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First published online 15 December 2008
doi: 10.1242/dev.025460
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1 Department of Molecular Biology and Biochemistry, Simon Fraser University,
8888 University Drive, Burnaby, BC, V5A 1S6 Canada.
2 Division of Cell Regulation Systems, Post-Genome Science Center, Medical
Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan.
* Author for correspondence (e-mail: everheye{at}sfu.ca)
Accepted 13 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Hipk/Hipk2, Wnt/Wg signaling, β-catenin
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The evolutionarily conserved Wnt/Wingless (Wg) signaling pathway is
involved in diverse biological processes, including determination,
proliferation, migration and differentiation during embryonic development and
adult homeostasis (reviewed in Clevers,
2006
). Inappropriate activation of Wnt-dependent gene expression
in mammals can lead to numerous cancers, and loss of Wnt pathway activity also
profoundly affects development. The ability of this pathway to control
different developmental events in a temporally and spatially specific manner
requires coordination between numerous regulators. Canonical Wnt signaling
controls cell fate by regulating transcription of target genes
(Cadigan and Nusse, 1997).
Wingless (Wg), a secreted glycoprotein, is the best characterized of the seven
Drosophila Wnt ligands, and initiates the canonical pathway by
binding to the Frizzled2 (Fz2) and LRP5/6/Arrow co-receptors
(Bhanot et al., 1996). This
leads to the activation of Dishevelled, which then inhibits the activity of
the destruction complex composed of Axin, glycogen synthase kinase 3β
(GSK-3β)/Zw3 and adenomatous polyposis coli (APC)
(Ikeda et al., 1998;
Kishida et al., 1998;
Polakis, 1997). As a result,
cytosolic Drosophila β-catenin called Armadillo (Arm)
accumulates and enters the nucleus to interact with a Tcf/Lef
(Drosophila Tcf) family transcription factor to promote target gene
expression (van de Wetering et al.,
1997). In the absence of Wg signaling, the Axin/GSK-3β/APC
complex promotes the proteolytic degradation of Arm
(Aberle et al., 1997;
Willert et al., 1999;
Yost et al., 1996), whereas
transcriptional co-repressors bind to Tcf and repress transcription
(Cavallo et al., 1998;
Roose et al., 1998).
The Nemo-like kinase family (Nlk) of protein kinases can regulate
activation of Tcf/Lef target genes
(Ishitani et al., 1999;
Rocheleau et al., 1999;
Zeng and Verheyen, 2004). In
Drosophila, Nemo (Nmo) inhibits Drosophila Tcf activity, and
is itself a transcriptional target of the Wg pathway
(Zeng and Verheyen, 2004).
Recently Homeodomain-interacting protein kinase 2 (Hipk2) was proposed to
participate in a kinase cascade to activate Nlk during the regulation of the
Myb transcription factor (Kanei-Ishii et
al., 2004). We thus sought to identify whether this regulation was
perhaps more general and whether Drosophila Hipk played a role in
regulating Nmo, and thus also Wg signaling. We rapidly learned that Hipk
exerts a positive effect on Wg signaling, distinct from Nmo, which we have
more fully characterized using the developing wing as a model system.
The patterning of the adult wing blade is a tightly regulated process
involving numerous essential signaling pathways, including Wg, Notch, EGFR and
TGFβ, making it an excellent tissue in which to examine regulatory and
epistatic relationships between many genes involved in patterning. The adult
wing blade possesses five longitudinal veins (LI-LV) that extend proximally to
distally. These are connected by the anterior cross vein (ACV) and posterior
crossvein (PCV). The Wg pathway acts at several stages of wing patterning and
growth (reviewed by Martinez Arias,
2003). Wg is expressed along the dorsal/ventral boundary, which in
imaginal discs is a stripe bisecting the wing imaginal disc, and in adult
wings gives rise to the wing margin and bristles that surround the edge of the
wing blade. Loss of wg can lead to loss of the entire wing, to wing
to notum transformations, to wing notching or to loss of bristles along the
entire wing margin (Phillips and Whittle,
1993; Couso et al.,
1994). Wg also promotes proliferation in the wing disc and ectopic
Wg can induce outgrowths from the ventral surface of the wing
(Phillips and Whittle, 1993;
Diaz-Benjumea and Cohen,
1995).
