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© 2017. Published by The Company of Biologists Ltd.

The phosphatase Pgam5 antagonizes Wnt/-Catenin signaling in

embryonic anterior-posterior axis patterning

Verena Rauschenberger1*, Dominic B. Bernkopf2*, Sabrina Krenn1, Kowcee Jalal2, Jens

Heller1, Jürgen Behrens2, Marc Gentzel3,4*, Alexandra Schambony1*§

1 Biology Department, Developmental Biology, Friedrich-Alexander University Erlangen-

Nuremberg, 91058 Erlangen, Germany

2 Experimental Medicine II, Nikolaus-Fiebiger-Centre, Friedrich-Alexander University

Erlangen-Nuremberg, 91054 Erlangen, Germany

3 Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany

4 Center for Molecular and Cellular Bioengineering, Molecular Analysis - Mass

Spectrometry, TU Dresden, 01307 Dresden

* these authors contributed equally

§ corresponding author

key words: Pgam5, Wnt, Dvl2, phosphatase, Xenopus, anterior-posterior patterning

http://dev.biologists.org/lookup/doi/10.1242/dev.144477Access the most recent version at First posted online on 15 May 2017 as 10.1242/dev.144477

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Abstract

The scaffold protein Dishevelled is a central intracellular component of Wnt signaling

pathways. Various kinases have been described that regulate and modulate Wnt signaling

through phosphorylation of Dishevelled. However, besides the general protein phosphatases

1 and 2 (PP1 and PP2), no specific protein phosphatases have been identified. Here, we

report on the identification and functional characterization of the protein phosphatase Pgam5

in vitro and in vivo. Pgam5 is a novel antagonist of Wnt/-Catenin signaling in human cells

and Xenopus embryogenesis. In early development, Pgam5 is essential for head formation

and establishing and maintaining the Wnt/-Catenin signaling gradient that patterns the

anterior-posterior body axis. Inhibition of Wnt/-Catenin signaling and developmental

function depend on Pgam5 phosphatase activity. We show that Pgam5 interacts with

Dishevelled2 and that Dishevelled2 is a substrate of Pgam5. Pgam5 mediates a marked

decrease of Dishevelled2 phosphorylation in the cytoplasm and in the nucleus as well as

decreased interaction between Dishevelled2, Tcf1 and -Catenin, indicating that Pgam5

regulates Dishevelled function upstream and downstream of -Catenin stabilization.

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Introduction

Wnt signaling pathways play a major role in axis determination, patterning, morphogenetic

movements and differentiation during embryonic development. In Xenopus laevis, the

relocalization of maternal components of the Wnt/-Catenin pathway at fertilization defines

the future dorsal side of the embryo and induces the formation of Spemann’s organizer

(reviewed in (Hikasa and Sokol, 2013). During gastrulation, an anterior–posterior (a-p)

gradient of Wnt/-Catenin activity that patterns the a-p axis is established by posterior

expression of Wnt ligands and anterior endomesoderm-derived, secreted antagonists such as

Dickkopf1 (Dkk1)1 and Cerberus as well as ectodermal, intracellular antagonists including

Amer2 (Glinka et al., 1998; Glinka et al., 1997; Kazanskaya et al., 2000; Kiecker and Niehrs,

2001; Pfister et al., 2012; Piccolo et al., 1999). Wnt/-Catenin signaling is functionally linked

and tightly balanced with the activity of -Catenin-independent Wnt pathways. The latter are

key regulators of mesodermal and neural convergent extension movements that position cells

within the tissue and shape the a-p body axis (reviewed in Wallingford, 2012).

