1 Howard Hughes Medical Institute, Columbia University, New York, NY 10032,
USA
2 Department of Genetics and Development, Columbia University, New York, NY
10032, USA
3 Department of Biochemistry and Molecular Biophysics, Columbia University, New
York, NY 10032, USA
Author for correspondence (e-mail:
greenwald{at}cancercenter.columbia.edu)
Accepted 5 September 2005
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SUMMARY |
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Key words: Notch, Ras, Endocytosis, C. elegans
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Introduction |
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Crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways is important
for proper VPC patterning. In P5.p and P7.p, the presumptive 2° VPCs,
activation of LIN-12 results in the expression of multiple negative regulators
of the EGFR-Ras-MAPK pathway, restricting the effects of the inductive signal
to P6.p (Berset et al., 2001;
Yoo et al., 2004
). In P6.p,
the presumptive 1° VPC, the EGFR-Ras-MAPK pathway leads to the
transcription of lateral signal genes (Chen
and Greenwald, 2004
), but expression of these genes in P6.p is not
sufficient to activate LIN-12 in neighboring VPCs. Instead, LIN-12 must be
downregulated in P6.p in response to EGFR-Ras-MAPK activation in order for
lateral signaling to occur: the LIN-12 intracellular domain has a
`downregulation targeting sequence' (DTS; see
Fig. 1B), and if LIN-12 is
stabilized in P6.p, by removing the DTS, lateral signaling is compromised
(Shaye and Greenwald,
2002
).
The DTS contains a di-leucine sorting motif, and we have proposed that
endocytic trafficking of LIN-12 is altered upon EGFR-Ras-MAPK activation,
leading to degradation. Indeed, we found that if the DTS is removed, LIN-12 is
not internalized efficiently and instead accumulates on the apical surface of
the VPCs. Furthermore, modified LIN-12 trafficking in response to
EGFR-Ras-MAPK activation is likely to depend on the activity of at least one
trans-acting factor transcribed in response to activation of Ras, as
degradation of LIN-12 does not occur when the function of the SUR-2/Mediator
transcription activator complex is removed
(Shaye and Greenwald,
2002).
Endocytic downregulation has been well studied as a mechanism for
attenuating the activity of activated receptors (reviewed by
Sorkin and Von Zastrow, 2002).
Downregulation of LIN-12 is a novel variation on this theme, as LIN-12 is
downregulated as a consequence of activating a different signaling pathway.
LIN-12 is likely to undergo a generic trafficking process, involving a series
of protein sorting decisions that occur at different points in the endocytic
pathway (Fig. 1C) (reviewed by
Katzmann et al., 2002
;
Sorkin and Von Zastrow, 2002
;
Bonifacino and Traub, 2003
). At
the plasma membrane, receptors can be retained at the cell surface or be
sorted into invaginating endocytic vesicles for internalization. In early
endosomes, receptors destined to be recycled to the plasma membrane are sorted
from those marked for downregulation, which are trafficked to late endosomes.
En route to, or at, late endosomes, receptors marked for downregulation
undergo a crucial sorting step known as multivesicular endosome (MVE) sorting
(reviewed by Katzmann et al.,
2002
; Gruenberg and Stenmark,
2004
). From late endosomes, downregulated receptors are trafficked
to the lysosome, where they are degraded.
|
Here we address how and why LIN-12 is downregulated, through the analysis of mutations within and near the DTS and other engineered forms of LIN-12, and the identification and analysis of trans-acting factors that mediate downregulation. Our results suggest that LIN-12 is degraded via the MVE sorting pathway, that the extracellular domain of LIN-12 is the agent that inhibits lateral signaling, and that internalization without degradation is sufficient to relieve this inhibition.
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Materials and methods |
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Feeding RNAi
For alx-1(RNAi), we cloned the cDNA insert from clone pB8-1 (see
above) into the double-T7 promoter plasmid pPD129.36
(Timmons et al., 2001) to
generate plasmid p402. For wwp-1(RNAi), we cloned the cDNA insert
from clone yk1104a10 (kindly provided by Y. Kohara) into pPD129.36 to generate
plasmid p416. For lacZ(RNAi) we used plasmid pXK10
(Karp and Greenwald, 2003
).
