doi: 10.1101/gad.230402.
The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling
Affiliations
- PMID: 12183362
- PMCID: PMC186448
- DOI: 10.1101/gad.230402
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The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling
Genes Dev.
.
Abstract
Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.
Figures
Domain structure and biochemical interactions
of Diversin. (A) Schematic representation of the domain
structure of mouse Diversin and of various deletion constructs. Numbers
represent amino acids of Diversin. (B) Coimmunoprecipitation
analyses in transfected Neuro2A cells, showing that the central domain
of Diversin (white in A) interacts with CKIε (top).
The C-terminal domain (black in A) confers interaction with
Conductin (middle). Similar interactions were seen with Axin.
Expression of the indicated proteins was verified by Western blotting
(bottom). (C,D) Yeast two-hybrid and
coimmunoprecipitation analyses showing that Diversin interacts with the
GSK3β-binding domain of Conductin and Axin. For constructs see
Behrens et al. (1998). (+) Growth of yeast on selection medium, (−)
absence of growth. The conductin ΔGSK-bd is as in C
(conductin lacking amino acids 343–396). (E)
Coimmunoprecipitation analyses of Diversin, Conductin, and GSK3β
complexes. Full-size Conductin is able to dimerize through the
DIX-domain (Hsu et al. 1999; data not shown) and allows the formation
of a triple complex that contains Diversin, Conductin, and GSK3β
(a). Conductin1–465 and the GSK3β-binding domain of
Conductin (Conductin GSK-bd) cannot dimerize and triple complexes with
Diversin and GSK3β are not observed (b,c). The
conductin GSK-bd is as in C (amino acids 343–465; Behrens et
al. 1998). (d) Note that amounts of Conductin GSK-bd similar
to those in c are able to efficiently coprecipitate GSK3β.
Schematic representations of the interactions in (a–d) are
shown at right. For immunoprecipitation experiments,
6 × 105 cells per 10-cm culture dish were transfected with
5 μg of the indicated cDNAs (in the pCDNA vector, Invitrogen) and
cultured for 48 h, followed by lysis and immunoprecipitation (Behrens
et al. 1998). Polyacrylamide gel electophoresis and Western blot were
performed with 1/5 of the immunoprecipitate from 1-ml lysate, 1/50 of
the lysate was used as expression control. Concentrations of the
commercial antibodies were as suggested by the manufacturers.
Diversin inhibits Wnt signaling in cell culture and
in Xenopus embryos. (A) Diversin promotes degradation
of β-catenin in 293 cells, as analyzed by Western blot analysis of
cytoplasmic β-catenin. Cells were mildly lysed using 0.1% Triton-X
100, and amounts of cell lysates were normalized using Shp2.
(Top, left) Transfection of increasing amounts (2 and
5 μg) of Diversin cDNA destabilized endogenous β-catenin and
blocked β-catenin that is induced by transfection of 1.5 μg of
Dishevelled cDNA. (Bottom, left) Diversin inhibits
the stabilization of β-catenin induced by transfection of increasing
amounts (0.25–2 μg) of Dishevelled cDNA. (Right) The
activity of Diversin is blocked by addition of 20 μM MG132, and is
observable up to 16 h after transient transfection. Dishevelled (1.5
μg) and Diversin (3 μg) were transfected, and expression was
monitored by Western blotting (data not shown). The proteasome
inhibitor MG132 was added 2 h before cell lysis.
(B,C) Diversin blocks Dishevelled- and Wnt-induced
transcription of a Lef/Tcf-dependent luciferase reporter gene in a
dose-dependent manner. A total of 1–3 μg of Diversin cDNA and 1.5
μg of Dishevelled or Wnt3a cDNA were cotransfected in 293 cells with
the TOP (gray bars) or the control FOP reporter (white bars).
(D) Analysis of the functionally important domains of Diversin
in Lef/Tcf-dependent transcriptional assays. Full-length Diversin and a
fragment able to interact with CKIε and Axin/Conductin inhibit
transcription. Expression of similar levels of Diversin and Diversin
fragments were verified by Western blot analysis. Reporter assays were
performed as in B and C. (E) Epistasis
analysis of Diversin in Xenopus embryos. Ventral injection of
1 ng of Dishevelled, CKIε, (dn)GSK3β, or β-catenin mRNA induced
secondary body axes (percent axis duplication shown). Coinjection of 3
ng of Diversin mRNA blocked axis duplication by Dishevelled and CKIε,
but not dn-GSK3β or β-catenin.
Diversin recruits CKIε to the β-catenin
degradation complex. (A) Diversin promotes association of
endogenous CKIε with Conductin. The Conductin-binding domain of
Diversin did not mediate this interaction. Note that the Diversin
constructs (lanes a,f) are as in Fig. 1A.
