Dishevelled Proteins Lead to Two Signaling Pathways
REGULATION OF LEF-1 AND c-Jun N-TERMINAL KINASE IN MAMMALIAN
CELLS*
Lin
Li
§¶,
Huidong
Yuan
¶,
Wei
Xie
,
Junhao
Mao
,
Ann M.
Caruso
,
Andrew
McMahon
,
Daniel J.
Sussman**, and
Dianqing
Wu

From the
Department of Pharmacology and Physiology
and Department of Oncology, University of Rochester, Rochester, New
York 14642-8711, § Shanghai Institute of Biochemistry,
Chinese Academy of Science, Shanghai, People's Republic of China,
Harvard University, Cambridge, Massachusetts 02163, and
** Department of Obstetrics and Gynecology, University of Maryland,
Baltimore, Maryland 21250
 |
ABSTRACT |
Dishevelled (Dsh/Dvl) proteins are known to
mediate Wnt signaling by up-regulating
-catenin levels and
stimulating T cell factor (TCF)/LEF-1-dependent
transcription. We have identified a new Dvl-mediated signaling pathway
in that mouse Dvl proteins, when expressed in COS-7 cells, stimulate
c-Jun-dependent transcription activity and the kinase
activity of the c-Jun N-terminal kinase (JNK). The DEP domain of Dvl1
is essential for JNK activation. By contrast, all three conserved
domains of Dvl, including DIX, PDZ, and DEP, are required for
up-regulation of
-catenin and for stimulation of LEF-1-mediated
transcription in mammalian cells. Thus, Dvl can lead to two different
signaling pathways. Furthermore, the small G proteins of Cdc42 or Rac1,
which are involved in JNK activation by many stimuli, do not appear to
play a major role in Dvl-mediated JNK activation, because the dominant
negative mutants of Cdc42 and Rac1 could not inhibit Dvl-induced JNK
activation. This suggests that Dvl may activate JNK via novel pathways.
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INTRODUCTION |
The Wnt family of secretory glycoproteins plays important roles in
embryonic induction, generation of cell polarity, and specification of
cell fate (see reviews, Refs. 1-6). Genetic studies in
Drosophila revealed the involvement of Dishevelled (Dsh),
armadillo (Arm), and pangolin (Pan, DTcf) in Wingless (Wg) signaling. A
genetic order of these signal transducers has been established in which Wg acts through Dsh to inhibit Zw-3, thus relieving the suppression of
Arm by Zw-3, with a net result of up-regulation of Arm (1-6). Recent
results suggest that the Frizzled (Fz) family of cell surface proteins
might function as the receptors for Wnt, because the null mutation of
the Drosophila fz gene has similar phenotypes as some Dsh
mutants (7-10). In addition, heterologous expression of Dfz2
(Drosophila fz homolog) in cells that normally do not express Dfz2 and do not respond to Wg reconstituted the responsiveness to Wg (11).
The Wnt signaling mechanism appears to be conserved in mammals. In
addition to the existence of a large number of Wg homologs, there are
mammalian homologs for Fz, Dsh, Zw-3, Arm, and Pan. More than eight
mammalian Fz homologs have been cloned and sequenced (12, 13). Although
studies using immunostaining suggest that various Fz may bind to
various Wnt proteins (11), it is not clear whether Fz by itself is
sufficient to transduce signals. In addition, there is little
information about the specificity in interactions between different Fz
proteins and Wnt proteins, because affinities cannot be measured
without purified Wnt proteins. However, there appears to be certain
selectivity in the interactions between Fz and Wnt proteins, because
Wnt-5A-induced axial duplication in Xenopus requires Fz-5
(14).
