dishevelled
Dishevelled acts in both wingless and frizzled signaling. It is interesting to note that many frizzled proteins, including Frizzled2, the putative Wingless receptor, contain a Ser/Thr-X-Val motif at their C-terminal end: this motif has been shown to interact with PDZ (or DHR) domains in a variety of proteins (Gomperts, 1996). DSH contains a PDZ domain, suggesting a direct interaction of DSH with Frizzled2 (Klingensmith, 1994). Unlike DFZ2, there is no canonical PDZ consensus binding site in the C-terminus of Frizzled.
Wingless signaling
generates a hyperphosphorylated form of DSH, which is associated with a membrane fraction.
Overexpressed DSH becomes hyperphosphorylated in the absence of extracellular WG and
increases levels of the Armadillo protein, thereby mimicking the wg signal (Yanagawa, 1995). DSH functions upstream, prior to the action of shaggy/ zw3, a serine-thronine kinase (Siegfried, 1994). armadillo is downstream of shaggy.
Dishevelled is phosphorylated by shaggy/zeste signaling in response to wingless. Overexpressed dsh results in an increase in cytoplasmic Armadillo. Deletion of the C-terminal basic domain of DSH protein has no effect on ARM accumulation. Mutation of the GLGF/DHR motif modifies DSH affect on ARM. (Yanagawa, 1995).
shotgun (DE-cadherin] transcription level is regulated through the Wingless pathway. Drosophila genetic studies suggest that in the Wingless (Wg) signaling pathway, the segment polarity
gene products, Dishevelled (Dsh), Zeste-white 3 (Zw-3), and Armadillo (Arm), work sequentially; wg
and dsh negatively regulate Zw-3, which in turn down-regulates Arm. To biochemically analyze
interactions between the Wg pathway and Shotgun, which binds to Arm, three proteins (Dsh, Zw-3, and Arm) were overexpressed in the Drosophila wing disc cell line (clone 8), which responds
to Wg signal. Dsh overexpression leads to accumulation of Arm primarily in the cytosol and elevation of
Shotgun at cell junctions. Overexpression of wild-type and dominant-negative forms of Zw-3
decreases and increases Arm levels, respectively, indicating that modulation in Zw-3 activity negatively
regulates Arm levels. Overexpression of an Arm mutant with an amino-terminal deletion elevates
Shotgun protein levels, suggesting that Dsh-induced Shotgun elevation is caused by the Arm
accumulation induced by Dsh. Moreover, the Dsh-, dominant-negative Zw-3-, and truncated
Arm-induced accumulation of Shotgun protein is accompanied by a marked increase in the
steady-state levels of Shotgun mRNA, suggesting that transcription of shotgun is activated by
Wg signaling. In addition, overexpression of shotgun elevates Arm levels by stabilizing Arm at
cell-cell junctions (Yanagawa, 1997).
Dishevelled protein is targeted by Casein kinase II.
Immunoprecipitated Dsh protein is associated with Casein
Kinase II. Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated
by CkII in vitro and in vivo, suggesting that CkII is at least one of the kinases that phosphorylates Dsh.
Overexpression of frizzled2, a Wingless receptor, also stimulates phosphorylation of Dsh, Dsh-associated
kinase activity, and association of CkII with Dsh. It is not known whether association of CkII with unphosphorylated Dsh occurs first, or whether phosphorylation of Dsh promotes association with CkII. Unphosphorylated Dsh clearly has some affinity for CkII, however, in vivo phosphorylated Dsh is associated with more CkII than is underphosphorylated Dsh. This suggests a model in which CkII can bind with low affinity to underphosphorylated Dsh and effect its phosphorylation. The phosphorylated Dsh then has a higher affinity for CkII, leading to an increase in the amount of Dsh-CkII complex. Phosphorylation of Dsh in response to the Wg signal, leads to the phosphorylation of Dsh but this is insufficient for the transduction of the Wg signal to Armadillo.
Thus the function of the phosphorylation of Dsh by CkII is unknown (Willert, 1997).
Wingless (Wg) treatment of the Drosophila wing disc clone 8 cells leads to Armadillo (Arm) protein elevation, and this effect has been used as the basis of in
vitro assays for Wg protein. Previously analyzed stocks of Drosophila Schneider S2 cells could not respond to added Wg, because they lack the Wg
receptor, Frizzled2. However, a line of S2 cells obtained from another source express both Frizzled-2 and Frizzled. Thus,
this cell line was designated as S2R+ (S2 receptor plus). S2R+ cells respond to addition of extracellular Wg by elevating Arm and Shotgun protein levels and by
hyperphosphorylating Dsh, just as clone 8 cells do. Moreover, overexpression of Wg in S2R+, but not in S2 cells, induces the same changes in Dsh, Arm,
and DE-cadherin proteins as induced in clone 8 cells, indicating that these events are common effects of Wg signaling, which occurs in cells expressing
functional Wg receptors. In addition, unphosphorylated Dsh protein in S2 cells is phosphorylated as a consequence of expression of Frizzled-2 or
mouse Frizzled-6, suggesting that basal structures common to various frizzled family proteins trigger this phosphorylation of Dsh. S2R+ cells are as
sensitive to Wg as are clone 8 cells, but theycan grow in simpler medium. Therefore, the S2R+ cell line is likely to prove highly useful for in vitro analyses of
Wg signaling (Yanagawa, 1998).
Thus expression of Dfz2 or Mfz6 induces phosphorylation of Dsh in S2 cells and a
small proportion of Dsh protein is phosphorylated in S2R+ and clone 8 cells. These results suggest that
expression of frizzled family proteins induces the basal phosphorylation of Dsh. In this regard, Casein kinase 2 (CK2), which binds to the PDZ domain of Dsh, is known to be the major
kinase responsible for phosphorylation of Dsh upon Dfz2 overexpression in S2 cells. Therefore, CK2
may take part in the basal phosphorylation of Dsh in Dfz2/S2, Mfz6/S2, clone 8 and S2R+ cells not
stimulated with soluble Wg. In addition, Frizzled
overexpression leads to translocation of Dsh from cytoplasm to plasma membrane. Overexpression of rat frizzled-1 has been shown to result in recruitment of
Xwnt-8 and XDsh to the plasma membrane in Xenopus embryos. Thus, it is possible that Dfz2 or Mfz6
expression induces translocation of at least a part of Dsh to the plasma membrane in S2 cells and that
this Dsh translocation in some way stimulates Dsh phosphorylation by CK2. However, it is not clear
whether CK2 also participates in Wg-induced hyperphosphorylation of Dsh or whether other kinase(s)
are activated by the binding of Wg to Dfz2 in clone 8, S2R+, and Dfz2/S2 cells and that these other kinases induce the
hyperphosphorylation. In view of the reports indicating association (probably indirect) between frizzled
family proteins and Dsh and the binding of Dsh to CK2, it is attractive to speculate that Wg binding
induces aggregation of Fz2 receptors, which, in turn, brings the Dsh-CK2 or other kinase complexes
close together, and this aggregation stimulates the Dsh phosphorylation by CK2 or other kinases in
these Dsh-kinase complexes. This could explain how Wg induces Dsh hyperphosphorylation in clone
8, S2R+, and Dfz2/S2 cells. However, it is noteworthy that Dfz2 overexpression leads to marked
phosphorylation of Dsh, but not to elevation of Arm, in S2 cells, indicating that phosphorylation of
Dsh, at least by Dfz2 overexpression, cannot activate the Wg signaling pathway by itself. Clearly,
further detailed experiments are necessary to evaluate the function of Dsh phosphorylation in Wg
signaling (Yanagawa, 1998 and references).
In Drosophila embryos the protein Naked cuticle (Nkd) limits the effects of the Wnt signal Wingless (Wg) during early segmentation.
nkd loss of function results in segment polarity defects and embryonic death, but how nkd affects Wnt signaling is unknown. Using
ectopic expression, it has been found that Nkd affects, in a cell-autonomous manner, a transduction step between the Wnt signaling components
Dishevelled (Dsh) and Zeste-white 3 kinase (Zw3). Zw3 is essential for repressing Wg target-gene transcription in the absence of a Wg
signal, and the role of Wg is to relieve this inhibition. Double-mutant analysis shows that, in contrast to Zw3, Nkd acts to restrain signal transduction when the Wg
pathway is active . Yeast two hybrid and in vitro experiments indicate that Nkd directly binds to the basic-PDZ region of Dsh. Specially
timed Nkd overexpression is capable of abolishing Dsh function in a distinct signaling pathway that controls planar-cell polarity. These results suggest that Nkd acts
directly through Dsh to limit Wg activity and thus determines how efficiently Wnt signals stabilize Armadillo (Arm)/ß-catenin and activate downstream
genes (Rousset, 2001).
The Drosophila eye is composed of mechanosensory bristles
present at vertices of ommatidia. Bristle formation is suppressed near the circumferential margin of the eye, and the degree
of suppression is least at the extreme dorsum of the head, typically
0-2 ommatidial diameters in width. Wg signaling, active at the circumference of
the developing eye where wg is expressed, is responsible for
this suppression of peripheral bristle formation. To assay the function of
nkd in eye bristle formation, the EGUF/hid method was used to make homozygous mutant nkd eyes in nkd/+ animals. In this technique, Flp-mediated
recombination between a chromosome mutant for nkd and a
chromosome harboring both recessive and dominant cell-lethal mutations
is specifically induced in the eye using the eyeless promoter.
During eye development, the only cells surviving are those that have
lost the cell-lethal chromosome through recombination, producing an eye
homozygous mutant for nkd. Examination of eyes mutant for the
strong allele nkd7E89 reveals, at the dorsum of the
eye, consistent eye bristle suppression 3-5 ommatidial diameters away
from the margin, with occasional closer bristles. This result
suggests that endogenous nkd regulates interommatidial bristle
suppression by antagonizing the effects of endogenous Wg in cells
farther than one cell diameter away from the Wg source (Rousset, 2001).
To determine how Nkd impinges on the Wg pathway, the
ability of Nkd to block the action of the positive regulators Wg, Dsh,
and Arm was tested. To do so, advantage was taken of a Drosophila eye
misexpression system. Production of Wg in a
subset of photoreceptor cells throughout the eye using a
sevenless promoter transgene (P[sev-wg]) prevents
formation of interommatidial bristles in a paracrine fashion;
otherwise, the eye is normal. Previous Nkd misexpression experiments did not indicate whether Nkd blocks Wg synthesis, Wg distribution, or cellular responses to received Wg. To distinguish between these possibilities, the GAL4/UAS binary expression system was used to evaluate the effect of
Nkd (UAS-nkd) on Wg-mediated eye bristle suppression. Misexpression of Nkd alone using multiple repeats of the eye-specific glass (gl) enhancer (GMR) to drive the yeast transcription factor GAL4 (P[GMR-GAL4]) has no visible effect on eye development. However, the combination of
sev-wg with nkd misexpression results in nearly
complete suppression of the P[sev-wg]-induced bristle-loss phenotype. Nkd misexpression did not alter the levels or distribution of Wg antigen, indicating that Nkd is probably blocking signaling events downstream from Wg (Rousset, 2001).
The effect of Nkd on the downstream Wg pathway components Dsh and
Arm was also tested using the GMR-GAL4 system. Dsh
misexpression (UAS-dsh) produces small, bristle-less eyes
devoid of ommatidia. Nkd strongly suppresses the Dsh
misexpression eye phenotype, restoring numerous bristles and ommatidia. If the Dsh misexpression eye phenotype is Wg-dependent, its
suppression by Nkd could be due to Nkd acting on Wg rather than on Dsh
or other downstream components. Previous work suggests that the Dsh
misexpression eye phenotype is Wg-independent. To confirm the Wg-independence of the Dsh phenotype, a dominant-negative form of Dfz2 (UAS-GPI-Dfz2) was coexpressed with either sev-wg or UAS-dsh.
UAS-GPI-Dfz2 effectively suppresses sev-wg-induced
bristle loss in the eye. Coexpression of UAS-GPI-Dfz2 and UAS-Dsh results in some eye necrosis, but it has negligible effects on the UAS-dsh eye phenotype. These results confirm that the Dsh misexpression effect in the eye is Wg-independent. Therefore, rescue of the UAS-dsh phenotype by Nkd is not an indirect effect due to suppression of Wg activity (Rousset, 2001).
GMR-driven expression of UAS-armS10, a
constitutively activated form of arm, also produces bristle
loss and failure of proper ommatidial development. Nkd coexpression had no effect on the Arm misexpression phenotype. Dsh and Arm misexpression phenotypes are not
affected by simultaneous expression of UAS-lacZ, indicating
that suppression of the dominant eye phenotypes by Nkd was not due to
GAL4 titration. The ability of Nkd to block effects of
Wg and Dsh but not Arm suggests that Nkd is acting at the level of, or
downstream from, Dsh but not downstream of Arm (Rousset, 2001).
The relationship between Nkd and Zw3 could not be determined by a
similar suppression test because both proteins are negative regulators
of Wg. In addition, the subtlety of the nkd phenotype in the
eye made this tissue unsuitable for analyzing the epistasis between
nkd and zw3. Instead, Zw3/Gsk3ß was overproduced in
nkd mutant embryos using genetic and mRNA injection methods:
Heat shock promoter (hsp70)-controlled GAL4 was used to drive
Zw3 production, or injections with Xenopus gsk3ß
mRNA. nkd mutants lack ventral denticle belts and are
considerably smaller than wild-type embryos. Overproduction
of Gsk3ß or Zw3 in nkd mutants results in partial to almost
complete restoration of denticle belts and restoration of more normal
embryo size. Because Zw3 restores denticles
to nkd mutants, Zw3 cannot act genetically upstream of the
defect in nkd mutants (i.e., by stimulating nkd
function) in the linear Wg pathway. Nkd therefore is likely to act
upstream of, or in a pathway parallel to, Zw3 and downstream from, or
at the level of, Dsh (Rousset, 2001).
