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First published online 12 December 2007
doi: 10.1242/dev.013540
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1 The F. M. Kirby Neurobiology Center, Children's Hospital Boston, Department of
Neurology, Harvard Medical School, Boston, MA 02115, USA.
2 Department of Pediatrics and Medicine, University of California, San Diego, La
Jolla, CA 92093-0627, USA.
3 Samuel Lunenfeld Research Institute, 600 University Avenue, Toronto, Ontario,
M5G 1X5, Canada.
4 Department of Biochemistry and Cell Biology, Center for Developmental
Genetics, State University of New York, Stony Brook, Stony Brook, NY 11794,
USA.
Author for correspondence (e-mail:
xi.he{at}childrens.harvard.edu)
Accepted 15 October 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Axin, Dishevelled, Frizzled, Gsk3, Lrp6, Wnt
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Logan and Nusse, 2004). The
mechanism of Wnt/β-catenin signaling, however, remains to be fully
defined. In the absence of Wnt stimulation, the β-catenin protein is
sequentially phosphorylated by casein kinase 1
(CK1; also known
as CSNK1A1 - Human Gene Nomenclature Database, and Csnk1a1 - Mouse Genome
Informatics) and Gsk3 within a protein complex that is assembled by the
scaffolding protein axin, and is thereby ubiquitinated and degraded. Upon Wnt
stimulation, β-catenin phosphorylation and degradation is inhibited,
thereby resulting in elevated β-catenin protein levels and
β-catenin-dependent transcriptional activation
(Clevers, 2006;
Logan and Nusse, 2004). Two
types of cell surface receptors, a member of the frizzled (Fz) serpentine
receptor family and a single-span transmembrane receptor LDL receptor-related
protein 6 (Lrp6) or closely related Lrp5, mediate Wnt signaling at the plasma
membrane (He et al., 2004;
Pinson et al., 2000;
Tamai et al., 2000;
Wehrli et al., 2000).
Biochemical studies indicate that Wnt may induce Fz-Lrp6 complex formation
(Semenov et al., 2001;
Tamai et al., 2000), and
experimentally induced Fz-Lrp6 proximity appears to be sufficient to stimulate
Wnt/β-catenin signaling (Cong et al.,
2004; Holmen et al.,
2005; Liu et al.,
2005; Tolwinski et al.,
2003).
We previously demonstrated a phosphorylation-dependent mechanism for Lrp6
activation (Tamai et al.,
2004). We and others showed that Wnt activates Lrp6 by inducing
Gsk3 and CK1-mediated phosphorylation of PPPSPxS motifs within the Lrp6
intracellular domain (Davidson et al.,
2005; Zeng et al.,
2005). We showed that Gsk3 phosphorylation of the PPPSP
motif appears to be the primary Wnt-inducible event, which primes the
subsequent phosphorylation of xS in the PPPSPxS motif by CK1
(Wei et al., 2007;
Zeng et al., 2005;
Davidson et al., 2005). The
dually phosphorylated PPPSPxS motifs provide docking sites for axin
(Tamai et al., 2004;
Zeng et al., 2005). The
association of the axin complex with the phosphorylated Lrp6 is believed to
lead to (via an as yet unknown mechanism) inhibition of β-catenin
phosphorylation and activation of β-catenin signaling
(Mao et al., 2001;
Tamai et al., 2004;
Zeng et al., 2005). The
involvement of Gsk3 and CK1 in both Wnt pathway activation (via Lrp6
phosphorylation) and inhibition (via β-catenin phosphorylation) is
surprising and implies intricate regulatory mechanisms. In the meantime, the
biochemical nature by which the Fz protein and its downstream cytosolic
partner dishevelled (Dvl or Dsh) protein exert their obligatory roles in Wnt
signal transduction has been obscure. In this study, we demonstrate that the
Fz, Dvl and axin proteins are important components that control Wnt-induced
Lrp6 phosphorylation by Gsk3.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
Flag-tagged mouse Dvl2 and the deletion mutants
(Habas et al., 2001),
HA-tagged human GSK3 (Zeng et al.,
2005), HA-tagged human Fz5
(Habas et al., 2001),
Flag-tagged mouse axin (Liu et al.,
2002) in the pCS2+ vector were previously described. The Fz
mutants m11-12 (P267L and I353A), m14-15 (deleting the cytoplasmic tail from
the fourth amino acid residue adjacent to the seventh transmembrane domain)
were generated by PCR-mediated mutagenesis or truncation in the pCS2+Fz5
vector. The fragments containing the mutations replaced the corresponding
fragments in the DKK1-Fz5 (Holmen et al.,
2005). The Axin L396Q mutant was generated by PCR-mediated
mutagenesis. All mutations were confirmed by DNA sequencing. Details of the
plasmids are available from the authors upon request. Flag-tagged Shisa
(Yamamoto et al., 2005) (also
known as TMEM46 - Human Gene Nomenclature Database) was kindly provided by
Shinichi Aizawa (Riken, Japan). Myc-tagged DKK1-Fz5 and DKK3-Fz5
(Holmen et al., 2005) were
kindly provided by Bart O. Williams (Grand Rapids, MI). The CAAX-motif derived
from K-Ras (DGKKKKKSKTKCVIM) was fused C-terminal to the axin-derived GID and
the GID-LP mutant (Hedgepeth et al.,
1999) to allow myristylation. The lentiviral-based shRNA vectors
against mouse Dvl3 and Axin2 were purchased from Sigma (Mission TRC shRNA
Target Set).