Most Wnt signaling cascades share a subset of effector proteins including Frizzled (Fzd)

receptors, Dishevelled (Dvl) proteins, -Arrestins (Arrb) and protein kinases such as Casein

Kinase 1 (CK1) and Glycogen Synthase Kinase 3 (GSK3, (Niehrs, 2012; Schulte et al.,

2010; Seitz et al., 2014). In unstimulated cells, free -Catenin is rapidly degraded. The core

-Catenin destruction complex consists of -Catenin, Adenomatous Polyposis Coli protein

(APC), Axin, the kinases CK1 and GSK3 and the Ubiquitin ligase TrCP. In the absence of

a Wnt stimulus, N-terminal phosphorylation of -Catenin targets it for ubiquitinylation and

subsequent proteasomal degradation (reviewed in (Stamos and Weis, 2013). The Wnt/-

Catenin pathway is typically activated by binding of Wnt ligands to a receptor complex

composed of Fzd and LRP5/6 as co-receptor. Receptor activation triggers phosphorylation of

LRP5/6 and the recruitment of Axin, Dvl, Casein Kinases and GSK3 to the receptor

complex. These events lead to inhibition of -Catenin degradation and the accumulation of -

Catenin in the cytoplasm and translocation into the nucleus (Hsu et al., 1998; Schneider et al.,

1 In general we use the nomenclature recommended for Xenopus genes and proteins

(http://www.xenbase.org/gene/static/geneNomenclature.jsp), i.e. gene and RNA symbols

small and italic, protein symbols first letter upper case and regular. Only when specifically

referring to the human genes and proteins, the nomenclature suggested by the HUGO gene

nomenclature committee (HGNC (Gray et al., 2015)), i.e. gene and RNA symbols all

capitalized and italic, protein symbols all capitalized and regular, will be used.

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1996). -Catenin then binds to LEF/TCF transcription factors, acts as a transcriptional

transactivator and thus regulates expression of -Catenin responsive genes (reviewed in

(Clevers and Nusse, 2012; Nusse, 2012).

Dvl becomes highly phosphorylated in response to Wnt stimuli (Bryja et al., 2007; Lee et al.,

1999; Takada et al., 2005; Yanagawa et al., 1995), which is considered a key event in Wnt

signal transduction. Multiple kinases have been found to phosphorylate Dvl and to play a role

in Wnt signal transduction including but not limited to the serine/threonine kinases CK1,

Casein kinase 2 (CK2), Protein Kinase C (PKC), Hypoxia-induced protein kinase (HIPK),

Receptor-interacting protein kinase 4 (RIPK4), MARK2/Par1b and MARK3/Par1a (Huang et

al., 2013; Kühl et al., 2001; Ossipova et al., 2005; Peters et al., 1999; Shimizu et al., 2014;

Sun et al., 2001; Willert et al., 1997). Despite the large number of kinases that phosphorylate

Dvl, little is known about Dvl dephosphorylation, except for the inhibitory effects of the

ubiquitous protein phosphatase 1 (PP1C, (Shimizu et al., 2014) and putatively more context

specific regulation by protein phosphatase 2 (PP2) regulatory subunits (Hannus et al., 2002;

Yamamoto et al., 2001). Here, we have identified the protein phosphatase Pgam5 as a novel

binding partner of Dvl2. Pgam5 belongs to the phosphoglycerate mutase family, but in

contrast to other members of this family, Pgam5 acts as protein serine-threonine phosphatase

(Takeda et al., 2009). We demonstrate that Pgam5 is a potent antagonist of Wnt/-Catenin

signaling and we have identified Dvl2 as one target of Pgam5-mediated dephosphorylation.

Pgam5 inhibits -Catenin stabilization and interaction of -Catenin, Tcf1 and Dvl2. In vivo,

Pgam5 is required for the formation of an a-p gradient of -Catenin activity, a-p patterning

and head formation in Xenopus laevis.

Results

The protein phosphatase Pgam5 is a novel interactor of Dvl2/Arrb2 complexes

We hypothesized that an Arrb2/Dvl2 protein complex might provide a hub for protein-protein

interactions, which are distinct from interactions of the single proteins. Therefore, we have

analyzed the interactome of Arrb2 as bait and co-expressed Dvl2 by quantitative mass

spectrometry. Besides known Dvl interactors such as Casein Kinases or CAMK2 (Bernatik et

al., 2011; Cong et al., 2004a; Gentzel et al., 2015; Kishida et al., 2001; Klein et al., 2006), we

have identified the phosphatase PGAM5 as a novel protein interaction partner. In the reverse

co-immunoprecipitation experiment with Dvl2 as bait and Arrb2 co-expression, PGAM5

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recovery relative to the bait was increased 66-fold as compared to the Arrb2 bait, indicating

that PGAM5 binds to Dvl2 rather than Arrb2. Similarly, more interacting kinases were

detected at higher abundances. The relative abundances of the kinases in each experiment

were within the same order of magnitude as PGAM5 abundance and support the assumption

that PGAM5 interacts with Dvl2 and only indirectly with Arrb2 (Figure 1A).

