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Mice lacking RIPK4 die at birth with fused external orifices that result
from defective epidermal differentiation (1). In humans, RIPK4 muta-
tions cause autosomal recessive Bartsocas-Papas syndrome characterized
by severe defects in face, skin, and limb development (2, 3). To investi-
gate RIPK4 signaling, we used a PathwayFinder PCR Array to deter-
mine gene expression changes in human embryonic kidney 293T cells
after transfection with RIPK4. Upregulation of Wnt target genes such as
CCND1, LEF1, JUN, Myc, and TCF7 (fig. S1) prompted us to compare
RIPK4 transfection to Wnt3a treatment in human PA-1 teratocarcinoma
cells. RIPK4 but not the related kinases RIPK1, RIPK2, and RIPK3
caused transcriptional changes similar to those caused by Wnt3a (Fig.
1A), upregulating AXIN2, APCDD1, GAD1, and NKX1-2 (fig. S2).
RIPK4 also activated a Wnt-dependent TOPbrite luciferase reporter in
293 cells (Fig. 1B).
Wnt signaling stabilizes β-catenin in the cytosol, thereby facilitating
its interaction with TCF transcription factors to drive Wnt-dependent
gene expression (4). 293T cells overexpressing RIPK4 contained more
cytosolic β-catenin than did cells transfected with empty vector, RIPK1,
RIPK2, or RIPK3, but they did not contain more CTNNB1 mRNA (Fig.
1C). Therefore, RIPK4 may inhibit β-catenin protein degradation as Wnt
signaling does. The kinase activity of RIPK4 appeared to be critical for
its Wnt-like effects because catalytically inactive mutant RIPK4-K51R
neither activated the TOPbrite reporter
nor altered cytosolic β-catenin levels
(fig. S3). RIPK4 interaction with PKC-
δ or PKC-β was neither sufficient nor
required for increased cytosolic β-
catenin because the K51R mutation did
not prevent PKC binding (fig. S4a), and
knockdown of both PKC isoforms had
no effect on β-catenin accumulation by
wild-type RIPK4 (fig. S4b). Bartsocas-
Papas syndrome RIPK4 point mutants
(I81N, I121N) and a truncation mutant
(S376X) did not alter the amount of
cytosolic β-catenin (Fig. 1D), implying
that RIPK4 signaling to β-catenin may
be relevant to mammalian development.
We investigated whether RIPK4
was required for Wnt signaling in vari-
ous cell lines. RIPK4 knockdown in
293T, ovarian A2780, and ovarian
COV434 cells reduced Wnt3a-induced
accumulation of β-catenin, TOPbrite
luciferase activation, and transcription
of AXIN2 and APCDD1 (Fig. 1E; fig.
S5), whereas Wnt3a signaling in pan-
creatic PANC-1, kidney 786-O, and
breast Hs578T cells was not compro-
mised by RIPK4 knockdown (fig. S6).
Therefore, the contribution of RIPK4 to
To explore a role for RIPK4 during
Xenopus laevis Ripk4 in the ventral-
vegetal cells of Xenopus embryos
where ectopic expression of Wnt path-
way agonists causes axis duplication
(5). Ripk4 alone did not produce a sec-
ondary axis, but it synergized with sub-
threshold amounts of Xenopus Wnt8
(Xwnt8), such that a full secondary axis
developed in approximately 25% of embryos (Fig. 2A). Ripk4 overex-
pression in dorsal-marginal cells, like expression of Wnt agonists (6),
broadened the region expressing the dorsal organizer gene Chordin (Fig.
2B). Catalytically inactive Ripk4-K52R had no effect (Fig. 2B). To test
whether Ripk4 was necessary for Wnt signaling in vivo, we suppressed
Ripk4 expression in Xenopus embryos with translation-blocking morpho-
linos and measured expression of Wnt target gene Xnr3 in animal cap
cells (7). Xnr3 induction by Xwnt8 was reduced (Fig. 2C and fig. S7),
suggesting that RIPK4 is necessary for optimal Wnt pathway activation
in this region. Absence of Xbra expression in the animal cap samples
excluded the possibility that Xnr3 derived from contaminating meso-
dermal tissue. Ripk4 morpholinos also anteriorized the neural tube and
caused a posterior shift in the hindbrain marker gene Engrailed2 (Fig.
