Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA
Author for correspondence (e-mail:
irvine{at}waksman.rutgers.edu)
Accepted 25 September 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: fringe, Notch, Fucose, Glycosylation, O-fucosyltransferase, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The O-linked fucose monosaccharide transferred to protein by OFUT1
can be elongated by ß1,3N-acetylglucosaminyltransfrases encoded by Fringe
genes (Fig. 1B)
(Bruckner et al., 2000;
Moloney et al., 2000a
). In
contrast to the general positive requirement for OFUT1 in Notch signaling,
glycosylation by Fringe exerts a positive influence on Delta-Notch signaling,
but a negative influence on Serrate-Notch signaling
(Fleming et al., 1997
;
Panin et al., 1997
;
Klein and Martinez Arias,
1998
; Papayannopoulos et al.,
1998
; Bruckner et al.,
2000
; Hicks et al.,
2000
; Moloney et al.,
2000a
). The influence of Fringe on Notch signaling has been best
studied in the Drosophila wing (reviewed by
Irvine and Vogt, 1997
). Both
Fringe and Serrate are expressed by dorsal wing cells
(Irvine and Wieschaus, 1994
;
Couso et al., 1995
;
Diaz-Benjumea and Cohen,
1995
), whereas Delta and Notch are initially broadly expressed
(Fehon et al., 1991
;
Doherty et al., 1996
;
de Celis and Bray, 1997
).
Fringe facilitates Notch activation in dorsal cells by potentiating their
ability to respond to Delta (Panin et al.,
1997
). At the same time, by inhibiting Serrate signaling, Fringe
limits Serrate to signaling back across the dorsoventral (DV) boundary to
ventral cells (Fig. 1E)
(Fleming et al., 1997
;
Panin et al., 1997
;
Klein and Martinez Arias,
1998
). Notch activation promotes Serrate and Delta expression in
the wing (de Celis and Bray,
1997
; Panin et al.,
1997
), and although the initial expression of Notch ligands does
not require Notch activation, maintenance of their expression does.
Consequently, Serrate and Delta become restricted to the DV boundary, where
their expression is maintained by a positive feedback loop.
Genetic and cell culture studies indicate that OFUT1 and Fringe function
specifically on the receiving side of the Notch pathway, rather than on the
signaling side (Panin et al.,
1997; Bruckner et al.,
2000
; Hicks et al.,
2000
; Okajima and Irvine,
2002
; Sasamura et al.,
2003
). Biochemical studies indicate that Notch is a substrate for
the glycosyltransferase activities of OFUT1 and Fringe
(Bruckner et al., 2000
;
Moloney et al., 2000a
;
Moloney et al., 2000b
;
Okajima and Irvine, 2002
).
These results implicate the glycosylation of Notch as the mechanism by which
Fringe and OFUT1 exert their effects. Studies of proteins involved in blood
clotting and fibrinolysis led to a proposed consensus site for
O-fucosylation, C2XXGG(S/T)C3, in which S/T is
the modified amino acid, and C2 and C3 are the second
and third cysteines, respectively, of the EGF domain
(Harris and Spellman, 1993
).
According to this consensus sequence, 11 of the 36 EGF repeats in
Drosophila Notch could potentially be O-fucosylated
(Moloney et al., 2000b
).
However, other studies have indicated that the original consensus sequence is
too narrow (Wang and Spellman,
1998
; Panin et al.,
2002
; Shao et al.,
2003
). Based on a broader consensus sequence,
C2XXX(G/A/S)(T/S)C3, 23 of the 36 EGF repeats of
Drosophila Notch could potentially be O-fucosylated
(Fig. 1A). The presence of
O-fucose is a prerequisite for glycosylation by Fringe, but
additional structural constraints that influence Fringe-dependent elongation
of O-fucose on particular EGF domains also appear to exist
(Shao et al., 2003
).
Confirmation that Notch is the substrate that accounts for the effects of OFUT1 and Fringe on Notch signaling requires the identification of functional sites of modification. Additionally, it remains unclear whether the different effects of OFUT1 versus Fringe, or the effects of Fringe on Delta versus Serrate, are mediated through the same or distinct sites of O-fucosylation. The large number of potential sites poses a challenge to the identification of biologically relevant sites. Nonetheless, prior studies of Notch suggest two possible regions as potentially important sites of O-fucosylation.