Pgam5 is highly conserved among vertebrates. Due to its allotetraploidy, the Xenopus laevis

genome comprises two pgam5 genes designated pgam5.S and pgam5.L. These denominations

should not be confused with human PGAM5-S and PGAM5-L (Lo and Hannink, 2008),

which describe two protein isoforms derived from the same gene by alternative splicing. The

last exon of the shorter human transcript variant (Ensemble accession number

ENST00000317555.6) is not present in Xenopus tropicalis or Xenopus laevis pgam5 genes.

Consistently, all Xenopus Pgam5 proteins correspond to the longest human PGAM5 isoform

PGAM5-L (Supplementary Figure 1). In this study, either Xenopus laevis Pgam5.S or human

PGAM5-L were used and in the following are designated Pgam5 or PGAM5 respectively.

The Pgam5 family shares characteristic features including an N-terminal mitochondrial

targeting sequence and the Pgam domain with a catalytic Histidine residue in the active

center, which are also present in Xenopus Pgam5 proteins. Overall, Xenopus laevis Pgam5

and Xenopus tropicalis Pgam5 are 91% identical. Xenopus laevis Pgam5 shares 66%, 65%

and 60% identical residues with its mouse, human and zebrafish orthologs respectively

(Supplementary Figure 1).

We validated the functional proteomics results by co-immunoprecipitation and Western Blot

analysis (Figure 1B and Supplementary Figure 2A). Pgam5 co-precipitated with Dvl2

irrespective of the co-expression of Arrb2 (Figure 1B). If present, Arrb2 also strongly co-

precipitated with Dvl2, as expected from our previous work (Bryja et al., 2007). By contrast,

only low amounts of Pgam5 co-precipitated with Arrb2 (Supplementary Figure 2A), which

recapitulated our results from quantitative mass spectrometry. Together, these findings

support the conclusion that Pgam5 interacts with Dvl2 and only indirectly with Arrb2. We

further confirmed this interaction by co-immunoprecipitation of endogenous PGAM5 and

ARRB2 with endogenous DVL2 from HEK293T cell lysates (Figure 1C). Relative

quantification of band intensities for PGAM5 showed that PGAM5 was enriched five-fold in

DVL2 immunoprecipitates as compared to control IgG. Next, we have mapped the region

within the Dvl2 protein that is required for Pgam5 binding to amino acids 334-428 between

the PDZ and DEP domains. The DIX and DEP domain as well as the C-terminus were

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dispensable for Pgam5 binding. Interestingly, the strongest binding was observed when the

PDZ domain and the interdomain region between DIX and PDZ domain were also present,

indicating that these contribute to the Pgam5 binding interface (Figure 1D and

Supplementary Figure 2B).

Dvl is highly phosphorylated upon Wnt stimulation (Bryja et al., 2007; Lee et al., 1999;

Takada et al., 2005; Yanagawa et al., 1995) therefore, we evaluated whether Dvl2 might be a

substrate of Pgam5. Knock-down of PGAM5 was sufficient to induce hyperphosphorylation

of endogenous DVL2 in human cells (Figure 2A), indicating that PGAM5 indeed

dephosphorylates DVL2. Consistently, overexpression of PGAM5, but not the phosphatase-

deficient mutant PGAM5-H105A (Takeda et al., 2009), suppressed phosphorylation of

overexpressed DVL2 (Figure 2B) and reduced WNT3A-induced phosphorylation of

endogenous DVL2 (Supplementary Figure 2C). To further confirm that DVL2 is a substrate

of PGAM5, we added recombinant GST-PGAM5 to whole cell lysates (Figure 2C) and

observed dose-dependent dephosphorylation of DVL2. Moreover, affinity-purified DVL2

was dephosphorylated by recombinant PGAM5 to the fully dephosphorylated state

(Supplementary Figure 2D), confirming that PGAM5 directly dephosphorylates DVL2.