The eye misexpression results suggest that Nkd antagonizes Wg
signaling at the level of, or downstream from, Dsh. Loss-of-function dsh clones reveal that Dsh acts autonomously in
Wg-responsive cells, suggesting that Nkd
must also act in Wg-responsive cells. Indeed, previous observations in
fly embryos suggest an initial requirement for nkd in cells
receiving the Wg signal. Because eye development allows fairly easy
production of sharply bounded clones, the eye was chosen to assess the
cell autonomy of Nkd action. Marked clones of Nkd-misexpressing cells
were produced in developing eyes and the range of Nkd action on
sev-wg was monitored (Rousset, 2001).
The flip-on GAL4 system was used to make random clones of cells misexpressing both Nkd and a cell-autonomous marker, green fluorescent protein (GFP), in eyes with excess Wg (sev-wg eyes). All clones examined showed suppression of bristle loss, with the suppression consistently within or immediately adjacent to GFP misexpression clones. No bristles were present outside the clones, indicating a local action of Nkd. To address whether Nkd was acting only in bristle precursor cells, and hence cell-autonomously, those cells were specifically marked with antisera against the Cut nuclear protein in pupal eye discs. In the vicinity of Nkd misexpression clones, there was a perfect correlation between GFP and Cut-labeled cells: all Cut-positive bristle precursor cells expressed GFP and hence Nkd; no GFP-negative/Cut-positive cells were found. These results suggest that Nkd acts within Cut-positive bristle precursor cells to antagonize the inhibitory effects of Wg on bristle cell differentiation (Rousset, 2001).
Cuticles derived from embryos lacking wg activity
(wg, dsh, or arm) have nearly continuous
fields of denticles, whereas HS-wg embryos, or those mutant
for the negative regulator zw3, secrete naked cuticle. Wg misexpression and double-mutant analyses show that Wg acts sequentially through Dsh, Zw3, and Arm. Embryos doubly mutant for wg and zw3 (zw3;wg), as well as zw3;dsh embryos, resemble zw3 embryos, whereas zw3;arm embryos resemble arm embryos, indicating that zw3 acts downstream from dsh and upstream of arm. Mutations in either nkd or zw3 give rise to a naked cuticle phenotype, with posterior expansion of en expression and ectopic wg expression in the developing embryo. However, in contrast to the naked cuticle phenotype of the zw3; wg embryo, the
wg;nkd embryo has a wg-like phenotype, indicating a dependence on Wg for the naked cuticle phenotype of nkd mutants (Rousset, 2001 and references therein).
To clarify the relationship between Nkd and other Wg pathway components
in the embryo, embryos were made doubly mutant for nkd and
dsh or arm, using both genetic means and RNA
interference (RNAi). Whereas the nkd gene is strictly zygotic, dsh has a maternal contribution that must be removed via germ-line clones to obtain the embryonic dsh phenotype. Females heterozygous for
nkd and carrying dsh germ-line clones were crossed to
males heterozygous for nkd.
Embryos derived from crosses using different combinations of
nkd and dsh alleles were counted and grouped
according to their cuticle phenotypes. The expected Mendelian ratios are
37.5% wild type (3/8), 37.5% dsh (3/8), 12.5% nkd
(1/8), and 12.5% dsh;nkd (1/8). Only three phenotypes could
be detected -- wild type, dsh, and nkd -- indicating that
the dsh;nkd mutants exhibit one of these phenotypes or die
before secreting cuticle. Whereas the observed percentages for the
wild-type and nkd categories are very close to the expected
percentages (38.2% and 14.3%, respectively), the percentage of
dsh embryos is significantly higher (47.5%), suggesting that
the dsh;nkdmutant resembles the dsh mutant (Rousset, 2001).
To confirm the dsh;nkd phenotype, RNAi experiments were performed. Injection of nkd double-stranded RNA (dsRNA) into
wild-type embryos efficiently mimics nkd loss of function:
76% of the injected embryos develop with greatly
reduced denticles compared to wild-type embryos. The majority of these mutant embryos (69%) show an intermediate to strong nkd cuticle phenotype, the others showing a weak expressivity characterized by a loss of only
a few denticles. RNAi was also attempted with dsh,
but, in contrast to nkd, both the penetrance and the
expressivity of the dsh phenotype are very weak. Increasing the dsh dsRNA concentration has little
effect, producing only a fusion between belts A4 and A5 in <5% of the
injected embryos and ruling out the utility of nkd and
dsh double injections. Instead nkd dsRNA
was injected into dsh embryos derived from germ-line clones. Half of the
collected embryos are wild type due to rescue by the paternal
X-chromosome. To score only
nonrescued dsh mutant embryos, females carrying
germ-line clones were crossed to males carrying an X-chromosome GFP balancer and
GFP-negative embryos were scored after eliminating GFP embryos. Injection of
nkd dsRNA into dsh embryos had no effect on the
dsh phenotype, confirming
that dsh;nkd double mutants resemble dsh embryos (Rousset, 2001).
The null allele armYD35 was used to generate arm;nkd double-mutant embryos. Embryos homozygous for this allele have a strong arm
phenotype, even without making germ-line clones. Male heterozygotes for
the strong alleles nkd7H16 or
nkd7E89 were crossed to females heterozygous for
armYD35 and nkd7H16. Since these crosses generate a majority of wild-type embryos (a ratio of nine wild type to seven mutants), only cuticles from unhatched embryos were counted; these unhatched embryos are expected to be mutant for arm (ratio 3:7, 42.9%), nkd (3:7,
42.9%), and arm;nkd (1:7, 14.3%). Like the dsh;
nkd embryos, the arm;nkd mutants do not exhibit a
distinct phenotype. The results show that the nkd phenotype is found at the expected frequency (42.6%), but the arm phenotype is over-represented (57.4%
instead of 42.9%), indicating that this category also contains the
arm;nkd embryos. Therefore, the arm;nkd mutant is
covered with denticles and resembles arm embryos (Rousset, 2001).
The double-mutant analysis indicates that the nkd phenotype
occurs only if wg, dsh, and arm genes are
active, confirming the requirement for Wg signaling to generate the
nkd phenotype. Zw3 constitutively represses Wg target-gene transcription, and
the role of Wg is to overcome this inhibition. These results indicate that Nkd, in contrast, is required to oppose Wg signal. Removal of nkd in the absence of wg, dsh, or arm has little effect on cuticle phenotype.
Accordingly, increased levels of Nkd do not modify the wg
mutant cuticle. The negative influence of Nkd could
be mediated by inhibition of Dsh activity, stimulation of Zw3 activity,
or by interactions with unknown pathway components. To test whether Nkd
can directly interact with known Wg signaling components, yeast
two-hybrid and in vitro binding assays were used (Rousset, 2001).
Expression in yeast of full-length Nkd protein fused to the GAL4
DNA-binding domain (GB-Nkd) does not activate transcription by itself. When GB-Nkd was coexpressed with Dsh fused to the activation domain of GAL4 (GAD-Dsh), strong
ß-galactosidase activity was detected, indicating an interaction
between Nkd and Dsh. The interaction between Dsh and Nkd was confirmed using
coimmunoprecipitation and glutathione-S-transferase (GST) pull-down assays. Three protein association assays indicate that Nkd and Dsh can directly interact, in keeping with the epistasis results that suggested a role for Nkd at the level of Dsh or Zw3 (Rousset, 2001).
Production of Nkd-GFP fusion protein in larval salivary glands
reveals striking colocalization with endogenous Dsh, stained with an
anti-Dsh antibody, indicating that the two proteins may also interact in vivo. However, an association between the two proteins was not detected using coimmunoprecipitation experiments with embryo extracts or lysates from Drosophila cell lines. The negative results may be due to protein complex dynamics, accessibility to antibodies, low levels of the complex in fly cells, as well as possible modes of regulation of the interaction, which are currently being investigated (Rousset, 2001).
The Dsh protein contains three defined domains: DIX, PDZ, and DEP. The DIX (Dishevelled, Axin)
domain shares homology with the C-terminal part of D-Axin, the PDZ
(PSD-95, Dlg, Zo-1) domain is a modular region involved in
protein-protein interactions, and the DEP (Dsh, Egl-10, Pleckstrin)
domain is usually found in signaling proteins, although its role
remains unclear. In addition, a stretch of basic residues is present
between the DIX and PDZ domains. Using both the yeast two-hybrid system
and GST pull-down experiments, the region in Dsh that binds Nkd was
defined. These results indicated that the central region of Dsh
containing the basic sequence and the PDZ domain is sufficient for
binding Nkd. The PDZ domain of Dsh is necessary for the
interaction with Nkd but, in contrast to proteins such as casein kinase
I or Frat1, it cannot efficiently bind Nkd by itself (Rousset, 2001).
Dsh is a branchpoint connecting two distinct signaling pathways in
Drosophila development: the Wg pathway and the planar cell polarity pathway (PCP). nkd mutant clones have normal planar cell polarity; so there is no detectable normal role for nkd in the PCP pathway. If
Nkd affects Dsh during Wg signaling, as the data suggest, then
appropriately timed overexpression of Nkd might be able to specifically
alter Dsh function in PCP signaling. Nkd was tested for its ability to
interfere with PCP signaling during a time when Fz and Dsh are not
appreciably participating in Wg signaling. Timed overexpression of Nkd
at 24 h after puparium formation (APF) produces adult flies with wing
hair polarity defects that are indistinguishable from those seen in
dsh1 mutant adults. dsh1 is an adult
viable allele of dsh that harbors a missense mutation in the
C-terminal DEP domain. Genetic tests have shown dsh1 to be a null allele for PCP signaling. The Nkd overexpression
polarity pattern is reproducible and qualitatively distinct from that
produced by complete loss of function of other known PCP mutants,
including fz or prickle (pk). The Nkd overexpression defect is also different from those associated with Fz or Dsh overexpression (Rousset, 2001).
The PCP phenotype associated with Fz overexpression is sensitive to the
dose of dsh. To determine whether Nkd
could similarly titrate Dsh from PCP signaling induced by Fz
overexpression, Fz and Nkd were simultaneously expressed. Indeed,
overexpressed Nkd suppresses the effects of excess Fz.
Neither excess Nkd nor decreased nkd dosage modifies the wing
bristle polarity of dsh1 mutant flies. The results suggest that Nkd can specifically interfere with Dsh function in planar cell polarity and that this effect requires wild-type Dsh protein (Rousset, 2001).
That Nkd can act through Dsh has important implications for the dynamic
control of Wg/Wnt signaling. By acting upstream of the ß-catenin
degradation machinery, Nkd may determine how effectively a given dose
of Wnt causes ß-catenin accumulation and target-gene activation and
thereby influences the sensitivity of a cell to a given amount or type
of Wnt ligand. The kinetic and dynamic parameters of the feedback loop
involving Wg, Dsh, and Nkd may play key roles in controlling the
duration and extent of signaling activity. Tight regulation of this
feedback loop is clearly important for normal Drosophila
embryonic development, and in various animals it may be subject to
spatial and temporal adjustments during evolution or during disease
progression. Future experiments will test how the interaction between
Nkd and Dsh affects responses to Wnt signals during development and may
provide insight into Wnt-associated tumor progression (Rousset, 2001).
Wnt signaling regulates ß-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by ß-catenin and the other by Jun kinase (JNK). A Dsh-associated kinase has been purified from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the ß-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signaling through ß-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the ß-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-ß-catenin signaling, indicating a link between two important developmental pathways (Sun, 2001).
Dsh is progressively phosphorylated in Drosophila during embryonic development. To identify the kinase that phosphorylates Dsh, various domains of Dsh were expressed as glutathione S-transferase (GST)-fusion proteins and tested for their ability to bind the kinase from Drosophila embryos. Only the GST fusion protein containing the middle domain, DM, but not the N-terminal or C-terminal domain of Dsh, has a high affinity for the kinase. The Dsh-associated kinase activity that is precipitated with GST-DM increases as Dsh becomes progressively phosphorylated during development. Similarly, there is a kinase activity present in Dsh immunoprecipitates. An in-gel kinase experiment has shown that the kinase activity, eluted from Dsh immunoprecipitates, is associated with two major bands and a minor band on a polyacrylamide gel. These bands of 110 kDa, 64 kDa and 130 kDa (minor band) are the kinase and its major fragment. The region on Dsh that interacts with the kinase was mapped more precisely to a 36 amino acid segment, DM5, which is N-terminal to the PDZ domain. Importantly, this region in Dsh is well conserved among Drosophila, C. elegans, Xenopus and mammals (Sun, 2001).
Using GST-DM5 precipitation and in vitro phosphorylation as an assay to monitor the Dsh-associated kinase activity, the kinase was purified 60,000-fold from Drosophila embryos and several peptide sequences were then used to clone the cDNA of this kinase. The cDNA clones encoded a protein kinase that is highly homologous to the C. elegans protein PAR-1 (85% identity in the kinase domain and 42% identity overall), which is known to regulate embryo polarity but whose function in Wnt signaling has not been explored (Sun, 2001).
To determine whether dPAR-1 and Dsh form a complex in vivo, endogenous Dsh was immunoprecipitated from Drosophila cells with an affinity purified anti-Dsh antibody and dPAR-1 was detected in the immunocomplex by Western blot using anti-dPAR-1 antibody. These data indicate that dPAR-1 is the kinase responsible for the activity that co-purified with Dsh in the GST pull-down experiment. The PAR-1 protein, prepared by in vitro translation, strongly phosphorylates the middle region of Dsh in vitro. Co-expression of Dsh with wild-type dPAR-1, but not kinase-negative dPAR-1 (dPAR-1 KN), in NIH3T3 cells results in a reduction in Dsh mobility on SDS-PAGE. These data indicate that dPAR-1 directly phosphorylates Dsh in vitro and in vivo (Sun, 2001).
If PAR-1 acts in Wnt signaling, PAR-1 activity might be expected to change in response to Wnt. Drosophila clone-8 cells were stimulated with conditioned medium containing Wingless and it was confirmed that Dsh becomes phosphorylated, and Armadillo is stabilized. The kinase activity of dPAR-1 was also measured under the same conditions. Several experiments have shown that soluble Wg stimulates dPAR-1-specific activity. Wg treatment does not increase the amount of dPAR-1 protein that interacts with GST-DM5. The increased dPAR-1 activity correlates with enhanced Dsh phosphorylation and elevation of Arm levels in Clone-8 cells (Sun, 2001).