Molecular cloning also revealed several mammalian Dsh homologs (Dvl),
including three from the mouse (15-17). Amino acid sequence comparison
of all known Dsh/Dvl molecules across species revealed several highly
conserved regions. Most notable is the one located in the central part
of the molecule referred to as the disc-large homology region or PDZ
domain, which was found in a number of proteins including PSD-95, ZO-1,
and Discs-large (18). Studies have shown that the PDZ domain in PSD-95
can bind to a C-terminal motif of four amino acids
(Xaa-Thr/Ser-Xaa-Val) (19). However, ligands for the PDZ domain of
Dsh/Dvl remain unknown. At the C-terminal side of the PDZ domain is
located a DEP (Dishevelled, egl-10, and
pleckstrin) domain. Similar DEP motifs are also found in a number of other proteins (20). The N-terminal conserved domain shares
homology to a newly identified protein, Axin, which also plays an
important role in Wnt signaling (21-24). This N-terminal domain is
referred to as the DIX (Dishevelled and Axin)
domain. Although a mouse knock-out of Dvl-1 did not display any of the dramatic developmental phenotypes exhibited by many of the Wnt knock-outs (5), behavioral and neurological abnormalities were observed
(25). Despite great interest, the molecular mechanism by which Dsh/Dvl
is involved in signal transduction of Wnt is largely unknown,
especially in mammalian systems. One study using cultures of
Drosophila cells showed that overexpression of Dsh can
induce elevation of the Arm levels and that the N-terminal portion of
Dsh, containing the DIX and PDZ domains, is required for such activity
(26).
The mammalian homologs to Zw-3 and Arm are
GSK-3
1 and
-catenin,
respectively. GSK was found to form a complex with
-catenin and the
product of adenomatous polyposis coli (APC) gene (27, 28). Recent
evidence indicates that Axin and its homolog, Conductin, may also be
part of the complex (22-24). GSK was shown to phosphorylate APC and
destabilize the complex (5). APC appears to play a role in promoting
the degradation of
-catenin via ubiquitination pathways (29), and a
similar role was also proposed for Conductin (24). APC was originally
identified as a tumor suppressor gene (30), and mutations in APC that
are associated with human colorectal cancers appear to lose the ability
to destabilize
-catenin (27).
-Catenin has been shown to bind
high mobility group box transcription factors of the TCF-LEF-1 family.
Complexes between
-catenin and Tcf/LEF-1 have strong transcriptional
activity on reporter gene constructs containing the Tcf-LEF-1
recognition sequences (31-33).
Genetic studies in Drosophila and Caenorhabditis
elegans suggested that there might exist Wnt signaling pathways
independent of
-catenin and TCF/LEF-1 (5). Drosophila
fz and dsh genes function in the tissue polarity
pathway, which regulates cell orientation. However, wg,
arm (
-catenin homolog), and dtcf do not appear
to be involved in this pathway, in which a number of other genes,
fuzzy, inturned, rhoA, and
bsk (DJNK) are involved (5, 34). Thus, Dsh
appears to mediate two pathways of different functions. Mammalian JNK
belongs to the superfamily of mitogen-activated protein kinases (see
reviews, Refs. 35 and 36). JNK phosphorylates the N terminus of
proto-oncogene c-Jun and its related transcription factor ATF2, leading
to stimulation of the transcriptional activity. The best-characterized
pathways leading to JNK activation in mammalian cells are those
mediated by small GTP-binding proteins Rac and Cdc42 (37, 38). Rac and
Cdc42 belong to the Rho family, which also includes RhoA (39).
Mammalian RhoA, however, is a weak activator of JNK but a strong
activator of serum response factor (SRF) (37, 38, 40).
To determine whether Dvl can interact with JNK- and RhoA-linked
pathways in mammalian cells, we tested the effects of ectopically expressed Dvl on c-Jun- and SRF-mediated transcription activity. We
found that Dvl can stimulate c-Jun-mediated but not SRF-mediated transcription of the reporter genes, suggesting that Dvl may regulate c-Jun. This is confirmed by the observation that Dvl activates JNK.
Moreover, we also found that the DEP domain of Dvl-1 is required for
JNK activation. By contrast, all three conserved domains, including
DIX, PDZ, and DEP, are required for up-regulation of
-catenin and
for stimulation of LEF-1-mediated transcription.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfection, and Luciferase Assay--
COS-7 and
NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum at 37 °C under 5%
CO2. For transfection, cells (5 × 104
cells/well) were seeded into 24-well plates the day before
transfection. Cells were transfected with 0.25 µg of DNA/well for
COS-7 cells and 0.5 µg of DNA/well for NIH3T3 cells using
LipofectAMINE Plus (Life Technologies, Inc.), as suggested by the
manufacturer. The transfection was stopped by switching to normal
growth medium after 3 h. Cell extracts were collected 24 h
later for luciferase assays, kinase assays, and Western analysis.