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Pgam5 is required for head induction in Xenopus laevis embryos

To investigate the role of Pgam5 in embryonic development, we have first analyzed the

temporal and spatial expression pattern of pgam5 in Xenopus laevis embryos. We detected

predominantly one mRNA corresponding to the pgam5.S homeolog, whereas pgam5.L was

expressed only very weakly or not at all (Supplementary Figure 3A). Accordingly, we have

focused on pgam5.S for the following experiments.

Pgam5 was expressed maternally and zygotically throughout embryonic development (Figure

3A and Supplementary Figure 3B-D). In cleavage and blastula stages, pgam5 RNA was

detected in the entire animal hemisphere (Figure 3A, i and ii, Supplementary Figure 3C).

Expression was restricted to the ectodermal layer in the late blastula (Figure 3A, ii’). During

gastrulation, pgam5 expression extended vegetally with progressing blastopore closure and

was detected in the entire ectoderm by mid-gastrulation albeit with a bias towards the animal-

dorsal region (Figure 3A, iii and iii’ and Supplementary Figure 3C and D). Ectodermal

expression remained biased towards the anterior neuroectoderm throughout neurulation,

which became more prominent in later stages (Figure 3A, iv, iv’, v and v’). Post-neurulation,

the highest levels of pgam5 RNA were detected in the central nervous system, the eye and the

otic vesicle as well as in the migrating neural crest and later in branchial arches (Figure 3A,

vi and vii and Supplementary Figure 3C and D).

Pgam5 was knocked-down using two translation-blocking morpholino-oligonucleotides

(MOs) that target non-overlapping sequences in pgam5.S RNA (Supplementary Figure 4A

and 4C). MO1 is predicted to bind to pgam5.L RNA with two mismatches and therefore

supposedly also blocks translation of pgam5.L (Supplementary Figure 4B). Injection of either

Pgam5 MO1 or Pgam5 MO2 in both blastomeres at the two-cell stage resulted in strongly

reduced or absent head structures in approximately 40% of the embryos, which was not

observed in embryos injected with a control MO (Figure 3B and Supplementary Figure 4D).

The phenotype was rescued by co-injection of a morpholino-insensitive Xenopus pgam5

RNA or by human PGAM5 RNA (Figure 3B and Supplementary Figure 4D), which

confirmed the functional conservation of Pgam5 between human and frog and the specificity

of the observed knock-down phenotype. Moreover, co-injection of RNA encoding the

phosphatase-deficient mutant PGAM5-H105A failed to rescue the Pgam5 MO phenotype,

suggesting that phosphatase activity of Pgam5 is required for its developmental function

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(Figure 3B). Co-injection of PGAM5 RNA with the control MO in both dorso-animal

blastomeres of eight-cell-stage embryos resulted in targeted overexpression. About 20% of

the embryos showed a moderate enlargement of anterior structures, consistent with Pgam5 as

a positive regulator of head development (Figure 3B). However, 18% also showed a small

head phenotype similar to PGAM5 morphant embryos. Targeted overexpression of PGAM5-

H105A also resulted in 15% embryos with smaller heads and in addition, overexpression of

wild-type or mutant PGAM5 caused shortened anterior-posterior body axes in a subset of the

embryos (Figure 3B).

Pgam5 has been functionally implicated in apoptosis, necroptosis and mitophagy, although it

apparently acts as positive or negative regulator depending on the experimental context

(Chen et al., 2014; Ishida et al., 2012; Lenhausen et al., 2016; Lu et al., 2016; Moriwaki et

al., 2016; Wang et al., 2012). Increased cell death in the anterior ectoderm might lead to a

loss of head structures, while inhibition of cell death in early Xenopus embryos results in an

expansion of neuroectoderm (Offner et al., 2005; Yeo and Gautier, 2003). After Pgam5 MO

injection we neither observed overall increased cell death assayed using Acridine Orange, nor

increased apoptosis indicated by cleaved-Caspase 3-positive cells (Supplementary Figure 5A

and B). At the dose used here, overall survival rates were also comparable to control

injections (Supplementary Figure 5C). Therefore, we concluded that the Pgam5 loss-of-

function phenotype described above was neither caused by increased cell death in general nor

by specific loss of anterior cells.