Hipk2 is a member of a conserved family of serine/threonine kinases.
Vertebrate species possess four Hipk proteins (Hipk1-4) that have evolved
distinct functions (reviewed by Rinaldo et
al., 2007). Singly mutant Hipk1 and Hipk2 mice are viable, whereas
double mutant mice die before birth (Isono
et al., 2006). Drosophila possesses a single Hipk
ortholog (which has been referred to as both Hipk and Hipk2)
(Choi et al., 2005;
Link et al., 2007) that shares
extensive sequence homology within the kinase domain with members of the
vertebrate family.
Vertebrate Hipk2 has been the most extensively studied member of the family
(reviewed by Calzado et al.,
2007; Rinaldo et al.,
2007). Biochemical studies have identified a growing list of Hipk2
interactors, including proteins involved in transcriptional regulation,
chromatin remodeling and key components of evolutionarily conserved signaling
pathways. However, the biological investigation of these interactions in
multicellular organisms has been minimal. In vivo examination of murine Hipk2
protein function has thus far revealed a role in neurogenesis and homeotic
transformation of the skeleton (Isono et
al., 2006; Wiggins et al.,
2004; Zhang et al.,
2007). Studies in Drosophila using transgenic flies
expressing Hipk transgenes have uncovered a role for Hipk in regulating the
global corepressor Groucho (Choi et al.,
2005). Using loss-of-function mutant analyses, we have also
identified a role for Hipk in promoting Notch signaling during
Drosophila eye development (Lee
et al., 2008).
In this study, we present an analysis of the function of Hipk in Drosophila canonical Wg signaling. Genetic studies show that ectopic hipk can rescue phenotypes owing to loss-of-function wg alleles or inhibition of the pathway with a dominant-negative Fz2 receptor. Immunohistochemical studies show that hipk positively regulates expression of Wg targets, and that Hipk can act to stabilize cellular levels of the Arm protein in wing discs. Wnt reporter assays show that both Drosophila Hipk and mouse Hipk2 can promote the Wnt-responsive Topflash reporter. In addition, we have found that Hipk/Hipk2 can promote the stabilization of Arm/β-catenin in cell culture and in vivo. Our results suggest that Hipk is a positive regulator of the Wg pathway that refines Wg activity during wing development. Our findings suggest that these roles may be conserved across species.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
UAS-hipk (Lee et al.,
2008), UAS-ArmS2/Cyo and
UAS-ArmS10 (Pai et
al., 1997), UAS-DaxinA2-4
(Willert et al., 1999),
UAS-Dfz2N33/Cyo (Zhang and
Carthew, 1998), UAS-nmoC5-1e
(Verheyen et al., 2001),
UAS-hipk RNAi (Vienna Drosophila RNAi Center, National Institute of
Genetics), nmoDB24 (Zeng et al., 2004),
nmoadk2 (Verheyen et
al., 2001), hs-Flp22; GFP, FRT79/TM6B,
hipk3/TM6B, hipk4, FRT79/TM6B. All
wild-type flies are w1118, and all crosses were performed
according to standard procedures at 25°C. In assays examining interaction
between two UAS transgenes, control crosses were performed with
UAS-lacZ to rule out suppressive effects caused by titration of Gal4.
To generate hipk loss-of-function clones, hsflp.22; GFP
FRT79/TM6B females were crossed to hipk4 FRT79/TM6B
males and progeny were collected for 24 hours and heat shocked at 38°C for
90 minutes at 48 AEL. Wing imaginal discs were dissected from late
third-instar larvae for immunohistochemistry, and adult flies were collected
for phenotypic analyses.
Wing mounting
Adult wings were dissected in 100% ethanol followed by mounting in Aquatex
(EM Science).