Luciferase assays were performed using Boehringer Mannheim Constant
Light luciferase assay kit as instructed. Cell lysates were first taken
for determining fluorescence intensity emitted by green fluorescence
protein (GFP) proteins in a Wallac multi-counter, which is capable of
counting fluorescence and luminescence. Then, luciferase substrate was
added to the cell lysates, and luciferase activities were determined by
measuring luminescence intensity using the same counter. Luminescence
intensity was normalized against fluorescence intensity.
Construction of Expression Plasmids and Mutagenesis--
The
wild type and deletion mutants of mouse Dvl1 were generated by
polymerase chain reaction with the mouse Dvl-1 cDNA as the template
using the high fidelity thermostable DNA polymerase Pfu
(Stratagene, CA). Myc epitope tags (EQKLISEEDL) were introduced to the
C termini of the full-length and mutant Dvl molecules. The expression
of Wnt1, wild type Dvls, and Dvl-1 mutant was driven by a
cytomegalovirus promoter. All constructs were verified by DNA
sequencing. The LEF-1 reporter gene construct was kindly provided by
Dr. Grosschedl (University of California at San Franscisco).
Elk- and c-Jun-reporter gene systems were purchased from Strategene,
CA. For the construction of the reporter gene plasmid, SRE derivative,
SRE.L, was synthesized exactly as described in (Hill et al.
(40)). SER.L was inserted in front of a TK minimal promoter-luciferase
gene. Lef-1 and the Lef reporter gene plasmid were provided by Rudolf Grosschedl.
In Vitro JNK Assay--
Cells expressing JNK-HA were lysed with
the lysis buffer containing 1% Nonidet P-40, 137 mM sodium
chloride, 20 mM Tris, pH 7.4, 1 mM
dithiothreitol, 10% glycerol, 10 mM sodium fluoride, 1 mM pyrophosphate, 2 mM sodium vanadate, and
CompleteTM protease inhibitors (Boehringer Mannheim, used
as the instructions suggest). The cell lysates were precleared with 20 µl of protein A/G-Sepharose beads (Santa Cruz Biotech, CA) for
0.5 h at 4 °C and then incubated with 1 µl of monoclonal
anti-HA tag antibody (Berkeley Antibody Company, CA) and 20 µl of
protein A/G-Sepharose beads for 3.5 h on ice. The immunocomplexes
were pelleted and washed 3 times with cold lysis buffer in the absence
of protease and phosphatase inhibitors and twice with cold kinase
buffer containing 25 mM HEPES, pH 7.4, 10 mM
MgCl2, and 1 mM dithiothreitol. The kinase
reactions were performed for 30 min at 30 °C in the presence of 10 µCi of [
-32P]ATP, 10 µM ATP, and an
excess of the substrate, c-Jun-GST. The reactions were terminated by
the addition of 4× SDS sample buffer. The samples were boiled and
loaded on 12% SDS-PAGE gels. The results were visualized and
quantified using a PhosphorImager (Molecular Dynamics).
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RESULTS |
Regulation of c-Jun-mediated Transcription by Dvl-1--
To test
whether Dvl is involved in JNK- and RhoA-linked pathways, the effects
of overexpressed Dvl on the activities of c-Jun (regulated by JNK) and
SRF (regulated by RhoA) were examined. Activation of SRF was determined
by cotransfection of the Dvl cDNA with a luciferase reporter
construct containing a mutated c-Fos serum response element, SRE.L,
upstream of the reporter gene. Because only SRF binds to SER.L,
SRE.L-mediated production of luciferase mainly depends on the activity
of SRF (40). Regulation of c-Jun was studied using the PathDetect
signal transduction reporting systems from Stratagene. In this system,
transcription of the luciferase reporter gene depends on activation of
c-Jun. We also tested the effect of Dvl on Elk (regulated by
Ras)-dependent transcriptional activity using the same
method as for c-Jun. Positive controls and specificity of activation of
these reporting systems were shown in Fig. 1; the activated form of
Cdc42 activates SRF and c-Jun, whereas the activated forms of Ras and
mitogen-activated protein kinase kinase are potent activators of Elk.
These results are consistent with previous findings (37, 38, 40). When Dvl was tested in these reporting systems, we found that it could significantly activate c-Jun-mediated transcription but not SRF or
Elk-mediated transcription (Fig. 1).
These data suggest that Dvl may activate c-Jun.

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Fig. 1.
Transcriptional regulation by Dvl-1.