To examine whether PAR-1 is required in the Wnt pathway, endogenous PAR-1 activity was suppressed by expressing a kinase-negative PAR-1 (hPAR-1Balpha KN). Chinese hamster ovary (CHO) cells were used because good expression from transfected DNA can be achieved and these cells have a well-characterized response to Wnt. Three hallmarks of Wnt activity were measured: Dsh phosphorylation, ß-catenin stabilization and transcriptional activation. Wnt treatment of CHO cells retards the mobility of Dvl proteins (mammalian homologs of Dsh) on SDS-PAGE, and phosphatase treatment increases the mobility of the Dvl band, thereby confirming that Dvl is phosphorylated in response to Wnt. The hPAR-1Balpha KN suppresses Wnt-mediated phosphorylation of endogenous Dvl proteins, as shown by the reduced amount of a retarded Dvl band. This result is consistent with the data that PAR-1 phosphorylates Dsh in vitro and in cells. Furthermore, both human and Drosophila PAR-1 KN strongly suppress Wnt-induced ß-catenin stabilization. The kinase-negative forms of hPAR-1A, hPAR-1B and hPAR-1C all strongly suppress Wnt-mediated transcriptional activation (measured by LEF1/TCF reporters) in a dose-dependent manner. Importantly, co-expression of wild-type hPAR1 can override the suppression mediated by hPAR-1 KN, indicating that hPAR-1 KN affects Wnt signaling by specifically blocking the effects of endogenous PAR-1 in cells. However, hPAR-1 KN is unable to inhibit the transcriptional activation induced by overexpression of ß-catenin, consistent with PAR-1's role in regulation of Dsh function upstream of ß-catenin (Sun, 2001).
All three human PAR-1 homologs strongly potentiated the responses to Wnt or Dvl3 in CHO cells. The hPAR-1 proteins alone do not activate the signaling pathway but require co-expression of either Wnt or Dvl, indicating that there is synergy between hPAR-1 and other components of the Wnt pathway. The specificity of these responses was verified by their dependency on the co-expression of the LEF1 transcription factor, which is required for Wnt signaling. Furthermore, the effects of hPAR-1 were suppressed by Axin, a negative regulator of the Wnt pathway that acts downstream of Dsh. As predicted, hPAR-1 overexpression does not alter the gene response induced by overexpression of ß-catenin, consistent with the idea that PAR-1 regulates Wnt signaling at a step upstream of Axin and ß-catenin (Sun, 2001).
Dsh also activates an alternative pathway that culminates in the stimulation of JNK in mammalian cells and controls of cell polarity in Drosophila and in vertebrates. Expression of Dvl in NIH3T3 cells induces JNK activation. Interestingly, hPAR-1 strongly suppresses the ability of Dvl3 to activate JNK in NIH3T3 cells. This inhibitory effect depends on the kinase activity of hPAR-1, because a mutation in hPAR-1, which is expected to inactivate its kinase activity (hPAR-1 KN), leads to a loss of its inhibitory effect on JNK activation. Together, these data indicate that PAR-1 promotes the participation of Dsh in the Wnt-ß-catenin pathway but suppresses its function in the JNK pathway, thereby acting as a switch for the downstream responses mediated by Dsh protein (Sun, 2001).
Several studies have shown that Wnt signaling controls axis specification during Xenopus embryogenesis. Ectopic ventral expression of Wnt before the onset of zygotic transcription results in duplication of the dorsal axis: this duplication can be blocked by dominant-negative forms of Frizzled, Dsh and CKIepsilon. Likewise, interference with negative regulators of the Wnt pathway, such as GSK3ß or Axin, by ventral expression of dominant negatives also induces axis duplication. In order to further the understanding of the function of PAR-1 in vertebrates, whether PAR-1 KN can alter Wnt signaling was investigated by injecting PAR-1 RNA into Xenopus embryos. Ventral blastomere injection of XWnt8 RNA results in significant axis duplication, as expected. Co-injection of human or Drosophila kinase-negative PAR-1 RNA significantly inhibits XWnt8-induced axis duplication in injected embryos. Both human and Drosophila kinase-negative PAR-1 can exert functional effects on the Wnt pathway in Xenopus axis duplication experiments (Sun, 2001).
To examine the function of dPAR-1 in Wg signaling in Drosophila embryos, double-stranded RNA-mediated interference (RNAi) was used to generate a dpar-1 loss-of-function phenotype. The ventral epidermis of a wild-type embryo is covered with alternating smooth cuticle and denticle belts. The Wg pathway is required to specify the fate of the epidermal cells that secrete smooth cuticle. RNAi of dpar-1 causes localized ectopic denticle formation and fusion of denticle belts, resembling the wg RNAi phenotype previously described. A similar wg phenotype is also generated with RNAi using double-stranded RNAs derived from different regions of the dpar-1 transcript (Sun, 2001).
Whether Dsh itself or perhaps other proteins bound to Dsh are the most important substrates of PAR-1 remains to be determined. Previous reports have shown that CKIepsilon, a positive regulator of the Wnt pathway, also interacts with Dsh. However, CKIpsilon seems to exert its major effect on more downstream components of the pathway. GBP/Frat (GSK3-binding protein) also interacts with Dsh and has a positive role in the Wnt pathway. Detailed biochemical studies will be required to elucidate the precise function(s) of PAR-1 and its functional interactions with Dsh and other regulators of Dsh in Wnt signaling (Sun, 2001).
Planar cell polarity signaling in Drosophila requires the receptor Frizzled and the cytoplasmic proteins Dishevelled and Prickle. From initial, symmetric subcellular distributions in pupal wing cells, Frizzled and Dishevelled become highly enriched at the distal portion of the cell cortex. A Prickle-dependent intercellular feedback loop is described that generates asymmetric Frizzled and Dishevelled localization. In the absence of Prickle, Frizzled and Dishevelled remain symmetrically distributed. Prickle localizes to the proximal side of pupal wing cells and binds the Dishevelled DEP domain, inhibiting Dishevelled membrane localization and antagonizing Frizzled accumulation. This activity is linked to Frizzled activity on the adjacent cell surface. Prickle therefore functions in a feedback loop that amplifies differences between Frizzled levels on adjacent cell surfaces (Tree, 2002).
Using a glutathione-S-transferase (GST) pull-down assay, it was found that full-length, in vitro translated PkSple (one of the three alternatively spliced forms of Prickle) binds to a GST-Dsh fusion protein. To determine the domain dependency of this interaction, domains of Pk were tested for binding to individual Dsh domains. A Pk construct containing just the conserved PET and LIM domains that are common to all Pk isoforms (PkPETLIM), but not either domain individually, is sufficient for binding to full-length Dsh. Furthermore, of the three defined Dsh domains, DIX, PDZ, and DEP, it was found that PkPETLIM binds a construct containing the DEP domain but not other Dsh domains. The binding between Pk and Dsh was confirmed, and the domain specificity was verified, using a yeast two-hybrid assay. PkPETLIM fused to a DNA binding domain interacts specifically with the Dsh DEP domain but not with the other domains of Dsh. It is concluded that Pk, via its conserved PET and LIM domains, binds the Dsh DEP domain (Tree, 2002).
Since the Dsh DEP domain is required for membrane localization, it was hypothesized that Pk antagonizes Fz activity by interfering with Dsh membrane localization. To test this, it was necessary to find conditions in which Pk activity is uncoupled from Fz/Dsh activity in the adjacent cell. Fz-dependent Dsh localization was therefore reconstituted in cultured cells. In U20S cells, transfected GFP-tagged Dsh (Dsh::GFP) can be seen in punctate patches in the cytoplasm. On cotransfection with Fz, Dsh::GFP is translocated to the cell membrane, as was seen in a similar frog animal cap assay. To test whether the physical interaction between Pk and Dsh affects Dsh membrane localization, Fz, Dsh::GFP, and Pk were cotransfected. Dsh is localized in the cytoplasm in 90% of the cotransfected cells, resembling cells transfected with Dsh::GFP alone. In contrast, Pk lacking the PET domain is severely impaired in its ability to block Dsh membrane localization. Pk, therefore, interferes with Dsh membrane association in this heterologous system and by extension may interfere with Dsh membrane localization at the proximal boundary of pupal wing cells. Since Dsh is required to generate asymmetric localization of Fz, it is suggested that blocking Dsh membrane localization also inhibits the accumulation of Fz at the proximal boundary (Tree, 2002).
Fz activity on the distal side of the cell is linked to the accumulation of Pk on the proximal side of the adjacent cell and that accumulation of Pk suppresses Fz/Dsh localization at the proximal cell cortex. In this way, Fz and Pk appear to function in a feedback loop rather than a linear signaling pathway. The ordering of these members of the PCP pathway was analyzed through epistasis analysis. The loss-of-function phenotypes of pkpk-sple, dsh, and fz are very similar, suggesting that they activate the same signaling mechanism. However, these similarities make classical genetic epistasis tests difficult to interpret. In previous work, Fz overexpression phenotypes have been used to show that Fz signaling requires Dsh. These results were interpreted as implying that the PCP genes form a linear pathway with Dsh acting downstream of Fz. Consistent with this is the finding that Dsh localization requires Fz protein. In contrast, other observations do not fit this model. For example, localization of Fz requires Dsh, a presumed 'downstream' element of the pathway. Fz localization also requires the other presumed downstream elements Pk, the 7 cadherin Flamingo (Fmi), and novel TM protein Van gogh/Strabismis (Vang/Stbm) (Tree, 2002).
To clarify these results, several further epistasis tests were performed. Dsh was found to be required to generate a Fz overexpression phenotype and Fz is required to produce a Dsh overexpression phenotype. In addition, Pk is required for the Dsh and Fz overexpression phenotypes. These results argue against a simple linear pathway but are consistent with Fz, Dsh, and Pk participating in a feedback loop (Tree, 2002).
Evidence is provided that Fmi acts with Pk on the proximal side of the cell. Like Pk, overexpression of fmi using ptc-Gal4 causes hairs to point toward the midline of the wing. fmi overexpression clones show distal-domineering nonautonomy, phenocopying fz loss-of-function and pk overexpression clones. Thus, it is possible that Fmi could be acting similarly to Pk in feedback amplification of PCP signaling. Fmi is likely to be present at both sides of the cell, but it may act with Pk at the proximal side to suppress Fz signaling. However, Fmi must have an additional function since, within and surrounding fmi clones, very little Pk and Dsh localize to cell boundaries. In addition to functioning with Pk to suppress Fz/Dsh activity, it is suggested that Fmi could have a role in stabilizing the Fz complex at the membrane, without which no signaling occurs (Tree, 2002).
Whether differences in Pk activity between adjacent cells affect the localization of Dsh and Fz was examined. pk was overexpressed in the posterior of the wing (using UAS-pk, engrailed-Gal4), and Dsh and Fz localization were assessed near the edge of the pk overexpression domain. Dsh and Fz were relocalized to the antero-posterior cell boundaries, indicating that accumulation of Dsh and Fz occurs at interfaces between cells expressing high and low levels of Pk. Furthermore, within the posterior domain, Dsh and Fz are seen to accumulate to higher levels than in the anterior, consistent with a role for Pk in promoting PCP signaling. Two conclusions are drawn from these observations: (1) Dsh and Fz localization occur preferentially at boundaries between cells where one cell expresses high levels and the other expresses lower levels of Pk; (2) providing high levels of Pk amplifies Fz signaling, as measured by the increased accumulation of both Fz and Dsh at cell peripheries. In the wild-type, therefore, Pk at the proximal side of the cell drives Fz and Dsh accumulation at the distal side of the adjacent cell. Conversely, providing high levels of Fz induces higher levels of Pk accumulation (Tree, 2002).
Since Pk accumulates at boundaries between cells expressing different levels of Fz, and Dsh accumulates at boundaries between cells expressing different levels of Pk, it is suggested that Fz and Dsh on one side and Pk on the opposite side of an intercellular boundary form a self-organizing complex. Indeed, evidence is found for this in the posterior, pk-overexpressing domain of the same wings. In the posterior, Dsh and Fz accumulate in discrete patches around the cell peripheries. Simultaneous staining for Pk, Dsh, and Fmi, or Fz and Fmi, reveals that all four proteins colocalize to these patches. Thus, Pk, Dsh, Fmi, and Fz form self-organizing complexes bridging adjacent cells (Tree, 2002).
Various models have been proposed for the distribution of PCP information within Drosophila epithelia. Regardless of which model is correct, it is likely that this cue induces only a slight asymmetry within each cell. In the Drosophila wing, this initial small asymmetry may result in slightly higher levels of Fz accumulation or activation on the distal than on the proximal side of cells. However, this initial level of asymmetry is insufficient to polarize the cytoskeletal architecture of the cell and is undetectable as assessed by Dsh localization. Fz and Dsh localization subsequently become highly polarized through the action of a feedback amplification loop. Although the mechanism for such a feedback loop has not been demonstrated, it has previously been speculated that Fz activity on distal cell surfaces might promote expression of an inhibitory Wnt molecule that would suppress Fz activity on the adjacent cell surface. This study demonstrates suppression of Fz activity on adjacent cell surfaces but finds that it is mediated through asymmetric localization of Pk, which antagonizes Fz/Dsh accumulation by blocking cortical Dsh localization (Tree, 2002).