Immunoprecipitations were performed as described in Fig. 1. A scheme of
Diversin linking CKIε to the Axin/Conductin-GSK3β complex is shown
at right. (B) Hybrid molecules that contain the
catalytic domain of CKIε and the carboxy-termini of murine or
zebrafish Diversin inhibit Dishevelled-induced Lef/Tcf-dependent
transcription. cDNAs that encode the individual domains have no effect.
The catalytic activity of CKIε is essential, as shown by a
kinase-inactive fusion construct or addition of 50 μM CKI-7, a
specific CKI inhibitor. The indicated cDNAs (3 μg) were transfected
into 293 cells either with Dishevelled (1.5 μg) cDNA (gray bars) or
transfected alone (white bars). Reporter assays are as described in
Fig. 2, hybrid constructs are specified in Materials and Methods.
(C) Diversin cooperates with Frat-1 in displacing GSK3β from
Axin/Conductin complex. Expression of Frat-1 (1.5 μg) in 293 cells
activates Lef/Tcf-dependent transcription. Cotransfection of Diversin
cDNA (3 μg) enhances this further. The C-terminal, but not the
central domain of Diversin is responsible for this activity. Reporter
assays were performed as in Fig. 2. Gray bars represent the TOP and
white bars the control FOP reporters. A cooperative effect of Diversin
and Frat-1 was also observed in axis duplication of Xenopus
embryos. For this, Diversin (3 ng) and Frat-1 mRNA (1 ng) were injected
ventrally into the 4–8-cell embryo. Note that limited amounts of
Frat-1 mRNA were injected to visualize cooperation. A schematic model
of the action of Diversin in displacing GSK3β from the Axin/Conductin
complex, which leads to enhanced signaling of β-catenin, is presented
at right.
Diversin affects the formation of the dorsal
organizer in zebrafish embryos. (A–D) In situ hybridization
of diversin in zebrafish embryos at different developmental
stages. Diversin is expressed ubiquitously (B) in the
blastomeres at the 4–8-cell stage and (C) in shield-stage
embryos. Pronounced expression in the yolk-blastoderm interphase is
indicated by arrows. (D) Specific expression of
diversin in ganglial layers of the retina, otic vesicles, the
roof plate of the brain, and in the forebrain-midbrain (arrowhead) and
midbrain-hindbrain boundaries (arrow) at 72 h post fertilization (hpf).
A shows the sense control. (E–P) Down-regulation of
Diversin by the injection of antisense morpholinos leads to a
dorsalization of zebrafish embryos and the appearance of an enlarged or
ectopically localized dorsal organizer. (E) Wild-type embryo
at 30 hpf (dorsal up, arrowhead indicates ventral tail fin).
(F,G) Embryos at 30 hpf after injection with 4–6 ng
of Diversin antisense morpholino oligonucleotides (divMO) that display
class 3 (F, arrowhead indicates missing ventral tail fin) and
class 4 dorsalization (G, arrowhead indicates wound-up trunk).
(H,I,K,L) In situ hybridization of
gsc at the shield stage (animal view, dorsal to the right;
arrowheads indicate the size of the gsc expression domain;
arrow indicates ectopic gsc expression). (H)
Uninjected control; (I,K) embryo injected with
Diversin MOs; (L) embryo coinjected with Diversin MOs and
mouse Diversin mRNA. (M,N) In situ hybridization of
tbx6 at 70% epiboly (lateral view, anterior up, dorsal to the
right). (M) Control; (N) embryo injected with
Diversin MOs; note the expansion of the tbx6 expression
domain. (O,P) In situ hybridization of
pax2.1 at the 5-somite stage (lateral view, anterior to the
left, dorsal up; arrows indicate the midbrain-hindbrain boundary
region). (O) Control; (P) embryo injected with
Diversin MOs; note the ventral expansion of the pax2.1
expression domain. (Q–Z) Overexpression of Diversin
ventralizes zebrafish embryos. (Q,R) Strongly
ventralized embryos (Q, class 3, and R, class 4) at
30 hpf after injection with full-length Diversin mRNA;
(S,T) In situ hybridization of embryos at shield
stage for gsc (arrowheads in S). (S)
Uninjected control. (T) Embryo injected with Diversin mRNA
loses gsc expression at the dorsal side.
(U,V) In situ hybridization of embryos at 70%
epiboly stained for eve1 in the ventral mesoderm (arrowheads)
and gsc in the prechordal plate (arrow). (U)
Uninjected control. (V) Embryo injected with Diversin mRNA
shows an expansion of the eve1 expression domain and looses
gsc expression at the dorsal side. (W–Z) Diversin
acts downstream of Wnt and upstream of β-catenin, as shown on
injected embryos by in situ hybridization with gsc that marks
the dorsal organizer. (W) Embryo injected with Wnt8 mRNA;
(X) embryo coinjected with Diversin and Wnt8 mRNA;
(Y) embryo injected with β-catenin mRNA; (Z) embryo
coinjected with Diversin and β-catenin mRNA. Arrowheads indicate
lateral borders of gsc expression domains, arrow indicates
ectopic gsc expression. All embryos are shown as lateral views
(dorsal to the right and animal pole up).