|
;
Zeng et al., 2005). Other
antibodies were used according to manufacturer's instructions: mouse
monoclonal antibodies anti-β-catenin (BD Transduction Laboratories),
anti-β-actin and Flag (M2, Sigma), anti-Gsk3 (4G-1E, Upstate
Biotechnology), anti-Dvl3 (Sc-8027, Santa Cruz Biotechnology), anti-transferin
receptor (TfR) (Zymed), rabbit polyclonal anti-Dvl2 (Sc-13974, Santa Cruz),
anti-Myc (A14, Santa Cruz), anti-Frizzled 5 (Upstate Biotechnology). The
rabbit polyclonal anti-Dvl1 antibody was kindly provided by Lin Mei (Augusta,
GA).
Cells, transfection, reporter assay, lentivirus production and infection, Wnt induction and immunoblotting
Dvl1-/-;Dvl2-/- MEFs were derived from a
mouse Dvl1 and Dvl2 double knockout embryo
(Wang et al., 2006). Stable
Dvl1-/-;Dvl2-/- MEFs expressing the mouse Dvl2
and the various mutants were established by puromycin-selection after
cotransfection of the Dvl2 expression vectors with pBABE-puro plasmids. Mouse
ES cells for Gsk3-/-,
Gsk3β-/- and the double knockout
Gsk3-/-;Gsk3β-/-
(Doble et al., 2007) and the
mouse Axin-/- (Zeng et
al., 1997) have been described previously. HEK293T, L cells and
MEFs were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin-glutamine (PSA; Invitrogen). ES cells were cultured in
the Knockout DMEM (Invitrogen) supplemented with 15% FBS, 1% PSA,
103 Units/ml of ESGRO (Chemicon) on a MEF feeder layer for
passages. ES cells were separated from the feeder cells by three passages onto
gelatin-coated plates for a 0.5-hour incubation to eliminate the attached MEF
cells. Fugene6 (Roche) was used for all cell transfections. For induction of
Lrp6 phosphorylation, cells were treated for 1 hour with Wnt3a CM (collected
from mouse L cell stably expressing Wnt3a; ATCC) as previously described
(Tamai et al., 2004;
Zeng et al., 2005). The
lentiviruses encoding the shRNA against mouse Dvl3 and Axin2 were produced in
HEK293T cells by cotransfection with the packaging plasmids. The conditioned
media containing the viral particles were collected and filtered through 0.4
µm syringe filters. The MEFs or ES cells were incubated with the
virus-containing conditioned media for 6 hours, followed by fresh media for 3
days before Wnt treatment. Luciferase assays were carried out in duplicate in
HEK293T cells in 12-well plates as previously described
(Tamai et al., 2004;
Zeng et al., 2005) and the
results were shown as fold induction from multiple experiments, presented as
mean ± s.d. Cell extracts for western blotting were collected in a
lysis buffer containing 50 mM Hepes (pH 7.4), 1.5 mM EDTA, 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 0.5
mM DTT and a cocktail of protease inhibitors. Cytosolic β-catenin was
prepared using hypotonic buffer as described previously
(Liu et al., 2002).