Immunohistochemical staining and microscopy
Antibody staining was performed according to standard protocols. The
following primary antibodies were used: 1:50 mouse-anti-Achaete, 1:200 mouse
anti-Armadillo (concentrated supernatant; Developmental Studies Hybridoma
Bank), 1:400 mouse anti-Distalless (Duncan
et al., 1998), 1:2000 rabbit β-galactosidase (Cappel) and
1:1000 mouse Myc (Sigma-Aldrich). The secondary antibodies were used as
follows: anti-mouse CY3 (Molecular Probes), anti-rabbit CY3 (Jackson
Immunolabs) and anti-mouse HRP (Jackson Immunolabs). All secondary antibodies
were used at 1:200 dilution. Wing imaginal discs were mounted in 70%
glycerol.
Cell culture and in vitro biochemical assays
HEK293T cell culture, protein expression, immunoprecipitations and kinase
assays, with Drosophila Hipk, were performed according to Zeng et al.
(Zeng et al., 2007). The Hipk
kinase dead construct (HA-Hipk-KD, EGFP-Hipk KD)
(Choi et al., 2005) contains a
K221R mutation within the catalytic ATP-binding site. For experiments using
expression plasmids encoding mammalian cDNA, HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.
Cells were then transiently transfected with Polyethylenimine MW 25000
(Polysciences). For co-immunoprecipitation assay, cells in 100 mm diameter
plates were transfected with the expression plasmids encoding mammalian cDNA
(12 µg). For reporter assays, cells in six well 35 mm diameter plates were
transfected with the expression plasmids encoding mammalian cDNA (1 µg).
Details on the generation of truncation constructs can be provided on
request.
Transcription assays
HEK293T and HeLa cells were cultured in six-well (35 mm diameter) plates
and transiently transfected with Polyfect (Qiagen) or Polyethylenimine MW
25000 (Polysciences) according to manufacturer's manual. Cells were
transfected at 24 hours after seeding with the TOPFLASH or FOPFLASH reporter
gene plasmids along with each expression vector as indicated.
Drosophila S2R+ cells were seeded (1x106) in
six-well plates and maintained at 25°C in Schneider's Drosophila
medium supplemented with 10% heat-inactivated FCS (Invitrogen) and 125
µg/ml hygromycin B (Sigma). Before transfection, cells were re-seeded in S2
conditioned media or Wingless-conditioned Schneider's medium. Cells were
transfected with 40 ng of Drosophila Hipk or 0.4 µg of mouse
Hipk2, using the Effectene transfection reagent (Qiagen) according to the
manufacturer's instructions. Eight hours post-transfection, the induction of
genes under the control of the metallothionein promoter was performed by
supplementing the medium with CuSO4 at a final concentration of 0.5
mM. For both mammalian and Drosophila cells, total DNA concentration
was kept constant by supplementation with empty vector DNAs. Luciferase assays
were performed with the Dual Luciferase Reporter assay system (Promega)
according to manufacturer's instructions and as described by Korinek et al.
(Korinek et al., 1997). The
renilla luciferase pRL-CMV or pRL-EF vector was used for normalizing
transfection efficiencies. The values shown are the average of one
representative experiment in which each transfection was performed in
duplicate or triplicate as noted.
Protein stability assay
For examination of protein stability, HEK293T and HeLa cells were
transfected in 60 mm plates with 1.5-2 µg per construct and empty vector
was added to maintain a constant concentration of DNA. Cells were treated with
25 µg/ml cyclohexamide after 24 hours of transfection and harvested at the
indicated times after treatment. Whole cell lysates were analyzed by SDS PAGE
electrophoresis and immunoblotted with appropriate antibodies.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Loss of zygotic hipk resulted in pupal or larval
lethality, a finding recently also described by Link et al.
(Link et al., 2007).
Whole-mount in situ hybridization reveals that hipk is expressed
broadly in a non-uniform pattern in multiple stages of development, including
all imaginal discs (data not shown). Removal of the maternal contribution
caused embryonic lethality characterized by twisted embryos and head holes,
showing that hipk is an essential gene for Drosophila
development (W.L., unpublished). In our analyses we focused on the most severe
allele, hipk4, which causes early larval lethality, or
pupal lethality in trans to hipk3
(Lee et al., 2008). Given the
embryonic lethality caused by loss of maternal Hipk, we speculate that
maternally contributed Hipk perdures and obscures its requirement at later
stages and impacts the severity of mutant phenotypes. We used the FLP/FRT
technique to generate mutant somatic clones to examine the requirements for
Hipk in patterning adult structures (Xu
and Rubin, 1993).
