IQGAP1 Functions as a Modulator of Dishevelled Nuclear Localization in Wnt Signaling
et al. (2013) IQGAP1 Functions as a Modulator of Dishevelled Nuclear Localization in Wnt Signaling. PLoS
ONE 8(4): e60865. doi:10.1371/journal.pone.0060865
IQGAP1 Functions as a Modulator of Dishevelled Nuclear Localization in Wnt Signaling
Toshiyasu Goto 0
Atsushi Sato 0
Masahiro Shimizu 0
Shungo Adachi 0
Kiyotoshi Satoh 0
Shun- ichiro Iemura 0
Tohru Natsume 0
Hiroshi Shibuya 0
Masaru Katoh, National Cancer Center, Japan
0 1 Department of Molecular Cell Biology, Medical Research Institute, Tokyo Medical and Dental University , Bunkyo-ku, Tokyo , Japan , 2 Biomedicinal Information Research Center, National Institutes of Advanced Industrial Science and Technology , Kohtoh-ku, Tokyo , Japan
Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.
. These authors contributed equally to this work.
Wnt signaling plays important roles in multiple developmental
events during embryogenesis , . Canonical Wnt signaling is
initiated by binding of the Wnt ligand to the cell-surface Frizzled
and transmembrane LRP complex. This leads to the membrane
recruitment and activation of Dishevelled (DVL), which
inactivates the APC/Axin/GSK-3 complex in the cytoplasm,
responsible for the degradation of -catenin , . As a result, -catenin
accumulates in the cytoplasm, translocates to the nucleus and
associates with Tcf transcription factors, which activate the Wnt
target genes , . In Xenopus, Wnt signaling accompanied by
catenin nuclear localization at the dorsal side is an important for
axis formation during early embryogenesis . Ventral
overexpression of Xwnt-8, -catenin and DVL2 induces a secondary axis
and promotes expression of Wnt target genes, such as Siamois, Xnr3
and Xtwn .
There are three DVL isoforms, DVL1, DVL2 and DVL3 ,
, which are well-conserved among various organisms. Each
isoform plays a similar role in the canonical Wnt pathway, but
have different sensitivities to Wnt stimulation . DVL contains
three conserved regions known as the DIX, PDZ and DEP
domains , . Both the DIX and PDZ domains are necessary
for canonical Wnt inactivation of -catenin degradation , .
In contrast, the DEP domain does not affect canonical signaling,
but is involved in the planar cell polarity (PCP) pathway .
DVL plays an additional role in the Wnt signaling pathway, by
localizing to the nucleus and binding a complex containing
catenin and Tcf, which in turn activates Wnt target genes in the
nucleus . The subcellular localization of DVL, either on the
cell membrane or in the nucleus, is important for understanding its
function in Wnt signaling.
IQGAP1 contains multiple protein-interacting domains: the
CH (calponin homology) domain binds to F-actin, the WW
domain binds to ERK2, the IQ repeat motifs bind to calmodulin
and myosin light chain, and the Ras GAP-like domain binds to
Cdc42 and Rac1 . IQGAP1 is also known to bind to
Ecadherin and -catenin, and is involved in cytoskeltal
reorganization and cell adhesion , . On the other hand, IQGAP1
stimulates -catenin-mediated transcriptional activation34. The
subcellular localization of IQGAP1 varies in several cultured cells,
and analysis of its domains indicates that IQGAP1 may be
localized in the cytoplasm, cell membrane and nucleus . These
subcellular localizations are presumably linked to its cellular
functions. There are also three isoforms of IQGAP: IQGAP1,
IQGAP2 and IQGAP3. Their subcellular localizations suggest
both similarities and differences in function . Each isoform has
a different role, and in some cases IQGAP1 has an opposite
function to IQGAP2 , . The Xenopus xIQGAP1 and
xIQGAP2 genes have been isolated  and shown to be involved
in cadherin-mediated cell adhesion , . We also isolated
xIQGAP3 and have generated antisense morpholino
oligonucleotides based on these sequences.
In the present study, we identified IQGAP1 as a novel
DVLbinding protein. Binding between IQGAP1 and DVL2 mutually
contributed to their nuclear localization. The depletion of
endogenous IQGAP1 in Xenopus embryos suppressed secondary
axis induction and expression of Wnt target genes. These results
reveal a novel role for IQGAP1 in modulating the subcellular
localization and transcriptional activation of components of the
Wnt signaling pathway.
Materials and Methods
All animal experiments were performed under the ethical
guidelines of Tokyo Medical and Dental University, and animal
protocols were reviewed and approved by the animal welfare
committee of the Tokyo Medical and Dental University.