NIH3T3 cells were cotransfected with 0.15 µg of SRE.L-luciferase
reporter plasmid, 0.15 µg of GFP expression construct, and 0.2 µg
of LacZ, Dvl-1, activated Ras mutant Ras61L, or activated
Cdc42 (A); 0.15 µg of the Elk-luciferase reporter plasmid,
0.15 µg of GFP expression construct, and 0.2 µg of LacZ,
Dvl-1, Ras61L, or activated mitogen-activated protein kinase kinase
(MEK) B; 0.15 µg of c-Jun-luciferase reporter
plasmid, 0.15 µg of GFP expression construct and 0.2 µg of
LacZ, Dvl-1, Ras61L, or activated Cdc42 (C). One
day later, cells were lysed, and GFP levels and luciferase activities
were determined. The luciferase activities presented are normalized
against the levels of GFP expression. Each experiment was carried out
in triplicate, and error bars represent S.D.
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Activation of JNK by Dvl--
It has been shown that JNK
phosphorylates and activates c-Jun (35). Thus, activation of JNK by
mouse Dvl proteins was examined. Monkey kidney COS-7 cells were
transfected with cDNA-encoding mouse Dvl-1 and JNK-HA. JNK protein
immunoprecipitated via an HA tag from cells coexpressing Dvl-1 showed
significantly higher activity in phosphorylating a JNK substrate than
that from cells coexpressing the control
-galactosidase
(LacZ) (Fig. 2). The expression of mouse Dvl-1 is shown in Fig. 2C. Heterologous
expression of mouse Dvl-2 and 3 (12, 13) could also activate JNK (Fig. 2). Activation of JNK was also observed when Dvl-1 was expressed in
NIH3T3 cells (data not shown).

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Fig. 2.
Activation of JNK by mouse Dvl proteins.
A, schematic representation of the wild type mouse Dvl-1 and
its mutants. Domains that are highly conserved among Dsh/Dvl molecules
from different species are striped. The summary of JNK
activation by mouse Dvl-1 and its mutants is tabulated at the right.
B and C, Cos-7 cells were transfected with 0.1 µg of JNK-HA and 0.15 µg of lacZ, wild type Dvl, or
various Dvl-1 mutants as indicated in the figure. HA-tagged JNK was
immunoprecipitated by anti-HA antibodies, and the abilities of JNK to
phosphorylate the c-Jun-GST fusion protein were determined. The results
were visualized (B) and quantified (C) using a
PhosphorImager. Each experiment was carried out in triplicate, and
error bars represent S.D. The results were repeated at least
three times. D, the expression levels of JNK-HA, Myc-tagged
Dvl-1, and its mutants were determined by Western analysis using
anti-HA antibodies and anti-Myc tag antibodies, respectively.
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C-terminal Portion of Dvl-1 Is Required for JNK Activation--
To
determine which portions of the Dvl-1 molecule are responsible for
activation of JNK, truncation mutants of mouse Dvl-1 were generated.
Amino acid sequence homology analysis of mammalian and
Drosophila Dsh/Dvl molecules suggests that these molecules can be roughly divided into three parts, each of which contains one
highly conserved domain (Fig. 2A). Therefore, three mutants were generated by deleting each of the conserved domains separately. The mutants are: N, an N-terminal truncation mutant; C, a C-terminal truncation mutant; and PDZ, in which the PDZ domain was deleted (Fig.
2A). The ability of these Dvl-1 mutants to activate JNK was
tested by coexpression with JNK-HA in COS-7 cells. The expression of
the Dvl-1 mutants is shown in Fig. 2C. Both N and PDZ were able to activate JNK (Fig. 2), whereas C could not (Fig. 2). This result suggests that the C-terminal region of Dvl-1 may be involved in
regulation of JNK.
DEP Domain Is Required for JNK Activation--
To test whether the
C-terminal portion of Dvl is sufficient for JNK activation, the
C-terminal 320 amino acids of mouse Dvl-1, designated as C1, were
expressed in COS-7 cells (Fig.
3C) and tested for JNK
activation. As shown in Fig. 3, C1, which contains the DEP domain, is
sufficient for activation of JNK. Two additional Dvl-1 deletion
mutants, which are depicted in Fig. 3C, were generated to
further delineate the sequences that are important for JNK regulation.