RNAi plasmids were transformed into HT115 bacteria and used for RNAi as
described by Timmons and Fire (Timmons and Fire, 2001), except that IPTG was
omitted from the overnight seed culture and the plates contained 60 µg/ml
AMP and 6 mmol/l IPTG. Synchronized starved L1-stage arIs82
hermaphrodites were placed on bacterial lawns and grown at 25° until the
mid-L3 stage (about 18 hours). Larvae were then fixed and stained as described
below.
Immunofluorescence and image analysis
For lines expressing mutant proteins from lin-12 genomic
constructs (Figs 2,
3), eggs were collected and
grown for 42 hours at 20°C. For lines carrying egl-17p-driven
constructs (Fig. 6), eggs were
collected and grown for 32 hours at 25°C. Fixation, staining and image
acquisition were essentially as described
(Shaye and Greenwald, 2002),
except that the monoclonal antibody MH27 (Developmental Studies Hybridoma
Bank, USA), which recognizes AJM-1, an apical component of the adherens
junction (Priess and Hirsh,
1986
; Koppen et al.,
2001
), was used at a dilution of 1:600. For measurements of
endocytic puncta in LIN-12(+)::GFP and LIN-12(KtoA)::GFP images, we analyzed
single confocal sections from five individual animals. Measurements were done
with ImageJ software (National Institutes of Health, USA).
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Results |
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Other amino acids contribute to the activity of characterized di-leucine
motifs, including upstream negatively charged amino acids and serine/threonine
residues that can be phosphorylated to modulate the activity of these signals
(Bonifacino and Traub, 2003).
We compared the sequence of region 1 from C. elegans LIN-12 with the
corresponding sequence of GLP-1, the other C. elegans Notch protein,
which can functionally substitute for LIN-12
(Fitzgerald et al., 1993
), and
with LIN-12 and GLP-1 homologs for C. briggsae and C.
remanei (Fig. 1D). In
addition to the conserved leucine residues, we identified conserved serines
and a non-conserved threonine as potential sites of phosphorylation, and
conserved lysine residues as potential sites of ubiquitination.
LIN-12(S/TtoA)::GFP and LIN-12(KtoA)::GFP mutants effectively rescued the
sterility and lethality of lin-12(0) mutants
(Table 1), but had different
effects on downregulation.
LIN-12(S/TtoA)::GFP, like LIN-12(LLtoAA)::GFP, appears to accumulate mostly
at the apical membrane (Fig.
2G,H), suggesting that these residues are also important in
regulating internalization. Some LIN-12(S/TtoA)::GFP hermaphrodites display
mislocalization to the basolateral plasma membrane
(Fig. 2G, arrowheads),
suggesting that the Ser/Thr residues within the DTS may also modulate
post-internalization trafficking. Internalization is not affected by mutation
of the lysine residues; instead, LIN-12(KtoA)::GFP showed a striking
accumulation in large and irregularly shaped intracellular vesicles
(Fig. 2I,J arrowheads). We
measured the area of these vesicles (see Materials and methods), and found
that LIN-12(+)::GFP accumulated in vesicles that measured on average 0.250
µm2 (±0.078, n=66). By contrast,
LIN-12(KtoA)::GFP accumulated in vesicles that measured on average 0.442
µm2 (±0.227, n=122). This difference in size was
highly significant (P=2.8x1010, two-tailed,
unpaired Student's t-test). Therefore, we believe that
LIN-12(KtoA)::GFP accumulates in endocytic compartments distinct from those
where internalized LIN-12(+)::GFP is seen. These large vesicles may be late
endosomes and/or lysosomes, as these compartments tend to be significantly
larger and more pleiomorphic than earlier compartments in the endocytic system
(Gruenberg and Stenmark, 2004;
Patton et al., 2005
). Such
accumulation of LIN-12(KtoA)::GFP would be consistent with a block in MVE
sorting, which depends on ubiquitination and occurs at late steps in the
endocytic pathway.
|
If these residues were phosphorylated during the process of internalization and downregulation, then we would expect to see an effect of these mutations on subcellular localization or the promotion of downregulation in cells where we usually do not see it. Indeed, the phospho-mimicking mutations appeared to enhance internalization of LIN-12 from the apical plasma membrane, as LIN-12(S/TtoD)::GFP appeared to accumulate mostly in internal vesicles (Fig. 3A,B, white arrowheads). Additionally, even though these mutations did not promote downregulation in cells other than P6.p, we observed conspicuous GFP accumulation at what appeared to be the basolateral, instead of the apical, plasma membrane in the daughters of P5.p/P7.p (Fig. 3A,B, yellow arrowheads; compare with Fig. 2A,B).