Diversin affects the formation of the dorsal
organizer in zebrafish embryos. (A–D) In situ hybridization
of diversin in zebrafish embryos at different developmental
stages. Diversin is expressed ubiquitously (B) in the
blastomeres at the 4–8-cell stage and (C) in shield-stage
embryos. Pronounced expression in the yolk-blastoderm interphase is
indicated by arrows. (D) Specific expression of
diversin in ganglial layers of the retina, otic vesicles, the
roof plate of the brain, and in the forebrain-midbrain (arrowhead) and
midbrain-hindbrain boundaries (arrow) at 72 h post fertilization (hpf).
A shows the sense control. (E–P) Down-regulation of
Diversin by the injection of antisense morpholinos leads to a
dorsalization of zebrafish embryos and the appearance of an enlarged or
ectopically localized dorsal organizer. (E) Wild-type embryo
at 30 hpf (dorsal up, arrowhead indicates ventral tail fin).
(F,G) Embryos at 30 hpf after injection with 4–6 ng
of Diversin antisense morpholino oligonucleotides (divMO) that display
class 3 (F, arrowhead indicates missing ventral tail fin) and
class 4 dorsalization (G, arrowhead indicates wound-up trunk).
(H,I,K,L) In situ hybridization of
gsc at the shield stage (animal view, dorsal to the right;
arrowheads indicate the size of the gsc expression domain;
arrow indicates ectopic gsc expression). (H)
Uninjected control; (I,K) embryo injected with
Diversin MOs; (L) embryo coinjected with Diversin MOs and
mouse Diversin mRNA. (M,N) In situ hybridization of
tbx6 at 70% epiboly (lateral view, anterior up, dorsal to the
right). (M) Control; (N) embryo injected with
Diversin MOs; note the expansion of the tbx6 expression
domain. (O,P) In situ hybridization of
pax2.1 at the 5-somite stage (lateral view, anterior to the
left, dorsal up; arrows indicate the midbrain-hindbrain boundary
region). (O) Control; (P) embryo injected with
Diversin MOs; note the ventral expansion of the pax2.1
expression domain. (Q–Z) Overexpression of Diversin
ventralizes zebrafish embryos. (Q,R) Strongly
ventralized embryos (Q, class 3, and R, class 4) at
30 hpf after injection with full-length Diversin mRNA;
(S,T) In situ hybridization of embryos at shield
stage for gsc (arrowheads in S). (S)
Uninjected control. (T) Embryo injected with Diversin mRNA
loses gsc expression at the dorsal side.
(U,V) In situ hybridization of embryos at 70%
epiboly stained for eve1 in the ventral mesoderm (arrowheads)
and gsc in the prechordal plate (arrow). (U)
Uninjected control. (V) Embryo injected with Diversin mRNA
shows an expansion of the eve1 expression domain and looses
gsc expression at the dorsal side. (W–Z) Diversin
acts downstream of Wnt and upstream of β-catenin, as shown on
injected embryos by in situ hybridization with gsc that marks
the dorsal organizer. (W) Embryo injected with Wnt8 mRNA;
(X) embryo coinjected with Diversin and Wnt8 mRNA;
(Y) embryo injected with β-catenin mRNA; (Z) embryo
coinjected with Diversin and β-catenin mRNA. Arrowheads indicate
lateral borders of gsc expression domains, arrow indicates
ectopic gsc expression. All embryos are shown as lateral views
(dorsal to the right and animal pole up).
Diversin activates the JNK branch of the
Wnt-signaling pathway. (A) Coimmunoprecipitation analyses of
CKIε, Diego and Diversin, which show that both Diego and Diversin
interact with CKIε. Note that CKIε was transfected here.
Immunoprecipitations were performed as described in Fig. 1. Note equal
expression of Diego and Diversin. (B) Transfection of 1- and
3-μg Diversin cDNA activate transcription from a JNK-dependent
luciferase reporter gene. Diversin (1–3 μg) also enhances
Dishevelled (1.5 μg) and Wnt11 (1.5 μg)-induced JNK activity. Wnt-1
(1.5 and 3 μg) do not activate JNK-dependent, but activate
Lef/Tcf-dependent transcription (inset). The indicated cDNAs
were transfected into 293 cells, and MEKK (3 μg) served as control.