The human and Xenopus DVL, IQGAP isoforms were amplified
by RT-PCR from cDNA templates prepared from HEK 293 cells
and Xenopus embryos, respectively, and were subcloned into the
pRK5 and pCS2+ vectors. Each truncated mutant was
constructed by PCR and contained the following amino acid sequences.
hDVL1-1: 1486 aa, hDVL1-2: 476671 aa, hIQGAP1-1: 1669
aa, hIQGAP1-2: 631951 aa, hIQGAP1-3: 6311657 aa,
hIQGAP1-4: 9011060 aa, hIQGAP1-5: 10671154 aa,
hIQGAP1-6: 10321116 aa, xDVL2-DIBR: 1509 aa, xDVL2-IBR:
510736 aa, xIQGAP1-DDBR: 1856 and 10611657 aa,
xIQGAP1-DBR: 8571060 aa. We made GFP constructs of
xDVL2, xIQGAP1 and their truncated mutants by conjugating
with the GFP sequence at the C-terminus.
The sequences of the primer pairs were as follows. In Figure S1,
xDVL1: Forward 59-CCAGCATAGCGAAGGTAGTA-39;
Reverse 59-TACACCTTGCTCCCGATCTT-39. xDVL2: Forward
59-TCAGACTCACTACCAGATCC-39. xDVL3: Forward
59-CATGCGGAAGGATTGTCTAC-39. xIQGAP1: Forward
59TCAATGCTGTGTGTGTCTGC-39. xIQGAP2: Forward
59-AACATCTTCATCACGGCGAC-39. xIQGAP3: Forward
59-TGCTGTGTAATTGAGGGACG-39. Ornithine decarboxylase (ODC): Forward
59TCCATTCCGCTCTCCTGAGCAC-39. IIn Figure S5,
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): Forward
59-CATGAGGTCCACCACCCTGTTGCTG-39. Axin2: Forward
59-ATGACACTGCTGATGGTGGTGGTGC-39. TGF2: Forward
59CAGAAGTTGGCATTGTACCCTTTGG-39. Mouse IQGAP1:
Forward 59-AAGTTTGACGTGCCTGGTGA-39; Reverse
59GGTATCTGTTCTTTGGGTCC-39. xIQGAP1: Forward
Embryo handling and morpholino oligonucleotides
Capped mRNAs were synthesized from linearized vectors using
the mMessage Machine kit (Ambion). The morpholino
oligonucleotides (MO) (Gene Tools, LLC) used here were
(xIQGAP3-MO). The specificity of each MO was confirmed by its
ability to inhibit the translation of FLAG-tagged mRNAs
containing the targeted site with or without 5-mismatched
sequences. MO (10 ng) and FLAG-tagged mRNAs (100 pg) were
co-injected with -globin-FLAG mRNA (100 pg) as loading control
into the animal poles of 4-cell stage embryos, and the injected
animal caps were dissected at stage 10. Lysates from the animal
caps were subjected to Western blotting with anti-FLAG antibody
(M2, Sigma) (Fig. S1C).
MOs and mRNAs were injected into four animal blastomeres at
the 4-cell stage for dissection of animal caps or into two dorsal or
ventral blastomeres at the 4-cell-stage for quantitative RT-PCR
analysis and observation of embryo phenotypes. Animal cap
explants of the injected (10 pg mRNA of each GFP fused
construct) embryos were dissected at the early gastrula stage
(st.10), and fixed for DAPI staining as previously reported .
We counted the number of cell that has fluorescence signals. When
the fluorescence signal overlapped with DAPI staining was similar
and brighter than un-overlapped fluorescence signal in counted
cells, we defined such cells as nuclear localized cells. If nuclear
fluorescence signals were not clear, we used ImageJ software (NIH)
and measured the strength of brightness of fluorescence signals to
define nuclear localized signals or not. The ratio of nuclear
localized cells in total counted cells was computed for every
explant and the average of ratio was taken with six explants in 3
independent experiments. Dorsal or ventral sectors of the injected
embryos were dissected at st.10, and total RNA was extracted for
RT-PCR analysis. The cytoplasmic and nuclear fractions were
prepared as described with modifications .
Total RNA was prepared using TRIzol (Invitrogen). cDNA
synthesis was carried out using Moloney murine leukemia virus
reverse transcriptase (Invitrogen). Quantitative PCR was
performed with an Applied Biosystems 7300 Real-Time PCR Cycler
(ABI) using THUNDERBIRD SYBR qPCR Mix (TOYOBO).
The sequences of the primer pairs were as follows. Ornithine
decarboxylase (ODC): Forward
59-AATGAAGATGCTGACTGGCAAAAC-39. Siamois: Forward
59-TGTTGACTGCAGACTGTTGA39. Xnr3: Forward 59-CTTCTGCACTAGATTCTG-39; Reverse
59-CAGCTTCTGGCCAAGACT-39. Xtwn: Forward
59GTGCCGATGGTAGGAAATGATC-39. Xenopus embryonic
ODC was used for normalization of cDNA samples.