A series of gain-of-function alleles of Notch, called
Abruptex (NAx) are caused by missense
mutations that map to EGF repeats 24, 25, 27 and 29
(Kelley et al., 1987;
de Celis and Bray, 2000
). These
mutations cause a ligand-dependent, hyperactivation of Notch
(Kelley et al., 1987
;
Heitzler and Simpson, 1993
;
de Celis and Garcia-Bellido,
1994
; de Celis and Bray,
2000
). Formally then, a normal function of this region of Notch
appears to be to negatively regulate Notch activation. Ectopic expression
experiments suggest that this might occur because
NAx alleles impair autonomous inhibition
(de Celis and Bray, 2000
).
Genetic interactions between NAx alleles and
fringe mutations have also led to the suggestion that
NAx alleles might affect the sensitivity of Notch
to Fringe (de Celis and Bray,
2000
; Ju et al.,
2000
), which has itself been suggested to act through autonomous
inhibition (Irvine and Vogt,
1997
; Sakamoto et al.,
2002
). The observations that both O-fucose glycans and
NAx alleles influence the activation of Notch by
its ligands, and that the NAx region overlaps an
array of potential sites of O-fucosylation
(Fig. 1A), identify this as a
region of interest.
Studies of Notch-ligand binding suggest EGF12 as another potentially
relevant site of O-fucosylation. In a cell aggregation assay, two EGF
repeats, EGF11 and EGF12, were found to be necessary and sufficient for
interaction with Delta (Rebay et al.,
1991). Notably, EGF12 is one of only three EGF repeats that
contains an O-fucose consensus site in all Notch receptors
(Fig. 1A), and studies of
murine Notch1 have demonstrated that it is a substrate for O-FucT-1 and Fringe
(Shao et al., 2003
). Moreover,
both Fringe and OFUT1 can influence Notch-ligand binding
(Bruckner et al., 2000
;
Shimizu et al., 2001
;
Okajima et al., 2003
;
Sasamura et al., 2003
).
In order to begin to assess the significance of these potential sites of O-fucosylation on Notch, we eliminated evolutionarily conserved O-fucose sites in EGF12 and the NAx region. Mutation of EGF12 results in a dramatic change in Notch activation in vivo, and in its physical interactions with Serrate in vitro. Our results underscore the importance of negatively regulating Notch-ligand interactions, and suggest a novel mechanism by which such regulation might occur.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Drosophila stocks and crosses
Multiple insertions of each UAS-N construct were created.
Designations and chromosomes for lines analyzed are UASNEGF12f[M4a]
(second chromosome), UAS-NEGF12f[M4b] (third),
UAS-NEGF12f[M2] (third), UAS-NEGF24f[a] (first),
UASNEGF24f[b] (second), UAS-NEGF24f[c] (third),
UAS-NEGF26f[a] (second), UAS-NEGF26f[b] (third),
UAS-NEGF26f[c] (third), UASNEGF24+26f[a] (second),
UAS-NEGF24+26f[b] (second), UASNEGF31f[a] (second),
UAS-NEGF31f[b] (third) and UAS-N.865 (second). We also
examined the previously described insertions UASN[8] and
UAS-N[13] (Doherty et al.,
1996). Initial analysis of these lines was conducted by crossing
them to ptc-GAL4, and examining wing imaginal discs and adult wings.
The approximate level of Notch expression generated by each line was
determined by anti-Notch staining; the phenotypes described are from lines
with similar levels of Notch. In most cases independent insertions gave
similar results. All phenotypes described are from crosses at 25°C.
Stocks for ligand mutant clones and ectopic Notch expression were: y w hs-Flp[122]; ptc-Gal4 UAS-lacZ; FRT82B Ubi-GFP;
UAS-N.865; FRT82B Serrev2-11 / TM6b; and
UAS-N.865; FRT82B Dlrev10 e / TM6b.
Stocks for generating MARCM clones (Lee
et al., 2000; Struhl and
Greenwald, 2001
) were:
y w hs-Flp[122] tub-Gal4 UAS-GFP:nls; UAS-NEGF12f[M4a]; FRT82B tub-Gal80/TM6b;
y w hs-Flp[122] tub-Gal4 UAS-GFP:nls; UAS-N.865; FRT82B tub-Gal80/TM6b;
FRT82B Serrev2-11 / TM6b;
FRT82B Dlrev10 e / TM6b;
FRT19A N55e11/FM7; UAS-NEGF12f[M4a];
FRT19A N55e11/FM7; UAS-N.865; and
FRT19A tub-Gal80 hs-Flp[122];UAS-GFP; tub-Gal4/TM6b.
Other Gal4 and UAS transgenes used, and corresponding FlyBase ID numbers
(http://flybase.bio.Indiana.edu/),
are ptc-Gal4 (FBti0002124), da-GAL4 (FBtp0001168),
Ay-GAL4 (FBti0009983), UAS-GFP (FBti0003040),
UAS-lacZ (FBtp0000355), and UAS-Fringe 22a
(Kim et al., 1995).