However, phospho-mimicking mutations are not sufficient to promote downregulation in VPCs where the EGFR-Ras-MAPK pathway and SUR-2/Mediator are not active. This observation could indicate that a Ser/Thr kinase that phosphorylates the LIN-12 DTS is not a target, or at least not the only target, of sur-2. Alternatively, the phospho-mimicking mutations may functionally bypass the requirement for sur-2, but the basolateral localization of this mutant protein in P5.p and P7.p precludes effective downregulation. We address these questions below.
LIN-12(S/TtoD)::GFP downregulation is dependent on sur-2
If phosphorylation of serine/threonine residues were the only step
regulated by a target of the EGFR-Ras-MAPK pathway, then we would expect that
the phospho-mimicking mutations could bypass the requirement for
sur-2. However, LIN-12(S/TtoD)::GFP was not downregulated in
sur-2() hermaphrodites
(Fig. 3C,D,E). Thus, the
EGFR-Ras-MAPK pathway does not solely regulate a kinase that modifies these
residues to mediate LIN-12 downregulation. There may be at least one other
factor regulated by sur-2 required for downregulation. Alternatively,
the putative kinase that phosphorylates the LIN-12 DTS may also phosphorylate
another trans-acing factor required for downregulation. We note that
the LIN-12(S/TtoD)::GFP that persisted in the 1° lineage of
sur-2() hermaphrodites accumulated mostly at the basolateral
membrane (Fig. 3C,D, yellow
arrowheads), as in P5.p and P7.p daughters in a wild-type background
(Fig. 3A,B). This suggests that
the failure to downregulate LIN-12(S/TtoD)::GFP in P5.p and P7.p in wild-type
hermaphrodites is not due to the basolateral localization of this protein, but
instead that this re-localization is a consequences of reduced or absent
sur-2 activity in these cells.
|
|
We note that alx-1(RNAi) hermaphrodites resemble sur-2
mutant hermaphrodites, in that LIN-12 is internalized but not degraded
(Shaye and Greenwald, 2002),
raising the possibility that alx-1 is a target of sur-2.
However, a GFP::ALX-1 translational reporter (kindly provided by B. Grant)
appeared to be highly and ubiquitously expressed, including in all the VPCs
and their descendants (data not shown), suggesting that alx-1 is not
a transcriptional target of the EGFR-Ras-MAPK pathway. GFP::ALX-1 appeared to
accumulate in very large and pleiomorphic vesicular structures in the VPCs,
which seem likely to be C. elegans MVEs
(Fig. 4C).
WWP-1 is required for LIN-12 degradation, but not internalization
Our analysis of the DTS suggests the existence of a kinase that can
phosphorylate the serine/threonine residues of the DTS and a ubiquitin ligase
that targets the DTS-flanking lysines. The yeast two-hybrid screen did not
yield predicted serine/threonine kinases or ubiquitin ligases, so we
considered a candidate gene approach.
Unfortunately, there are approximately 230 predicted serine/threonine
kinases in C. elegans (WormBase release WS144, searched for Interpro
motif IPR002290), and it would be prohibitively difficult to test them all for
failure of downregulation. Although there are also many potential ubiquitin
ligases, we focused on the Nedd4 family of E3 ubiquitin ligases
(Ingham et al., 2004), because
Suppressor of Deltex [Su(dx)], a Drosophila Nedd4-like protein, was
known to be a negative regulator of Notch
(Cornell et al., 1999
), and the
mouse Nedd4-like protein Itch was also known to ubiquitinate mouse Notch1
(Qiu et al., 2000
;
McGill and McGlade, 2003
).
Analysis of the C. elegans genome for proteins with the domain
structure of Nedd4-family proteins (Fig.
4D) identified Y65B4BR.4 (WWP-1) (see also
Huang et al., 2000
) as the
apparent ortholog of Su(dx) and Itch (Fig.
4E).