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;
Couso et al., 1994;
Rulifson et al., 1996).
We next studied the effects of ectopic expression of hipk using
the Gal4-UAS system (Brand and Perrimon,
1993). We observed phenotypes that suggested that hipk is
involved in promoting Wg signaling. Expression of one copy of hipk in
the central domain of the imaginal wing discs with omb-Gal4
(Fig. 5C) induced the formation
of an additional wing margin and outgrowths emanating from the distal most tip
of the ventral surface of the wing (Fig.
1D,E). Expression of two copies of UAS-hipk
enhanced this phenotype and caused the outgrowths to extend further distally
from the ventral plane of the wing (Fig.
1F). These effects phenocopied the effects observed in ectopic
wg-expressing clones
(Diaz-Benjumea and Cohen,
1995). Similarly, expression of UAS-hipk in the wing
(Fig. 2B) phenocopies the
ectopic venation pattern seen both upon ectopic expression of activated Arm
(ArmS10) with bs-Gal4
(Fig. 2C) and in
nmoDB24/nmoadk2 mutants
(Fig. 2D). Although control of
wing vein patterning is not generally attributed to Wg signaling, we and
others have observed ectopic venation upon elevated Wg signaling. For example,
ectopic expression of constitutively active Arm by en-Gal4 in the
posterior region of the wing, or ubiquitously with 69B-Gal4 or
MS1096-Gal4, leads to disturbed and ectopic venation
(Greaves et al., 1999;
Lawrence et al., 2000).
Moreover, loss-of-function clones of sgg/zw3 (encoding the fly
homolog of GSK3β, a component of the destruction complex) induce the
formation of ectopic veins (Ripoll et al.,
1988). The results of these phenotypic analyses of hipk
are surprising because they demonstrated that Nmo and Hipk did not act in
concert to inhibit Wg signaling. Rather, hipk mutant and
gain-of-function phenotypes suggest a role in promoting the Wg pathway
Hipk can rescue inhibition of Wg signaling in the wing
To explore whether Hipk is a positive regulator of the Wg pathway, as
suggested by our initial analyses, genetic interaction studies were performed.
During early wing development, Wg specifies the wing and disruption of
signaling during the second larval instar induces a wing-to-notum
transformation (Fig. 2E)
(Morata and Lawrence, 1977;
Ng et al., 1996). Only 14% of
wg1/wg1-17 transheterozygotes (n=72)
develop a normal pair of wings and misexpression of hipk in this
genetic background suppressed the formation of the ectopic notum and restored
the structure of the wing (Fig.
2F) in 41% of flies (n=39). Expressing a
dominant-negative form of the Fz2 receptor Dfz2N33 ubiquitously in
the wing with 69B-Gal4 caused a severe loss of wing margin phenotype
in both the anterior and posterior regions of all wings examined
(Fig. 2G; n=41)
(Zhang and Carthew, 1998).
This phenotype was entirely rescued with 100% penetrance (n=47) upon
hipk co-expression (Fig.
2H). Altogether, these interactions indicated that hipk
can counteract the effects of inhibiting the Wg pathway, and, more
importantly, that elevating the levels of Hipk can compensate for the reduced
Wg signal.
Consistent with these observations, we find that removal of one dose of hipk can enhance phenotypes owing to inhibited Wg activity. Expression of the Dfz2N33 along the anteroposterior boundary using dpp-Gal4 induced a mild wing notching phenotype (Fig. 2I, 47%, n=15). Heterozygosity for hipk4 enhanced the loss of wing tissue as seen by the formation of moderate (27.8%, n=36) to severely truncated wings (Fig. 2J, 30.6%), which were rarely observed in dpp>Dfz2N33 adults (6.7%, n=15). Similarly, heterozygosity for hipk3 enhanced the severity of the wing notching induced by expression of Axin with sd-Gal4 (Fig. 2K,L). Thus, either a loss or a gain of hipk demonstrates that Hipk plays a positive role in transmission of the Wg signal.