Cell binding assay
Cell binding assays were conducted as described previously
(Bruckner et al., 2000;
Okajima et al., 2003
).
Antibody staining
Antibody staining was conducted as described previously
(Panin et al., 1997), using as
primary antibodies: mouse anti-WG (4D4, DSHB), rat anti-ELAV (DSHB), rabbit
anti-Notch (E. Giniger and M. Young), mouse anti-SXL (M8, DSHB), goat
anti-ß-galactosidase (Biogenesis), rabbit
anti-ß-galactosidase (ICN), rat anti-Serrate
(Papayannopoulos et al., 1998
)
and rabbit anti-MYC (Santa Cruz).
Sequence comparisons
The conservation of O-fucose sites was assessed by comparing the
sequences of: Notch from Drosophila melanogaster (P07207),
Lucilia cuprina (AAC36151), Boophilus microlus (AAN06819),
Lytechinus variegatus (7512075), Takifugu rubripes
(BAA20535), Danio rerio (18859115), Halocynthia roretzi
(7522619), Brachiostoma floridae (12057020) and Xenopus
laevis (1709335); Notch1 from Mus musculus (6679092), Rattus
norvegicus (6093542) and Homo sapiens (27894368); and Notch2
from Homo sapiens (24041035), Mus musculus (20138876) and
Rattus norvegicus (13242247).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A Notch overexpression assay
To assay the activity of altered Notch proteins, they were expressed under
the control of the ptc-Gal4 driver. This drives expression in a
stripe of cells along the anteroposterior (AP) compartment boundary, with
highest expression in cells at the boundary, and lower expression farther
away. Notch signaling was then assessed by examining the expression of Notch
targets in the developing wing imaginal disc, such as Wingless (WG). WG is
normally expressed along the DV border of the wing in response to Notch
activation (Fig. 1E,F)
(Diaz-Benjumea and Cohen,
1995; Kim et al.,
1995
; Rulifson and Blair,
1995
). WG also serves as a marker of position in developing wing
discs, because it is expressed in two rings in the wing hinge that do not
depend on Notch (Phillips and Whittle,
1993
).
Notch protein is normally expressed throughout the wing disc
(Fehon et al., 1991).
Nonetheless, when wild-type Notch is overexpressed, ectopic Notch activation
can be induced (Fig. 2A). The
pattern of Notch activation induced is instructive with regard to the
mechanisms that regulate Notch. First, ectopic Notch activity is only observed
near the DV boundary. This is consistent with it being due to an elevated
response to Notch ligands, which are expressed preferentially in cells near
the DV boundary during most of wing development
(Couso et al., 1995
;
Diaz-Benjumea and Cohen, 1995
;
Doherty et al., 1996
;
de Celis and Bray, 1997
).
Second, ectopic Notch activity is observed in cells that are along the
posterior edge of the ptc expression stripe. This suggests that cells
in which Notch is overexpressed can respond to Notch ligands expressed by
neighboring cells outside the ptc stripe, but that their ability to
signal is inhibited. This is presumably due to cis interactions
between Notch and its ligands (Jacobsen et
al., 1998
). Third, although weak ectopic Notch activation can
sometimes be observed in ventral cells, Notch activation is only consistently
observed in dorsal cells (Fig.
2A). As Serrate is expressed exclusively by dorsal cells in the
wing, this observation suggests that although both Delta-to-Notch and
Serrate-to-Notch signaling can be enhanced, Serrate-to-Notch signaling is much
more sensitive to Notch overexpression in the wing.
|
Normally, Fringe blocks Serrate-to-Notch signaling in dorsal cells. The ability of Serrate to signal when Notch is overexpressed implies that excess Notch can partially overcome the inhibitory effect of Fringe, presumably because at least some Notch is no longer sufficiently glycosylated. To examine this possibility, Fringe and Notch were co-expressed under ptc-Gal4 control. Indeed, co-expression of Fringe with Notch can inhibit ectopic Notch activation in dorsal cells (Fig. 2B).