Pgam5 antagonizes Wnt/-Catenin signaling and contributes to anterior-posterior axis

patterning in Xenopus

Head induction and anterior patterning of the neuroectoderm in vertebrates require the

inhibition of Wnt/-Catenin signaling (reviewed in (Niehrs, 2010)). Notably, the Pgam5

morphant phenotype was highly reminiscent of impaired head induction reported after

blocking of the Wnt-antagonist Dkk1 (Glinka et al., 1998). Anterior overexpression of Dkk1

in Pgam5 morphant embryos restored head development similar to co-injection of MO-

insensitive pgam5 RNA (Figure 4A), indicating that elevated Wnt/-Catenin signaling

underlies the Pgam5 morphant phenotype. Indeed, we observed higher amounts of

dephosphorylated, active -Catenin as well as increased Dvl2 phosphorylation in Pgam5

morphant embryos (Figure 4B). We next analyzed the expression levels of the Wnt/-Catenin

target genes xnr3, msgn1 and hoxd1 by quantitative RT-PCR in Pgam5 morphant embryos.

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All three genes were upregulated confirming overall increased Wnt/-Catenin activity in

Pgam5 deficient embryos (Figure 4C and D). Notably, mRNA levels of dkk1 were not

changed (Figure 4D), indicating that the Pgam5 knock-down phenotype was not due to

repression of dkk1 expression. PGAM5 knock-down in human cells enhanced the activation

of the TOP-Flash reporter (Korinek et al., 1997) by WNT3A in HEK293T and C2C12 cells

(Figure 4E and Supplementary Figure 6A) and induced -Catenin stabilization and

expression of the feedback target gene AXIN2 ((Leung et al., 2002; Lustig et al., 2002),

Supplementary Figure 6B). Taken together, these results supported a role of Pgam5 as

antagonist of Wnt/-Catenin signaling in Xenopus development and human cells.

The a-p axis is patterned by a gradient of Wnt/-Catenin signaling activity, which is

established at mid-gastrula stages (Kiecker and Niehrs, 2001). We have visualized this

gradient in dorsal explants of stage 11.5 embryos that were dissected into the anterior,

midpart and posterior thirds. Active, dephoshorylated -Catenin (ABC) was low in the

anterior third and increased in mid and posterior parts of control explants (Figure 5A),

reflecting graded Wnt/-Catenin signaling activity. By contrast, in Pgam5 depleted embryos

the anterior levels of active -Catenin were significantly increased and no gradient of active

-Catenin was observed (Figure 5A). These results strongly suggested that the disruption of

graded Wnt/-Catenin activity underlies the head phenotype observed after Pgam5 knock-

down. Consistently, anterior-posterior patterning of the neuroectoderm was modulated by

Pgam5 knock-down or overexpression. Pgam5 knock-down shifted the expression of the

forebrain marker otx2 and more pronouncedly of the hindbrain marker krox20 anteriorly

(Figure 5B). Again, co-injection of human PGAM5 RNA rescued the phenotype while

PGAM5-H105A RNA did not. When PGAM RNA was co-injected with the control MO,

resulting in anterior overexpression of PGAM5, both markers were shifted posteriorly

(Figure 5B) as would be expected by stronger inhibition of Wnt/-Catenin signaling. By

contrast, overexpression of the phosphatase-dead mutant PGAM5-H105A in embryos

injected with control MO induced an anterior shift of both markers (Figure 5B). In addition,

we observed downregulation of krox20 in about 15% of the embryos injected with PGAM5-

H105A RNA, indicating more severe defects induced by this mutant.