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Hipk can promote Arm stabilization
In the absence of Wg pathway activation, the destruction complex targets
Arm for degradation. Upon Wg signaling, Arm is stabilized and accumulates in
the cytoplasm, serving as a measure of Wg activity. In wild-type wing discs,
cytoplasmic Arm is stabilized in cells adjacent to the DV boundary in which Wg
is active (Peifer et al.,
1994) (Fig. 4A). We
determined the status of stabilized Arm in hipk mutant clones. We
found that Arm is reduced in hipk clones at the DV boundary (arrows
in Fig. 4B-D). These mutant
cells showed a reduction of cytoplasmic Arm (Fig.
4B''-4D''),
whereas the adherens junction pool of Arm appeared normal. A similar decrease
in Arm is seen in discs expressing hipk RNAi
(Fig. 4E).
We then assessed whether Hipk could promote Arm stabilization by ectopically expressing Hipk using omb-gal4. Endogenous Arm protein levels are expanded in omb>hipk wing discs (Fig. 5B), compared with wild type (Fig. 5A). Consistent with these observations, western blot analysis of omb>hipk imaginal discs revealed higher levels of Arm than lysates obtained from wild-type larvae (Fig. 9A). These results suggested that elevated levels of Hipk can promote more Arm stabilization even in regions of the wing disc receiving lower levels of Wg signaling and that the stabilized Arm is active for Wg signaling, as it can induce target gene expression.
Hipk inhibits the degradation of Arm
To further address the role of Hipk in Arm stabilization, the consequences
of reducing hipk were assessed on the stability of a series of
UAS-Arm constructs expressed throughout the wing pouch in
hipk3/hipk4 mutant wing discs. The Arm
constructs UAS-ArmS2 and UAS-ArmS10
(Pai et al., 1997) have been
used to dissect the regulatory mechanisms of Arm localization and stability in
the embryo (Tolwinski and Wieschaus,
2001) and wing (Bajpai et al.,
2004). UAS-Myc-ArmS2 (encoding Myc-tagged
full-length Arm) was expressed in a broad domain in the central region of the
wing pouch and in the ventral periphery using omb-gal4
(Fig. 5C). Wing discs stained
with anti-Myc antibody showed stabilization of Myc-ArmS2 along the
DV boundary and the dorsal hinge primordia, similar to what was seen with
endogenous Arm (Fig. 5D). In
hipk mutant discs expressing omb>myc-ArmS2, we
observed a failure to accumulate Arm protein (arrow in
Fig. 5E), suggesting that in
these discs Arm protein is not efficiently stabilized owing to the reduction
of Hipk.
Expression of a UAS-Myc-ArmS10 construct lacking the
N-terminal target sites of the destruction complex induced a wg
gain-of-function phenotype characterized by ectopic bristles within the wing
blade (Couso et al., 1994;
data not shown). omb>UAS-ArmS10 discs show stabilized
Myc-tagged ArmS10 throughout the omb expression domain
(Fig. 5F). The accumulation of
the degradation-resistant form of Arm is unchanged in discs lacking
hipk (Fig. 5G). These
data show that Hipk promotes the stabilization of wild-type Arm, whereas
degradation-resistant Arm bypasses the need for Hipk-promoted
stabilization.
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We next investigated whether β-catenin was required for the
interaction between Lef1 and Hipk2. Hipk2 binds a Lef1
N mutant, lacking
the β-catenin binding domain, suggesting that β-catenin is not
required for the interaction between Lef1 and Hipk2
(Fig. 6C, lane 4). Furthermore,
Hipk2 co-immunoprecipitated with Lef1C, a construct lacking the
conserved DNA binding HMG box, suggesting this region was also dispensable for
the Lef1/Hipk2 interaction (Fig.
6C, lane 6). Although the β-catenin and Hipk2 interaction
does not require Lef1, we observe that complex formation is enhanced in the
presence of Lef1 (Fig. 6C,
lanes 8, 9).