Antibodies and cell lines
The following antibodies were used for immunoprecipitation
and/or Western blotting analysis: Horseradish peroxidase
conjugated anti-mouse IgG (GE); Horseradish peroxidase conjugated
anti-rabbit IgG (GE); anti-FLAG (M2 and F7425, Sigma);
antiMYC (9B11, Cell Signaling); anti-DVL1 (3F12 and Q-25, Santa
Cruz); anti-IQGAP1 (H-109, Santa Cruz); anti-beta-tubulin
(sc58884, Santa Cruz); anti-histone-H3 (sc-10809, Santa Cruz). We
used following cell lines: HEK 293 cells , HEK 293T cells
, NIH3T3 cells , L cells (CRL-2648, ATCC), L Wnt3A
cells (CRL-2647, ATCC). Recombinant human Wnt3A (R&D
Systems; 20 ng/ml) or four day Wnt-3A conditioned medium
from L-Wnt-3A cells was used for Wnt stimulation of cultured
cells. The growth medium for each cell type is described by
American Type Culture Collection.
Protein identification by LC-MS/MS analysis
FLAG-human DVL1 was expressed in HEK 293 cells, and
DVL1 and associated proteins were recovered from cell extracts
by immunoprecipitation with anti-FLAG antibody. The
DVL1associated complexes were digested with Axhromobacter protease I,
and the resulting peptides were analyzed using a nanoscale
LCMS/MS system, as described previously .
IQGAP associates with DVL
To identify novel proteins that may bind to DVL, we performed
a high-throughput analysis of proteins that co-immunoprecipitated
with mouse DVL1 in HEK 293 cells using direct nanoflow liquid
chromatography-coupled tandem mass spectrometry . We
identified several known DVL-binding proteins, such as CK1 ,
CK2 , Strabismus , Par1 , Axin  and PP2C .
In addition, we identified IQGAP1 as a candidate protein that
may physically interact with DVL1. An interaction between
ectopically expressed IQGAP1 and DVL1 was confirmed in HEK
293 cells (Fig. 1A). Immunoprecipitation analysis using each
protein antibody also confirmed the existence of an endogenous
IQGAP1 and DVL1 complex in HEK 293T cells, and their
interaction was increased by Wnt stimulation (Fig. 1B). In
vertebrates, three isoforms of IQGAP and DVL have been
identified: IQGAP1, IQGAP2 and IQGAP3, and DVL1, DVL2
and DVL3. We confirmed that each IQGAP isoform also bound
to each DVL isoform (Fig. 1C-1E). To determine the region in
IQGAP1 responsible for binding to DVL1, several truncated
mutants of IQGAP1 were examined in co-precipitation assays. We
found that the region between the C-terminal IQ repeat domain
and the N-terminal Ras GAP-like domain of IQGAP1 (termed
DBR; Dishevelled Binding Region) was responsible for binding to
DVL1 (Fig. 1F and 1G) Conversely, the C-terminus of DVL1
(termed IBR; IQGAP Binding Region) is necessary for binding to
IQGAP1 (Fig. 1H and 1I). Both the IBR and DBR are
wellconserved among the three DVL and IQGAP isoforms,
respectively. The amino acid sequence similarities of IBR and DBR
among the three DVL and IQGAP isoforms were as follows, the
similarity of IBR between DVL1 and DVL2 is 47.1%; the
similarity of IBR between DVL1 and DVL3 is 55.7%; the
similarity of IBR between DVL2 and DVL3 is 53.7%; the
similarity of DBR between IQGAP1 and IQGAP2 is 82.4%; the
similarity of DBR between IQGAP1 and IQGAP3 is 76.8%; the
similarity of DBR between IQGAP2 and IQGAP3 is 72.8%. We
conclude that IQGAP1 can associate with DVL in mammalian
IQGAP1 determines the nuclear localization of DVL
Whereas membrane-localized DVL functions to inhibit
degradation of cytoplasmic -catenin in the canonical Wnt pathway
, , nuclear-localized DVL is required, together with
nuclear -catenin, for transactivation of the downstream targets of
Wnt signaling . To analyze DVL and IQGAP1 functions, we
used the systems of Xenopus embryos. All isoforms of DVL and
IQGAP are conserved well among vertebrates. The transcripts of
their Xenopus homologues were expressed during early embryonic
stages and were equivalently expressed at early gastrula stages (Fig.
S1A and S1B). We subcloned their cDNA and generated their
antisense morpholino oligo nucleotides (Fig. S1C). To examine
how IQGAP affects DVL localization in Wnt signaling, we
investigated the subcellular distribution of DVL fused to green
fluorescent protein (GFP) in Xenopus embryonic cells. Fluorescence
produced by the DVL2 (xDVL2)-GFP fusion appeared as a
punctate pattern in the cytoplasm (Fig. 2A, left panel). DVL has
been reported to be recruited to the plasma membrane by the
Frizzled receptors in the Wnt pathway . We confirmed that
xDVL2-GFP accumulated in the plasma membrane when
coexpressed with Xenopus frizzled 7 (Xfz7) (Fig. 2A, center panel).