Xenopus laevis embryo manipulations and immunofluorescent staining in animal pole explants
Xenopus embryo injection and animal pole explant assays were
performed as described previously (Tamai
et al., 2000). For immunostaining, in vitro transcribed mRNAs for
Flag-axin, human Fz5 and Xenopus Dishevelled (Xdsh), Xdsh
N, or
Xdsh-GFP were injected into animal pole cells at the 2-4 cells stage. Animal
pole explants were dissected at stage 9.5-10 and fixed in 4% paraformaldehyde
for 1.5 hours. The explants were washed and blocked with 2% goat serum in PBST
(PBS with 0.2% Triton X-100) and were subsequently incubated with anti-Flag M2
(for Axin) and anti-Fz5 primary antibodies overnight at 4°C. Secondary
antibodies used in the experiment were goat-anti-rabbit or mouse IgG
conjugated with Cy5, Alexa Fluor 568 or Alexa Fluor 488 (Molecular Probes).
The explants were mounted and examined by Zeiss LSM510 confocal
microscopy.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
probably Fz-Lrp6 complex formation, we suspect that Fz may function in
regulating Lrp6 phosphorylation. Given that there are nine Fz genes in the
mammalian genome (Huang and Klein,
2004; Logan and Nusse,
2004), we first employed Shisa, a specific Fz antagonist
(Yamamoto et al., 2005). Shisa
is an endoplasmic reticulum (ER) resident protein that functions to retain Fz
proteins in the ER, thereby preventing Fz from trafficking to the plasma
membrane (Yamamoto et al.,
2005). We found that Shisa expression in HEK293T cells inhibited
Wnt3a-induced LRP6 phosphorylation, and as expected, β-catenin
stabilization (Fig. 1A). We
further generated a mouse L cell line in which two highly expressed Fz
proteins, Fz2 and Fz7, were stably knocked down using shRNAs (J-C.H.,
unpublished). Wnt3a-induced Lrp6 (endogenous) phosphorylation and
β-catenin stabilization was significantly diminished in these cells
(Fig. 1B). Expression of human
Fz5 (FZ5) in these cells rescued Wnt3a-induced Lrp6 phosphorylation
(Fig. 1C). These results
suggest that Fz function is required for Wnt-induced Lrp6 phosphorylation.
|
; Tamai et al.,
2000). Indeed, various molecular means to bring Fz and Lrp6 into
proximity can activate the β-catenin pathway in the absence of any Wnt
stimulation (Cong et al.,
2004; Liu et al.,
2005; Tolwinski et al.,
2003; Holmen et al.,
2005). We found that such induced Fz-Lrp6 proximity is sufficient
to promote LRP6 phosphorylation (Fig.
2A). Dickkopf-1 (DKK1), but not the related DKK3, is a high
affinity antagonistic ligand for LRP6
(Bafico et al., 2001;
Mao et al., 2001;
Semenov et al., 2001). A
DKK1-Fz5 fusion protein, which fuses DKK1 in frame with the amino terminus of
Fz5 thus forcing Fz5 to associate with LRP6 in the absence of the Wnt ligand
(Holmen et al., 2005),
synergized with LRP6 to activate β-catenin signaling as monitored via the
TCF/β-catenin reporter assay (Holmen
et al., 2005) (Fig.
2B). By contrast, neither Fz5 nor a DKK3-Fz5 fusion protein
exhibited such activity (Holmen et al.,
2005) (Fig. 2B).
Importantly, we found that DKK1-Fz5, but not Fz5 or DKK3-Fz5, induced LRP6
phosphorylation without any Wnt stimulation
(Fig. 2A). These results
suggest that the Fz-LRP6 complex formation is sufficient to trigger LRP6
phosphorylation.
We further employed the DKK1-Fz5 fusion protein to examine whether the
signaling function of Fz is involved in LRP6 phosphorylation. We generated two
Fz5 mutants: one contained substitutions in the first and second intracellular
loops (m11-12) and the other had a truncation of the carboxyl-terminal region
(m14-15; Fig. 2C). Identical or
similar mutations in Fz5 and other Fz proteins have been shown to inactivate
Fz signaling capability (Cong et al.,
2004; Povelones et al.,
2005; Umbhauer et al.,
2000). The DKK1-Fz5 fusion protein harboring each of these
mutations showed diminished ability to induce LRP6 phosphorylation
(Fig. 2D) and to synergize with
LRP6 to activate β-catenin signaling
(Fig. 2E). Therefore Fz
signaling is critical for LRP6 phosphorylation.