Hipk phosphorylates Arm
In vitro kinase assays were performed to determine whether Hipk
phosphorylates components of the transcriptional complex. We found that Hipk
phosphorylates Arm, but not Drosophila Tcf
(Fig. 7A, lane 1; data not
shown). The kinase activity was crucial for this event, as a kinase dead Hipk
protein (Hipk KD) was unable to phosphorylate Arm
(Fig. 7A, lane 2). We next
tested the ability of Hipk to phosphorylate Arm truncations
(Fig. 7A, lanes 4, 11;
Fig. 7B). We find that Hipk can
phosphorylate a number of Arm truncations, namely Arm-N (encoding only the N
terminus, Fig. 7A, lane 11),
Arm N (missing the N terminus, data not shown) and Arm C
(missing most of the C terminus, Fig.
7A, lane 4), but not Arm-R (composed of just the central repeat
region, data not shown), suggesting that phosphorylation sites map to both the
N and C termini of Arm. Hipk proteins are known to phosphorylate numerous
targets on their substrates, and further detailed analyses will reveal the
functional significance of each of these phosphorylation events.
Hipk enhances Arm/Tcf-mediated transcription
We examined whether Hipk affected expression of the Tcf-responsive Topflash
transcriptional reporter (Korinek et al.,
1997). Transfection of HEK293T cells with Hipk enhanced the
transcriptional activity induced by Drosophila Tcf/Arm nearly tenfold
compared with transfection with Drosophila Tcf and Arm alone
(Fig. 8A, lanes 2, 3). This
effect was not observed upon transfection with Hipk KD
(Fig. 8A, lane 4), suggesting
that the kinase activity of Hipk is required for this effect. We also assessed
the combined effect of Nmo and Hipk on Topflash. As expected, expression of
the Tcf antagonist Nmo suppressed Topflash activation
(Fig. 8A, lane 5). This
inhibitory effect was partially relieved when HEK293T cells were
co-transfected with Hipk and Nmo (Fig.
8A, lane 6).
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Hipk and Hipk2 are functionally conserved
Although Hipk enhances Topflash in HEK293T cells and genetically promotes
Wg signaling, transfection of Hipk2 led to an inhibition of Topflash in
HEK293T cells (data not shown) (Wei et
al., 2007). This effect of Hipk2 may be attributed to additional
mammalian Hipk homologues or to the cellular context. To investigate this, we
performed similar assays with Hipk2 in HeLa cells and Drosophila S2R+
cells. Transfection of Hipk2 in HeLa cells led to an enhancement of Topflash
compared with control cells that were solely treated with Wnt3a-
(Fig. 8B, lanes 5, 6) or
Wnt1-conditioned media (Fig.
8B, lanes 9, 10). Hipk2 KD did not cause an increase in the
transcriptional response, indicating this effect is kinase dependent
(Fig. 8B, lanes 7,11).
Strikingly, addition of fly Hipk to mammalian cells also enhanced the
transcriptional response (Fig.
8B, lanes 8, 12). Consistent with the HeLa cell data, Hipk2 was
able to induce Topflash in the presence or absence of Wg-conditioned media in
Drosophila S2R+ cells (Fig.
8C, lanes 3, 6). These experiments revealed that Hipk2 and Hipk
perform conserved functions in multiple cellular contexts.
Hipk promotes Arm stability in vivo and in vitro
Our data suggested that Hipk is required to stabilize both endogenous and
overexpressed full-length Arm. We confirmed this by western blotting of
protein extracts from omb>hipk wing discs and observed
dramatically elevated levels of Arm protein
(Fig. 9A), consistent with the
elevated levels of Arm seen in discs in
Fig. 5B. Arm and β-catenin
protein levels are also increased in S2R+ cells
(Fig. 9B) and HeLa cells
(Fig. 9C) transfected with
Hipk2, respectively.
Further cell culture assays were performed to confirm this role.
Degradation of Arm was assessed in HEK293T cells expressing HA-Arm that were
treated with the protein synthesis inhibitor, cycloheximide (Chx)
(Abou Elela and Nazar, 1997).