A model is proposed for a Pk-dependent feedback mechanism that functions across each P-D cell interface to amplify the difference between the initial levels of Fz/Dsh activity. Pk and Dsh are initially distributed nearly symmetrically, but in largely nonoverlapping distributions around the cells' apices. As a result of an initial, slight asymmetry in one or more components, Pk suppresses Fz accumulation on the proximal side of the cell by antagonizing Dsh cortical localization. Dsh association with the cortex is required for asymmetric Fz accumulation. Reduced proximal Fz accumulation then decreases Pk activity on the adjacent, distal side of the neighboring cell, allowing even greater Dsh cortical localization and Fz accumulation. Conversely, Pk localization is induced by Fz localization on the neighboring cell. The result is the highly asymmetric distribution of Fz, Dsh, and Pk observed at 30 hr APF. This mechanism is bidirectional, constituting a negative feedback loop that is predicted to be unstable; once any asymmetry is initiated, high levels of Fz/Dsh are promoted on one side of the boundary and low levels on the other. Fmi is proposed to play two roles. The localization of all components to the apical cell cortex depends on Fmi, which is thought to be on both sides of the boundary. Since Fmi overexpression mimics Pk overexpression, it is proposed that Fmi is activated selectively on the proximal side of the cell, where it works with Pk to suppress Fz/Dsh activity. In essence, the function of this regulatory loop depends on the balance of forces regulating Dsh membrane association on either side of the boundary. Fz recruits Dsh to the cell cortex, and Pk blocks this recruitment. Once an initial asymmetry is induced, these forces become different on either side of the boundary, and the feedback mechanism amplifies the differences (Tree, 2002).
Because fmi overexpression produces polarity phenotypes very similar to those seen with pk overexpression, it is likely that Fmi activity is also polarized across cell boundaries and participates in blocking Fz/Dsh accumulation on the distal side of the interface. However, Fmi has been proposed to exist on both sides of the boundary, and Fmi is required for the efficient localization of Fz, Dsh, and Pk to boundaries. Fmi, therefore, appears to have an additional, complex-stabilizing function. Diego (Dgo), an ankyrin repeat protein, is required for PCP signaling and localizes to P-D cell boundaries, though it has not been possible to resolve on which side of the boundary Diego is found. Dgo may function as a scaffold for assembly of PCP-signaling components (Tree, 2002).
The core PCP protein Van Gogh (Vang, also known as Strabismus [Stbm], a transmembrane protein, is likely to be involved in the feedback amplification mechanism. In a vang/stbm mutant background, Fz is localized symmetrically to the membrane in the same manner as in a pk null background, and vang/stbm has been proposed to function downstream of pk. Interestingly, in Xenopus and zebrafish, Stbm binds Dsh and seems to toggle its activity between the canonical Wnt and a PCP-like signaling pathway. Stbm antagonizes canonical Wnt signaling and activates a PCP-like pathway that regulates convergent extension and induces JNK signaling. It is proposed that both fly and vertebrate Vang/Stbm may function together with Pk, facilitating PCP signaling by antagonizing Dsh activity (Tree, 2002).
The feedback amplification mechanism described here provides a clue to understanding the long-standing problem of domineering nonautonomy. Loss-of-function clones of fz, vang/stbm, and to a lesser extent pk induce polarity phenotypes in neighboring wild-type tissue. The feedback loop model predicts that loss of Fz will disrupt the localization of these components in the neighboring distal cells, causing their polarity to reverse. Furthermore, if the immediate clone neighbors have reversed polarity, this could then cause the reversal to propagate over a distance. This phenomenon is observed near the lateral borders of a fz clone, where Pk accumulation is seen to run parallel to the clone, even at a distance from the mutant cells. Thus, the reversal in Pk localization may underlie, in part, the domineering nonauontomy observed within wild-type tissue distal to fz clones (Tree, 2002).
Once asymmetrically localized, the Fz/Dsh complex must direct reorganization of the cytoskeleton by both determining the location for prehair initiation and by limiting the number of prehairs to one. Rho and Rho-associated kinase directly regulate myosins to limit the number of prehairs. The recently described Formin homology protein, Daam1, links Dsh to Rho during vertebrate PCP-like signaling, and a Drosophila homolog may function similarly. However, further studies will be required to understand how localized Fz and Dsh orient prehair initiation (Tree, 2002).
Planar polarity decisions in the wing of Drosophila involve the assembly of asymmetric protein complexes containing the conserved receptor Frizzled. This study analyses the role of the Van Gogh/strabismus gene in the formation of these complexes and in determination of cell polarization. The Strabismus protein becomes asymmetrically localized to the proximal edge of cells. In the absence of strabismus activity, the planar polarity proteins Dishevelled and Prickle are mislocalized in the cell. Strabismus binds directly to Dishevelled and Prickle and is able to recruit them to membranes. Furthermore, the putative PDZ-binding motif at the C terminus of Strabismus is not required for its function. A two-step model is proposed for assembly of Frizzled-containing asymmetric protein complexes at cell boundaries. First, Strabismus acts together with Frizzled and the atypical cadherin Flamingo to mediate apicolateral recruitment of planar polarity proteins including Dishevelled and Prickle. In the second phase, Dishevelled and Prickle are required for these proteins to become asymmetrically distributed on the proximodistal axis (Bastock, 2003).
The subcellular localiaation of Stbm protein was investigated during wing morphogenesis using both a Stbm-YFP (Stbm yellow fluorescent protein) expressing transgene and using specific antibodies raised against Stbm. During the third instar stage, Stbm-YFP in the wing pouch localizes unevenly around apicolateral cell boundaries. Based on its molecular homology as a multi-pass transmembrane protein, it is assumed that Stbm is present in the outer cell membrane. At 18 hours of pupal life, a similar pattern is seen, Stbm-YFP still being distributed patchily in an apicolateral ring. By 24 hours, there is preferential distribution of Stbm-YFP to proximodistal cell boundaries; this distribution is clearly present at 28 hours and persists until at least 32 hours, which corresponds to the time of trichome initiation. The pattern seen with Stbm antibodies confirms that Stbm-YFP is a faithful reporter of Stbm protein distribution (Bastock, 2003).
The timecourse and distribution of Stbm broadly fits that described for other planar polarity proteins such as Fmi, Fz, Dsh and Pk-Sple. Consistent with this, good colocalization is found between Stbm-YFP and other polarity proteins. The localization of Stbm-YFP to the adherens junction zone was confirmed by costaining for Armadillo distribution. Conversely, Stbm-YFP shows no overlap with the distribution of Discs-Large, which is localized in the septate junction region. Mosaic analysis revealed that Stbm-YFP becomes preferentially distributed to the proximal edges of cells with no appreciable accumulation at distal edges (Bastock, 2003).
The three putative multipass transmembrane proteins Fmi, Fz and Stbm all play important roles in the first step of localizing planar polarity proteins to the apicolateral adherens junction zone. It is thought that Fmi acts at the top of the hierarchy in this process, since, in its absence, negligible amounts of any planar polarity proteins become apicolaterally localized. Stbm is also key, because, in its absence, both Fz and Fmi recruitment are reduced. Additionally, Stbm is also required for Dsh apicolateral recruitment and for efficient localization of Pk to membranes. Fz is not significantly required for apicolateral recruitment of Fmi, but is partly needed for apicolateral localization of Stbm and is absolutely required for apicolateral localization of Dsh. Hence, in the absence of Fmi, Fz or Stbm, one or more planar polarity proteins do not become apicolaterally localized and the process of asymmetric localization on the proximodistal axis does not occur (Bastock, 2003).
An important question is which of these factors are directly binding together, in the process of apicolateral recruitment. So far no direct protein interactions have been reported for Fmi, although it is tempting to speculate that Fmi might bind directly to Fz and Stbm in the process of apicolateral recruitment. However, Fz is able to recruit Dsh to membranes in a heterologous cell type, suggesting that these factors directly interact. In addition, vertebrate Stbm and Dsh homologs have been shown to directly interact. Direct interactions are shown between Drosophila Stbm and Dsh, and Stbm and Pk. This suggests a model in which Dsh and Pk both become apicolaterally localized as a result of direct interactions with Fz and Stbm. Notably, in the absence of Stbm, Pk accumulates in the cytoplasm, suggesting that its interaction with Stbm is important for regulating its level in the cell in addition to its subcellular localization (Bastock, 2003).
At the stage when the planar polarity proteins are apicolaterally localized, but prior to the stage when they are asymmetrically localized on the proximodistal axis of the wing, it is possible that they are present in either 'symmetric' or 'asymmetric' complexes assembled across cell-cell boundaries. If the complexes were symmetric, then Fmi, Fz, Stbm, Pk and Dsh would all be present in a complex together on the same side of the cell-cell boundary. Such symmetric complexes would then subsequently evolve into asymmetric complexes, with Fz/Dsh at distal cell edges and Stbm/Pk at proximal cell edges and Fmi on both sides. Alternatively, the initial apicolateral complexes formed could be asymmetric, with Fz/Dsh always on the opposite side of the cell-cell boundary from Stbm/Pk. These asymmetric complexes would initially be randomly oriented relative to the axes of the wing, but would gradually become aligned to the proximodistal axis. The possibility is favored that planar polarity protein complexes are initially symmetric, since Stbm directly interacts with Dsh and these molecules colocalize during earlier stages of wing development. However, it has been reported that Pk and Dsh-GFP do not precisely colocalize in early pupal wings: this observation supports the early presence of asymmetric complexes (Bastock, 2003).
After the apicolateral recruitment of planar polarity proteins, over a number of hours their localization alters such that they become asymmetrically distributed on the proximodistal axis of the wing. Although Dsh and Pk play negligible roles in the apicolateral recruitment of proteins, both are required for this subsequent proximodistal redistribution. Since overexpression of both factors leads to the accumulation of polarity proteins at apicolateral cell boundaries, it is suggested that they both function to promote the assembly and/or stabilization of protein complexes. Removal of the function of the planar polarity gene dgo also blocks asymmetric proximodistal localization but not apicolateral localization of other polarity proteins. Furthermore, overexpression of Dgo causes an accumulation of other polarity proteins at cell boundaries similar to that seen when Dsh and Pk are overexpressed. Therefore, it is proposed that Dsh and Pk act together with Dgo in the assembly of asymmetric complexes (Bastock, 2003).
Recently, it has been proposed that the function of Pk in asymmetric complex assembly is to antagonize Dsh localization to membranes. This model is mechanistically attractive, in providing an explanation for the formation of asymmetric complexes in which Dsh and Pk are found on opposite sides of cell-cell boundaries. However, it is found that in the presence of Stbm, Dsh and Pk will colocalize at the same membranes. Furthermore, it was not possible to show an effect of overexpressing Pk on the association of Fz and Dsh at membranes. In addition, high level Pk expression in vivo does not cause Dsh to lose its membrane localization but instead appears to increase levels of Dsh at the membrane. Resolution of these issues will require a more detailed understanding of the composition and properties of the protein complexes involved (Bastock, 2003).
Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is ß-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, ß-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble ß-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3ß [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of ß-catenin. In Wnt signaling, this complex is inhibited; as a consequence, ß-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. This study shows that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling (Cliffe, 2003).
To identify further components of the Wingless pathway that are required for Axin relocation to the membrane, Axin-GFP was examined in various mutants. In sgg mutants, there are no significant changes in the subcellular distribution of the Axin-GFP dots, and their relocation to the plasma membrane in +Wg cells appears normal. Likewise, the few residual GFP-Axin in +Wg cells of APC double mutants are associated with the plasma membrane. Thus, neither GSK3 nor APC are required for relocation of Axin-GFP to the plasma membrane. Interestingly however, none of the Axin-GFP dots are associated with the plasma membrane in dsh mutants; Wingless is still expressed in these mutants at this stage). This is the case even if Wingless is coexpressed with Axin-GFP in these mutants. Thus, Dsh is the most downstream-acting component of the Wnt pathway that is required for the relocation of Axin-GFP to the plasma membrane (Cliffe, 2003).
It was asked whether overexpressed Dsh may mediate additional relocation. In wing discs, GFP-Dsh is associated with apicolateral regions of the plasma membrane whether or not Wingless signals. In the embryo, GFP-Dsh is expressed very weakly, and it can only be detected in late stages when Wingless has ceased to signal in the epidermis. In these -Wg cells, GFP-Dsh is detectable in the cytoplasm throughout the embryo and forms occasional dots; it is also weakly associated with the plasma membrane. Notably, overexpressed Dsh causes additional relocation of GFP-Axin from the cytoplasm to the plasma membrane. Most embryos show wide zones of membrane-associated Axin-GFP spanning +Wg cells that alternate with narrow zones of cytoplasmic Axin-GFP dots coincinding with -Wg cells, but, occasionally, the membrane relocation is seen throughout the embryo. This suggests that the additional Dsh-mediated relocation depends on low levels of Wingless signaling (Cliffe, 2003).
Overexpressed Dsh results in a partially naked cuticle, with only small denticles remaining. Thus, Dsh may inhibit endogenous Axin by relocating it to the plasma membrane. Consistent with this, limited restoration of naked cuticle is seen if Axin-GFP is coexpressed with Dsh, and an abundance of small denticles are seen that are sparse in wg mutants or in cuticles expressing Axin-GFP alone. As in the case of Wingless, this suppression of the activity of Axin-GFP is mild, suggesting that the putative limiting component is upstream of Dsh. This component may be Arrow, given that Arrow can bind to Axin and that an interaction between these two components in mammalian cells is induced by Wnt signaling (Cliffe, 2003).
Therefore, a possible model is that the Axin complex and Dsh are associated with the same vesicles, which may be recycling endocytic vesicles. Dsh may target these vesicles constitutively to the plasma membrane, where the Axin complex can interact potentially with Wnt receptors. This complex may be retained at the plasma membrane as a result of a Wnt-induced interaction between Axin and LRP/Arrow, and this retention may allow its subsequent inactivation. It is noted that LRPs are thought to recycle to the plasma membrane through endocytic vesicles, like their rapidly recycling LDL receptor relative. Recycling vesicles may thus provide a platform for APC-mediated assembly of the Axin complex and may convey this complex to the plasma membrane for inactivation by Wnt receptors (Cliffe, 2003).
Epithelial planar cell polarity (PCP) is evident in the cellular organization of many tissues in vertebrates and invertebrates. In mammals, PCP signalling governs convergent extension during gastrulation and the organization of a wide variety of structures, including the orientation of body hair and sensory hair cells of the inner ear. In Drosophila melanogaster, PCP is manifest in adult tissues, including ommatidial arrangement in the compound eye and hair orientation in wing cells. PCP establishment requires the conserved Frizzled/Dishevelled PCP pathway. Mutations in PCP-pathway-associated genes cause aberrant orientation of body hair or inner-ear sensory cells in mice, or misorientation of ommatidia and wing hair in Drosophila. This study provides mechanistic insight into Frizzled/Dishevelled signalling regulation. The ankyrin-repeat protein Diego binds directly to Dishevelled and promotes Frizzled signalling. Dishevelled can also be bound by the Frizzled PCP antagonist Prickle. Strikingly, Diego and Prickle compete with one another for Dishevelled binding, thereby modulating Frizzled/Dishevelled activity and ensuring tight control over Frizzled PCP signalling (Jenny, 2005).