Next we investigated, if overexpression of Pgam5 suppressed Wnt/-Catenin activity. In

Xenopus embryos, ectopic ventral activation of Wnt/-Catenin signaling induces a secondary

body axis (McMahon and Moon, 1989). Here, we injected Xenopus wnt8 RNA into both

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ventral blastomeres of four-cell stage embryos, which resulted in full or partial axis

duplication in more than 90% of the embryos (Figure 6A). Co-expression of human PGAM5

partially suppressed axis duplication induced by Wnt8. In particular, the percentage of

complete secondary axes was reduced from 54% to 18%. PGAM5-H105A showed only weak

inhibition of axis duplication (Figure 6A), confirming that phosphatase activity is required for

Pgam5 activity as Wnt/-Catenin antagonist.

Dorsal inhibition of Wnt/-Catenin activity in early Xenopus embryos prevents formation of

Spemann’s organizer and therefore results in ventralized embryos characterized by the lack

of dorsal structures such as the dorsal fin, the neural tube, notochord or somites (Heasman et

al., 2000; Montross et al., 2000). After injection of PGAM5 RNA into both blastomeres of

two-cell stage embryos, i.e. global overexpression of PGAM5, we observed different degrees

of ventralization marked by the progressive loss of the dorsal fin, somites and dorsal head

structures (Figure 6B). In contrast to wild-type PGAM5, PGAM5-H105A overexpression

caused small or absent heads in around 40% of the embryos, while the dorsal fin and somites

were well formed in the trunk (Figure 6B). These phenotypes were reminiscent of the loss-of-

function phenotypes (Figure 3B), indicating that PGAM5-H105A might act as a dominant-

negative mutant in Xenopus embryos. Next, we investigated whether overexpression of

Pgam5 was sufficient to decrease nuclear -Catenin levels in Xenopus embryonic fibroblasts.

In WNT3A stimulated cells nuclear -Catenin was clearly visible. Overexpression of GFP-

tagged Pgam5 resulted in a significant decrease of nuclear -Catenin (Figure 6C), which

further confirmed our findings obtained in Xenopus embryos.

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Pgam5 acts downstream of -Catenin stabilization

In order to further characterize the mechanism underlying Pgam5-mediated inhibition of

Wnt/-Catenin signaling, we asked whether Pgam5 inhibited Wnt/-Catenin signaling

upstream or downstream of -Catenin stabilization. Overexpression of either human PGAM5

or Xenopus Pgam5 antagonized TOP-Flash activation induced by LRP6, Dvl2 or stabilized -

Catenin in HEK 293T cells (Figure 7A and Supplementary Figure 7A and B). PGAM5 also

suppressed Wnt/-Catenin signaling in the APC-deficient SW480 colon carcinoma cell line,

in which -Catenin signaling is constitutively active (Korinek et al., 1997), to a similar extent

as ectopic AXIN (Supplementary Figure 7C). Consistently, Pgam5 also blocked -Catenin

induced axis duplication in Xenopus laevis embryos (Supplementary Figure 7D). These

results indicated that PGAM5 interferes with Wnt/-Catenin signaling downstream of -

Catenin stabilization and suppresses -Catenin-dependent transcription.

Transcriptional regulation of Wnt/-Catenin target genes requires the interaction of -

Catenin with transcription factors of the Lef/Tcf family (Behrens et al., 1996; Molenaar et al.,

1996). Overexpression of Pgam5 suppressed WNT3A-induced activation of the TOP-Flash

reporter in the presence of Tcf1-Flag (Figure 7B) indicating that Pgam5 might interfere with

Tcf1--Catenin binding or transcriptional activation. Immunoprecipitation of Tcf1-Flag from

unstimulated cells showed a basal interaction with endogenous -Catenin, which was

increased by WNT3A stimulation. Pgam5 overexpression reverted the effect of WNT3A and

reduced Tcf1--Catenin interaction back to basal levels (Figure 7C). Notably, we also

observed co-precipitation of Pgam5 with Tcf1, indicating that Pgam5 was able to bind to one

or more components of the transcription factor complex. Next, we overexpressed the

stabilized mutant -Catenin 4ST/A (Yost et al., 1996) together with Tcf1. Binding of -