In these cells, addition of Hipk prolonged the stability of Arm
(Fig. 9D). Similar results were
obtained in HeLa cells (data not shown). The presence of HA-Hipk KD partially
promoted the accumulation of Arm levels, but not to the extent seen with Hipk
WT.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Hipk and Nmo exert differential effects on Wg signaling
Nlk is a conserved antagonist of the Lef1/β-catenin transcriptional
complex. Our research has shown that Nmo is also an inducible antagonist of
the Wg signal in the developing Drosophila wing (Zeng et al., 2004).
It was previously reported that Wnt1 induces the activation of a putative
Tak1-Hipk2-Nlk kinase cascade to promote the degradation of the Myb
transcription factor (Kanei-Ishii et al.,
2004). We sought to delineate the physiological relevance of this
potential kinase cascade; specifically, we determined whether these
interactions played a role in the regulation of the Wg pathway. Our data
reveal that in Drosophila Nmo and Hipk do not form a kinase cascade
in this context, rather they exert opposing effects on the same pathway,
probably through distinct mechanisms.
Hipk proteins promote Tcf/Lef1-mediated transcription
The Wg morphogen can bring forth a spectrum of biological processes.
Sustaining maximal levels of signaling could be accomplished through the
amplification and enhancement of the signal in the wing margin. In support of
this model, we found that overexpression of Hipk expands the expression of Wg
targets such as Dll, Sens and Ac. Transcriptional assays reveal that both
Drosophila Hipk and mouse Hipk2 enhance the transcriptional activity
of Tcf and Lef1, respectively, in a kinase-dependent manner. These findings
strongly suggest that Hipk and Hipk2 function to enhance the activity of the
transcriptional complex to promote the Wg/Wnt signal.
Hipk stabilizes Arm
Accumulation of stabilized Arm is paramount to effective Wg signaling.
Failure to escape the destruction complex results in Arm degradation and
inhibition of Tcf-mediated gene activation. Thus understanding the regulation
of Arm is central to the global understanding of how the Wg signal is
modulated. In our studies, we reveal a role for Hipk in Arm stabilization.
This feature is highlighted by the loss of stabilized Arm in hipk
mutant clones. Additionally, in hipk mutant discs, overexpressed
wild-type Arm fails to accumulate, despite its expression in domains of high
Wg signaling. These findings demonstrate that Hipk plays an important role in
Arm stabilization. Hipk may reduce the ability of Arm to interact with
destruction complex components or may increase the nuclear retention of Arm.
In the absence of Hipk, either of these scenarios would give the destruction
complex more access to Arm. In agreement with such a model, we find that
increasing Hipk activity in the wing surpasses the inhibitory effects of the
degradation machinery and expands the perimeter of stabilized Arm.
Furthermore, we have found that the presence of Hipk or Hipk2 in cell culture
stabilizes Arm/β-catenin. Thus, the enhanced transcriptional activity is
probably due to the elevated availability of Arm protein.
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).
Elaborate regulatory networks in the cytoplasm and nucleus are vital to
maintaining appropriate levels of β-catenin. It is well documented that
phosphorylation in the N terminus of β-catenin is crucial for its
negative regulation (reviewed by Daugherty
and Gottardi, 2007). A chain of phosphorylation events begins when
Casein Kinase I (CKI) primes β-catenin for successive modifications by
GSK-3β (Amit et al., 2002;
Liu et al., 2002;
Yanagawa et al., 2002).
Central to this event is Axin, which provides a scaffold for APC, CK1,
GSK-3β and β-catenin (Amit et
al., 2002; Hart et al.,
1998; Hinoi et al.,
2000; Ikeda et al.,
1998). N-terminally phosphorylated β-catenin is ubiquitinated
by βTrCP ubiquitin ligase and targeted for degradation via the
proteasome.
Wnt signaling promotes the accumulation of β-catenin; however, some of
the mechanisms governing this process remain enigmatic. Although
overexpression of wild-type β-catenin/Arm is unable to overcome the
effects of the degradation machinery
(Mohit et al., 2003;
Pai et al., 1997),
Wnt-stimulated β-catenin can resist the activity of the destruction
complex. Although achieving stabilized pools of β-catenin represents the
core goal of the Wnt pathway, high levels of β-catenin are not always
coupled with elevated transcription (Guger
and Gumbiner, 2000; Staal et
al., 2002). For example, in Xenopus, alanine substitution
of one of the GSK3 target residues leads to elevated β-catenin levels,
without causing an increase in Tcf-mediated transcription
(Guger and Gumbiner, 2000).
