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The postsynaptic density 95/disc-large/zona occludens protein syntenin directly interacts with frizzled 7 and supports noncanonical Wnt signaling.
Luyten A
,
Mortier E
,
Van Campenhout C
,
Taelman V
,
Degeest G
,
Wuytens G
,
Lambaerts K
,
David G
,
Bellefroid EJ
,
Zimmermann P
.
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Wnt signaling pathways are essential for embryonic patterning, and they are disturbed in a wide spectrum of diseases, including cancer. An unresolved question is how the different Wnt pathways are supported and regulated. We previously established that the postsynaptic density 95/disc-large/zona occludens (PDZ) protein syntenin binds to syndecans, Wnt coreceptors, and known stimulators of protein kinase C (PKC)alpha and CDC42 activity. Here, we show that syntenin also interacts with the C-terminal PDZ binding motif of several Frizzled Wnt receptors, without compromising the recruitment of Dishevelled, a key downstream Wnt-signaling component. Syntenin is coexpressed with cognate Frizzled during early development in Xenopus. Overexpression and down-regulation of syntenin disrupt convergent extension movements, supporting a role for syntenin in noncanonical Wnt signaling. Syntenin stimulates c-jun phosphorylation and modulates Frizzled 7 signaling, in particular the PKCalpha/CDC42 noncanonical Wnt signaling cascade. The syntenin-Frizzled 7 binding mode indicates syntenin can accommodate Frizzled 7-syndecan complexes. We propose that syntenin is a novel component of the Wnt signal transduction cascade and that it might function as a direct intracellular link between Frizzled and syndecans.
Figure 1. Syntenin interacts with Fz in a PDZ-dependent mode. (A) Sequences of the last 25 cytosolic amino acids of all human Fz and Fz
7 mutants; overview of syntenin binding to these peptides as detected in overlay; nd, not determined. C-terminal PDZBMs are in bold. For
Fz 1, 2, and 7, the membrane proximal PDZBM for Dsh is also present in the 25 last amino acids, and it is indicated in gray. (B) Overlays
illustrating syntenin interaction with Fz. GST-Syndecan-2 cytoplasmic domain (WT) was used as a positive control, and GST or GSTSyndecan-
2 PDZBM (cytoplasmic domain deleted for the last 2 amino acids) were used as negative controls. Note the interaction of syntenin
with Fz 7, 3, and 8 last 25 amino acids, and the lack of interaction with Fz 7 T/A and Fz 7 T/A V/A mutants. The quality and concentration
of the fusion proteins were controlled in Coomassie as shown at the bottom. (C) Respective roles of the PDZ domains of syntenin in Fz
interaction. Structure of syntenin and coordinates of the amino acids that define the different domains are shown on the right. The relevant
GST fusions were overlayed with recombinant proteins containing different combinations of the PDZ domains of syntenin as indicated. Note
that Fz 7 interacts preferentially with the PDZ1 domain, whereas syndecan 2, Fz 3, and 8 interact preferentially with the PDZ2 domain of
syntenin. The quality and concentration of the fusion proteins were controlled in Coomassie as shown at the bottom. (D) Interaction of
syntenin and syntenin PDZ domains with Fz 7 cytoplasmic domain in surface plasmon resonance. RU, response units. Note that the binding
relies primarily on the PDZ1 domain of syntenin. (E) Coimmunoprecipitation of endogenous syntenin with eYFP-tagged Fz 7 cytoplasmic
domain. MCF-7 cells overexpressing the Fz 7 cytoplasmic domain fused N-terminally to eYFP were extracted with detergent. The cell lysate
was immunoprecipitated with anti-eYFP antibodies and protein G beads before immunoblotting (right lanes, IP Fz7) with anti-eYFP to detect
the Fz 7 fusion (top) or anti-syntenin antibodies (bottom). The cell lysate was used as positive control (left), in the negative control the
anti-eYFP antibodies were omitted (middle). Note that endogenous syntenin coimmunoprecipitates with the eYFP-Fz 7 fusion (right bottom
lane).
Figure 2. Syntenin is recruited by Fz 7 and stimulates c-jun phosphorylation.
Confocal micrographs of MCF-7 cells overexpressing
eGFP-syntenin alone (A), overexpressing eGFP-syntenin together
with Fz 7 (B), or overexpressing eGFP-syntenin together with an Fz
7 carrying a mutated C-terminal PDZBM (T/A) (C). (D) Results are
expressed as the mean percentage of cells where the syntenin fluorescence
was concentrated at the plasma membrane; bars represent
standard deviations. Note that the recruitment of eGFP-syntenin to
the plasma membrane relies on Fz 7 and on the integrity of its
C-terminal PDZBM. (E) Cell lysates originating from HEK293T cells
transfected as indicated on top were tested for c-jun expression and
c-jun phosphorylation (ser-63). Actin was used as a loading control.
Note that syntenin addition stimulates c-jun phosphorylation in a
concentration dependent manner.
Figure 3. (A) Sequence alignment of the human syntenin (Husyntenin) with the three orthologues found in X. laevis (Xsyntenin-a, -a’, and
-b). (B) Percentage of identity between the different domains of the various syntenins. Note the extensive conservation of the PDZ domains.