We depleted wwp-1 activity by RNAi and examined the effect on LIN-12(+)::GFP accumulation (Materials and methods). About 62% of wwp-1(RNAi) hermaphrodites failed to downregulate LIN-12(+)::GFP (Fig. 5E,I), as opposed to 14% of lacZ(RNAi) hermaphrodites (Fig. 5A,I). This difference was highly statistically significant (P=3.3x104, two-tailed, unpaired Student's t-test), indicating that wwp-1 is required for LIN-12 downregulation. Additionally, LIN-12(+)::GFP was still able to accumulate in intracellular puncta in wwp-1(RNAi) hermaphrodites (Fig. 5E, arrowheads). These results lead us to conclude that wwp-1 is required for LIN-12 downregulation at a post-internalization step. A GFP::WWP-1 translational reporter, in which GFP is fused in frame to the amino terminus of WWP-1 in a genomic context, was widely expressed and appeared to accumulate in the cytoplasm of all VPCs and their descendants (data not shown), suggesting that wwp-1 is unlikely to be a transcriptional target of the EGFR-Ras-MAPK pathway.
Lateral signaling is normal in wwp-1(RNAi) and alx-1(RNAi) hermaphrodites: correlation between subcellular localization and lateral signal inhibition
If lateral signaling occurs, P5.p and P7.p undergo three rounds of
division, and their descendants stain with the adherens junction marker
AJM-1 (see Fig. 5B). If
lateral signaling fails, then P5.p and P7.p undergo one round of division and
their daughters join the hypodermal syncytium, so they do not stain with
AJM-1 (see Fig. 5H).
Deletion of the LIN-12 DTS results in a concomitant failure of downregulation
and lateral signaling (Fig.
5G,H) (Shaye and Greenwald,
2002
). By contrast, wwp-1(RNAi) and alx-1(RNAi)
hermaphrodites displayed significant persistence of LIN-12 without compromised
lateral signaling (Fig.
5C-F,I). Internalization appeared to be normal, but
post-internalization trafficking and degradation appeared to be affected,
suggesting that the crucial event that allows lateral signaling to occur is
clearance of LIN-12 from the surface. The experiments presented in subsequent
sections address why persistent LIN-12 may inhibit lateral signaling at the
cell surface, but not after internalization.
|
To test this model, we expressed a constitutively active form of LIN-12
that cannot be downregulated and assessed whether lateral signaling is
compromised. We activated LIN-12 by truncating its ectodomain (see
Struhl and Adachi, 2000) and
stabilized it in P6.p by removing the DTS. We expressed this form,
LIN-12(
E
DTS)::GFP, under the control of the egl-17
promoter, which is continuously expressed in P6.p after inductive signaling
(Burdine et al., 1998
;
Shaye and Greenwald, 2002
;
Yoo et al., 2004
). We found
that LIN-12(
E
DTS)::GFP was not downregulated
(Fig. 6A,I), and did not cause
a lateral signaling defect (Fig.
6B,I), indicating that inhibition of lateral signaling is not
caused by activation of LIN-12 in P6.p. GFP staining was found mostly in the
nucleus (Fig. 6A',A"),
consistent with the expected constitutive transmembrane cleavage and signal
transduction of this form. In addition, we found that
LIN-12(
E
DTS)::GFP did not appear to affect the 1° fate of
P6.p (Fig. 6B), suggesting that
a presumptive 1° cell is refractory to activated LIN-12.
As activated LIN-12 did not cause lateral signal inhibition, we considered the possibility that the extracellular domain of LIN-12, which was deleted from the activated form, inhibits the function of the DSL proteins expressed in P6.p. We made LIN-12(extra)::TM::GFP, which consists of the extracellular and transmembrane domains of LIN-12 and a cytosolic GFP tag (Fig. 6J). When this protein was expressed in P6.p, it was not downregulated (Fig. 6C,J), and appeared to accumulate mostly at the apical plasma membrane, as it was mostly restrained to the apical domain demarcated by AJM-1 (Fig. 6C',C"). LIN-12(extra)::TM::GFP caused a highly penetrant lateral signaling defect (Fig. 6D,J). Therefore, persistence of the LIN-12 extracellular domain is sufficient to cause lateral signal inhibition. The addition of region 1 to LIN-12(extra)::TM::GFP restored downregulation and relieved lateral signal inhibition (Fig. 6E,F,K), indicating that the mechanism by which LIN-12(extra)::TM::GFP inhibits lateral signaling is related to the inhibition caused by persistence of full-length LIN-12.