Single O-fucose sites in the Abruptex region do not
influence Notch signaling
The induction of Notch activation observed when Notch is overexpressed
allows the assessment of mutant forms of the Notch receptor for their ability
to respond to ligand expressed by neighboring cells. At the same time, the
limited ectopic activation of Notch also enables the identification of
O-fucose sites that normally inhibit Notch activation. Expression of
Notch proteins that cannot be O-fucosylated in EGF24, EGF26 or EGF31,
as well as of an EGF24/EGF26 double mutant, all result in an ectopic
activation of Notch that is similar to that generated by wild-type Notch
(Fig. 3A-D). This induction of
ectopic Notch activation implies that they are all functional Notch receptors
that can respond to Notch ligands. At the same time, the absence of additional
Notch activation implies that they remain subject to the same regulatory
influences that limit normal Notch activation.
|
The pattern of Notch activation again suggests explanations as to its basis. First, the observation that Notch is activated throughout the ptc-stripe rather than just at its edge implies that cells overexpressing N-EGF12f can participate in signaling with neighboring cells. Second, the observation that a substantial increase in Notch activity is observed in dorsal cells but not in ventral cells implies that the ability of Serrate to signal to Notch is specifically increased, and/or the ability of Fringe to inhibit Serrate-to-Notch signaling is impaired. Third, the observation that Notch is activated throughout the wing pouch, rather than only near the DV border, is consistent with the observation that the ectopic Notch activation is sufficient to maintain the expression of Notch ligands.
To examine these inferences, we again examined the influences of Fringe and
Notch ligands. Strikingly, co-expression of Fringe with N-EGF12f does not
result in any noticeable decrease in Notch activation in dorsal cells
(Fig. 3F). This suggests that
mutation of the O-fucose site in EGF12, which is a substrate for
Fringe (Fig. 1D)
(Shao et al., 2003), renders
Notch insensitive to the inhibitory effect of Fringe on Serrate-to-Notch
signaling.
To confirm this, we assayed Notch activation in cells that simultaneously
expressed N-EGF12f and were mutant for Serrate or Delta. We
accomplished this using an adaptation of the MARCM method
(Lee et al., 2000), in which
the recombination event that creates mutant cells simultaneously results in
the loss of a transcriptional repressor, Gal80, and hence allows Gal4-driven
expression from UAS transgenes. UAS-Notch was expressed under the
control of a tubulin-Gal4 (tub-Gal4) driver in these
experiments. Although tub-Gal4 drives expression at lower
levels than ptc-Gal4, the results of ectopic Notch expression are
qualitatively similar. When wild-type Notch is overexpressed, Notch activation
is only observed along the edge of Notch expression, and only in cells near
the DV boundary (Fig. 4B). By
contrast, when N-EGF12f is overexpressed, Notch activation is no longer
restricted to the edge of Notch expression, and can occur anywhere in the
dorsal wing (Fig. 4A).
|
Unexpectedly however, we found that Delta is also required for the enhanced
Notch activation of N-EGF12f expression
(Fig. 4E). As the expression of
Notch ligands in the wing is maintained by a feedback loop, we reasoned that
both ligands might be required in N-EGF12f- expressing cells to generate the
levels of Notch activation required to maintain their own expression. Although
for simplicity Delta is generally portrayed as a ventral to dorsal signal in
the wing (Fig. 1E), Delta is
actually expressed to some degree on both sides of the compartment border
(Doherty et al., 1996;
de Celis and Bray, 1997
). Prior
analysis of the genetic requirements for Delta suggested that
Delta expression in dorsal and ventral cells might be partially
redundant, as clones of cells that are mutant for Delta and span the
compartment boundary can result in substantial loss of wing tissue, but
Delta mutant clones that are exclusively dorsal or ventral have
milder effects (Doherty et al.,
1996
; de Celis and Bray,
1997
). To investigate the possibility that dorsally-expressed
Delta could contribute to the maintenance of Serrate expression, we examined
Serrate expression in Delta mutant clones. Ventral or dorsal
Delta mutant clones cause only slight decreases in Serrate
expression, but large Delta mutant clones that span the DV boundary
can eliminate Serrate expression (Fig.
4H). The loss of expression is non-autonomous within Delta mutant
clones, and cell autonomous within Notch mutant clones
(Fig. 4H,I), consistent with
the inference that Serrate is a direct target of Notch signaling. Thus, we
suggest that Delta is best thought of not as a signal from ventral cells, but
rather a signal to dorsal cells, which can originate on either side of the
compartment border.