Dvl is an indispensable component downstream of Fz in Wnt signaling
(Wallingford and Habas, 2005).
Given that the Fz mutants we employed that are incapable of promoting Lrp6
phosphorylation are also defective in binding and/or recruiting Dvl to the
plasma membrane (Cong et al.,
2004; Wong et al.,
2003), we suspect that Dvl may act downstream of Fz in mediating
Wnt-induced Lrp6 phosphorylation. Because of functional redundancy of the
three mammalian Dvl genes (Wang et al.,
2006), we generated primary mouse embryonic fibroblast (MEF) cells
that have drastically reduced Dvl function. MEFs derived from the
Dvl1 and Dvl2 double knockout mice
(Dvl1-/-;Dvl2-/-) (see Fig. S1 in the
supplementary material) were infected with lentiviruses expressing shRNAs
against Dvl3. The four shRNAs exhibited different efficiency in
knocking down the Dvl3 protein level (Fig.
3A). Importantly, Wnt3a-induced Lrp6 phosphorylation was
significantly diminished in cells expressing each of the four shRNAs, and the
residual Lrp6 phosphorylation was directly proportional to the remaining
amount of Dvl3 protein (Fig.
3A). These results show that Dvl function is required for
Wnt-induced Lrp6 phosphorylation.
|
).
The DIX domain is critical for Wnt/β-catenin signaling, whereas the
requirement for the PDZ and DEP domains appears to be somewhat variable from
different studies (reviewed by Wallingford
and Habas, 2005; Wharton,
2003). We note that these studies employed different Dvl proteins
and/or different Dvl deletion mutants, which may account, in part, for the
observed differences. Re-expression of the wild-type Dvl2 rescued Wnt-induced
Lrp6 phosphorylation (lanes 1-6, Fig.
3B). The Dvl2DEP mutant, which lacks the DEP domain
(Habas et al., 2001), was also
able to rescue Wnt-induced Lrp6 phosphorylation (compare lanes 1-4 with lanes
13-16, Fig. 3C). However,
neither the Dvl2DIX mutant, which lacks the DIX domain
(Habas et al., 2001), nor the
Dvl2PDZ mutant, which lacks the PDZ domain
(Habas et al., 2001), was able
to do so (compare lanes 1-4 with lanes 5-8 and lanes 9-12,
Fig. 3C). Therefore the Dvl DIX
and PDZ domains, but not the DEP domain, appear to be involved in promoting
Lrp6 phosphorylation.
Earlier studies have documented that the Dvl DIX domain is involved in
direct binding to axin (Fagotto et al.,
1999; Kishida et al.,
1999; Li et al.,
1999; Salic et al.,
2000; Smalley et al.,
1999), possibly via the ability of the DIX domain to form dynamic
aggregates or polymers (Schwarz-Romond et
al., 2007a; Schwarz-Romond et
al., 2007b), and that the Dvl PDZ domain may mediate association
with Fz (Fujii et al., 2007;
Shan et al., 2005;
Wong et al., 2003). Thus one
possible scenario is that Fz recruits Dvl, which in turn, via its DIX domain,
recruits axin to promote Lrp6 phosphorylation. We first examined the validity
of such a sequential recruitment model using Xenopus embryonic
explants, which have been previously used to demonstrate Fz recruitment of
Xenopus Dishevelled (referred to here as Xdsh)
(Rothbacher et al., 2000;
Umbhauer et al., 2000;
Yang-Snyder et al., 1996). In
these embryonic cells, axin, when overexpressed alone or together with Xdsh,
showed diffuse cytoplasmic staining (Fig.
4A,B). As was shown previously, Xdsh-GFP was recruited to the
plasma membrane in the presence of human Fz5
(Fig. 4D, compared with
Fig. 4B). Axin remained largely
cytoplasmic when human Fz5 was coexpressed
(Fig. 4C). In the presence of
human Fz5 plus Xdsh-GFP, however, axin became prominently
plasma-membrane-bound in a pattern that was indistinguishable from that of
Xdsh-GFP (Fig. 4D). Either the
wild-type Xdsh or Xdsh-GFP was able to mediate human Fz5 recruitment of axin
to the plasma membrane (Fig.