Depletion of xIQGAP1 by antisense morpholino oligonucleotides
(xIQGAP1-MO) did not affect the membrane localization of
xDVL2-GFP (Fig. 2A, right panel). These results suggest that
xIQGAP1 is not involved in the plasma membrane localization of
Stimulation by Wnt ligands is known to increase the nuclear
localization of DVL . When xDVL2-GFP was co-expressed
with Xwnt-8 in animal cap cells, nuclear GFP fluorescence was
increased (Fig. 2B, third and fourth panels, 2D, lanes 1, 3),
whereas GFP was mainly localized in the cytoplasm with or
without Xwnt-8 (Fig. 2B, first and second panels, 2C). However,
injection of xIQGAP1-MO decreased nuclear fluorescence
generated by co-expression of xDVL2-GFP and Xwnt-8 (Fig. 2B, fifth and
sixth panels, 2D, lanes 3, 4). We also confirmed that the amounts
of xDVL2-MYC protein in the nuclear fractions of animal cap
cells were reduced by depletion of xIQGAP1 (Fig. 2E). Expression
of xIQGAP1-GFP resulted in fluorescence localized mainly to the
cytoplasm, but nuclear fluorescence was increased by
co-expression of Xwnt-8 (Fig. 3A, first and second panels, 3B, lanes 1,3).
Depletion of xDVL2 led to a decrease in nuclear fluorescence
generated by the co-expression of xIQGAP1-GFP and Xwnt-8
(Fig. 3B, lanes 3, 4). Moreover, we found that depletion of all three
xDVLs (xDVL1, xDVL2 and xDVL3) reduced severely the
nuclear localization of xIQGAP1-GFP in Wnt-stimulated cells
(Fig. 3A, third and fourth panels, 3C, lanes 3, 4 and 3D). These
results suggest that xIQGAP1 and xDVL2 play a crucial role in
each other nuclear accumulation, depending on Wnt signaling.
We next examined whether a physical interaction between
xIQGAP1 and xDVL2 is required for their nuclear localization.
We generated a fusion of GFP to xDVL2-DIBR, a truncated
version of xDVL2 lacking the IBR domain, and observed a
punctate fluorescence pattern in the cytoplasm, similar to that seen
with xDVL2-GFP (Fig. S2A). The proportion of fluorescence
found in the nucleus was also similar to xDVL2-GFP (Fig. S2B).
However, co-expression of Xwnt-8 did not alter the proportion of
GFP fluorescence found in the nucleus, (Fig. S2B), suggesting that
the ability of xIQGAP1 to promote nuclear localization of xDVL2
requires the IBR domain in xDVL2. Consistent with this, a fusion
of just the IBR domain to GFP (xDVL2-IBR-GFP) was localized
predominantly in the nucleus (Fig. S2A and S2C). We next fused
GFP to xIQGAP1-DDBR, which deletes the DBR domain of
xIQGAP1. This xIQGAP1-DDBR-GFP fusion was also localized
mainly in the cytoplasm (Fig. S2A), and the proportion of
fluorescence found in the nucleus was less than that observed with
full-length xIQGAP1-GFP (Fig. S2D). Co-expression of Xwnt-8
also did not affect the nuclear localization of
xIQGAP1-DDBRGFP (Fig. S2D). A fusion of just the DBR domain to GFP
(xIQGAP1-DBR-GFP) was localized mainly in the nucleus (Fig.
S2A and S2E). We further investigated the effects of
overexpression of xIQGAP1 or xDVL2 on the nuclear localization of
xDVL2-GFP or xIQGAP1-GFP, respectively. Nuclear localization
of xDVL2-GFP was increased by the expression of xIQGAP1 and
xIQGAP1-DBR (Fig. S3A). Meanwhile, expression of
xIQGAP1DDBR suppressed the nuclear localization of xDVL2-GFP induced
by co-expression of Xwnt-8 (Fig. S3A). In contrast, the nuclear
localization of xIQGAP1-GFP was not affected by the expression
Figure 1. IQGAP associates with DVL. (A) Interaction between ectopically-expressed hIQGAP1 and hDVL1 in HEK 293 cells. Immunoprecipitates
(IP) obtained using anti-FLAG antibody were subjected to Western blotting (WB) with the indicated antibodies. +, present; -, absent. (B) Interaction
between endogenous hIQGAP1 and hDVL1 in HEK 293T cells. The cultured cells were stimulated with recombinant human Wnt3A for 6 hours (right
panels). (CE) Interaction between ectopically-expressed hIQGAP1, hIQGAP2, hIQGAP3 and hDVL isoforms in HEK 293T cells. (C) hIQGAP1. (D)
hIQGAP2. (E) hIQGAP3. (F) A schematic of the domains of hIQGAP1 and truncated constructs. (G) Interactions between ectopically-expressed hDVL1
and truncated hIQGAP1 constructs. (H) Interactions among ectopically-expressed hIQGAP1 and truncated hDVL1 constructs. (I) A schematic of the
domains of hDVL1 and truncated constructs.