Planar cell polarity (PCP) signaling generates subcellular asymmetry along an axis orthogonal to the epithelial apical-basal axis. Through a poorly understood mechanism, cell clones that have mutations in some PCP signaling components, including some, but not all, alleles of the receptor frizzled, cause polarity disruptions of neighboring wild-type cells, a phenomenon referred to as domineering nonautonomy. A contact-dependent signaling hypothesis, derived from experimental results, is shown by reaction-diffusion, partial differential equation modeling and simulation to fully reproduce PCP phenotypes, including domineering nonautonomy, in the Drosophila wing.
This work suggests that Fz does not require a Wnt ligand in PCP signaling but that its activity is regulated by interactions between neighboring cells and differential levels of the cytoplasmic mediators Pk and Dsh. The sufficiency of this model and the experimental validation of model predictions reveal how specific protein-protein interactions produce autonomy or domineering nonautonomy (Amonlirdviman, 2005).
As the understanding of cellular regulatory networks grows, system behaviors resulting from feedback effects have proven sufficiently complex so as to preclude intuitive understanding. The challenge now is to show that enough of a network is understood to explain such behaviors. Using mathematical modeling, the sufficiency of a proposed biological model is shown and its properties studied, to demonstrate that it can explain complex PCP phenotypes and provide insight into the system dynamics that govern them (Amonlirdviman, 2005).
Many epithelia are polarized along an axis orthogonal to the apical-basal axis. On the Drosophila adult cuticle, each hexagonally packed cell elaborates an actin-rich hair that develops from the distal vertex and points distally. Genetic analyses have identified a group of PCP proteins whose activities are required to correctly polarize these arrays. The domineering nonautonomy adjacent to cell clones mutant for some, but not other, PCP genes has not yet been adequately explained. For example, in the Drosophila wing, Van Gogh/strabismus (Vang; encoding a four-pass transmembrane protein) clones disrupt polarity proximal to the mutant tissue, whereas null frizzled (fz; encoding a seven-pass transmembrane protein) alleles disrupt polarity distal to the clone. Models to explain this phenomenon have often invoked diffusible factors, referred to as factor X or Z, because they have not yet been identified. It is proposed instead that the observed behaviors of known PCP proteins are sufficient to explain domineering nonautonomy (Amonlirdviman, 2005).
Fz and other PCP signaling components accumulate selectively on the distal or proximal side of wing cells. Evidence has been provided that these proteins function in a feedback loop that amplifies an asymmetry cue, which converts uniform distributions of PCP proteins into highly polarized distributions. The proposed feedback mechanism depends on several functional relationships. Fz recruits Dishevelled (Dsh; a cytoplasmic protein) to the cell membrane. In addition, Fz promotes the recruitment of Prickle-spinylegs (Pk; a LIM domain protein) and Vang to the cell membrane of the adjacent cell. Feedback is provided by the ability of Pk (and Vang) to cell-autonomously block Fz-dependent recruitment of Dsh. This feedback loop functions strictly locally, between adjacent cells. Global directionality is imposed through the agency of the novel transmembrane protein Four-jointed and the cadherins Dachsous and Fat (Ft). Widerborst, a regulatory subunit of protein phosphatase 2A, accumulates asymmetrically within each cell and is required to bias the feedback loop. Although the mechanism by which Ft biases the direction of the feedback loop is unknown, one possibility is that Ft may direct Widerborst distribution (Amonlirdviman, 2005).
However, it is not readily apparent that this biological model does not readily explain the complex patterns observed in fields of cells containing mutant clones, and it has been argued that it cannot account for some of the observed phenotypes. Indeed, progress in understanding PCP signaling has been hampered by an inability to deduce, given a particular signaling network hypothesis, definitive links between molecular genetic interventions and tissue patterning effects. For example, although it is apparent that removing Dsh or Fz would disrupt the feedback loop, it is not obvious how the feedback loop in adjacent wild-type cells responds, such that dsh mutant clones behave autonomously, whereas for most fz alleles, mutant clones behave nonautonomously. Interestingly, though, for some fz alleles, mutant clones produce an almost cell-autonomous phenotype. As another example, Pk overexpression promotes the asymmetric accumulation of Dsh and Fz, despite the role of Pk in the feedback loop as an inhibitor of Dsh membrane recruitment (Amonlirdviman, 2005).
A mathematical model has been developed based on the described feedback loop and an initial asymmetry input representing the global directional cue. Although mathematical modeling cannot prove the correctness of the underlying biological model, the ability of the mathematical model to capture the known behaviors of the system proves the feasibility of the biological model, provides testable hypotheses, and yields insight into the factors contributing to autonomy and nonautonomy (Amonlirdviman, 2005).
The features of the biological feedback loop model have been represented as a mathematical reaction-diffusion model that describes the concentrations of Dsh, Fz, Vang, and Pk throughout a network of cells. Although the mechanisms that underlie the local feedback loop are not fully understood, the essential logic of this feedback loop is preserved by representing these interactions as binding to form protein complexes (Amonlirdviman, 2005).
Inhibition of Dsh membrane recruitment by Pk and Vang is represented in the mathematical model as an increase in the backward reaction rate of reactions in which Dsh binds Fz (or Fz complexes) by a factor dependent on the local concentration of Pk and Vang. The specific mechanism for the introduction of the directional bias into the feedback loop network is not known. Two forms of a global biasing signal were therefore implemented, and the results using either of these models were similar. The resulting mathematical model consists of a system of 10 nonlinear partial differential equations. With a given set of model parameters, an array of cells could then be simulated, and the resulting hair pattern assigned on the basis of the final distribution of Dsh (Amonlirdviman, 2005).
The model parameters, including the initial protein concentrations, reaction rates, and diffusion constants, were not known, and so these parameters were identified by constraining them to result in specific qualitative features of the hair pattern phenotypes. A sensitivity analysis showed that the model results are not highly sensitive to the precise parameter values and suggests that the conclusions regarding the feasibility of the model are valid for considerable ranges of parameters (Amonlirdviman, 2005).
In simulated wild-type cells, Dsh and Fz localize to the distal membrane, and Vang and Pk localize to the proximal membrane, as is seen in vivo. Simulated clones of cells lacking fz function disrupt polarity in wild-type cells distal to the clones, whereas simulated clones lacking Vang function disrupt polarity on the proximal side of the clones. Simulated clones lacking dsh function result in the disruption of polarity within the mutant cells, but only show a mild effect outside of the clones. The nearly, though not fully cell autonomous, phenotype is similar to that which is observed experimentally. Clones lacking all pk function show only a subtle phenotype. Examination of protein distributions shows that the results are highly concordant with published observations. Similarly, simulated overexpression clones produce results closely mimicking observed experimental results. In simulations and in wings, relatively small clones lacking a global biasing signal show no phenotype, demonstrating that not all cells need to respond to the global directional signal for the feedback loop to cooperatively align all of the cells (Amonlirdviman, 2005).
Previously, it was found that Pk overexpression in the posterior wing domain enhances the accumulation of Fz and Dsh at cell boundaries, despite the observed ability of Pk and Vang to block Dsh recruitment. Consistent with these results, Dsh and Fz are seen in a simulation of this experiment to accumulate to higher levels in the region overexpressing pk than in the wild-type region, and they accumulate perpendicular to the wildtype orientation near the anterior-posterior boundary (Amonlirdviman, 2005).
The results suggested a mechanistic explanation for the difference between autonomous and nonautonomous fz alleles. Because the nearly autonomous fz alleles (fzJ22 and fzF31) have phenotypes similar to dsh clones, it is hypothesized that these alleles may be selectively deficient in complexing with Dsh, but normal in their ability to complex with Vang. Simulations of clones in which the interaction was disrupted between Dsh and Fz by reducing the corresponding forward reaction rates produced nearly cell autonomous polarity phenotypes (Amonlirdviman, 2005).
This hypothesis makes two easily testable predictions. (1) Fz autonomous proteins should be present in the membrane and should recruit Vang to the adjacent membrane, whereas Fz nonautonomous protein should not recruit Vang. It has previously been shown that GFP-tagged FzJ22, expressed in a wild-type background, is present at the apical cell cortex, but remains symmetrically distributed, a distribution in accordance with the simulation of this condition. Examining this further, it was found that in cells adjoining clones of the autonomous fzF31 allele, Vang is recruited to the boundary between wild-type and mutant cells, whereas substantially less Vang is recruited to those boundaries in cells adjoining clones of the nonautonomous fzR52 allele. Thus, Fzautonomous proteins recruit Vang to the opposing cell surface, whereas nonautonomous alleles do not. (2) Autonomous Fz proteins should fail to recruit Dsh. Indeed, it was found that both are substantially impaired in Dsh recruitment, though somewhat less impaired than the very strong, nonautonomous fzR52 allele. Thus, strong fz alleles, many of which fail to accumulate Fz protein, display no or severely impaired interaction with Dsh and Vang, whereas autonomous alleles have impaired interaction with Dsh, but retain substantial ability to recruit Vang to the adjacent membrane. Notably, simulated overexpression of Fz with impaired Dsh interaction also produced the correct polarity disruption in cells proximal to the clones (Amonlirdviman, 2005).
The Dsh1 protein produces nearly autonomous clones, and it carries a mutation in its DEP domain, which is required for membrane localization; autonomous fz alleles bear point mutations in the first cytoplasmic loop, suggesting these mutations may affect the same interaction. A low affinity interaction between the Dsh PDZ domain and a sequence in the cytoplasmic tail of Fz has been demonstrated. These data suggest that sequences in the Dsh DEP domain, and in the Fz first intracellular loop, are also important for Dsh membrane association. Thus, a regulated, bipartite, high affinity association of Dsh with Fz may be selectively disrupted in fzautonomous alleles (Amonlirdviman, 2005).
The ability of the mathematical model to simultaneously reproduce all of the most characteristic PCP phenotypes demonstrates the feasibility of the underlying biological model as a PCP signaling mechanism. Further, the mathematical model demonstrates how the overall scheme of the model -- a local feedback loop between adjacent cells amplifying an initial asymmetry -- can explain the autonomous and nonautonomous behavior of PCP mutant clones. Alternative models invoking diffusible factors have not been supported by the identification of such factors, and the contact-dependent intercellular signaling model more readily accounts for the slight nonautonomy of dsh and fzautonomous clones than do the diffusible factor models (Amonlirdviman, 2005).
The ability of the mathematical model to make predictions and provide a detailed picture of PCP signaling is limited by the lack of complete biological understanding. Although the validity of quantitative model predictions is subject to its assumptions and the set of features used in parameter identification, the model has allowed a direct connection of a biological model to the complex behaviors it is hypothesized to explain and to explore the implications of variations in the model (Amonlirdviman, 2005).
Wnt/ß-catenin signals orchestrate cell fate and behavior throughout the animal kingdom. Aberrant Wnt signaling impacts nearly the entire spectrum of human disease, including birth defects, cancer, and osteoporosis. If Wnt signaling is to be effectively manipulated for therapeutic advantage, how Wnt signals are normally controlled must first be understood. Naked cuticle (Nkd) is a novel and evolutionarily conserved inducible antagonist of Wnt/ß-catenin signaling that is crucial for segmentation in Drosophila. Nkd can bind and inhibit the Wnt signal transducer Dishevelled (Dsh), but the mechanism by which Nkd limits Wnt signaling in the fly embryo is not understood. This study shows that nkd mutants exhibit elevated levels of the ß-catenin homolog Armadillo but no alteration in Dsh abundance or distribution. In the fly embryo, Nkd and Dsh are predominantly cytoplasmic, although a recent report suggests that vertebrate Dsh requires nuclear localization for activity in gain-of-function assays. While Dsh-binding regions of Nkd contribute to its activity, a conserved 30-amino-acid motif, separable from Dsh-binding regions, was identified that is essential for Nkd function and nuclear localization. Replacement of the 30-aa motif with a conventional nuclear localization sequence rescued a small fraction of nkd mutant animals to adulthood. This studies suggest that Nkd targets Dsh-dependent signal transduction steps in both cytoplasmic and nuclear compartments of cells receiving the Wnt signal (Waldrop, 2006; full text of article).
This study reports a structure–function analysis of Drosophila Nkd. The finding that nkd mutants have elevated Arm/ß-catenin levels concomitant with broadened domains of Wg target gene expression is consistent with prior reports of Nkd targeting Dsh, an enigmatic Wnt signal transducer that acts upstream of ß-catenin degradation. Although Wnt-signal-induced Dsh accumulation has been observed in cultured cells, transgenic mice, and some cancers, and recent studies indicate that Dsh, like ß-catenin, can be degraded by the ubiquitin–proteasome pathway, the current data show that Nkd does not attenuate Wnt signaling in the embryo by significantly altering steady-state Dsh levels or distribution. If Nkd promotes Dsh degradation in the fly embryo, as has recently been proposed on the basis of overexpression of mammalian Nkd in cultured cells, it must act only on a subset of Dsh, perhaps the fraction engaged in signaling. Consistent with this idea, rare, punctate Nkd/Dsh colocalization was observed in embryonic ectodermal cells (Waldrop, 2006).
Several Nkd constructs with mutant or deleted Dsh-binding regions possessed a reduced but still substantial nkd rescue activity. Perhaps NkdΔR1S/GFPC, lacking both Dsh-binding regions, is able to target Dsh in vivo (and hence rescue a nkd mutant) by virtue of overexpression, through other low-affinity Nkd/Dsh-binding regions, or by as yet uncharacterized proteins that bridge Nkd to Dsh. Consistent with these possibilities, some NkdΔR1S/GFPC/Dsh colocalization was also observed (Waldrop, 2006).