4D,E). By contrast, XdshN
(Tada and Smith, 2000), which
lacks the DIX domain, did not mediate human Fz5 recruitment of axin
(Fig. 4F). These data support
the notion of the sequential recruitment of axin by Fz through Xdsh (Dvl), and
are consistent with previous and recent findings on the role of the DIX domain
in Dvl-axin association in mammalian cells
(Fagotto et al., 1999;
Kishida et al., 1999;
Li et al., 1999;
Salic et al., 2000;
Smalley et al., 1999;
Schwarz-Romond et al., 2007b).
We note, however, one caveat is that XdshN may be recruited less
effectively by Fz to the plasma membrane
(Rothbacher et al., 2000).
|
;
Tamai et al., 2004). We found
that Wnt3a-induced Lrp6 phosphorylation was significantly reduced in
Axin-/- mouse embryonic stem (ES) cells
(Fig. 5A). Since these cells
also express Axin2 (also known as Axil and conductin)
(Behrens et al., 1998;
Kikuchi, 1999)
(Fig. 5C), which can
functionally replace axin and thus shares a redundant function with axin
(Chia and Costantini, 2005), we
further employed shRNAs to knockdown Axin2 in these
Axin-/- cells. Multiple independent shRNAs against
Axin2 further reduced Wnt3a-induced Lrp6 phosphorylation
(Fig. 5B and see Fig. S2 in the
supplementary material), and the diminished Lrp6 phosphorylation level
correlated with the reduced amount of Axin2 mRNA expression upon
shRNA treatment (Fig. 5C).
These results suggest that axin function is required for Wnt-induced Lrp6
phosphorylation. The axin involvement in Lrp6 phosphorylation is likely
mediated by its ability to recruit Gsk3, as implied by the overexpression of
an axin mutant that lacks the Gsk3-binding domain
(Yamamoto et al., 2006).
Indeed the wild-type axin was able to enhance LRP6 phosphorylation by GSK3,
whereas the axin (L396Q) mutant, which corresponds to the zebrafish
masterblind mutation and is incapable of binding to GSK3 [because of
the leucine 396 to glutamine mutation
(Heisenberg et al., 2001;
van de Water et al., 2001)],
failed to promote LRP6 phosphorylation by GSK3
(Fig. 5D).
Our results therefore suggest that Gsk3, recruited via axin to the plasma
membrane, mediates Lrp6 phosphorylation and activation. Analogous to the
redundancy observed among Dvl genes and axin genes, Gsk3 and Gsk3β
appear to have redundant roles in Wnt3a-induced Lrp6 phosphorylation, which
was not significantly affected in either
Gsk3-/- or
Gsk3β-/- ES cells but was completely
abolished in
Gsk3-/-;Gsk3β-/-
ES cells (Fig. 5E). To
demonstrate an activating role of the membrane-associated Gsk3 in Wnt
signaling, we previously generated a membrane-tethered Gsk3 whose
overexpression activates β-catenin signaling in a manner that depends on
the PPPSP motifs in Lrp6 (Zeng et al.,
2005). However, genetic studies and pharmacological inhibitors
have not been able to reveal this activating role of Gsk3 in Wnt signaling,
probably because of Gsk3 phosphorylation of β-catenin that plays a key
negative role downstream of the pathway. We therefore attempted to inhibit
Gsk3 solely at the plasma membrane. We took advantage of a highly specific
Gsk3 inhibitory peptide, GID (Gsk3 interaction domain), which is a 25-amino
acid-residue peptide derived from Axin and has been shown to inhibit Gsk3
specifically in vivo (Hedgepeth et al.,
1999; Jiang et al.,
2005; Zhang et al.,
2003). GID expression in Xenopus embryos induced complete
axis duplication (Hedgepeth et al.,
1999) (and data not shown) and the expression of nr3, a
Wnt/β-catenin target gene (lanes 12-14,
Fig. 5F), indicating activation
of Wnt/β-catenin signaling as expected. Importantly, when GID was
targeted to the plasma membrane via the CAAX box from the K-Ras protein,
CAAX-GID inhibited wnt8-induced nr3 expression in a dose-dependent
fashion (compare lane 7 with lanes 8-10,
Fig. 5F), demonstrating that
the plasma membrane-tethered GID blocks Wnt signaling. As a specificity
control, we generated a membrane tethered GID-LP (CAAX-GID-LP), which harbors
a single amino acid residue change (leucine 396 to proline) in the GID peptide
and thus neither binds to nor inhibits Gsk3
(Jiang et al., 2005;
Zhang et al., 2003).