46.4%, lane 4: n = 477, 29.4%. P,0.01 [between lane 1 and lane 2], P,0.01 [between lane 3 and lane 4]. (E) Cytoplasmic and nuclear distribution of
xDVL2, xIQGAP1 and -catenin in animal cap cells. MYC-tagged xDVL2 mRNA (100 pg) was injected into the animal poles of 4-cell stage embryos, and
the injected animal caps were dissected at stage 10. Lysates from the animal caps were fractionated and subjected to Western blotting with indicated
antibodies. Each relative intensity was measured by ImageJ, and its relative ratio was calculated against Input with beta-tubulin for cytoplasm or with
Histone H3 for nuclear. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Students
t-test. P,0.1 [between lane 5 and lane 6], P,0.1 [between lane 7 and lane 8].
of xDVL2 or xDVL2-IBR (Fig. S3B). However, xDVL2-DIBR
suppressed the nuclear localization of xDVL2-GFP induced by
coexpression of Xwnt-8 (Fig. S3B). Moreover, immunoprecipitation
experiments showed that Wnt stimulation increased the
interaction between xDVL2 and xIQGAP1 in HEK 293T cells (Fig. 3E).
Taken together, these results suggest that a physical interaction
between xDVL2 and xIQGAP1 is required for their nuclear
localization induced by the canonical Wnt signaling pathway.
xIQGAP1 is necessary for the canonical Wnt pathway
To determine whether xIQGAP1 is also involved in the
canonical Wnt pathway during early development, we investigated
the effects of xIQGAP1 on the transactivation of Wnt target genes
and the secondary axis induction. Dorsal injection of an antisense
morpholino oligonucleotide against xIQGAP1 (xIQGAP1-MO)
reduced endogenous transcripts of the Wnt signal target genes
Siamois, Xnr3 and Xtwn (Fig. 4A). When xDVL2, Xwnt-8 or -catenin
mRNA was injected into the ventral sides of four-cell embryos, a
secondary axis was formed and Wnt signal target genes were
induced. The induction of the partial secondary axis and Wnt
target genes induced by Xwnt-8 was also suppressed by the
depletion of xIQGAP1 (Fig. 4B4D). Dorsal overexpression of
xIQGAP1 mRNA increased expression of Wnt target genes
(Fig. 4E). On the other hand, the overexpression or depletion of
xIQGAP2 showed opposite effects for Wnt target gene expression
(Fig. 4A, 4D and 4E). xIQGAP3 showed ambiguous effects on Wnt
target gene expression, especially expression of Siamois (Fig. 4A, 4D
and 4E). However, depletions of either xIQGAP2 or xIQGAP3
did not alter the partial secondary axis induction by Xwnt-8 (Fig. 4B
and 4C). These results suggest that xIQGAP1 is necessary for
Wnt-related early embryogenesis in a subtype-specific manner.
We also found that the suppressions of induction of Wnt target
genes and partial secondary axis by xIQGAP1-MO were rescued by
expression of wild-type xIQGAP1, but not by either
xIQGAP1DDBR or -DBR (Fig. 4F, 4G, S4AS4D). However, depletion of
xIQGAP1 did not affect the secondary axis formation induced by
Simaois, which is one of the Wnt signal target genes (Fig. 4H).
Moreover, we also observed the reduction of endogenous
IQGAP1 by the siRNA (siIQGAP1) suppressed the expression of
Wnt target genes induced by Wnt3A stimulation in cultured cells
(Fig. S5A). These results suggest that xIQGAP1 functions as an
intermediate molecule in the canonical Wnt signaling pathway in
early development promoting the nuclear localization of xDVL2.
The IQGAP binding region of xDVL2 is important for
canonical Wnt signaling
To further confirm whether the binding between IQGAP1 and
DVL is critical in the canonical Wnt pathway during early
development, we investigated the effects of xDVL mutants on the
transactivation of Wnt target genes and secondary axis induction.
Similar to our previous observation that injection of xDVL2-MO
did not affect severely nuclear localization of xIQGAP1 (Fig. 3B),
we also observed no reduction in Wnt target gene expression
induced at the ventral side by Xwnt-8 when xDVL2-MO was
coinjected (Fig. 5A). However, depletion of all three xDVLs reduced
nuclear localization of xIQGAP1, expression of the Wnt target
genes and suppressed formation of the secondary axis induced by
Xwnt-8 or -catenin (Fig. 3C, 3D, 5B, and 5C). These results suggest
that three xDVL genes act redundantly in the canonical Wnt
signal pathway. Suppression of secondary axis formation and Wnt
target gene expression caused by depletion of all three xDVLs
could be rescued by co-expression of wild-type xDVL2, but only
weakly by xDVL2-DIBR and barely by xDVL2-IBR (Fig. 5B, and
5C). Moreover, co-expression of xIQGAP1-DBR reduced the
expression of Wnt target genes induced by Xwnt-8, xDVL2 or
catenin in Xenopus embryos (Fig. 5D, S6A and S6B), and the
expression of xIQGAP1-DBR in cultured cells reduced the
expression of Wnt target gene induced by Wnt3A (Fig. S5B).