Three independent lines of investigation—evolutionary sequence comparisons, sequencing of lethal nkd alleles, and transgenic nkd rescue assays—pinpointed a 30-aa motif, separable from Dsh-binding regions, that is crucial for fly Nkd activity and nuclear localization. The comparable positions, identical sequence length, and similar predicted structure of insect and mammalian 30-aa motifs suggests that the family of Nkd proteins may inhibit Wnt signaling through a common mechanism. Given the small size and presumably simple α-helical structure of the 30-aa motif, it is unlikely to possess intrinsic catalytic activity but, in addition to its weak NLS activity, it could serve as a protein-docking motif (Waldrop, 2006).
In addition to several reports that have documented nucleo-cytoplasmic shuttling of ß-catenin, Axin, and APC, it is noteworthy that two recent reports revealed a potential role for Fz and Dsh in the nucleus. In response to Wg signaling at the fly neuromuscular synapse, the Fz2 C terminus was detected in puncta of postsynaptic muscle nuclei although not in ectodermal nuclei, so this report's significance to Nkd's action in ectoderm is unclear. Xenopus Dsh has a separable NLS and nuclear export sequence (NES), with the former required for 'signaling activity' in gain-of-function assays. However, a vertebrate Dsh construct with a mutant NES exhibited increased nuclear accumulation but no activity increase relative to that of wild-type Dsh, arguing against nuclear Dsh concentration—at least when it is overexpressed—being rate limiting for activity. Intriguingly, Dsh NES and NLS motifs seem to be conserved in D. melanogaster Dsh, but their significance remains to be investigated (Waldrop, 2006).
These data extend the still rudimentary knowledge of Nkd action in the fly embryo. The epistatic relationship between wg and nkd suggests that, in the absence of Wg ligand, the low levels of Nkd in a wg mutant (because Wg normally upregulates nkd transcription) inhibit spontaneous ligand-independent signaling through the Wnt receptor complex. Wg exposure promotes Arm accumulation and induction of target genes, including en, hh, and nkd. Nkd, synthesized in the cytoplasm, accumulates and targets an uncharacterized fraction of cytoplasmic Dsh. However, Nkd/Dsh binding alone is apparently insufficient to limit Wg signaling during stages 10–11, since Nkd uses its 30-aa motif to inhibit Arm accumulation, restrict Wg-dependent gene expression, and access the nucleus. Although it is possible that the 30-aa motif is required in the cytoplasm, and that the ability of the 30-aa motif to confer nuclear access is a consequence rather than a cause of activity, three lines of evidence support a nuclear role for Nkd: (1) a subpool of Nkd normally accumulates in the embryonic nuclei after stage 10; (2) the 30-aa motif, distinct from the Dsh-binding sequence, is necessary for both nuclear localization and activity and is sufficient to increase the activity of mouse Nkd1 when expressed in the fly; and (3) a heterologous NLS increased nuclear localization and nkd rescue activity of NkdΔ30aa (Waldrop, 2006).
While these experiments strongly suggest a role for Nkd in the nucleus, they do not reveal the nature of that role. Likewise, lacking insight into how Dsh transmits Wnt signals into the nucleus, the experiments thus far reveal neither the relevant subcellular location(s) of Nkd action nor a molecular mechanism by which Nkd inhibits Dsh activity. The punctate Nkd/Dsh colocalization that was observed in embryonic cytoplasm, and rarely, in nuclei, is consistent with Nkd either affecting Dsh nucleo-cytoplasmic transport or impinging directly on the chromatin of Wnt-responsive genes. The inability to observe increased nuclear Nkd or Dsh after treatment with a nuclear export inhibitor suggests that nuclear export of Nkd (and possibly Dsh) in the fly embryo (1) does not occur (e.g., if each protein were degraded in the nucleus following import); (2) occurs over a longer time period relative to proteins such as Lines that can rapidly shuttle between nucleus and cytoplasm; (3) is independent of CRM-1; or (4) like the presumed Nkd/Dsh interaction, involves only a fraction of the total pool of each protein. Future experiments will be required to distinguish among these possibilities (Waldrop, 2006).
The four-domain structure of both insect and vertebrate Nkd's argues that there once existed an ancient 'core' mechanism by which Nkd engaged Dsh to inhibit Wnt signaling. However, given the sequence divergence between insect and mammalian Nkds, their current mechanisms may share little similarity beyond Dsh binding. Recently, PR72 and PR130, two alternatively spliced B'' subunits of the multi-subunit enzyme protein phosphatase 2A (PP2A), were shown to associate with mammalian Nkd and to modulate its inhibitory effect on ectopic Wnt signaling. Since Dsh is phosphorylated by kinases such as CK1, CK2, and Par1 following Wnt stimulation, recruitment of phosphatases to Dsh by Nkd represents an attractive hypothesis to explain the inhibitory action of Nkd on Wnt signaling via Dsh. Consistent with this possibility, Nkd, PP2A, and Dsh kinases co-immunoprecipitated with vertebrate Dsh. However, unlike the vertebrate Nkd/PR72 interaction, thus no direct interactions by Y2H were detected between the fly PR72 homolog (CG4733) and full-length fly Nkd or any of the regions in Nkd, in particular the 30-aa motif, that are crucial for activity. Thus, regulation of Nkd activity by PR72/PR130 may be a derived, vertebrate-specific phenomenon—analogous in some ways to the effect of mammalian Nkd2 but not Nkd1 on intracellular TGF-α trafficking that may be distinct from the mechanism by which Nkd regulates Wg signaling in Drosophila (Waldrop, 2006).
In Drosophila, nkd is crucial for shaping gradients of Wnt activity, but is this role conserved in vertebrates? Mouse nkd genes are expressed during embryogenesis in dynamic patterns reminiscent of known Wnt gradients. A recent report described nkd1 mutant mice with a targeted deletion of exons 6 and 7 (encoding the EFX domain) but allowing in-frame splicing between exons 5 and 8, resulting in expression of a residual Nkd1 protein very much analogous to the NkdΔR1S/GFPC construct that lacks Dsh-binding sequences but retains three conserved motifs. Given that nkd1 is more broadly expressed than nkd2 during mouse development, it was surprising that nkd1–/– mice are viable and fertile, even though mutant mouse embryo fibroblasts show elevated Wnt reporter activity and homozygous male mice exhibit a sperm maturation defect. Although genetic redundancy between nkd1 and nkd2 could account for these observations, the results suggest an alternative hypothesis, namely that the residual protein produced in the reported nkd1 mutant mice, like EFX-deleted NkdΔEFX/GFPC and NkdΔR1S/GFPC constructs, has significant activity in vivo, despite the observation that a mutant mNkd1 protein lacking the EF hand is defective at blocking Wnt signaling in cultured cells. Resolution of this quandary awaits an investigation of strong loss-of-function mutations in each mammalian nkd gene. Given the broad involvement of Wnt/ß-catenin signaling in mammalian development and cancer, coupled with the similar loss-of-function phenotypes of fly nkd, axin, and apc homologs, it is hoped that these studies guide future investigations of vertebrate Nkd proteins as regulators of Wnt signaling and candidate tumor suppressor genes (Waldrop, 2006).
Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/β-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. The role of a Dsh-binding PCP molecule, Diego (Dgo), was studied in both Wnt-Fz/β-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/β-catenin signaling. The data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell (Wu, 2008).
It has suggested that the KTXXXW motif in Fz C-tails is important for the activation of Wnt-Fz/β-cat signaling targets, but conversely other data implied that this motif was dispensable for Wnt-Fz/β-cat signaling. This issue was addressed in a physiological context. Expressing Fz under control of the tubulin (tub)-promoter fully rescues Fz-activity in fz, fz2 double mutant flies with respect to both Wnt-Fz/β-cat and Fz–PCP signaling. Importantly no dominant phenotypes result from tub-fz expression and thus the tubulin promoter presumably drives the Fz transgenes close to endogenous levels. All Fz C-tail isoforms defective in the Dsh interaction motifs in the C-tail and third cytoplasmic loop (M469R) failed to rescue Wnt-Fz/β-cat signaling, indicating that the Dsh interacting sites are important for Wnt-Fz/β-cat signaling in vivo (Wu, 2008).
The function of the KTXXXW C-tail motif in PCP signaling has previously not been determined. All KTXXXW mutations tested in vivo in this study failed to rescue the fz− PCP mutant phenotypes, indicating that the Dsh interacting sites are required for both signaling pathways. These data suggest that there are no obvious differences in how Fz and Dsh interact with each other in the context of either pathway, and therefore that additional factors are likely involved to modulate the signaling outcome and to provide specificity (Wu, 2008).
Dgo is a core Fz/PCP signaling factor (Feiguin, 2001: Jenny, 2005). During the interactions of the core PCP factors, Fz, Dsh, and Dgo become localized to the distal end of pupal wing cells (or the R3-side of the R3/R4 border in the eye), suggesting that they form a functional complex (Axelrod, 2001; Das, 2004; Strutt, 2001). The localization of Dsh and Dgo depends on Fz (Axelrod, 2001; Das, 2004). Dgo localization also partially depends on Dsh and Dgo and Dsh interact physically (Jenny, 2005). Taken together, these data are consistent with the notion that Fz, Dsh, and Dgo are forming a functional complex during PCP signaling that promotes Dsh PCP-activity (Wu, 2008).
Co-expression of Dgo enhances Fz-mediated inhibition of Wnt-Fz/β-cat signaling in the wing. Furthermore, overexpressed Dgo sequesters more Dsh into the subapical junctional region (where PCP signaling takes place) in a Fz dependent manner, suggesting that a Dgo-Dsh association sequesters Dsh away from canonical Wnt-signaling. Thus, a Fz–Dsh–Dgo complex selectively acts in Fz/PCP signaling and is likely not active for the Wnt-Fz/β-cat pathway (Wu, 2008).
Does Dgo affect the levels of Wnt-Fz/β-cat signaling in a loss-of-function (LOF) scenario? Although dgo LOF alleles show only minor effects on Wnt-Fz/β-cat associated phenotypes, double mutant LOF combinations of dgo and nmo, a mild inhibitor of Wnt-Fz/β-cat signaling, show more robust defects (manifest in the observation that the nmoP allele, which does not show Wg GOF defects, in combination with dgo LOF alleles frequently displayed ectopic margin bristles). This indicates that in vivo Dgo can affect the levels of Wnt-Fz/β-cat signaling, but redundantly with other Wg-signaling inhibitors. It is interesting to note that while Dgo presumably acts at the level of Dsh, Nmo phosphorylates the nuclear transcription factors of the TCF family and thus inhibits their association with β-cat and/or the DNA (Wu, 2008).
How does Dgo negatively affect Dsh in Wnt-Fz/β-cat signaling? in vivo data suggest that Dgo acts mainly by sequestering Dsh away from the cytoplasmic and/or basolateral cell regions where Wnt-Fz/β-cat signaling is thought to take place. Thus, a Dgo influenced shift in Dsh subcellular localization, caused either by loss or excess of Dgo, makes the pathway sensitive to additional changes. When Dsh itself or other Wnt-Fz/β-cat signaling factors become more limiting, alteration of Dgo levels can have effects on Wnt-Fz/β-cat signaling strength (Wu, 2008).
Does Dgo affect overall Dsh levels? Studies with the vertebrate Dgo homologue Inversin have suggested that Inversin, the vertebrate Dgo homologue, can affect Dsh levels through ubiquitination and associated degradation in HEK 293T cells (Simons, 2005). It seemed thus possible that Dgo affected the overall Dsh levels: Dgo could stabilize Dsh at the subapical membrane but cause its destabilization in the cytoplasm. However, no evidence was seen for a destabilization mechanism in vivo or in HEK 293T cells. Thus, it seems that Dgo and Inversin do not share this biochemical property (Wu, 2008).
Diversin is a second Dgo-related vertebrate factor that can act as a repressor of Wnt-Fz/β-cat signaling (Schwarz-Romond, 2002; Simons, 2005). The Diversin and Dgo sequences C-terminal to the Ankyrin repeats do not share homologous domains, although clusters of high homology are present. Diversin is thought to inhibit Wnt-Fz/β-cat signaling through its interaction with Axin and CKIε (Schwarz-Romond, 2002). Dgo does not interact with Axin. Thus, it appears that both Diversin and Inversin can inhibit Wnt-Fz/β-cat signaling by (at least partially) different mechanisms from Dgo, suggesting that these features have diverged evolutionarily (Wu, 2008).
Taken together, the in vivo and cell culture data suggest that Dgo can negatively affect Wnt-Fz/β-cat signaling by trapping Dsh in a Fz/PCP specific complex that is inactive for canonical Wnt-Fz/β-cat signaling. Comparative analyses with Dgo, Inversin, and Diversin will be interesting to shed light on conserved mechanisms of action for these three related proteins (Wu, 2008).
Precise control of Wnt/β-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In Drosophila, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. This study identified a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-α3. The Nkd NLS is similar to the Importin-α3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-α3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. These results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/β-catenin signaling and broaden the understanding of signaling pathways that engage the nuclear import machinery (Chan, 2008).
A growing body of evidence indicates that the traditionally 'cytoplasmic' Wnt signal transducers Axin, Apc, and Dsh also act in the nucleus. In the cytoplasm, Apc promotes β-catenin degradation and regulates the cytoskeleton, but nuclear Apc can recruit transcriptional corepressors to Wnt target genes and, like Axin, escort β-catenin from the nucleus. In light of recent evidence that Axin/Dsh oligomers cross-link Wnt-bound receptors at the plasma membrane during signal activation, it remains unclear whether the nuclear roles for Axin or Dsh are similar to their cytoplasmic functions or whether they, like Apc, have novel nuclear functions (Chan, 2008).
Nkd is also a conserved Wnt signal regulator whose subcellular localization initially suggested a cytoplasmic site of action. These studies have shown that fly Nkd is composed of discrete motifs that confer membrane localization and binding to Dsh, as well as two NLSs. In addition to Nkd targeting an uncharacterized fraction of Dsh in the cytoplasm and/or at the plasma membrane, these data strongly support a nuclear role for Nkd, but whether Nkd inhibits Wnt signaling by altering nucleo-cytoplasmic transport of critical signaling components, such as Dsh or Arm, or by acting on the chromatin of Wnt target genes remains to be elucidated (Chan, 2008).