2D), a phenotype linked to reduced Wnt signaling (8). Two different
Ripk4 morpholinos caused this phenotype and it was reversed by injec-
tion of human RIPK4 (Fig. 2D, column 3), ruling out non-specific, off-
target effects of the morpholinos. Ripk4-K52R overexpression in dorsal-
anterior cells elicited similar changes to those caused by Ripk4 morpho-
linos (Fig. 2D, column 4), consistent with the kinase inactive mutant
interfering with signaling by endogenous Ripk4. Finally, Ripk4 or Xwnt8
overexpression had the opposite effect of Ripk4 depletion, expanding
and shifting Engrailed2 expression anteriorly (Fig. 2D, columns 5-6).
Phosphorylation of Dishevelled bPhosphorylation of Dishevelled by y
Protein Kinase RIPK4 Regulates Wnt Protein Kinase RIPK4 Regulates Wnt
XiaoDong Huang,1, 2 James C. McGann,3* Bob Y. Liu,4* Rami N. Hannoush,5*
Jennie R. Lill,6 Victoria Pham,6 Kim Newton,1 Michael Kakunda,2 Jinfeng Liu,7
Christine Yu,8 Sarah G. Hymowitz,8 Jo-Anne Hongo,9 Anthony Wynshaw-
Boris,10 Paul Polakis,4 Richard M. Harland,3 Vishva M. Dixit1†
1Departments of Physiological Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080,
USA. 2Molecular Diagnostics and Cancer Cell Biology, Genentech, Inc., 1 DNA Way, South San Francisco,
CA 94080, USA. 3Department of Molecular and Cell Biology and Center for Integrative Genomics,
University of California, Berkeley, CA 94720, USA. 4Cancer Targets, Genentech, Inc., 1 DNA Way, South
San Francisco, CA 94080, USA. 5Early Discovery Biochemistry, Genentech, Inc., 1 DNA Way, South San
Francisco, CA 94080, USA. 6Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, CA
94080, USA. 7Bioinformatics, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
8Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. 9Antibody
Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA. 10Department of
Pediatrics and Institute for Human Genetics, School of Medicine, University of California, San Francisco,
CA 94143, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: email@example.com
Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal
differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein
kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not
catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of
cytosolic β-catenin and a transcriptional program similar to that caused by Wnt3a.
In Xenopus embryos, Ripk4 synergized with coexpressed Xwnt8, whereas Ripk4
morpholinos or catalytic inactive Ripk4 antagonized Wnt signaling. RIPK4 interacted
constitutively with the adaptor protein DVL2 and, after Wnt3a stimulation, with the
coreceptor LRP6. Phosphorylation of DVL2 by RIPK4 favored canonical Wnt
signaling. Wnt-dependent growth of xenografted human tumor cells was
suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may
contribute to the growth of certain tumor types.
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Collectively, these experiments indicate that Ripk4 can regulate Wnt
signaling in vivo.
To investigate how RIPK4 regulates Wnt signaling, we monitored
RIPK4-induced accumulation of cytosolic β-catenin and activation of a
TOPbrite reporter gene after siRNA-mediated depletion of Wnt pathway
components. Loss of either the Wnt co-receptor LRP6 or the Dishevelled
adaptor proteins (DVL1-3) blocked the increase in cytoplasmic β-catenin
(fig. S8A) and decreased TOPbrite luciferase activity (fig. S8B). Endog-
enous RIPK4 co-immunoprecipitated with endogenous DVL2 from
293T cells, irrespective of Wnt3a treatment (Fig. 3A). A direct interac-
tion seems likely as in vitro translated DVL2 and RIPK4 interacted also
(fig. S8C). Wnt3a treatment induced interaction of LRP6 with RIPK4,
albeit not until 15 min after treatment (Fig. 3B). These findings are con-
sistent with RIPK4 acting at the level of the Wnt receptor complex.
We explored whether RIPK4 phosphorylated LRP6, DVL, or their
associated proteins. Finding no evidence of LRP6 phosphorylation, we
focused on the DVL proteins as potential RIPK4 substrates. When
DVL2 was co-expressed in 293T cells with RIPK4 or K51R catalytic
inactive mutant RIPK4, two phosphopeptides, GDGGIYIGS298IMK and
KYAS480GLLK, derived from the PDZ and DEP domains of DVL2,
respectively, were enriched in cells expressing wild-type RIPK4 (fig.
S9A). Both sites are conserved, being found in Xenopus, zebrafish,
mouse, and human DVL isoforms (fig. S9B).
