming proteins with RBP-J
is also
found in Epstein-Barr virus-transformed B lymphocytes (36). Since
viral transforming proteins often associate with and dysregulate
molecules which seem to have general functions in the control of normal
cell proliferation, it may be that RBP-J
has a critical role in T
cell transformation as well as in B cell transformation induced by
Epstein-Barr virus. For similar reasons, dysregulation of RBP-J
may
be involved in neoplasms beyond the lymphoid system, and abnormal
NOTCH1 signaling could be a factor in this process.
Based on our data, the ankyrin repeat region of NOTCH1 is important for
transactivation of promoters, whereas neither the strong RBP-J
binding site around amino acid residue Trp1767 (a domain
termed RAM23 by Tamura et al. (24)) nor nuclear localization of NOTCH1 is required. A major role for the ankyrin repeats in the
effects of NOTCH1 has been previously indicated by studies showing that
a point mutation in the fourth ankyrin repeat prevented transactivation
of RBP-J
(22) and inhibition of myogenesis (48) by NOTCH1, and that
a peptide consisting of the ankyrin repeats of glp-1 and immediate
flanking sequences was sufficient to produce a gain-of-function
phenotype during development in C. elegans (49). Assuming
that the basic mechanism of NOTCH signaling has been conserved during
vertebrate evolution, this latter finding supports the possibility that
association of RBP-J
with the ankyrin repeat region is involved in
and may be sufficient for some level of transactivation.
Our observation that the region containing Trp1767 and the
RAM23 domain (defined as amino acids 1758-1857) is not required for transactivation is consistent with experiments showing that murine ICN
lacking amino acids 1758-1818 inhibited myogenic differentiation of
NIH 3T3 cells (37), and that human ICN lacking amino acids 1758-1770
inhibited ganglion cell differentiation in the developing chick retina
(11). In the latter of these two studies, the effects of ICN were
mimicked by exposure of cells expressing the full-sized NOTCH1 receptor
to the NOTCH ligand DELTA, suggesting that Trp1767 is not
required for production of a bona fide NOTCH1 signal. Similarly, we
have recently observed that the inhibitory effect of ICNN1 (which
lacks amino acids 1758-1857) on myogenic differentiation of murine
C2C12 cells is mimicked by treatment of these cells with the ligands
produced by the human JAGGED and SERRATE
genes.3 Since it seems unlikely that
binding of RBP-J
to the RAM23 sequence of NOTCH1 is without
functional significance, alternative functions for this sequence other
than a direct role in activation of RBP-J
need to be considered. One
possible function for RAM23 would be to serve as a docking site for
RBP-J
with normal, full-sized NOTCH1. Upon binding of ligand to the
extracellular domain of NOTCH1, this pool of pre-associated RBP-J
could then be activated through a mechanism involving the ankyrin
repeat binding site.
The strong oncogenic and transactivating activity of membrane-tethered
forms of NOTCH1 (E) and the failure to detect processing of
E to
a nuclear polypeptide in either T6E T-ALL cells or transiently expressing 293 cells implies that nuclear localization is not required
for NOTCH1 oncogenesis or transactivation of the Cp1 promoter. This
interpretation is consistent with the observation that among normal
tissues in invertebrates and vertebrates, intranuclear NOTCH1 has been
detected in only a small population of post-mitotic retinal cells in
the rat (50). Hence, while intranuclear forms of NOTCH (7, 8, 51, 52)
and NOTCH1 (6, 37, 48) produce gain-of-function phenotypes in a variety
of assays, intranuclear localization may not be essential for
function.
The conclusion that nuclear localization of NOTCH1 is non-essential for
at least some functions of NOTCH1 conflicts with one model for NOTCH1
signaling based on observations made using a slightly different form of
E, termed
E-C, lacking two conserved extracellular cysteine
residues, Cys1685 and Cys1692. Unlike
E,
which contains these two cysteine residues, transient expression of
E-C is accompanied by proteolytic processing to smaller polypeptides
that localize to the nucleus and associate with RBP-J
, suggesting
that polypeptides derived from
E-C transactivate by direct physical
interaction with RBP-J
bound to DNA (22, 35). Failure to detect
similar processing of
E could be explained if Cys1685
and Cys1692 influence proteolytic processing but not
transactivation or oncogenesis per se. In support of this
possibility,
E-C-induced T cell tumors contain a series of lower
molecular weight polypeptides cross-reactive with NOTCH1 antibody that
are absent from
E-induced T cell tumors (6) and also show nuclear
staining with anti-NOTCH1 antibodies that is not observed in
E-induced tumors.3
E and
E-C are equally potent
oncoproteins (6), however, so the observed differences in proteolytic
processing and subcellular localization do not appear to affect
transforming activity.
In contrast to the nuclear translocation model for NOTCH1 signaling,
our data pertaining to NOTCH1 oncogenesis and transactivation are
generally consistent with an alternative signaling model based on
observations made in Drosophila. This model proposes that
transient association of Su(H) and NOTCH in the cytoplasm results in
transactivation through Su(H) without nuclear translocation of NOTCH
(53). One important additional factor in this activation pathway is
deltex, a protein found in Drosophila that binds the ankyrin
repeats of NOTCH (54). Deltex can displace Su(H) from the ankyrin
repeats and is a positive regulator of NOTCH signaling (55, 56),
indicating the existence of factors that promote dissociation of NOTCH
and Su(H) yet enhance transactivation. Conceivably, cell type-specific variation in expression levels of deltex-like factors could result in
stable association of ICN with RBP-J in some cell types
(e.g. I22 cells) but not others (e.g. transiently
expressing 293 cells). A critical question not explained by this model
is whether Su(H) activation stems from post-translational modification
or from association with unknown accessory factors. Further elucidation of the mechanism of NOTCH1 transactivation will require identification of mammalian accessory factors and additional investigation of the
effects of activated NOTCH1 on RBP-J
.
We gratefully acknowledge and appreciate the technical assistance of Vytas Patriubavicius.