Genetic epistasis can be a powerful method to infer the regulatory logic of signal transduction cascades. Because Nkd is a 'side-regulator' whose loss-of-function phenotype is dependent on intact Wg signaling, double-mutants between nkd and dsh or arm did not help discern at which level Nkd inhibits the linear Wg signaling pathway. However, Nkd overexpression suppressed the gain-of-Wg signaling phenotype caused by overexpression of Dsh but not that caused by overexpression of an N-terminally deleted and hence degradation-resistant Arm/β-catenin; taken together with the observation that Nkd binds Dsh, epistasis experiments have led to the conclusion that Nkd acted at the level of Dsh and not 'downstream' of Arm/β-catenin in Wg signaling. However, in view of the present data, Nkd might also act in the nucleus at or above the level of Arm/β-catenin. Unfortunately, overproduction of wild type Arm is without phenotypic consequence, presumably because of an excess capacity of the β-catenin 'destruction complex' to degrade ectopic Arm, thus preventing the making further conclusions at present about the epistatic relationship between Nkd and endogenous, degradation-sensitive Arm/β-catenin (Chan, 2008).
Despite the deletion of Dsh-binding sequences in otherwise wild type Nkd having only a minor effect on cuticle rescue activity, the present experiments further support the hypothesis that the Nkd/Dsh interaction is important for Nkd to inhibit Wg signaling. However, the experiments thus far do not clarify how the interaction is regulated in vivo or whether it must occur in the cytoplasm, nucleus, or both locations. Nevertheless, several lines of evidence indicate that a Nkd/Dsh interaction in the cytoplasm and/or near the plasma membrane is important for Nkd function: First, both proteins are predominantly cytoplasmic and/or membrane-associated. Second, punctate cytoplasmic Nkd/Dsh colocalization can be observed in embryos and in salivary gland. Third, the Dsh-binding EFX motif fused to GFP was predominantly cytoplasmic. Fourth, deletion of Dsh-binding sequences in Nkd promoted nuclear localization, consistent with Dsh anchoring Nkd in the cytoplasm. Fifth, deletion of both Nkd NLSs eliminated nuclear localization whether or not Dsh-binding sequences were present, but Dsh-binding sequences were required for Nkd activity (Chan, 2008).
How Nkd acts on Dsh in the cytoplasm to inhibit Wg signaling is not known. One possibility is that Nkd sequesters Dsh away from Fz and/or Axin during signal activation, freeing Axin to regenerate β-catenin destruction complexes. Alternatively, Nkd might target 'activated' Dsh, possibly the pool of Dsh bound to the Wnt receptor complex, for degradation; consistent with Nkd targeting only a fraction of Dsh is the minimal colocalization of the two proteins in embryos as well as the lack of any obvious changes in Dsh levels in nkd mutants. Since Nkd can block the gain-of-Wg signaling phenotypes caused by overexpression of the Dsh kinase CK1, a third possibility is that Nkd blocks CK1-dependent phosphorylation of Dsh via a steric mechanism, although the relationship between Dsh phosphorylation status and activity remains unclear. Future experiments should clarify this issue, because each of these hypotheses makes distinct predictions about phosphorylation status and associated proteins in a native Nkd/Dsh complex (Chan, 2008).
These studies also provide several lines of evidence that the Nkd/Dsh interaction is not sufficient for Nkd to inhibit Wg signaling, and that binding in the nucleus might also be required to fully antagonize Wg signaling. First, the Dsh-binding regions of Nkd when overexpressed blocked phenotypes induced by Dsh overexpression but had no nkd rescue activity. Second, (fly) Nkd and (vertebrate) Dsh have NLSs, although it is not yet known whether fly Dsh acts in the nucleus. Third, rare punctate Nkd/Dsh nuclear colocalization can be observed by confocal microscopy in fly embryos. Fourth, the SV40-NLS increased NkdΔ30aa/GFPC activity when Dsh-binding sequences were intact but reduced activity when Dsh-binding sequences were deleted. The possibility cannot be ruled out that the activity of NkdΔ30aaNLS/GFPC, some of which remains outside the nucleus despite the strong heterologous NLS, is due to cytoplasmic Nkd/Dsh interactions. Similarly, NkdΔR1SΔ30aaNLS/GFPC, which was exclusively nuclear in embryos, might lack activity because of its inability to bind and be retained by Dsh in the cytoplasm. While one must be cautious when inferring site(s) of protein action from subcellular localizations, these studies collectively suggest that fly Nkd is required at multiple locations in Wg-receiving cells (Chan, 2008).
The Nkd-D6 motif has been subject to intense selection pressure, as it is identical in Nkd from D. pseudoobscura, a fly species that diverged from D. melanogaster approximately one billion generations ago. Similarly, the 30 aa NLS is part of a 58 aa motif, and the EFX is part of a 91 aa motif, that are also identical in the two Drosophila species. Using yeast two hybrid, Nkd-EFX residues have been identified that are either dispensable or critical for NkdEFX/DshbPDZ interactions, suggesting that interactions between the EFX motif and proteins other than Dsh might enforce strict motif conservation. Although each NLS contributes to Nkd activity and nuclear localization, heterologous NLSs did not fully replace the function of each Nkd NLS in rescue assays, and in both cases in this work, a heterologous NLS was deleterious to protein function. Absolute conservation of each of these motifs implies that both the tertiary structure and every square angstrom of each motif's surface are necessary for species survival. These experiments suggest that each Nkd motif is required for distinct thresholds and/or duration of Wg signal inhibition: the N-terminal and 30 aa motifs were required for reduction of Arm levels by stage 10, whereas the Dsh-binding EFX and Importin-α3-binding D6 motifs were dispensable to reduce Arm levels but were required, either directly or indirectly, to fully repress en and/or wg transcription by stage 11. Since the deletion of two highly conserved motifs (EFX and D6) preserved the mutant Nkd protein's ability to reduce Arm levels during stage 10, it seems unlikely that these motifs will be shown to possess an intrinsic catalytic activity. The hypothesis is therefore favored that Nkd acts as an inducible protein scaffold, with each of the conserved motifs able to bind additional protein(s). Perhaps there exist distinct Nkd-complexes depending on the subcellular compartment, state of signal activation, or time following signal initiation (Chan, 2008).
Alignment of the Importin-α3-binding NLSs in Nkd and dHSF revealed several conserved residues. Interestingly, the dHSF-NLS has been shown to be bifunctional, suppressing dHSF trimerization in the absence of heat-shock, and in response to heat or other stress conferring Importin-α3-dependent dHSF nuclear translocation and transcriptional induction of heat-responsive genes such as hsp70. These data suggest that the Nkd D6-NLS is also bifunctional, conferring Importin-α3-dependent nuclear localization as well as possibly binding nuclear protein(s) that repress Wg target gene transcription in some cells through stages 10-11. While non-import (presumably scaffolding) functions for Importin-αs have been inferred from phenotypes observed with importin-α deficiency in flies and worms, all of the current experiments support the hypothesis that the Nkd/Importin-α3 interaction promotes nuclear localization. The central region of Importin-α consists of 10 alpha-helical 'Arm' repeats (so named because they were first identified in the Drosophila Arm protein) stacked to form a banana-shaped molecule, the concave side of which harbors a groove that binds basic residues within NLSs. At present, it is not possible based on primary sequence to predict which Importin-α a given NLS will bind, although both the NLS and its three-dimensional context (i.e., adjacent sequence) have been demonstrated to contribute to NLS/Importin-α specificity. Future experiments will investigate whether the residues conserved between Nkd and dHSF represent a consensus Importin-α3-specific binding motif (Chan, 2008).
Vertebrate Nkds have a conserved 30 aa motif between the EFX and C-terminal histidine-rich regions, but whether the vertebrate proteins act in the nucleus like fly Nkd is not known. In this regard, no obvious difference was observed between the subcellular localizations of mouse Nkd1 fused to C-terminal GFP (mNkd1GFPC) vs. a similar construct that lacks the 30 aa motif (mNkd1Δ30aa/GFPC) when the proteins were produced in cultured mammalian cells. However, no obvious difference was observed between fly Nkd and NkdΔ30aa localizations in Drosophila S2 cells, but the differences in localization and function of these two constructs when produced in nkd mutant embryos were dramatic. These findings illustrate the importance of investigating the subcellular localizations of mutant proteins in a native environment that lacks the endogenous wild type protein. It might therefore be interesting to examine the subcellular localization of vertebrate Nkd constructs in nkd-mutant mice or zebra fish just as has been done in Drosophila. More importantly, future experiments must address the critical question of how Nkd antagonizes Wnt/β-catenin signaling in each of the subcellular compartments to which it localizes (Chan, 2008).
The planar polarization of developing tissues is controlled by a conserved set of core planar polarity proteins. In the Drosophila pupal wing, these proteins adopt distinct proximal and distal localizations in apicolateral junctions that act as subcellular polarity cues to control morphological events. The core polarity protein Flamingo (Fmi) localizes to both proximal and distal cell boundaries and is known to have asymmetric activity, but the molecular basis of this asymmetric activity is unknown. This study examined the role of Fmi in controlling asymmetric localization of polarity proteins in pupal wing cells. Fmi was found to interact preferentially with distal-complex components, rather than with proximal components, and evidence is presented that there are different domain requirements for Fmi to associate with distal and proximal components. Distally and proximally localized proteins cooperate to allow stable accumulation of Fmi at apicolateral junctions, and evidence is presented that the rates of endocytic trafficking of Fmi are increased when Fmi is not in a stable asymmetric complex. Finally, evidence is provided that Fmi is trafficked from junctions via both Dishevelled-dependent and Dishevelled-independent mechanisms. A model is presented in which the primary function of Fmi is to participate in the formation of inherently stable asymmetric junctional complexes: Removal from junctions of Fmi that is not in stable complexes, combined with directional trafficking of Frizzled and Fmi to the distal cell edge, drives the establishment of cellular asymmetry (Strutt, 2009).
The differing ability of overexpressed Fmi to modulate Fz:Dsh and Stbm:Pk levels at junctions could be explained by a number of mechanisms. One likely hypothesis is that Fmi may require a cofactor for a robust interaction with Stbm, and that this cofactor is limiting when Fmi is overexpressed. Alternatively, Fmi may require posttranslational modification or a conformational change to interact with Stbm, and a factor needed for this modification is limiting. The cytoplasmic C-terminal tail of Fmi is a likely region to mediate an interaction with Fz:Dsh or Stbm:Pk; therefore, a truncated form of Fmi was constructed, in which this region is either absent or replaced with GFP (Strutt, 2009).
When overexpressed in pupal wing cells, FmideltaIntra is much more efficient at recruiting Fz and Dsh to junctions than full-length Fmi, an effect similar to that caused by removal of stbm or pk. Stbm is still reduced at junctions, although less than when full-length Fmi is overexpressed. This suggests that the C-terminal intracellular domain of Fmi is dispensible for the interaction of Fmi with Fz:Dsh and, importantly, that Fz:Dsh no longer have to compete with Stbm:Pk for access to Fmi (Strutt, 2009).
Interestingly, two isoforms of Fmi have been identified, one of which contains a PDZ binding motif (PBM) at its C terminus. It is possible that loss of the PBM alone could account for the failure of overexpressed Fmi or FmideltaIntra to associate with Stbm:Pk. However, this is unlikely, because Fmi that lacks the PBM can rescue the planar polarity phenotype of fmi mutants (Strutt, 2009).
Endogenous Fmi is thought to be localized on both proximal and distal cell boundaries. This was confirmed by expressing CFP-tagged Fmi at physiological levels in clones in pupal wings, and it was observed that levels of staining appear similar at each end of the cell, consistent with the homophilic-interaction model. Notably, expression of a GFP-tagged form of FmideltaIntra results in its preferential localization to distal cell edges, where Fz and Dsh also localize (Strutt, 2009).
Interestingly, junctional localization of FmideltaIntra-EGFP is not dependent on endogenous, full-length Fmi, suggesting that this molecule is still able to participate in homophilic interactions. Hence, the ability of FmideltaIntra-EGFP to functionally rescue the polarity phenotype of fmi null mutant clones was investigated. If FmideltaIntra-EGFP interacts preferentially with the distal Fz:Dsh complex, then Stbm recruitment to junctions inside clones would be compromised. Consequently, FmideltaIntra-EGFP:Fz complexes inside the clone would preferentially interact with Fmi:Stbm outside the clone, leading to a reversal in polarity on proximal clone edges. Importantly, this prediction is upheld, and fmi clones rescued with FmideltaIntra-EGFP exhibit weak proximal polarity inversions, such that trichomes point away from the clone, and polarity proteins are recruited to the clone boundary (Strutt, 2009).
Nevertheless, Stbm localizes asymmetrically inside the clone, although not always at the correct site, whereas in a fmi null mutant it lacks any asymmetric localization. Thus, FmideltaIntra-EGFP must retain some ability to interact with Stbm. To confirm this, the ability of full-length Fmi or FmideltaIntra-EGFP to interact with Fz and Stbm in Drosophila S2 cells was analyzed. In this assay, Fmi and FmideltaIntra-EGFP are recruited to sites of cell contact, as a result of homophilic interactions between their extracellular domains. Cotransfection of Fz or Stbm with either full-length Fmi or FmideltaIntra-EGFP in Drosophila S2 cells results in the recruitment of both to sites of cell contact (Strutt, 2009).
Interestingly, if S2 cells were transfected with either Fz or Stbm and then mixed, weak recruitment is also observed to sites of cell contact, arguing that their extracellular domains can interact independently of Fmi. Nevertheless, recruitment was weaker and less frequent than when Fmi was cotransfected, suggesting that Fmi:Fmi interactions are more important than Fz:Stbm interactions in stabilizing complexes between adjacent cells (Strutt, 2009).