CAAX-GID-LP did not block wnt8 induction of nr3 expression (compare
lane 7 with lanes 4-6, Fig.
5F). The protein expression level of CAAX-GID and CAAX-GID-LP was
comparable (data not shown). These data suggest that CAAX-GID blocks Wnt
signaling by inhibiting Gsk3 at the plasma membrane. The opposite properties
of GID and CAAX-GID (activating and inhibiting Wnt/β-catenin signaling;
compare lanes 7-10 with lanes 12-14, Fig.
5F) is consistent with the notion that phosphorylation of
β-catenin and of Lrp6 by Gsk3/axin occurs in cytosol and at the plasma
membrane, respectively, and therefore is spatially separated.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Cadigan and Liu, 2006) and our
demonstration of Fz-Dvl-axin recruitment to the plasma membrane, and that
mutations that disrupt Fz-Dvl, Dvl-axin, axin-Gsk3 interactions all perturb or
prevent Lrp6 phosphorylation, we suggest a working model that represents
important reinterpretations and rewiring of the Wnt signaling diagram,
particularly at the receptor initiation stage
(Fig. 6). In the Wnt-induced
Fz-Lrp6 receptor complex Fz recruits Dvl through the PDZ domain, and Dvl, via
its DIX domain and possibly multimerization
(Schwarz-Romond et al., 2007a)
recruits the axin-Gsk3 complex, thereby promoting Gsk3 phosphorylation of Lrp6
to initiate downstream signaling (Fig.
6). This model is fully compatible with the observation in
Drosophila embryos that Dsh is required for axin recruitment to the
plasma membrane upon Wingless signaling
(Cliffe et al., 2003), and
provide clarifications for the long standing enigma and apparently
contradicting views regarding the functional relationships among Fz, Lrp6 and
Dvl (Li et al., 2002;
Schweizer and Varmus, 2003;
Tolwinski et al., 2003;
Wehrli et al., 2000;
Cadigan and Liu, 2006;
He et al., 2004).
|
;
Tamai et al., 2004). Previous
genetic studies, analogous to those on Gsk3, have established that axin has a
key inhibitory function in Wnt/β-catenin signaling via promoting
β-catenin phosphorylation and degradation. This and our previous studies
(Zeng et al., 2005) together
suggest dual roles of the axin-Gsk3 complex in both activation (via Lrp6
phosphorylation) and inhibition (via β-catenin phosphorylation) of the
Wnt pathway. Fz and Dvl appear to control the balance between the positive and
negative functions of the axin-Gsk3 complex. Whether the same or distinct
axin-Gsk3 complexes carry out these opposing functions remains to be
determined. Our results also imply the existence of a positive feedback loop
between Lrp6 and axin, given that axin is required for Lrp6 phosphorylation,
and Lrp6 phosphorylation at the PPPSPxS motifs in turn provides docking sites
for axin (Tamai et al., 2004;
Zeng et al., 2005). This in
principle could ensure sustained phosphorylation of most or all of the PPPSPxS
motifs in Lrp6 in cis, and in trans if/when Lrp6 multimerizes
(Baig-Lewis et al., 2007;
Bilic et al., 2007;
Cong et al., 2004), thereby
leading to β-catenin signaling (Fig.
6B). Consistent with this possibility, Lrp6 phosphorylation and
β-catenin stabilization, once induced, appear to be long lasting as long
as Wnt stimulation exists (Khan et al.,
2007; Wei et al.,
2007). A recent study in Drosophila, using a fusion
protein between Fz and the Lrp6 ortholog Arrow, and an Arrow protein that can
dimerize/multimerize, argues that Fz-Arrow initiates whereas multimerized
Arrow amplifies Wingless/β-catenin signaling
(Baig-Lewis et al., 2007). Our
data provide a potential molecular basis for such an initiation amplification
phenomenon and further suggest two routes to recruit the axin complex to the
Wnt receptor complex. Thus Fz-Dvl recruits axin to initiate Lrp6
phosphorylation (initiation), whereas phosphorylated Lrp6 further recruits
axin to sustain and amplify Lrp6 phosphorylation (amplification;
Fig. 6).