(C) Diversin affects gastrulation movements in zebrafish
embryos. Diversin induces failure of convergence and extension (CE), or
affects dorso-ventral patterning, depending on the amounts of injected
mRNA. (D,E) Zebrafish embryos injected with low
amounts (0.1 ng) of Diversin and standard amounts (0.4 ng) of Diego
mRNA show a shortened body axis and a curled tail, typical for defects
in gastrulation movements. (F–I) In situ hybridization of
flattened embryos injected with either Diversin MO (1.5 ng), Diversin
mRNA (0.1 ng), or Diego mRNA (0.4 ng), respectively, at the 5–10
somite stage. Embryos are stained for myoD to visualize
somites. Note the undulation of the body axis, the compression of the
anterior-posterior axis, and the shortening of the intersomitic
distances in the injected embryos. In situ hybridization with
krox20 expressed in the hindbrain (see the two stripes at
left) shows that the embryos are not ventralized.
Diversin activates the JNK branch of the
Wnt-signaling pathway. (A) Coimmunoprecipitation analyses of
CKIε, Diego and Diversin, which show that both Diego and Diversin
interact with CKIε. Note that CKIε was transfected here.
Immunoprecipitations were performed as described in Fig. 1. Note equal
expression of Diego and Diversin. (B) Transfection of 1- and
3-μg Diversin cDNA activate transcription from a JNK-dependent
luciferase reporter gene. Diversin (1–3 μg) also enhances
Dishevelled (1.5 μg) and Wnt11 (1.5 μg)-induced JNK activity. Wnt-1
(1.5 and 3 μg) do not activate JNK-dependent, but activate
Lef/Tcf-dependent transcription (inset). The indicated cDNAs
were transfected into 293 cells, and MEKK (3 μg) served as control.
(C) Diversin affects gastrulation movements in zebrafish
embryos. Diversin induces failure of convergence and extension (CE), or
affects dorso-ventral patterning, depending on the amounts of injected
mRNA. (D,E) Zebrafish embryos injected with low
amounts (0.1 ng) of Diversin and standard amounts (0.4 ng) of Diego
mRNA show a shortened body axis and a curled tail, typical for defects
in gastrulation movements. (F–I) In situ hybridization of
flattened embryos injected with either Diversin MO (1.5 ng), Diversin
mRNA (0.1 ng), or Diego mRNA (0.4 ng), respectively, at the 5–10
somite stage. Embryos are stained for myoD to visualize
somites. Note the undulation of the body axis, the compression of the
anterior-posterior axis, and the shortening of the intersomitic
distances in the injected embryos. In situ hybridization with
krox20 expressed in the hindbrain (see the two stripes at
left) shows that the embryos are not ventralized.
Diversin activates the JNK branch of the
Wnt-signaling pathway. (A) Coimmunoprecipitation analyses of
CKIε, Diego and Diversin, which show that both Diego and Diversin
interact with CKIε. Note that CKIε was transfected here.
Immunoprecipitations were performed as described in Fig. 1. Note equal
expression of Diego and Diversin. (B) Transfection of 1- and
3-μg Diversin cDNA activate transcription from a JNK-dependent
luciferase reporter gene. Diversin (1–3 μg) also enhances
Dishevelled (1.5 μg) and Wnt11 (1.5 μg)-induced JNK activity. Wnt-1
(1.5 and 3 μg) do not activate JNK-dependent, but activate
Lef/Tcf-dependent transcription (inset). The indicated cDNAs
were transfected into 293 cells, and MEKK (3 μg) served as control.
(C) Diversin affects gastrulation movements in zebrafish
embryos. Diversin induces failure of convergence and extension (CE), or
affects dorso-ventral patterning, depending on the amounts of injected
mRNA. (D,E) Zebrafish embryos injected with low
amounts (0.1 ng) of Diversin and standard amounts (0.4 ng) of Diego
mRNA show a shortened body axis and a curled tail, typical for defects
in gastrulation movements. (F–I) In situ hybridization of
flattened embryos injected with either Diversin MO (1.5 ng), Diversin
mRNA (0.1 ng), or Diego mRNA (0.4 ng), respectively, at the 5–10
somite stage. Embryos are stained for myoD to visualize
somites. Note the undulation of the body axis, the compression of the
anterior-posterior axis, and the shortening of the intersomitic
distances in the injected embryos. In situ hybridization with
krox20 expressed in the hindbrain (see the two stripes at
left) shows that the embryos are not ventralized.
Schematic model of the action of Diversin in
β-catenin degradation. (A) Diversin recruits CKIε to the
Axin/Conductin complex, and this leads to priming phosphorylation of
β-catenin on Ser 45. Dimeric Axin/Conductin allows simultaneous
binding of Diversin/CKIε and GSK3β in the complex. (B)
GSK3β mediates subsequent phosphorylation of β-catenin on Thr 41,
Ser 37, and Ser 33. Phosphorylated Ser 37 and Ser 33 are binding sites
for the E3ubiquitin ligase β-TrCP (Amit et al. 2002), which promotes
degradation of β-catenin.
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