Catenin 4ST/A to Tcf1 was not further enhanced by WNT3A stimulation. Interestingly, the

amount of Pgam5 that was co-precipitated with Tcf1 and -Catenin 4ST/A was reduced by

WNT3A stimulation. However, overexpression of Pgam5 was still sufficient to reduce

interaction between -Catenin 4ST/A and Tcf1 (Figure 7D) both in the presence and absence

of exogenous WNT3A. Together with the observation that Pgam5-mediated inhibition of

reporter gene activation by stabilized -Catenin requires its phosphatase activity (Figure 7A),

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these results suggested that a substrate of Pgam5 is required to form or stabilize the -

Catenin/Tcf complex.

Pgam5 inhibits recruitment of Dvl2 to the Tcf1 complex

Above, we have identified Dvl2 as a substrate of Pgam5. In addition to its role in the Frizzled

receptor complex, Dvl has also been found in the nucleus where it interacts with the Tcf/-

Catenin transcription complex and has been shown to play a role in transcriptional regulation

(Gan et al., 2008; Itoh et al., 2005; Wang et al., 2015).

Therefore, we analyzed whether PGAM5 affected nuclear localization of DVL2. As

expected, treatment of HEK293T cells with WNT3A induced stabilization of cytosolic -

Catenin and an increase of -Catenin in the nuclear fraction (Figure 8A). In agreement with a

putative nuclear function of DVL2, we detected endogenous DVL2 in the nuclear fraction.

Interestingly, phosphorylated DVL2 was observed in the nuclear fraction only after WNT3A

stimulation (Figure 8A). PGAM5 overexpression blocked the WNT3A-induced increase of -

Catenin and phospho-DVL2 in both the cytoplasmic and nuclear fractions. By contrast,

PGAM5-H105A increased cytoplasmic -Catenin and DVL2 phosphorylation already in

unstimulated cells, but seemed to simultaneously inhibit nuclear accumulation of -Catenin

and phospho-DVL2 (Figure 8A).

The nuclear function of Dvl2 is still somewhat controversial, but evidence is accumulating

that Dvl2 can form complexes with Tcf, -Catenin and FOXK transcription factors (Gan et

al., 2008; Itoh et al., 2005; Wang et al., 2015). Therefore, we asked whether Pgam5 also

regulated the interaction between Dvl2 and Tcf or -Catenin. Dvl2 was co-

immunoprecipitated with Tcf1 and endogenous -Catenin irrespective of WNT3A

stimulation (Figure 8B, see also Figure 7C). Only one band of Dvl2 was detected in the co-

precipitates whereas additional, faster migrating bands were present in the lysate, suggesting

that Tcf1 preferentially interacted with phosphorylated Dvl2. The interaction between -

Catenin and Tcf1 was not further stimulated by WNT3A in the presence of exogenous Dvl2,

while Pgam5 strongly reduced binding of both Dvl2 and endogenous -Catenin to Tcf1.

When cells overexpressing Pgam5 were stimulated with WNT3A, we observed a moderate

increase of Tcf1-Dvl2 interaction and a concomitant decrease of Tcf1-Pgam5 binding

compared to unstimulated Pgam5 overexpressing cells (Figure 8B). This was in agreement

with a similar effect shown in Figure 7D and indicated that WNT3A stimulation at least

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partially reverted the inhibitory effect of Pgam5. In the inverse experiment, when Dvl2 was

immunoprecipitated, an interaction of Dvl2 with -Catenin but not with Tcf1 was detected in

unstimulated cells (Figure 8C). WNT3A stimulation induced binding of Tcf1 to Dvl2, which

was again inhibited by Pgam5 overexpression, whereas the interaction of Dvl2 with -

Catenin was apparently not affected (Figure 8C).

Together, these results confirmed the interaction of Dvl2 with Tcf1 and -Catenin and further

suggested that this interaction is phosphorylation-dependent and therefore sensitive to

Pgam5-mediated dephosphorylation of Dvl2.