N-EGF12f is a functional Notch receptor, but does not rescue DV
boundary formation
The experiments described above demonstrate that the ectopic activation
induced by N-EGF12f is ligand-dependent. However, these experiments were all
carried out in cells that retain endogenous Notch. To exclude the possibility
that Notch activation induced by expression of N-EGF12f is mediated through
endogenous Notch, we used the MARCM method to create clones of cells that
simultaneously expressed N-EGF12f and were mutant for a null allele of
Notch, N55e11. Such clones in the dorsal part of
the wing continued to display elevated Notch activity (14/15 dorsal clones;
Fig. 5B). Thus, N-EGF12f is a
functional Notch receptor, which can transduce ligand-dependent signals,
resulting in the expression of downstream target genes.
|
To explore the activity of N-EGF12f further, we tested its ability to
rescue Notch function during embryonic neurogenesis. Notch plays a key role in
limiting the number of cells that will adopt the neural fate (reviewed by
Baker, 2000). In the absence of
zygotic Notch function, early development proceeds normally because of the
maternal contribution of Notch, but by stage 11 a neurogenic phenotype is
observed, in which ectodermal cells on the ventral side of the embryo become
neural instead of epidermal. The neurogenic phenotype of Notch
mutants can be rescued by the expression of Notch under the control of a
heterologous promoter (Seugnet et al.,
1997
). ELAV, a nuclear protein expressed specifically in neurons
(Robinow and White, 1991
), can
be used to visualize neural fate. Notch mutant embryos display a
massive increase in ELAV staining (Fig.
6A), but this increase is suppressed by the expression of Notch
under da-Gal4 control (Table
1) (Seugnet et al.,
1997
). Expression of NEGF12f in Notch mutant embryos
under da-Gal4 control similarly rescues the neurogenic phenotype of
Notch mutant embryos (Table
1, Fig. 6B). This
observation further supports the conclusion that N-EGF12f is a functional
Notch receptor. Moreover, because Delta is the only ligand that functions
during embryonic neurogenesis, this rescue experiment indicates that N-EGF12f
can respond to Delta signaling.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
By contrast, the phenotype of N-EGF12f is consistent with that which would be expected of a Notch receptor that had lost a functional site of glycosylation by Fringe. Expression of N-EGF12f results in an ectopic activation of Notch in dorsal wing cells that is insensitive to Fringe, yet dependent upon endogenous ligand expression. Binding studies further show that Serrate is able to bind to this mutant form of Notch even in the presence of Fringe, which contrasts with the lack of detectable Serrate binding to wild-type Notch expressed in the presence of Fringe. Based on these observations, we conclude that EGF12 is an essential site for inhibition of Serrate-to-Notch signaling by the Fringe glycosyltransferase (Fig. 8).
|
The importance of additional O-fucose sites is further underscored
by the distinct consequences of removal of O-fucose only at EGF12 by
the S to A mutation, compared with removal of O-fucose at all sites
by Ofut1 mutation or RNAi. Using a cell aggregation assay, Rebay et
al. found that EGF11 and EGF12 of Notch have a key role in ligand binding
(Rebay et al., 1991). Deletion
of EGF11 and EGF12 prevents aggregation between Notch-expressing cells and
Delta-expressing cells, and a construct including only EGF11 and EGF12 of
Notch is able to confer Delta-binding activity upon cells, albeit with
decreased efficiency compared with full-length Notch. Although a role for
other EGF repeats in ligand binding has been suggested based on the
consequences of expressing fragments of Notch in the wing imaginal disc
(Lawrence et al., 2000
), and
by cell aggregation experiments with mutant Notch proteins
(Lieber et al., 1992
), EGF11
and EGF12 have generally been considered to be the key EGF domains for ligand
binding. However, because RNAi of Ofut1 in S2 cells indicates that
O-fucose is required on Notch for binding to its ligands
(Okajima et al., 2003
;
Sasamura et al., 2003
), yet
O-fucosylation of EGF12 is not required for ligand binding, other
O-fucosylated EGF domains must also be required for Notch-ligand
interactions. Thus, multiple sites are subject to O-fucosylation, but
with different phenotypic consequences.
Among the 15 Notch receptors with 36 EGF repeats in sequence databases, an average of 20 of the 36 EGF repeats contain potential sites for O-fucosylation. However, only three EGF repeats contain O-fucose sites in all of these 15 Notch receptors: EGF12, EGF26 and EGF27 (Fig. 1A). Thirteen other EGF domains contain sites that are somewhat conserved (i.e. an O-fucose site is found in that repeat in 11 or more of the 15 Notch protein sequences), including EGF24 (13/15 Notch receptors) and EGF31 (14/15 Notch receptors). These conserved sites for O-fucosylation cluster in an N-terminal region, and in a more C-terminal region centered around the NAx mutations (Fig. 1A). This general pattern of conservation - most Notch receptors have many sites, but only a few sites are absolutely conserved - suggests that at least some aspects of OFUT1 and Fringe regulation might be achieved through glycosylation of regions of Notch, rather than through glycosylation of specific EGF repeats. The lack of effect of mutation of individual, highly conserved EGF repeats in the NAx region is consistent with this suggestion, and experiments to analyze the consequences of mutation of arrays of O-fucose sites are in progress.