These results support the idea that binding between xDVL2 and
xIQGAP1 plays important roles for canonical Wnt signaling.
In the present studies, we show that IQGAP1 is necessary for
the nuclear localization of DVL in the canonical Wnt signaling
pathway. Previous studies have shown that nuclear localization of
DVL is necessary for the sequential activation of Wnt target genes
, . It has also been shown that the nuclear localization
signal (NLS) located between the PDZ and DEP domain of
xDVL2, and the nuclear export signal (NES) located at the
Cterminus are important for the nuclear localization and
transcriptional activation of Wnt target genes . Interestingly, we
observed that xDVL2-IBR, a truncated protein consisting of just
the IQGAP-binding region from the C-terminus of xDVL2,
localized predominantly in the nucleus, even though this region
contains an NES and not an NLS. Moreover, the nuclear
localization of xDVL2-DIBR-GFP, which contains an NLS, but
not an NES, did not increase with Wnt-8 stimulation any longer.
Although our findings suggest a new molecular mechanism
mediating xIQGAP1-dependent nuclear localization of xDVL2,
we could not positively state that our findings is independent of the
NLS or NES motifs within DVL2. Further studies need to clarify
the inconsistencies using same mutants.
In the canonical Wnt pathway, DVL is necessary for both the
inactivation of -catenin degradation in cytoplasm21 and the
activation of Wnt target genes by forming a complex containing
catenin and Tcf in nuclei . In the present study, we have
shown that IQGAP1 interacted with DVL and that the depletion
of IQGAP1 reduced the nuclear localization of DVL, while
IQGAP1 did not affect on the membrane localization of DVL
required for -catenin stability in cytoplasm. These results suggest
that IQGAP1 plays a role in the nuclear translocation of DVL in
the canonical Wnt pathway.
We showed that the nuclear localization of xIQGAP1 and
xDVL2 were increased by Wnt stimulation. In contrast, the
nuclear localization of xDVL2-DIBR-GFP and
xIQGAP1-DDBRGFP did not increase with Wnt stimulation, while over-expression
of xDVL2-DIBR or xIQGAP1-DDBR interfered with the nuclear
localization of xIQGAP1-GFP or xDVL2-GFP induced by Wnt
stimulation, respectively. Reduced expression of Wnt target genes
due to depletion of endogenous xIQGAP1 or xDVLs was barely or
weakly rescued by expression of xIQGAP1-DDBR or xDVL2-DIBR,
respectively. Conversely, xDVL2-IBR-GFP and
xIQGAP1-DBRFigure 3. Localization of xIQGAP1-GFP. (A) Nuclear localization of xIQGAP1-GFP in stage 10 Xenopus animal cap cells over-expressing Xwnt-8
and xDVL1, 2, 3-MO. GFP signals (left). DAPI staining of animal cap cells (center). Merge (right). (B, C)The ratio of cells that had nuclear fluorescence
signals. The following procedure is indicated in Figure 2C. (B) The ratio of nuclear localized xIQGAP1-GFP in cells injected with xDVL2-MO. Lane 1:
n = 388, 22.7%, lane 2: n = 361, 22.2%, lane 3: n = 616, 20.9%, lane 4: n = 534, 39.7%. P.0.1 [between lane 1 and lane 2], P,0.05 [between lane 3 and
lane 4]. (C) The ratio of nuclear-localized xIQGAP1-GFP in cells injected with xDVL1-MO, xDVL2-MO and xDVL3-MO in Xenopus animal cap cells at stage
10. Lane 1: n = 1424, 23.0%, lane 2: n = 1306, 21.6%, lane 3: n = 1702, 37.6%, lane 4: n = 1409, 16.6%. P.0.1 [between lane 1 and lane 2], P,0.01
[between lane 3 and lane 4]. (D) Cytoplasmic and nuclear distribution of xIQGAP1 in animal cap cells. MYC-tagged xIQGAP1 mRNA (100 pg) was
injected into the animal poles of 4-cell stage embryos. The following procedure is indicated in Figure 2C. P,0.1 [between lane 5 and lane 6], P,0.1
[between lane 7 and lane 8]. (E) Interaction between ectopically-expressed xDVL2 and xIQGAP1 in HEK 293T cells. The transfected cultured cells were
stimulated with recombinant human Wnt3A for 6 hours in lanes 3 and 4. The bars represent the IP/Input ratios of xDVL2-FLAG for each transfection.
Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Students t-test. P,0.1 [between
lane 2 and lane 4].