Apical localization of the LIN-12 extracellular domain is necessary for lateral signal inhibition
We assessed whether the DTS is sufficient to cause downregulation by adding
it to LIN-12(extra)::TM::GFP, and found that it was not
(Fig. 6G,L). However, we
noticed that this protein was mostly found outside the apical domain, and
instead appeared to accumulate on the basolateral membrane
(Fig. 6G',G"). Thus, the
DTS is not sufficient to promote downregulation, but appears to promote
basolateral re-localization or to inhibit apical localization. The
basolaterally localized LIN-12(extra)::TM::DTS::GFP protein is not degraded,
nor does it inhibit lateral signaling (Fig.
6H,L). These observations suggest that apical localization of the
LIN-12 extracellular domain is crucial for lateral signal inhibition.
We note that the subcellular localization of LIN-12(extra)::TM::DTS::GFP was similar to that of LIN-12(S/TtoD)::GFP, the phospho-mimicking mutant described above, in cells where sur-2 was not active (Fig. 3). This similarity leads us to speculate that the DTS is phosphorylated in LIN-12(extra)::TM::DTS::GFP, but this protein lacks other determinants required for degradation, such as the conserved lysines that may mediate ubiquitination.
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Discussion |
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Regulation of LIN-12 internalization
We have found that a di-leucine motif and serine/threonine residues in the
DTS are required for internalization, and that lysine residues near the DTS
are required for degradation (see below). A mutant form in which the DTS
serine/threonine residues have been mutated to alanine shows diminished
internalization of LIN-12 in all VPCs, suggesting that at least some of these
serine/threonine residues are likely to be constitutively phosphorylated to
promote basal internalization.
A mutant form in which the serine/threonine residues have been mutated to aspartate displayed enhanced internalization of LIN-12 from the apical plasma membrane in all VPCs and basolateral accumulation of LIN-12 in VPCs with reduced sur-2 activity, i.e. P5.p and P7.p in a wild-type background, or P5.p, P6.p and P7.p in a sur-2() background. We also found that the wild-type DTS was sufficient to cause basolateral accumulation when flanking sequences that mediate degradation were not present. This observation leads us to speculate that basolateral localization indicates a phosphorylated state of LIN-12 that would usually not be detected because such a protein is normally marked for degradation. Basolateral localization may represent an intermediate step in the normal downregulation process. If so, sur-2 may regulate the activity of a basolaterally located factor required for degradation of phosphorylated LIN-12, and thus this factor would not be found in P5.p and P7p. Alternatively, basolateral accumulation may be an aberrant consequence of the inability to degrade a phosphorylated LIN-12 in cells where sur-2 is not active. At this point, we are unable to distinguish between these possibilities.
The mutant form of LIN-12 in which the DTS serine/threonine residues had been mutated to aspartate was downregulated in P6.p, but was not ectopically downregulated in other VPCs. Furthermore, this form did not bypass the requirement for sur-2, as this protein accumulated in the basolateral membrane of P6.p in a sur-2() background. These observations suggest that phosphorylation of LIN-12 is not the limiting step in downregulation, and thus that the EGFR-Ras-MAPK pathway does not simply lead to transcription of a kinase that promotes LIN-12 phosphorylation. However, the nature of the link between EGFR-Ras-MAPK activation remains to be determined.
Regulation of LIN-12 post-internalization trafficking: MVE sorting and degradation
LIN-12 appears to be downregulated via MVEs. Although mutation of the
conserved lysines near the DTS does not affect internalization of LIN-12,
degradation in P6.p is blocked, and in all VPCs this mutant form accumulates
in large pleiomorphic internal vesicles. Furthermore, the ubiquitin ligase
WWP-1 and the MVE-associated factor ALX-1 are required for LIN-12 degradation
after internalization. As sur-2 mutants display a similar phenotype,
transcriptional targets of the EGFR-Ras-MAPK pathway may be involved in
directing LIN-12 to MVEs.
Mutating the conserved lysines near the DTS caused the `Multivulva' phenotype associated with constitutive LIN-12 activation (Table 1). This phenotype is consistent with an MVE sorting defect. If a transmembrane protein does not go through the MVE sorting step, then upon delivery to the lysosome its extracellular domain will be degraded whereas its intracellular domain will remain exposed to the cytosol. For LIN-12/Notch, the mechanism of signal transduction involves cleavage and release of the intracellular domain. Thus, if MVE sorting is disrupted, degradation of the extracellular domain in the lysosome could mimic ectodomain shedding, creating a substrate for Presenilin-dependent release of the intracellular domain of LIN-12, or perhaps would release the intracellular domain by an alternative mechanism.