When the Dvl mutant, C2, which encompasses the sequence downstream of
the DEP domain, was expressed in Cos-7 cells, it was unable to activate
JNK (Fig. 3). Because the only difference between C2 and C1 is the lack
of the DEP domain (Fig. 3C), the failure of C2 to activate
JNK suggests that the DEP domain is involved in JNK regulation. This
hypothesis is further confirmed by the observation that the Dvl mutant
N, when expressed in COS-7 cells, could activate JNK (Fig. 3).

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Fig. 3.
Activation of JNK by the DEP domain.
A, Cos-7 cells were transfected with 0.1 µg of JNK-HA and
0.15 µg of lacZ, wild type Dvl-1, or various Dvl-1 mutants
as indicated in the figure. JNK activities were determined as in Fig.
2. B, the expression levels of JNK-HA, Myc-tagged Dvl-1, and
its mutants were determined by Western analysis using anti-HA
antibodies and anti-Myc tag antibodies, respectively. C,
schematic representation of wild type mouse Dvl-1 and its mutants.
Legends are described in Fig. 2.
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There is a nonlethal dsh1 allele in Drosophila.
Homozygous dsh1 appears to cause only planar polarity but
not wg-like phenotypes (8, 41, 42). The mutation in
dsh1 was identified as the substitution of Met-438 for Lys
(34). Because the JNK pathway was implicated in regulation of planar
polarity, we tested whether this mutation affects JNK activation. A Dvl
mutant (DvlKM) containing the equivalent mutation was thus generated.
We found that DvlKM activated JNK as effectively as the wild type (Fig.
4). DvlKM and its wild type were even
compared at two different expression levels (Fig. 4). Thus, although
the dsh1 mutation (at residue 438) lies inside the DEP
domain, the residue Lys-438 does not appear to play a significant role
in JNK activation.

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Fig. 4.
Activation of JNK by DvlKM.
A, Cos-7 cells were transfected with 0.1 µg of JNK-HA,
0.15 µg of lacZ, or varying amounts of Dvl-1 and DvlKM as
indicated in the figure. LacZ was used to make the total
amount of DNA equal per transfection. JNK activity was determined as in
Fig. 2. B, the expression levels of JNK-HA, Myc-tagged
Dvl-1, and DvlKM were determined by Western analysis using anti-HA
antibodies and anti-Myc tag antibodies, respectively.
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All Three Conserved Dvl-1 Domains Are Required for Up-regulation of
-Catenin--
It is well established that Dsh can signal through
-catenin and TCF/LEF-1 (2, 4-7). A previous study indicated that
the C-terminal region of Drosophila Dsh, corresponding to
C1, was incapable of up-regulating the
-catenin levels in cultured
Drosophila cells (26). This finding, together with our
results that C1 is sufficient for JNK activation, suggests that Dvl can
lead to two different signaling pathways. Nevertheless, we investigated the effects of Dvl and its mutants on the
-catenin-TCF/LEF-1-linked pathway in mammalian cells. We first tested whether ectopic expression of Wnt-1 and Dvl-1 can up-regulate the levels of
-catenin in Cos-7
cells as Wg and Dsh do in Drosophila cells (11, 26). Cells
were cotransfected with cDNA encoding Wnt-1 or Dvl-1 along with
cDNA encoding Myc-tagged
-catenin and GFP for 24 h. Cytosol fractions were prepared from cotransfected cells and analyzed by
Western blotting using antibodies specific to the Myc tag and GFP. The
levels of cytosolic recombinant
-catenin proteins in cells
coexpressing Wnt-1 (Fig. 5A)
or Dvl-1 (Fig. 5B) are clearly higher than those
coexpressing the control LacZ. Moreover, coexpression of
GSK-3
significantly decreased the levels of cytosolic
-catenin (Fig. 5A). All of these results are consistent with previous
findings (5). The levels of coexpressed GFP were determined to ensure that the changes in the
-catenin levels are not because of
variations in transfection (Fig. 5, A and B).
Dvl-1 mutants including C, N, PDZ, and C1 were tested for their
abilities to up-regulate the
-catenin levels in COS-7 cells. As
shown in Fig. 5B, none of the mutants were able to affect
the levels of cytosolic
-catenin.

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Fig. 5.