Figure 4. Xsyntenin expressions during X. laevis early development
and Xsyntenin-a corecruitment with XDsh, by XFz 7, to the
plasma membrane of animal caps. (A) RNA extracts from embryos
at different stages were used to analyze Xsyntenin-a expression by
RT-PCR. Histone H4 amplification was used as an internal control.
(B–I) Xsyntenin-a, -a’, and b and XFz 3, 7, and 8 mRNA distributions
(purple) at different stages (st) of development. Hd, head; nt, neural
tube; pn, pronephros; ba; branchial arches; cg, cement gland. (J)
Syntenin-Fz 7 interaction is conserved in Xenopus. Hu Fz7 and XFz7
cytoplasmic domain sequences are shown on the right. The sequences
vary for two amino acids (underlined). Fz 7 cytoplasmic
domain from Xenopus or human, mutated (T/A) or not mutated in
the PDZBM, was overlayed with Xsyntenin-a (top). The quality and
concentration of the fusion proteins were controlled in Coomassie
(bottom). (K–U) Confocal micrographs of Xenopus animal caps at
stage 9 showing the subcellular distribution of XDsh-myc (K–M and
Q), Xkermit-myc (S and T), Xsyntenin-a-myc (N–P) or Xsyntenina-
HA (R and U) as indicated at the bottom. The caps originate from
embryos injected at two-cell stage with different combinations of
mRNAs encoding proteins indicated on each micrograph. Note the
translocation of XDsh to the plasma membrane upon XFz 7 and
XFz 7 T/A expression (compare M and L with K) and the translocation
of Xsyntenin-a upon XFz 7 but not XFz7 T/A expression
(compare O and P with N). Note also that although Xsyntenin-a
impairs Xkermit translocation (T), XDsh translocation is maintained
(Q).
Figure 5. Xsyntenin overexpression and down-regulation
block the elongation of activin-treated animal caps
and of whole embryos. (A–I) Pictures of stage 19 animal
cap explants, originating from embryos injected at two
cell stage with different mRNAs as indicated on top,
dissected at stage 9, and cultured in the absence or in
the presence of 50 ng/ml activin (as indicated at the
bottom). In situ hybridization with the mesodermal
marker cardiac actin (B, D, G, and I) shows that the
mesodermal differentiation is sustained in all activintreated
caps. Note the specific block of elongation induced
by high dose of X-syntenin-a mRNA (F–G). (J)
Results are expressed as the mean percentage of elongated
caps per condition, bars represent standard deviations.
(K–Q) Illustration of selected anomalies in X.
laevis embryos depleted for Xsyntenin-a, a’, and b. Embryos
injected with Xsyntenin MOs show a delayed
gastrulation (compare L with K), and they have a
shorter body-axis (compare N–O with M). Injections
were performed at the four-cell stage with 7, 5, or 10 ng
of each Xsyntenin MO or with 22 or 30 ng mismatch MO
in each blastomere. Embryos with body-axis length
60% (O) and embryos with body-axis between 60 and
80% (N) of the average length of noninjected controls
were pooled for quantitative analysis (Q). The percentage
of embryos showing delayed gastrulation (P) and
shortened body-axis (Q) was scored in three independent
experiments, with at least 80 embryos. Results are
expressed as the mean percentage of embryos showing
the defects at the stages indicated; bars represent standard
deviations.
Figure 6. Xsyntenin supports the XFz 7/
XPKC/XCDC42 branch of the noncanonical
Wnt signaling pathway. (A, C, and E) Pictures
of stage 19 animal cap explants originating
from embryos injected at two-cell stage with
different components as indicated on top, dissected
at stage 9, and cultured in the presence
of 50 ng/ml activin. Note that the block of
elongation observed with MOs for XFz 7 (A,
right image) is rescued by the coinjection of a
low dose of Xsyntenin-a RNA (C, middle picture).
Note that the block of elongation induced
by a high dose of Xsyntenin-a RNA is
rescued by the coinjection of RNA encoding a
DN form of the small GTPase XCDC42 (compare
E, middle, with Figure 5F). (B, D, and F)
Results are expressed as the mean percentage
of elongated caps per condition; bars represent
standard deviations. (G and H) Confocal
micrographs of Xenopus animal caps at stage 9
showing the subcellular distribution of
XPKC-GFP as indicated at the bottom. (G)
Comparison of the XPKC-GFP distribution
upon the overexpression of XFz 7 alone (left),
together with Xsyntenin-a-HA (middle), or together
with X-syntenin MOs (right). (H) Comparison
of the XPKC-GFP distribution upon
the overexpression of Xsyntenin-a-HA (top)
or the down-regulation of X-syntenins (bottom).
Note that Xsyntenin enhances the
plasma membrane recruitment of XPKC-
GFP.
Figure 7. Model for the role of syntenin scaffolding in noncanonical
Wnt signaling. Syntenin interacts with Fz 7 via its PDZ1 domain
and with Syndecan-4 (A) or Syndecan-2 (B) via its PDZ2 domain.
Syndecan and/or Fz 7 can recruit syntenin to the plasma membrane.
(A) Syndecan-4 interacts directly with PKC. Syndecan-4
triggers the activation of PKC. PKC functions upstream of CDC42
in noncanonical Wnt signaling. (B) Syndecan-2 might also activate
CDC42. See Discussion for details and references.
sdcbp (syndecan binding protein (syntenin)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, lateral view, animal up.
sdcbp (syndecan binding protein (syntenin)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, lateral view, anteriorright, dorsal up.
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