Relationship between Notch-ligand binding affinity and signaling
Notch ligands activate Notch receptors expressed by neighboring cells, but
inhibit Notch receptors expressed by the same cell. Elevated expression of the
Notch extracellular domain can also inhibit the ability of ligands to signal
to neighboring cells (Jacobsen et al.,
1998). Thus, one apparent consequence of the transmembrane nature
of Notch ligands is that Notch activation depends not simply on the ability of
ligand to bind receptor, but also on a competition between intracellular and
intercellular interactions. Previously, most attention has focused on the
impact of different levels of expression on this competition. But the balance
in this competition can also be shifted by adjusting the affinity between
Notch and its ligands. Indeed, even though most studies have focused on the
ability of Fringe to inhibit the response of a cell to Serrate, the ability of
cells to send a Serrate signal appears to be enhanced by co-expression with
Fringe (Panin et al., 1997
;
Klein and Martinez Arias,
1998
), which is consistent with the idea that decreasing
intracellular Serrate-Notch interactions increases the amount of Serrate
available to signal to neighboring cells.
Cell-based binding assays indicate that the O-fucose site in EGF12
is not just important for Fringe-dependent inhibition: even the presence of
the O-fucose monosaccharide at this site inhibits Serrate binding.
The presence of an inhibitory site of O-fucosylation in EGF12 was
unexpected given the general positive requirement for O-fucose in
Notch signaling. However, we suggest that it can be rationalized in terms of a
competition between intracellular and intercellular Notch-ligand interactions.
The competition model implies that it is important, at least in certain
contexts, for Notch not to bind too strongly to its ligands. One such context
is probably the DV boundary of the Drosophila wing, because Notch
ligands are expressed on both sides of the compartment boundary, and Notch is
activated on both sides of the compartment boundary. Thus, we suggest that
N-EGF12f is unable to rescue normal Notch activation at the DV boundary
because its increased affinity for ligands enhances intracellular binding to a
degree that interferes with the ability of a cell to send and receive Notch
signals. Notably, EGF12 is apparently essential for both intercellular and
intracellular Notch-ligand interactions
(Rebay et al., 1991;
Jacobsen et al., 1998
;
Lawrence et al., 2000
).
The highly conserved presence of an O-fucose site in EGF12 suggests that inhibition of ligand binding by the O-fucosylation of EGF12 might be of widespread importance. However, if O-fucosylation of EGF12 was constitutive, it would simply counteract the positive influence of O-fucosylation at other sites. If, by contrast, O-fucosylation of EGF12 was regulated, then differential O-fucosylation of EGF12 could occur, and could serve as a mechanism of Notch regulation. Intriguingly then, EGF12 is distinguished from other potential O-fucose sites by the presence of an acidic amino acid (E or D) at the -2 position relative to the O-fucose attachment site (Fig. 1C). None of the other EGF repeats in Notch contain an acidic amino acid at this position, yet 13/15 Notch receptor proteins contain an acidic amino acid at this position in EGF12. We do not yet know what fraction of Notch receptors in a cell are modified at any of the potential sites of O-fucosylation, but the presence of this conserved sequence difference suggests that EGF12 might be O-fucosylated under different conditions, or with a different efficiency, than other EGF domains, and hence that differential fucosylation of this site might serve as a regulatory mechanism.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Baker, N. E. (2000). Notch signaling in the nervous system. Pieces still missing from the puzzle. BioEssays 22,264 -273.[CrossRef][Medline]
Bruckner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406,411 -415.[CrossRef][Medline]
Chen, J., Moloney, D. J. and Stanley, P.
(2001). Fringe modulation of Jagged1-induced Notch signaling
requires the action of beta 4galactosyltransferase-1. Proc. Natl.
Acad. Sci. USA 98,13716
-13721.
Couso, J. P., Knust, E. and Martinez Arias, A. (1995). Serrate and wingless cooperate to induce vestigial gene expression and wing formation in Drosophila. Curr. Biol. 5,1437 -1448.[Medline]
de Celis, J. F. and Garcia-Bellido, A. (1994).
Modifications of the notch function by Abruptex mutations in Drosophila
melanogaster. Genetics
136,183
-194.
de Celis, J. F. and Bray, S. (1997). Feed-back
mechanisms affecting Notch activation at the dorsoventral boundary in the
Drosophila wing. Development
124,3241
-3251.
de Celis, J. F. and Bray, S. J. (2000). The
Abruptex domain of Notch regulates negative interactions between Notch, its
ligands and Fringe. Development
127,1291
-1302.
de Celis, J. F., Barrio, R., del Arco, A. and Garcia-Bellido, A. (1993). Genetic and molecular characterization of a Notch mutation in its Delta- and Serrate-binding domain in Drosophila. Proc. Natl. Acad. Sci. USA 90,4037 -4041.[Abstract]
Diaz-Benjumea, F. J. and Cohen, S. M. (1995).