GFP were mainly localized in the nuclei regardless of Wnt
stimulation. Moreover, xIQGAP1-DBR reduced expression of the
Wnt target genes induced by xDVL2, Xwnt-8 and -catenin. Taken
together, these results suggest that the domains mediating binding
between xIQGAP1 and xDVL2 play important roles in both their
nuclear localization and their Wnt-stimulated activities.
In vertebrates, DVL1, DVL2 and DVL3 have redundant
function in part , . Depletion of all three xDVLs; xDVL1,
xDVL2 and xDVL3, did reduce severely the nuclear localization
of xIQGAP1 rather than only xDVL2 depletion. Moreover,
induction of Wnt target genes and formation of the secondary axis
by Xwnt-8 or -catenin was suppressed by the depletion of all three
xDVLs, but not by the depletion of xDVL2 alone. However,
xDVL2 expression could rescue suppression of Wnt target genes
by the depletion of all three xDVLs. These results suggest that
xDVL1, xDVL2 and xDVL3 also function redundantly in Wnt
signaling involving xIQGAP1. On the other hand, we showed that
all IQGAP isoforms bound to each DVL isoform, nevertheless
only IQGAP1 was necessary for Wnt signaling. Previous report
also showed the functional differences, their subcellular
localization and the interaction with binding proteins among IQGAP
isoforms in many different cellular processes . Therefore,
unidentified binding molecules might cause the functional
secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in Figure B. (D) Quantitative RT-PCR
analysis of early dorsal Wnt target genes (n = 3). Xwnt-8 (0.5 pg) mRNA was ventrally co-injected with xIQGAP1 constructs: xIQGAP1 (400 pg),
xIQGAP1DDBR (1 ng), xIQGAP1-DBR (1 ng) mRNA. The following procedure is indicated in Figure 4A. P,0.01 [between lane 3 and lane 4], P.0.1 [between lane
3 and lane 5], P,0.05 [between lane 3 and lane 6].
differences among IQGAP isoforms. Further molecular analyses
will be needed to clarify the different roles of IQGAP isoforms.
Figure S1 Expression of Xenopus DVL and IQGAP1
isoforms and confirmation of the morpholino
specificity. Reverse transcriptionpolymerase chain reaction analysis was
performed using total RNA extracted from Xenopus embryos at
different stages of development and from different regions.
Ornithine decarboxylase (ODC) was used as an internal control. (A)
Temporal expression patterns. U, unfertilized eggs. The numbers
indicate developmental stages. (B) Spatial expression patterns.
Embryos were dissected at stage 10, and dissections were
performed as shown in the right panel. D, dorsal; Vn, ventral;
A, animal; M, marginal; Vg, vegetal; H, head. (C) Morpholino
(MO) (10 ng) and FLAG-tagged mRNAs (100 pg) were co-injected
with -globin-FLAG mRNA (100 pg) as loading control into the
animal poles of 4-cell stage embryos, and the injected animal caps
were dissected at stage 10. Lysates from the animal caps were
subjected to Western blotting with anti-FLAG antibody (M2,
Figure S2 Localization of xDVL2 and xIQGAP1 GFP
constructs in Xenopus animal cap cells at stage 10. (A)
GFP signals (left panels). DAPI staining (center panels). Merge
(right panels). xDVL2-DIBR-GFP (upper panels).
xDVL2-IBRGFP (second panels). xIQGAP1-DDBR-GFP (third panels).
xIQGAP1-DBR-GFP (bottom panels). (B-E) The ratio of cells
that had nuclear fluorescence signals. The average of ratio was
taken with six explants in 3 independent experiments (See
Materials and Methods). Error bars represent standard deviation
of the mean with six explants. Statistical significance was
determined by Students t-test. (B) The ratio of nuclear-localized
xDVL2-DIBR-GFP. Lane 1: n = 499, 22.4%, lane 2: n = 349,
23.8%. P.0.1 [between lane 1 and lane 2]. (C) The ratio of
nuclear-localized xDVL2-IBR-GFP. Lane 1: n = 740, 78.9%, lane
2: n = 420, 87.6%. P.0.1 [between lane 1 and lane 2]. (D) The
ratio of nuclear localized xIQGAP1-DDBR-GFP. Lane 1:
n = 1205, 13.0%, lane 2: n = 410, 14.6%. P.0.1 [between lane
1 and lane 2]. (e) The ratio of nuclear localized
xIQGAP1-DBRGFP. Lane 1: n = 1598, 80.6%, lane 2: n = 408, 92.7%. P,0.01
[between lane 1 and lane 2].