Recent reports have described `ligand-independent' activation of
Drosophila Notch in late endosomes. In these studies, overexpression
of the protein Deltex was shown to promote internalization and accumulation of
Notch in late endosomes, correlated with activation of Notch signaling. It was
suggested that such endosomal activation of Notch might represent a novel and
relevant mode of activating this pathway
(Hori et al., 2004). However,
our finding that an apparent block in MVE sorting can lead to LIN-12
activation suggests an alternative explanation for the effect of Deltex
overexpression: the enhanced internalization and endosomal accumulation of
Notch may saturate the MVE sorting machinery, so that some Notch is not
correctly internalized into MVE lumenal vesicles, leading to degradation of
the extracellular domain without concomitant degradation of the intracellular
domain.
|
Both of these mechanisms may be utilized in vertebrate Notch proteins. Sequence analysis of vertebrate Notch proteins shows an intriguing inverse correlation between the presence of a di-leucine based motif and a PPXY signal. The corresponding juxtamembrane regions of vertebrate Notch1 and Notch2 proteins have a segment that is strikingly similar to the LIN-12 DTS, including conserved flanking lysines (Fig. 7), but these proteins do not have a conserved PPXY signal at their C-termini (data not shown). By contrast, most vertebrate Notch3 proteins appear more divergent in this region (Fig. 7), but possess a PPXY signal at their C-termini (data not shown); it is curious that zebrafish Notch3 lacks the PPXY motif (data not shown), but has instead a canonical di-leucine motif (Fig. 7). These observations raise the possibility that the two modes of internalizing Notch proteins (di-leucine based versus ubiquitination via a PPXY motif) have been conserved in different vertebrate Notch proteins through evolution. Perhaps other modes exist as well, as vertebrate Notch4 does not seem to have either of these conserved motifs (data not shown). Mutational analysis of these potential internalization sequences in vertebrate Notch proteins will be necessary to test their roles.
Regulation of DSL ligand activity by LIN-12/Notch in the signaling cell
We previously proposed that the lateral signaling defect of sur-2
was caused by the failure to downregulate LIN-12
(Shaye and Greenwald, 2002).
Subsequently, it was found that dsl gene transcription is regulated
by sur-2 (Chen and Greenwald,
2004
), and here, we have found that internalized LIN-12, as is
seen in sur-2 mutants (Shaye and
Greenwald, 2002
), did not appear to inhibit lateral signaling.
Thus, persistence of LIN-12 does not appear to be the basis of the
sur-2 lateral signaling defect; rather, loss of lateral signaling in
sur-2 mutants is likely to result simply from the failure to
transcribe the lateral signal.
We observed that expression of a constitutively active LIN-12 that could not be downregulated in P6.p did not affect the fate of this cell or its ability to signal laterally, implying that the principal role of LIN-12 downregulation is to permit DSL ligands to activate LIN-12 in P5.p and P7.p in a paracrine mode, rather than to prevent autocrine LIN-12 activation in P6.p. However, this result also indicates that activation of the EGFR-Ras-MAPK pathway in P6.p causes it to become refractory to activated LIN-12, suggesting another potentially novel mode of crosstalk between EGFR-Ras-MAPK and LIN-12/Notch signaling in P6.p.
In certain gain-of-function mutants or ectopic overexpression situations in
Drosophila, Notch also appears to be able to inhibit the ability of
Delta to signal laterally (Heitzler and
Simpson, 1993; Jacobsen et
al., 1998
). These results were interpreted as suggesting the
formation of a DSL-Notch inhibitory complex, but whether such interactions
occur during normal Drosophila development is not clear. Our results
establish that inhibition of ligand activity occurs at the surface of the
signaling cell, consistent with a DSL-Notch inhibitory complex. Furthermore,
our results suggests that relief of this inhibition by internalization of
LIN-12 appears to be part of the normal mechanism for coordinating
EGFR-Ras-MAPK-mediated inductive signal and LIN-12-mediated lateral signaling.
Given that the ability of LIN-12/Notch proteins to inhibit DSL signaling
activity appears to be conserved, and the presence of conserved endocytic
sorting motifs in all Notch proteins, there may be other natural situations in
which inhibition of DSL ligands by endogenous LIN-12/Notch proteins, and
regulated relief of such inhibition, may be relevant to patterning cell
fates.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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