Our data also help to resolve an important discrepancy in the literature
regarding which Lrp6 phosphorylation event is regulated by Wnt. We have
demonstrated that PPPSP motif phosphorylation, which is carried out by the
redundant function of Gsk3 and Gsk3β
(Zeng et al., 2005) (this
study), is inducible upon Wnt stimulation
(Tamai et al., 2004). This
observation has been further supported by others
(Binnerts et al., 2007;
Bryja et al., 2007;
Khan et al., 2007) (J. Reiter,
personal communication) and our additional studies
(Wei et al., 2007;
Zeng et al., 2005) (this
study). We further showed that in the extended PPPSPxS motif, PPPSP
phosphorylation primes the phosphorylation of xS by CK1, thereby resulting in
Wnt induction of phosphorylation by both Gsk3 and CK1
(Zeng et al., 2005). By
contrast, one study suggested that PPPSP phosphorylation is constitutive
whereas CK1 phosphorylation is the main Wnt-inducible event
(Davidson et al., 2005). The
origin for this discrepancy remains unclear, but may be because of basal
levels of PPPSP phosphorylation variably detected in cell cultures as a result
of some autocrine Wnt signaling, and/or to different qualities of the
phospho-Lrp6 antibodies. Nonetheless, the data presented here further
illustrate Wnt-induced PPPSP phosphorylation is under the control by Fz, Dvl
and axin functions, thereby supporting the notion that Gsk3 phosphorylation of
Lrp6 is a major regulatory step upon Wnt stimulation. While this manuscript
was in preparation, it was reported that Wnt may induce Lrp6 aggregation and
phosphorylation at threonine 1479 (T1479) by CK1 in a Dvl-dependent manner,
although the mechanism by which CK1 is regulated by Dvl and the involvement of
Fz, axin and Gsk3 in the process were not addressed
(Bilic et al., 2007). We note
that such an `aggregation' model and our sequential recruitment/amplification
model may be different but not mutually exclusive. We should note, however,
that T1479, unlike the PPPSPxS motif, is not conserved in Drosophila
Arrow, and the significance of T1479 phosphorylation in the context of the
wild-type Lrp6 function remains to be established.
How Wnt stimulation leads to inhibition of β-catenin phosphorylation
remains unclear. Constitutively activated (N-terminal truncated) Lrp5/6, and a
single PPPSPxS motif when transferred to a heterologous receptor, are
constitutively phosphorylated (Tamai et
al., 2004) and are each sufficient to trigger β-catenin
signaling, probably in a Dvl-independent manner
(Li et al., 2002;
Schweizer and Varmus, 2003).
These observations suggest that binding of axin to the phosphorylated PPPSPxS
motifs in Lrp6 may be sufficient to cause inhibition of β-catenin
phosphorylation, and that the only function of Dvl in this pathway may be to
promote Lrp6 phosphorylation. However, Dvl overexpression can activate
β-catenin signaling in Xenopus egg extracts
(Salic et al., 2000) and in an
Arrow (Lrp6) mutant background
(Wehrli et al., 2000;
Kimelman and Xu, 2006), and
this signaling capability by overexpressed Dvl seems to correlate with the
propensity of Dvl to multimerize
(Schwarz-Romond et al.,
2007b). Whether Dvl, in addition to mediating Lrp6
phosphorylation, also directly inhibits β-catenin phosphorylation by
axin-Gsk3 under physiological conditions remains to be determined. Finally,
the observations that the axin-Gsk3 complex plays opposing functions in Lrp6
and β-catenin phosphorylation, and that Dvl interacts with axin in a
dynamic manner (Schwarz-Romond et al.,
2007a; Schwarz-Romond et al.,
2007b) support the emerging view that Wnt/β-catenin signaling
is a highly kinetic/dynamic process rather than simple on and off states, the
nature of which may require new analytical tools beyond traditional
biochemical methodologies to fully capture.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/367/DC1
|
ACKNOWLEDGMENTS |
|---|
N
vector, L. Mei for Dvl1 antisera and J. Reiter for communication. We
appreciate constructive suggestions from anonymous reviewers, and comments
from B. MacDonald. X.Z. is in part supported by a career development award
from the Children's Hospital Boston. H.H. is supported by a postdoctoral
fellowship from CIHR (Canada). This work is in part supported by grants from
NIH to X.H., who is a W. M. Keck Foundation Distinguished Young Scholar and a
Leukemia and Lymphoma Society Scholar. |
Footnotes |
|---|
Present address: Department of Genetics, Case Western Reserve University,
BRB 723, 10900 Euclid Avenue, Cleveland, OH 44106, USA
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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