Serrate signals through Notch to establish a Wingless-dependent organizer at
the dorsal/ventral compartment boundary of the Drosophila wing.
Development 121,4215
-4225.
Doherty, D., Feger, G., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N. (1996). Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10,421 -434.[Abstract]
Fehon, R. G., Johansen, K., Rebay, I. and Artavanis-Tsakonas, S. (1991). Complex cellular and subcellular regulation of Notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J. Cell Biol. 113,657 -669.[Abstract]
Fleming, R. J. (1998). Structural conservation of Notch receptors and ligands. Semin. Cell Dev. Biol. 9, 599-607.[CrossRef][Medline]
Fleming, R. J., Gu, Y. and Hukriede, N. A.
(1997). Serrate-mediated activation of Notch is
specifically blocked by the product of the gene fringe in the dorsal
compartment of the Drosophila wing imaginal disc.
Development 124,2973
-2981.
Haines, N. and Irvine, K. D. (2003). Glycosylation regulates the Notch signaling pathway. Nature Rev. Mol. Cell. Biol. 4,786 -797.[Medline]
Harris, R. J. and Spellman, M. W. (1993). O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3,219 -224.[Abstract]
Heitzler, P. and Simpson, P. (1993). Altered
epidermal growth factor-like sequences provide evidence for a role of Notch as
a receptor in cell fate decisions. Development
117,1113
-1123.
Hicks, C., Johnston, S. H., diSibio, G., Collazo, A., Vogt, T. F. and Weinmaster, G. (2000). Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat. Cell Biol. 2,515 -520.[CrossRef][Medline]
Irvine, K. D. and Wieschaus, E. (1994). fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79,595 -606.[Medline]
Irvine, K. D. and Vogt, T. F. (1997). Dorsal-ventral signaling in limb development. Curr. Opin. Cell Biol. 9,867 -876.[CrossRef][Medline]
Jacobsen, T. L., Brennan, K., Arias, A. M. and Muskavitch, M.
A. (1998). Cis-interactions between Delta and Notch modulate
neurogenic signalling in Drosophila. Development
125,4531
-4540.
Ju, B. G., Jeong, S., Bae, E., Hyun, S., Carroll, S. B., Yim, J. and Kim, J. (2000). Fringe forms a complex with Notch. Nature 405,191 -195.[CrossRef][Medline]
Kelley, M. R., Kidd, S., Deutsch, W. A. and Young, M. W. (1987). Mutations altering the structure of epidermal growth factor-like coding sequences at the Drosophila Notch locus. Cell 51,539 -548.[Medline]
Kim, J., Irvine, K. D. and Carroll, S. B. (1995). Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell 82,795 -802.[Medline]
Klein, T. and Martinez Arias, A. (1998).
Interactions among Delta, Serrate and Fringe modulate Notch activity during
Drosophila wing development. Development
125,2951
-2962.
Klein, T., Brennan, K. and Martinez Arias, A. (1997). An intrinsic dominant negative activity of serrate that is modulated during wing development in Drosophila. Dev. Biol. 189,123 -134.[CrossRef][Medline]
Lawrence, N., Klein, T., Brennan, K. and Martinez Arias, A.
(2000). Structural requirements for notch signalling with delta
and serrate during the development and patterning of the wing disc of
Drosophila. Development
127,3185
-3195.
Lee, T., Winter, C., Marticke, S. S., Lee, A. and Luo, L. (2000). Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25,307 -316.[Medline]
Lieber, T., Wesley, C. S., Alcamo, E., Hassel, B., Krane, J. F., Campos-Ortega, J. A. and Young, M. W. (1992). Single amino acid substitutions in EGF-like elements of Notch and Delta modify Drosophila development and affect cell adhesion in vitro. Neuron 9, 847-859.[Medline]
Micchelli, C. A., Rulifson, E. J. and Blair, S. S.
(1997). The function and regulation of cut expression on the wing
margin of Drosophila: Notch, Wingless and a dominant negative role for Delta
and Serrate. Development
124,1485
-1495.
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. et al. (2000a). Fringe is a glycosyltransferase that modifies Notch. Nature 406,369 -375.[CrossRef][Medline]
Moloney, D. J., Shair, L. H., Lu, F. M., Xia, J., Locke, R.,
Matta, K. L. and Haltiwanger, R. S. (2000b). Mammalian Notch1
is modified with two unusual forms of O-linked glycosylation found on
epidermal growth factor-like modules. J. Biol. Chem.