To determine if RIPK4 phosphorylated DVL2 directly, we purified
the kinase domain of RIPK4 (amino acids 1-300) from Sf9 insect cells
and tested its ability to phosphorylate the PDZ or DEP domains of
DVL2 in vitro. The wild-type RIPK4 kinase domain, but not the K51R
mutant, phosphorylated both domains, and mutation of DVL2 Ser298
and Ser480 prevented this phosphorylation (Fig. 3C). Antibodies recog-
nizing each DVL2 phosphorylation site detected wild-type DVL2 over-
expressed in 293T cells, but not DVL2 with the relevant serine mutated
to alanine (fig. S9C). Further confirming the specificity of the antibod-
ies, the wild-type DVL2 bands were not detected when the lysates were
treated with calf intestinal alkaline phosphatase (fig. S9D). In keeping
with DVL2 being a RIPK4 substrate, phosphorylation at Ser298 and
Ser480 increased dramatically when DVL2 was co-transfected with
RIPK4, but not RIPK4-K51R (fig. S9D).
When 293T cells were stimulated with Wnt3a, phosphorylation of
DVL2 at Ser298 and Ser480 increased transiently after 10 min (fig. S9E)
and this was attenuated by RIPK4 depletion (Fig. 3D and fig. S9E). To
determine if DVL2 phosphorylation was necessary for Wnt3a signaling,
we depleted Dvl3 from Dvl1−/−Dvl2−/− mouse embryo fibroblasts
(MEFs) to obtain DVL-null cells that were reconstituted with either
wild-type or phospho-site mutant S298A/S480A DVL2. Cells expressing
DVL2 S298A/S480A contained less cytosolic β-catenin after Wnt3a
treatment than did cells expressing wild-type DVL2 (fig. S9F), suggest-
ing that phosphorylation at one or both sites was necessary for maximal
Wnt3a treatment redistributes cytoplasmic DVL proteins into large
signaling complexes detected as punctate structures by immunofluores-
cence microscopy (9). Approximately 20% of HeLa cells expressed
DVL2-FLAG in puncta, whereas the rest contained cytoplasmic DVL2-
FLAG (Fig. 3E). Co-transfection of RIPK4-GFP increased the percent-
age of cells with DVL2 puncta to more than 75% (Fig. 3E and fig. S10),
suggesting that RIPK4 facilitates assembly of the DVL2 signalosome.
DVL2 S298A/S480A did not form more puncta when co-expressed with
RIPK4, whereas DVL2 containing a single Ser to Ala mutation behaved
like wild-type RIPK4 (Fig. 3E). These data indicate that RIPK4 phos-
phorylation at Ser298 and Ser480 promotes DVL2 signalosome assem-
Mutations that enhance Wnt signaling are found in various human
cancers (10), so we examined human tumors for overexpression of
RIPK4. Microarray data revealed increased RIPK4 mRNA in many ovar-
ian, skin, and colorectal tumors (Fig. 4A). RIPK4 protein and cytosolic
β-catenin were detected in several human ovarian adenocarcinomas,
whereas RIPK4 was less abundant and cytosolic β-catenin not detected
in most non-cancerous ovarian tissue samples (Fig. 4B and fig. S11A).
To determine if RIPK4 contributes to tumor growth by enhancing Wnt
signaling, we used a RIPK4 short hairpin RNA (shRNA) to deplete
RIPK4 from the Wnt-dependent human teratoma-derived NTERA-2
xenograft tumor model (11). We used colon HCT116 cells with an acti-
vating β-catenin mutation (12) as a control. Although RIPK4 depletion
from the NTERA-2 and HCT116 cells was incomplete (fig. S11B),
growth of the NTERA-2 cells in athymic nude mice was suppressed
(Fig. 4, C and D). By contrast, RIPK4 depletion had no effect on
HCT116 tumor growth. RIPK4 depletion from NTERA-2 tumors, but
not HCT116 tumors, decreased expression of two Wnt-responsive genes,
GAD1 and AXIN2 (fig. S11C), implying a critical role for RIPK4 in Wnt
signaling upstream of β-catenin stabilization.
We have identified a RIPK4 signaling mechanism that might explain
why its mutation in mammals causes severe developmental defects.
RIPK4 appears to be recruited to the LRP6 co-receptor and phosphory-
lates DVL proteins after Wnt stimulation, leading to maximal stabiliza-
tion of β-catenin and transcription of Wnt-responsive genes (fig. S12).
Mutant forms of RIPK4 associated with human Bartsocas-Papas syn-
drome are compromised in this signaling ability. RIPK4 expression is
restricted to vertebrates, indicating that phosphorylation of DVL proteins
in lower organisms might require a different kinase. Finally, the finding
that RIPK4-mediated Wnt signaling may promote human ovarian cancer
suggests that small molecule inhibitors of RIPK4 might hold therapeutic
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Acknowledgments: We thank K. O’Rourke, J. Huang, and the next-generation
sequencing and baculovirus groups for technical assistance. The transcriptome
sequencing data for PA1 cells have been submitted to the National Center for
Biotechnology Information, NIH, Gene Expression Omnibus (GEO)
(http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE43362.