Effects of Dvl-1 and its mutants on the
-catenin/LEF-1 pathway. A and B, COS-7
cells were transfected with 0.1 µg of Myc- -catenin ( -cat), 0.1 µg of GFP expression construct, and 0.05 µg of LacZ,
Wnt-l, GSK-3 , Dvl-1, or its mutants as indicated in the figure.
Cells were collected 24 h after transfection, and cytosolic
fractions were prepared. Equal amounts of cytosol were loaded to 10%
SDS-PAGE, and proteins were transferred to nitrocellulose membranes.
GFP and -catenin were detected by antibodies against GFP and the Myc
tag, respectively. C, cells were transfected with 0.075 µg
of LEF reporter plasmid, 0.15 µg of GFP expression plasmid, and 0.125 µg of Wnt-1, Dvl-1, GSK-3 , and/or -catenin ( -cat) in the
presence or absence of 0.025 µg of LEF-1 expression plasmid as
indicated in the figure. D, cells were transfected with
0.025 µg of LEF-1 expression plasmid, 0.075 µg of LEF reporter
plasmid, 0.15 µg of GFP expression plasmid along with 0.25 µg of
LacZ, Dvl-1, or its mutants as indicated. E,
cells were transfected with 0.025 µg of LEF-1 expression plasmid,
0.075 µg of LEF reporter plasmid, 0.15 µg of GFP expression plasmid
along with 0.25 µg of LacZ or varying amounts of Dvl or
DvlKM as indicated. LacZ plasmid was added to make the total
amount of DNA equal. One day later, cells were lysed, and GFP levels
and luciferase activities were determined. The luciferase activities
are presented as in Fig. 2. The expression levels of Dvl were
determined by Western blot analysis and shown in D.
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All Three Conserved Dvl-1 Domains Are Required for LEF-1-mediated
Transcriptional Regulation--
-Catenin has been shown to interact
with TCF/LEF-1 and regulate transcription (43). A reporter construct,
in which LEF response element was placed in front of a minimal promoter
and a luciferase gene, were used to assay LEF-1 activity. When this reporter construct was cotransfected with cDNAs encoding LEF-1 and
Wnt-1 or Dvl-1, there was a marked increase in luciferase activity
(Fig. 5C). The increase in luciferase activity was Wnt-1- or
Dvl-1- and LEF-1-dependent (Fig. 5C). This
suggests that the cells do not contain endogenous LEF-1 and that Dvl-1
activates reporter gene transcription through LEF-1. In addition, the
observation that
-catenin is able to stimulate luciferase activity
and that GSK-3
could inhibit Dvl-stimulated luciferase activity
(Fig. 5C) indicates that the LEF-1 activity in this assay
system can be regulated by
-catenin and GSK.
Dvl Mutants Were Tested in the LEF Reporter Gene Assay system. Dvl
mutants, including C, N, PDZ, and C1, did not show significant stimulation of the luciferase activity regardless of the presence (Fig.
5D) or absence of LEF-1 (data not shown). This result is consistent with the one from the assay of the
-catenin levels. Only
Dvl-N was able to stimulate LEF1-dependent transcription (Fig. 5D). The fact that Dvl-N can stimulate
LEF1-dependent transcription indicates that Dvl sequences
downstream of DEP are not required for activation of
LEF1-dependent transcription. The ability of DvlKM to
regulate the
-catenin-LEF-1 pathway was also investigated. We found
that DvlKM was as effective as the wild type Dvl in stimulation of
LEF-1-dependent transcription (Fig. 5E). Thus,
the dsh1 mutation did not affect its ability to regulate its
downstream effectors, including LEF-1 and JNK, under our experimental conditions.
Rac1 and Cdc42 Are Not Involved in Dvl-mediated JNK
Activation--
Cdc42 and Rac, members of the Rho family of small G
proteins, have been shown to mediate JNK activation by a number of
stimuli (35, 36). To determine whether Cdc42 or Rac1 also mediates JNK
activation by Dvl, Dvl-1 was coexpressed with the dominant negative
mutants of Cdc42 and Rac1, Cdc42N17 and Rac1N17, respectively. Neither
of the dominant negative mutants could significantly inhibit Dvl-induced JNK activation (Fig. 6,
A and B). To ensure that the dominant-negative
mutants of Cdc42 and Rac1 are effective in suppression of Cdc42 and
Rac-mediated JNK activation, we tested these two dominant negative
mutants in inhibition of EGF-induced JNK activation. EGF was previously
shown to activate JNK through Rac1 and/or Cdc42 (37). As shown in Fig.