275,9604
-9611.
Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228,151 -165.[CrossRef][Medline]
Okajima, T. and Irvine, K. D. (2002). Regulation of Notch signaling by O-linked fucose. Cell 111,893 -904.[Medline]
Okajima, T., Xu, A. and Irvine, K. D. (2003).
Modulation of Notch-ligand binding by Protein O-fucosyltransferase 1 and
Fringe. J. Biol. Chem.
278,42340
-42345.
Panin, V. M., Papayannopoulos, V., Wilson, R. and Irvine, K. D. (1997). Fringe modulates Notch-ligand interactions. Nature 387,908 -912.[CrossRef][Medline]
Panin, V. M., Shao, L., Lei, L., Moloney, D. J., Irvine, K. D.
and Haltiwanger, R. S. (2002). Notch ligands are substrates
for Protein O-fucosyltransferase 1 and Fringe. J. Biol.
Chem. 277,29945
-29952.
Papayannopoulos, V., Tomlinson, A., Panin, V. M., Rauskolb, C.
and Irvine, K. D. (1998). Dorsal-ventral signaling in the
Drosophila eye. Science
281,2031
-2034.
Phillips, R. G. and Whittle, J. R. (1993).
wingless expression mediates determination of peripheral nervous system
elements in late stages of Drosophila wing disc development.
Development 118,427
-438.
Rebay, I., Fleming, R. J., Fehon, R. G., Cherbas, L., Cherbas, P. and Artavanis-Tsakonas, S. (1991). Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67,687 -699.[Medline]
Robinow, S. and White, K. (1991). Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J. Neurobiol. 22,443 -461.[Medline]
Rulifson, E. J. and Blair, S. S. (1995). Notch
regulates wingless expression and is not required for reception of the
paracrine wingless signal during wing margin neurogenesis in Drosophila.
Development 121,2813
-2824.
Sakamoto, K., Ohara, O., Takagi, M., Takeda, S. and Katsube, K. (2002). Intracellular cell-autonomous association of notch and its ligands: a novel mechanism of notch signal modification. Dev. Biol. 241,313 -326.[CrossRef][Medline]
Sasamura, T., Sasaki, N., Miyashita, F., Nako, S., Ishikawa, H.
O., Ito, M., Kitagawa, M., Harigaya, K., Spana, E., Bilder, D. et al.
(2003). neurotic, a novel maternal neurogenic gene,
encodes an O-fucosyltransferase that is essential for Notch-Delta
interactions. Development
130,4785
-4795.
Seugnet, L., Simpson, P. and Haenlin, M.
(1997). Transcriptional regulation of Notch and Delta:
requirement for neuroblast segregation in Drosophila.
Development 124,2015
-2025.
Shao, L., Moloney, D. J. and Haltiwanger, R.
(2003). Fringe modifies O-fucose on mouse Notch1 at epidermal
growth factor-like repeats within the ligand-binding site and the abruptex
region. J. Biol. Chem.
278,7775
-7782.
Shi, S. and Stanley, P. (2003). Protein
O-fucosyltransferase 1 is an essential component of Notch signaling pathways.
Proc. Natl. Acad. Sci. USA
100,5234
-5239.
Shimizu, K., Chiba, S., Saito, T., Kumano, K., Takahashi, T. and
Hirai, H. (2001). Manic fringe and lunatic fringe modify
different sites of the Notch2 extracellular region, resulting in different
signaling modulation. J. Biol. Chem.
276,25753
-25758.
Struhl, G. and Greenwald, I. (2001).
Presenilin-mediated transmembrane cleavage is required for Notch signal
transduction in Drosophila. Proc Natl Acad Sci USA
98,229
-234.
Thomas, U., Jonsson, F., Speicher, S. A. and Knust, E.
(1995). Phenotypic and molecular characterization of SerD, a
dominant allele of the Drosophila gene Serrate.
Genetics 139,203
-213.
Wang, Y. and Spellman, M. W. (1998).
Purification and characterization of a GDP-fucose:polypeptide
fucosyltransferase from Chinese hamster ovary cells. J. Biol.
Chem. 273,8112
-8118.
Wang, Y., Shao, L., Shi, S., Harris, R. J., Spellman, M. W.,
Stanley, P. and Haltiwanger, R. S. (2001). Modification of
epidermal growth factor-like repeats with O-fucose. Molecular cloning and
expression of a novel GDP-fucose protein O-fucosyltransferase. J.
Biol. Chem. 276,40338
-40345.
Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonas, S. (1985). Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43,567 -581.[Medline]