The data suggest that Fz:Dsh and Stbm:Pk complexes differ in their ability to associate with Fmi. Whereas endogenous levels of Fmi result in the formation of asymmetric complexes with Fz:Dsh on one side of the boundary and Stbm:Pk on the other, overexpressing Fmi favors Fz:Dsh recruitment. Furthermore, a C-terminally deleted form of Fmi preferentially localizes distally with Fz, and overexpression of this form has an even greater preference for Fz:Dsh recruitment. Thus, the C terminus of Fmi is important in promoting the interaction with Stbm:Pk. The Fmi truncation data could be explained simply by the possibility that the C terminus of Fmi contains a direct binding site for Stbm; however, this fails to explain why overexpressed full-length Fmi prefers to recruit Fz:Dsh. It is therefore proposed that the association of Fmi with Stbm:Pk requires a limiting factor that is saturated by Fmi overexpression. The most plausible hypothesis is a requirement for a cofactor for Stbm:Pk binding, but other possibilities include saturation of the machinery for a posttranslational modification or a conformational change in Fmi (Strutt, 2009).
The data also suggest that Fmi itself needs to associate with both proximal and distal components in order to be stably localized to apicolateral junctions. Although it can form homophilic dimers between adjacent cell membranes in tissue culture, in pupal wings Fmi does not localize strongly to apical junctions and presumably fails to form stable homodimers in trans. Fz on one side of the junction and Stbm:Pk on the opposite side stabilize Fmi at junctions, most likely by promoting homophilic interactions or preventing internalization. However, Fmi appears to be capable of forming complexes with either distal or proximal components alone, but these complexes (particularly the proximal complex) are apparently less stable at junctions. Taken together with overexpression experiments, this would suggest that the most stable configuration is Fz:Fmi on one side of the boundary and Fmi*:Stbm:Pk on the other (where Fmi* denotes the modified form able to preferentially associate with Stbm:Pk) (Strutt, 2009).
In order for an asymmetric complex to be stabilized across junctions, the extracellular domains must somehow 'look' different. One possibility is that the Fz and Stbm extracellular loops interact - a view supported by S2 cell data. Alternatively, the Fmi extracellular domain, when associated with either Fz or Stbm:Pk, could undergo a conformational change that promotes homophilic Fmi interactions (Strutt, 2009).
An intriguing question is why clones of cells that overexpress Fmi behave like fz loss-of-function clones. It is suggested that within the clones, excess Fmi associates with the entire available pools of both Fz and Stbm. However, there is still a pool of uncomplexed Fmi that can associate with Fmi:Fz in adjacent wild-type cells, forming the relatively stable Fmi-Fmi:Fz configuration, thus causing polarity to be reversed on distal clone boundaries. In support of this model, an identical nonautonomous effect is seen when FmideltaIntra is overexpressed, which itself interacts only poorly with Stbm but presumably can interact with Fmi:Fz in adjacent cells outside the clone (Strutt, 2009).
Interestingly, Fmi accumulates in excess at junctions in a dsh, stbm double mutant, whereas Fz does not. Thus, although Fz acts to stabilize Fmi at junctions, Fmi does not always need to associate with Fz in a stoichiometric fashion in order to be stabilized. Perhaps as long as there is some Fz associated with Fmi, this may permit local stabilization of other Fmi molecules in cis. Alternatively, this excess accumulation of Fmi might simply represent
'unstable' Fmi homodimers that are no longer being removed from junctions by the actions of Dsh and Stbm (Strutt, 2009).
The composition of the complex with which Fmi is associated appears to be critical for determining the frequency and manner by which Fmi is turned over from the plasma membrane. Most compellingly, Fmi accumulates more strongly in an enlarged endosomal compartment in Rab7TN mutant tissue when stbm and fz are absent than when they are present. Thus, it is suggested that more Fmi is resident in the endocytic pathway when it is unable to form stable asymmetric complexes. Fmi:Fz puncta have been observed that are selectively trafficked to distal cell edges. In the current experiments, these puncta colocalize with YFP-Rab4, suggesting that Fmi and Fz are recycled back to the plasma membrane by a Rab4-dependent mechanism. Furthermore, the increased intracellular and junctional levels of Fz and Fmi in dor mutant clones suggests that in addition to being recycled to the plasma membrane, a significant fraction of internalized Fmi and Fz is also sent for degradation. It is formally possible that the intracellular accumulation of Fmi and Fz seen when lysosomal trafficking is blocked by loss of Rab7 or in dor clones is due to their being sent for degradation immediately after synthesis (e.g., if damaged or misfolded); however this is unlikely because newly synthesized Fmi-ECFP appears first at junctions before been seen in puncta (Strutt, 2009).
Stbm has not been observed in large intracellular puncta, but it seems likely that it is also internalized and recycled, possibly together with Fmi, although it must do so by alternative pathways involving smaller or more rapidly recycling particles that are not visible by confocal microscopy. Indeed, the data suggest a potential role for Dsh and Stbm in regulating junctional levels of Fmi. A stbm mutant alone results in a loss of Fmi from junctions, consistent with a need for Stbm in stabilizing Fmi in asymmetric complexes. In contrast, loss of Dsh and Stbm together increases Fmi levels at junctions, suggesting a role for Stbm in internalization. It is suggested that the outcome of any interaction of Stbm with Fmi is dependent upon whether Fmi is able to form stable homodimers with Fz on the opposite cell membrane. In a wild-type situation, one could envisage that Fmi forms stable homodimers in a Fz:Fmi-Fmi*:Stbm configuration, and that both Dsh and Stbm promote internalization of any Fmi that is not in this configuration, the majority of which is subsequently recycled back to the plasma membrane. In dsh mutants, there is reduced internalization, but the effect on Fmi levels is subtle; Fz and Stbm are still present to promote Fmi homodimer formation, and Stbm still promotes internalization of any unstable Fmi. In contrast, in stbm mutants, the number of less stable Fmi complexes (associating only with Fz) is greatly increased, favoring internalization by Dsh. Finally in dsh, stbm double mutants, Fmi is again less stable (associating only with Fz), but there is no Dsh- or Stbm-mediated internalization, leading to an overall increase of Fmi at junctions (Strutt, 2009).
How do Dsh and Stbm regulate Fmi levels at junctions? Stbm contains potential interaction motifs for the endocytic adaptor AP2, but their role has not been functionally tested. In addition, in vertebrate Wnt signaling, there is evidence that Dsh interacts with the endocytic adaptor protein β-arrestin and mu2 subunit of AP2 to mediate Wnt/Fz endocytosis and downregulation of Wnt signaling. Interestingly, in planar polarity this is no evidence that Dsh directly mediates internalization of Fz, but the data rather point to Dsh promoting Fmi internalization when it is not associated with Fz. Instead, the trafficking of Fmi together with Fz into the lysosomal pathway is Dsh independent (Strutt, 2009).
In summary, it is proposed that a number of mechanisms exist by which Fmi contributes to the generation of asymmetry at the molecular level. First, the characterization of the previously inferred asymmetry in Fmi activity indicates that Fmi normally prefers to bind to Fz and requires a limiting factor for association with Stbm:Pk. Second, Fmi stability at junctions is dependent on both Fz and Stbm:Pk, with the most stable form being Fz:Fmi bound to Fmi*:Stbm. Finally, it is proposed that entry of Fmi into the endocytic trafficking pathway is decreased if it is in a stable complex, and this is regulated either by Dsh and Stbm or independently of Dsh and Stbm, depending on whether it is associated with Fz (Strutt, 2009).
An outstanding question is how these mechanisms translate into cellular asymmetry, such that in any particular cell, heterophilic polarity complexes preferentially form with Fz:Dsh at the distal junctions, rather than having heterophilic complexes in both orientations. It is thought that the acquisition of cellular asymmetry is likely to be driven by directional trafficking of Fmi:Fz, although other models, such as a mechanism for preferential stabilization of Fmi:Fz interactions at the distal cell edge, are also possible. In addition, it seems likely that an amplification mechanism would be required, although the molecular mechanisms remain to be elucidated (Strutt, 2009).
While this manuscript was in preparation, another manuscript was published, in which Fmi was proposed to mediate an asymmetric and instructive signal between proximal and distal complexes to generate asymmetry, and thus does not act merely as a scaffold for Fz:Stbm interactions across membranes. It is argued that the current data do not provide evidence for a specific signaling function of Fmi. Instead, the hypothesis is favored that the composition of the proximal and distal complexes is distinct, and that heterophilic complexes are inherently more stable than homophilic complexes. Together, removal of unstable nonasymmetric complexes through increased endocytic turnover, in concert with directional trafficking and an unknown amplification mechanism, may be sufficient to generate asymmetry without the need to invoke a specific signaling function for any components of the complexes (Strutt, 2009).
Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, Drosophila Abl (dAbl) was identified as a modulator of Frizzled(Fz)/PCP signaling. dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg-Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz-PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, these results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz-Dsh/PCP signaling by Dsh phosphorylation (Singh, 2010).
Evidence is provided for a specific role of tyrosine
phosphorylation of Dsh by Abl family kinases in Fz/Dsh-PCP signaling. dAbl is required for R3/R4 fate specification. dAbl interacts with fz and
dsh genetically in PCP signaling. Biochemical experiments
indicate that dAbl binds Dsh and phosphorylates
it on Tyr473 within the DEP domain, which has been specifically implicated in PCP signaling and is largely dispensable for canonical Wnt/Wg signaling. The data further show that Abl kinases do not affect canonical Wnt signaling in either Drosophila or MEFs. Taken together, these data indicate that Abl family kinases positively regulate PCP signaling by affecting Dsh/Dvl family members via phosphorylation of Tyr473. Abl family kinases appear to provide a molecular gating mechanism to increase the capability of Dsh/Dvl proteins to signal via the Fz/Dsh-PCP pathway. These data suggest the possibility that this function of Abl family kinases might be conserved from flies to mammals, as similar effects were observed with mammalian Abl and Dvl family members (Singh, 2010).
Most Abl studies in mammalian cell culture have focused
on their role in tumor formation. Little is known
about Abl’s normal physiological roles during development,
except in the context of junctional stability and
cytoskeletal events. Previous studies in the
Drosophila eye established that dAbl is expressed dynamically
in all photoreceptors, and dAbl mutant flies have a rough eye phenotype with significant photoreceptor loss. Its potential role in cell fate
specification has not been addressed. A detailed
analysis of the dAbl eye phenotype was performed in the context
of PCP establishment and R3/R4 specification. It was demonstrated
that dAbl is required for specification of both
R3-R4 cells. Differential activation of Fz/PCP signaling
specifies R3 and leads to the activation of Notch signaling
in the neighboring R4 to induce its proper fate. The data suggest
that dAbl is required in R3 for fate specification via its
interaction with Dsh and positive input into Fz/PCP
signaling. This Abl function appears to be common to
Fz/PCP signaling in general, as Fz and dAbl also synergize
in PCP establishment in the wing. Moreover, dAbl phosphorylation
of DshTyr473 is essential for Dsh PCP function
in general. In vertebrates, Abl kinases affect Dvl
localization in MEFs, and Abl1-/-,Abl2-/- mice display
similar phenotypes as dvl1-/-,dvl2-/- mice, with severe
open neural tube defects in 9.5-day embryos. The
requirement of dAbl in R4 specification remains obscure,
as Fz/PCP signaling is not required in R4 (Singh, 2010).
A likely explanation derives from studies of dAbl in noncanonical Notch signaling, where dAbl has been suggested to act downstream from Notch. Although the role of Notch in the developing eye has focused on canonical Su(H)-dependent Notch activity, it is quite likely that Abl could modulate Notch signaling activity in R4. Further work will be needed to explain the role of dAbl in R4 fate specification and associated Notch signaling (Singh, 2010).
Dsh contains three highly conserved domains and a stretch of basic residues and several serine/threonine-rich regions between the DIX and PDZ domains,
as well as a proline-rich region downstream from the PDZ domain, which encodes a class I consensus sequence for an SH3-binding protein. Many proteins that have been shown to bind Dsh/Dvl bind to Dsh in the PDZ domain. Abl binding, however, maps to the proline-rich region of Dsh just C-terminal to the PDZ domain, while the PDZ domain alone showed no binding. Different Dsh domain requirements are known for canonical and PCP signaling. While the DIX domain functions exclusively in canonical Wnt signaling, the DEP domain is required for PCP signaling and, in particular, stable membrane association. The results indicate that dAbl phosphorylates Dsh at Tyr473 (and possibly other Tyr residues in the DEP/C-term region). Phosphorylation of Dsh-Tyr473 is unique, as dAbl is a tyrosine kinase and all previously analyzed Dsh kinases have been serine/threonine kinases (Singh, 2010).
Wnt signaling is important in diverse physiological processes and, when deregulated, often leads to disease states. Studies in model organisms have unraveled two conserved pathways, now referred to as canonical Wnt/Wg signaling and Wnt-Fz/PCP signaling. The canonical Wnt signal is transduced via Fz family receptors (along with the LRP5/6 coreceptors), leading to Dsh-Axin complex formation, which in turn causes the stabilization of cytoplasmic β-catenin and allows gene transcription. In Fz/PCP signaling, Dsh is recruited to the membrane in an Axin- and LRP5/6-independent manner and acts on different downstream effectors, depending on the cellular context. The mechanism of pathway-specific Dsh 'activation' is poorly understood, and, similarly, the question as to how the individual pathways are specifically activated at the level of either Fz or Dsh remains unresolved. This study highlights the importance of Abl in the context of Fz/PCP signaling at the level of Dsh/Dvl. At the level of both Abl mutants as well as the DshY473F phosphorylation mutant, canonical Wnt/Wg signaling remains unaffected, while PCP signaling is defective. As the signal that activates dAbl in this context is not known, it is possible that Abl acts in a permissive manner in PCP signaling. In conclusion, this study provides evidence for Dsh tyrosine phosphorylation and a role of Abl in PCP signaling; further studies will be needed to establish a full framework for the regulation of Abl in PCP signaling and in the biology of Dsh (Singh, 2010).
Biological Overview
| Evolutionary Homologs
| Developmental Biology
| Effects of Mutation
| References
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