6, C and D, the Cdc42 dominant negative mutant
suppressed EGF-induced JNK activation by more than 50%, and the Rac1
mutant almost completely abolished EGF-induced JNK activation as
reported (37). Therefore, Cdc42 and Rac1 in COS-7 cells are unlikely to
play a significant role in Dvl-induced JNK activation in COS-7 cells.
RhoA, another member of the Rho family, has been shown by many
laboratories to lack the ability to activate JNK in mammalian cells
(37, 38, 40), which we also confirmed (data not shown). However, RhoA
was suggested to genetically interact with JNK in Drosophila
(10). Thus, we tested the dominant negative mutant of RhoA, RhoAN19,
for its ability to inhibit Dvl-1-mediated JNK activation. As shown in
Fig. 5A, RhoAN19 failed to inhibit Dvl-1-induced JNK
activation.

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Fig. 6.
Involvement of small G proteins. COS-7
cells were transfected with 0.1 µg of JNK-HA, 0.05 µg of Dvl-1, and
0.1 µg of dominant negative mutants of Rac1* (RacN17), Cdc42*
(CdcN17), Rho* (RhoN19) or LacZ. The JNK activities are
shown in A, and the expression of JNK and Dvl-1 is shown in
B. To test the inhibition of EGF-induced JNK activation by
the dominant negative mutants of Rac1, Cdc42, and RhoA, COS-7 cells
were transfected with 0.1 µg of JNK-HA, 0.05 µg of LacZ,
and 0.1 µg of the small G protein mutants. The JNK activities were
determined 30 min after the addition of 10 nM EGF
(C). The expression of JNK-HA was shown in D. JNK
kinase assays were carried out in triplicate, and error bars
represent S.D.
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DISCUSSION |
In this report, we have described a new signal transduction
pathway for Dvl. Dvl activates JNK and regulates
c-Jun-dependent gene transcription in addition to its
ability to regulate the
-catenin and LEF-1 pathways. Moreover, we
found that the DEP domain is required for JNK activation. Our findings
agree with two recent reports (10, 34) using genetic epistasis analysis in Drosophila showing that Dsh interacts with
Drosophila JNK. JNK was shown to be required for correct
establishment of planar polarity in Drosophila epidermal
tissues, which does not require Arm or Pan. In addition, it was shown
that Dvl activates JNK in mammalian cells (34). Our findings are also
largely consistent with the study (26) showing that deletion of the
conserved domains of Drosophila Dsh including DIX and PDZ
resulted in the loss of the ability in up-regulation of the Arm levels.
However, there is an apparent discrepancy with regard to the role of
the DEP domain in up-regulation of
-catenin. In contrast to our
findings, the study by Yanagawa et al. (26) suggested that
deletion of the part of Drosophila Dsh corresponding to C1
including the DEP domain could still up-regulate the levels of Arm.
This discrepancy may result from the use of different assay systems in
cells from different species.
The Drosophila dsh1 allele shows strong planar
polarity phenotypes (8, 41, 42). However, the dsh1 allele
does not appear to affect the Wg signaling pathways (8, 41, 42). The
mutation in dsh1 was identified, and Dsh 1 was shown to have
less potency in JNK activation than wild type Dsh 1 (34). However, our
data indicate that there are no significant differences between DvlKM and Dvl in JNK activation or in activation of
LEF-1-dependent transcription (Figs 4 and 5). In our
experiments, the effects of DvlKM (the Dvl-1 mutation equivalent to
dsh1) and Dvl-1 were compared at the same expression levels
of Dvl-1 and DvlKM, whereas Boutros et al. (34) had
significantly higher levels of wild type Dsh as compared with Dsh 1. If
our findings are correct, there may be several possible explanations to
the genetic data showing that the dsh1 allele only affects
the JNK pathway but not the
-catenin pathway (8, 41, 42). 1) The
dsh1 mutation may play different roles in Dvl-1 and Dsh; 2)
The mutation may disrupt the coupling of Dvl to its upstream regulators
(such as receptors) that are involved only in regulation of the JNK
pathway. Thus, only the JNK pathway but not the
-catenin pathway is
affected; 3) The mutation may partially reduce the coupling of Dvl to
its upstream regulators that are involved in both the
-catenin and JNK pathways. However, there may still be sufficient activity to
maintain normal functions required for the
-catenin pathway but not
enough to maintain the functions required for the JNK pathway, thus
leading solely to a planar polarity phenotype.