Figure S3 Effects of over-expression of xIQGAP1 and
xDVL2 constructs on the nuclear localization of
xDVL2GFP and xIQGAP1-GFP. The ratio of cells that had nuclear
fluorescence signals. The average of ratio was taken with six
explants in 3 independent experiments (See Materials and
Methods). Error bars represent standard deviation of the mean
with six explants. Statistical significance was determined by
Students t-test. (A) The ratio of nuclear-localized xDVL2-GFP
in cells expressing various xIQGAP1 constructs: xIQGAP1,
xIQGAP1-DDBR or xIQGAP1-DBR mRNA. Lane 1: n = 1038,
22.1%, lane 2: n = 495, 31.1%, lane 3: n = 698, 26.5%, lane 4:
n = 262, 55.7%, lane 5: n = 1171, 41.8%, lane 6: n = 655, 52.7%,
lane 7: n = 611, 27.7%, lane 8: n = 520, 61.0%. P,0.1 [between
lane 1 and lane 2], P.0.1 [between lane 1 and lane 3], P,0.01
[between lane 1 and lane 4], P,0.01 [between lane 5 and lane 6],
P,0.01 [between lane 5 and lane 7], P,0.01 [between lane 5 and
lane 8]. (B) The ratio of nuclear localized xIQGAP1-GFP in cells
expressing various xDVL2 constructs: xDVL21, xDVL21-DIBR or
xDVL2-IBR mRNA. Lane 1: n = 801, 22.0%, lane 2: n = 726,
23.3%, lane 3: n = 765, 21.4%, lane 4: n = 1223, 22.2%, lane 5:
n = 1171, 36.5%, lane 6: n = 362, 37.6%, lane 7: n = 641, 22.3%,
lane 8: n = 549, 33.2%. P.0.1 [between lane 1 and lane 2], P.0.1
[between lane 1 and lane 3], P.0.1 [between lane 1 and lane 4],
P.0.1 [between lane 5 and lane 6], P,0.01 [between lane 5 and
lane 7], P.0.1 [between lane 5 and lane 8].
Figure S4 The effects of xIQGAP isoforms and xIQGAP1
mutated constructs. (A, C) Quantitative RT-PCR analysis of
early dorsal Wnt target genes (n = 3). xIQGAP1-MO (15 ng) and
xDVL2 (50 pg) or -catenin (20 pg) mRNA were ventrally
coinjected with xIQGAP1 constructs: xIQGAP1 (400 pg),
xIQGAP1DDBD (400 pg), xIQGAP1-DBD (400 pg) mRNA. RNAs from
dissected ventral sectors of injected embryos were extracted at
stage 10. RNAs from dissected dorsal and ventral sectors of
uninjected embryos were used as controls. The value obtained for
each gene was normalized to the level of ODC (ornithine
decarboxylase). The value of dorsal sectors was set to 100 and
other values were computed. Error bars represent standard
deviation of the mean in three experiments. Statistical significance
was determined by Students t-test for each marker gene. The
highest P values in three marker genes were chosen as a
representative. (A) P,0.05 [between lane 3 and lane 4], P,0.05
[between lane 4 and lane 5], P,0.05 [between lane 5 and lane 6],
P,0.05 [between lane 5 and lane 7]. (C) P,0.05 [between lane 3
and lane 4], P,0.05 [between lane 4 and lane 5], P,0.05
[between lane 5 and lane 6], P,0.05 [between lane 5 and lane 7].
(B, D) The ratio of injected embryos exhibiting a partial secondary
axis. The numbered lanes indicate the injected mRNAs and MOs
consistent with the numbering in Figure A and C, respectively.
Figure S5 The effects of IQGAP1 on the Wnt target genes
in cultured cells. RT-PCR analysis of Wnt target genes in
NIH3T3 cells. The transfected cultured cells were stimulated with
the Wnt-3A conditioned medium from L-Wnt-3A cells for
24 hours. The condition medium from L cells was used for
unstimulated control. GAPDH was used for normalization of
cDNA samples. (A) siRNAs were transfected. (B) xIQGAP1 or
xIQGAP1-DBD was transfected.
Figure S6 The effects of xDVL2 and xIQGAP1 mutated
constructs. (A, B) Quantitative RT-PCR analysis of early dorsal
Wnt target genes (n = 3). xDVL2 (50 pg) or -catenin (20 pg) mRNA
were ventrally co-injected with xIQGAP1 constructs: xIQGAP1
(400 pg), xIQGAP1-DDBD (1 ng), xIQGAP1-DBD (1 ng) mRNA.
The following procedure is indicated in Figure S4A. (A) P,0.1
[between lane 3 and lane 4], P,0.05 [between lane 3 and lane 5],
P,0.05 [between lane 3 and lane 6]. (B) P,0.05 [between lane 3
and lane 4], P.0.1 [between lane 3 and lane 5], P,0.05 [between
lane 3 and lane 6].
We thank M. Lamphier for critical reading of the manuscript.
Conceived and designed the experiments: HS . Performed the experiments:
TG AS MS SA KS. Analyzed the data: TG AS MS SA KS. Contributed
reagents/materials/analysis tools: SI TN. Wrote the paper: HS TG.
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