Thus, further posttranslational modifications of β-catenin are necessary
to potentiate its signaling activity.
Furthermore, phosphorylation can affect β-catenin stability by
affecting protein-protein interactions that regulate protein turnover and
activity. Phosphorylation of β-catenin by Cdk5 inhibits APC binding to
β-catenin (Munoz et al.,
2007; Ryo et al.,
2001), whereas phosphorylation by CK2 promotes β-catenin
stability and transcriptional activity
(Song et al., 2003).
Regulation of β-catenin/Arm-mediated transcription
Recent advances have begun to unravel the molecular complexity that
controls β-catenin-mediated transcription within the nucleus. Upon
pathway activation, Tcf recruits Arm to the enhancers of Wg-responsive genes
where Arm forms multiple transcriptional complexes along its length
(Hoffmans et al., 2005;
Kramps et al., 2002;
Thompson et al., 2002;
Hecht et al., 1999;
Mosimann et al., 2006).
Formation of these transcriptional units is needed for the transmission of the
Wg/Wnt signal. Recent studies have shown that phosphorylation (distinct from
the N-terminal phosphorylation that triggers β-catenin destruction) may
modulate its ability to recruit these co-factors (reviewed by
Daugherty and Gottardi, 2007).
We have found that the Hipk-dependent stabilized form of Arm is
transcriptionally active and induces the expression of Wg targets, suggesting
modification by Hipk may promote protein interactions.
Models for the role of Hipk in Wg/Wnt signaling
APC and the cell-adhesion molecule E-cadherin compete with Tcf for
overlapping binding sites on β-catenin
(Hulsken et al., 1994;
von Kries et al., 2000).
Competition between proteins may play an important role in the regulation of
the Wnt signaling pathway (reviewed by Xu
and Kimelman, 2007). We propose that Hipks may promote the
stability of Arm/β-catenin by excluding further interactions with other
proteins, including those that antagonize Arm/β-catenin. Given that Hipks
can also bind to Tcf/Lef1, we predict that these proteins may act
synergistically to displace the inhibitory partners of β-catenin. In
agreement with such a role, we observe that Lef1 enhances the interaction
between Hipk2 and β-catenin, and these interactions may insulate
β-catenin from components of the degradation machinery.
Although Hipk phosphorylates Arm, the functional significance of this
modification has yet to be determined. Hipk might facilitate the interactions
between Arm and its transcriptional co-factors, as Hipk2 phosphorylation has
been shown to affect gene regulation by modifying the composition of various
transcriptional complexes (reviewed by
Calzado et al., 2007). Hipk may
also enhance the formation of the β-catenin/Tcf transcriptional complex
by inducing a conformational change and/or reducing the affinity of possible
inhibitors for β-catenin through the phosphorylation of β-catenin.
Recently, it has been reported that Hipk2 could antagonize
β-catenin/Tcf-mediated transcription in a kinase-independent manner
(Wei et al., 2007). Although
these data appear in conflict with our findings, we have observed that the
effect of Hipk2 on transcription is very cell type- and target gene-dependent,
suggesting Hipk2 function is affected by its cellular context, most probably
owing to the availability of targets and co-factors.
The dynamic localization of Hipk2 in the nucleus, nucleoplasm and in
cytosolic speckles suggests that the protein may carry out distinct roles in
each site (reviewed by Calzado et al.,
2007; Rinaldo et al.,
2007). Given the growing list of interacting proteins, it is
tempting to speculate that specific Hipk function is determined in part
through its particular localization. It is also possible that Hipk/Hipk2 may
act as a scaffolding protein, bringing together multiple binding partners.
Ongoing biochemical studies will further uncover the molecular significance of
these interactions. Hipk proteins are emerging as important components of
multiple signaling networks. Our studies describe the roles of Hipk and Hipk2
as Wnt/Wg regulators and shed light on the regulatory mechanisms governing
this conserved pathway.
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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