Although Drosophila RhoA may be involved in JNK activation
(although there is lack of biochemical data), mammalian RhoA is clearly
not involved in JNK activation (35, 36). Mammalian RhoA is involved in
the regulation of SRF via yet-to-be identified pathways (39, 40). In
mammals, JNK was found to be regulated by two other small G proteins
belonging to the Rho family, Cdc42 and Rac. Although Cdc42 and Rac were
shown to mediate JNK activation by many growth factor receptors and G
protein-coupled receptors (35, 36), they are not involved in all JNK
activation pathways (35). For instance, they are not involved in
ultraviolet, toxin anisomycin, tyrosine kinase c-Abl and Pyk2, or
TNF-induced JNK activation (35). Although mammalian and
Drosophila cells are conserved with regard to activation of
JNK by Dvl, different signaling pathways are apparently used in JNK
activation by Dvl/Dsh. Our data indicate that Rac1 and Cdc42 are not
involved in Dvl-mediated JNK activation. Our results as well as others
showed that RhoA is irrelevant in JNK activation in mammals. The fact
that Dvl-1 does not stimulate SRF-dependent transcription
(Fig. 1A) further supports the idea that Dvl-1 does not
regulate RhoA in mammalian cells. It is certainly of great interest to
determine the mechanism by which Dvl regulates JNK in mammalian cells.
Wg signals through Dfz2, Dsh, Arm (
-catenin), and Pan (TCF) in
Drosophila (5). Although there is no evidence for the
involvement of Wg in the Fz-Dsh-JNK pathway, Wnt-like molecules are
speculated to be involved (5, 34). We tested if mammalian Wnts, Wnt-1, and Wnt5a, can induce JNK activation in a paracrine paradigm by expression of Wnt in COS-7 cells. We found that both Wnt-1 and Wnt-5a
gave 2 to 3-fold stimulation of JNK activity (data not shown). However,
we were unable to obtain direct evidence to demonstrate that Wnt
activates JNK via Dvl. We have tried all of our Dvl mutants, but none
of them was able to block JNK activation by Wnt-1 or Wnt-5a (data not
shown). We also tested the effect of Fz in JNK activation. A recent
report demonstrating the requirement of Fz-5 in Wnt-5a-induced axial
duplication in Xenopus implies that Fz-5 may serve as a
receptor for Wnt-5a (14). However, coexpression of Fz-5 with Wnt-5a did
not potentiate JNK activation by Wnt-5a in COS-7 cells (data not
shown). This suggests that Fz-5 alone may not be sufficient for
mediating the effect of Wnt-5a on JNK activation. Alternatively, Wnt-5a
may use different receptors for JNK activation.
 |
ACKNOWLEDGEMENTS |
We thank Jeremy Nathans for the human Fz5
cDNA, Alan Hall for C3 transferase expression plasmid, Silvio
Gutkind for Cdc42V12, Cdc42N17, Rac1N17, RhoN19, JNK-HA, and AFT2-GST
expression plasmids, and Rudolf Grosschedl for LEF-1 expression plasmid
and reporter construct. We also thank Huiping Jiang and Mark Betz for
reading the manuscript.
 |
FOOTNOTES |
*
This work is supported by National Institutes of Health
Grants GM53162 and GM54167 and a grant from the National Heart
Association (to D. W.) and by National Institutes of Health Grant
CA63929 (to D. J. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
First two authors contributed equally.

To whom correspondence should be addressed. Tel.: 716-275-2029;
Fax: 716-244-9283.
 |
ABBREVIATIONS |
The abbreviations used are:
GSK, glycogen
synthase kinase;
APC, adenomatous polyposis coli;
Arm, armadillo;
Dsh/Dvl, Dishevelled;
Fz, Frizzled;
GFP, green fluorescence protein;
JNK, c-Jun N-terminal kinase;
LacZ,
-galactosidase;
Pan, pangolin/DTcf;
SRE, serum response element;
SRF, serum response factor;
Wg, wingless;
Zw-3, zeste-white3: HA, hemagglutinin;
EGF, epidermal
growth factor..
 |
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