26, 873 (2010).
Materials and Methods
Figs. S1 to S12
01 November 2012; accepted 23 January 2013
Published online 31 January 2013
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Fig. 1. Stimulation of the canonical Wnt pathway by RIPK4. (A) Heat maps show gene expression patterns determined by RNA
sequencing in PA-1 cells transfected with empty vector, RIPK1, RIPK2, RIPK3, or RIPK4 for 48 hours, or treated with 200 ng/mL
Wnt3a for 16 hours. Left: hierarchical clustering of variance-stablized expression values for genes with substantial changes in the
treatment groups compared with the vector group. Right: Pearson’s correlation coefficients between samples. (B) Expression of a
TOPbrite luciferase reporter in 293 cells transfected with the constructs indicated and then cultured in the absence or presence of
Wnt3a for 5 hours. Error bars represent the s.e.m. of triplicate measurements. Results are representative of 3 independent
experiments. (C) Cytosolic β-catenin protein abundance (upper panel) and CTNNB1 gene expression (lower graph) in 293T cells
transfected with RIPK4. Error bars represent the s.e.m. of triplicate measurements. (D) Cytosolic β-catenin in 293T cells transfected
with Bartsocas-Papas syndrome RIPK4 mutants. (E) Cytosolic β-catenin in A2780 or COV434 cells transfected with control (Ctrl) or
RIPK4 siRNAs for 60 hours and then treated with vehicle or Wnt3a for 2 hours.
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Fig. 2. Modulation of Wnt signaling in Xenopus by Ripk4. (A) Secondary axis formation in Xenopus embryos injected ventral-
vegetally with Ripk4 (1 ng), Xwnt8 (1 pg), or both. 269 embryos were examined in 3 independent experiments. Representative
embryos are shown. (B) Length of Chordin expression along the blastopore lip of embryos injected at the 4-cell stage with the RNAs
indicated. Bars represent the mean ± s.e.m of 108 embryos. Vegetal views of representative embryos are shown. (C) RT-PCR
analyses show the effect of Ripk4 morpholinos on Xwnt8-induced expression of Xnr3 in Xenopus animal cap cells. Control reactions
contained RNA from stage 10.5 Xenopus embryos with or without reverse transcriptase (RT). Amplification of epidermal keratin and
ornithine decarboxylase (ODC) confirmed RNA integrity. (D) Engrailed2 expression (blue) following blastomere injection at the two-
cell stage (Ripk4-MO1, Ripk4-MO2, sorted by fluorescence of co-injected fluorescein tracer) or the four-cell stage (all others).
Injected side is on the right with anterior on top. Red staining is due to nuclear β-galactosidase tracer.
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Fig. 3. Phosphorylation of DVL by RIPK4 promotes canonical Wnt signaling. (A) In 293T cells, endogenous RIPK4 co-
immunoprecipitated with endogenous DVL2 in the presence or absence of 200 ng/mL Wnt3a for 30 min. Control (Ctrl) and RIPK4
siRNAs confirmed RIPK4 antibody specificity. (B) Endogenous RIPK4 co-immunoprecipitated with endogenous LRP6 in 293T cells
treated with Wnt3a. (C) In vitro kinase assays using the RIPK4 kinase domain and the DVL2 DEP (or PDZ) domain as substrate.
(D) RIPK4-dependent phoshorylation of endogenous DVL2 in 293T cells treated with Wnt3a for 10 min. Non-specific bands (*). (E)
Effect of RIPK4-GFP on DVL2-FLAG cellular distribution in HeLa cells. Cells containing DVL2 puncta were enumerated by counting
250 cells per condition. Scale bars: 10 μm.
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Fig. 4. Delayed growth of Wnt-dependent xenograft tumors depleted of RIPK4. (A) Box and whisker plots show RIPK4 mRNA
abundance measured by microarray in human colorectal, ovarian, and melanoma samples. N: non-cancerous tissues; C: cancer. P-
values were derived from Student’s t tests. (B) Western blots show increased RIPK4 and cytosolic β-catenin in human ovarian
adenocarcinomas compared to non-cancerous ovarian tissue samples. (C and D) NTERA-2 and HCT116 cells were transduced
with lentiviral particles encoding RIPK4 or control (Ctrl) shRNAs and injected subcutaneously into athymic nude mice.
Representative tumor-bearing mice and their dissected tumors after 47 days are shown (C). Graphs show tumor volumes (D); error
bars represent the s.e.m. n=10).
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