Discussion

Various different kinases have been identified that phosphorylate Dishevelled proteins

(Bernatik et al., 2011; Cong et al., 2004a; Gentzel et al., 2015; Kishida et al., 2001; Klein et

al., 2006; Kühl et al., 2001; Ossipova et al., 2005; Peters et al., 1999; Shimizu et al., 2014;

Sun et al., 2001; Willert et al., 1997). As counterparts, to date only the rather universal

phosphatases PP1 and PP2 have been described (Carmen Figueroa-Aldariz et al., 2015;

Shimizu et al., 2014). Here, we have identified the phosphatase Pgam5 as interactor of Dvl2-

Arrb2 protein complexes and negative regulator of Wnt/-Catenin signaling.

In gastrula and post-gastrula stage Xenopus laevis embryos, pgam5 was expressed throughout

the ectoderm but biased towards the anterior-dorsal ectoderm and the anterior neural tube.

Consistent with this expression pattern, we showed that Pgam5 plays a role in head formation

and a-p patterning. Low anterior Wnt/-Catenin activity is required for the induction of

anterior fates in the dorsal ectoderm and, in combination with the inhibition of BMP

signaling, for head induction (Glinka et al., 1997; Glinka et al., 1998). Consistently, heads

were small or absent in Pgam5 morphant embryos. Considering that Pgam5 interacts with

Dvl2, a role of Pgam5 in Wnt signaling could be assumed. However, these phenotypes would

also be explained by a loss of anterior cells (Offner et al., 2005). Necroptosis promoting and

protective roles as well as pro- and anti-apoptotic functions of Pgam5 have been described

(Ishida et al., 2012; Lenhausen et al., 2016; Lu et al., 2016; Moriwaki et al., 2016; Zhou et

al., 2012). However, we have not detected any local or global changes in cell death or

apoptosis in Pgam5 morphant Xenopus embryos, indicating that regulation of cell death does

not represent the major early developmental function of Pgam5 in Xenopus.

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Our results demonstrate that Pgam5 indeed acts as antagonist of Wnt/-Catenin signaling in

vivo. We observed increased amounts of active -Catenin as well as elevated expression

levels of maternal and more pronouncedly of cygotic Wnt/-Catenin target genes in Pgam5

morphant embryos. Moreover, the Pgam5 knock-down phenotype was rescued by

overexpression of the Wnt antagonist Dkk1 (Glinka et al., 1998; Mao et al., 2001; Semenov

et al., 2001). Notably, dkk1 expression was not affected in Pgam5 morphant embryos, which

excluded an indirect effect via transcriptional regulation of dkk1.

Patterning of the a-p axis and head induction depend on a gradient of Wnt/-Catenin activity

present at mid-gastrula stages (Kiecker and Niehrs, 2001). Indeed, this gradient was flattened

and elevated to the higher posterior levels of active -Catenin in Pgam5 morphant embryos.

Consistently, Pgam5-depletion shifted the expression of forebrain and hindbrain markers

anteriorly, further confirming a role of Pgam5 in anterior-posterior patterning and head

induction in Xenopus development.

Unlike our observations in Xenopus development, no significant developmental phenotype

has been reported for Pgam5 knock-out mice except for a general reduction in size and body

mass and increased post-natal lethality (Lu et al., 2014; Moriwaki et al., 2016). Interestingly,

we also observed increased lethality when higher doses of Pgam5 MO were injected, which

might correspond to the observations in Pgam5 knock-out mice. The apparent lack of

developmental phenotypes in the mouse is likely due to redundant mechanisms such as

intracellular inhibition of Wnt/-Catenin signaling by ICAT (Inhibitor of -Catenin and TCF,

(Satoh et al., 2004)), the contribution of other signaling pathways and transcriptional control

of head inducers (reviewed in (Arkell and Tam, 2012)) or other compensatory mechanisms.

Although it must be assumed that redundant players are also active in Xenopus a-p patterning

and head induction, the contribution of individual proteins may vary as it is also observed e.g.

for the contribution of individual Wnt ligands to neural crest induction in Xenopus and

mouse (reviewed in (Barriga et al., 2015)).

Antagonism of Wnt/-Catenin signaling by Pgam5 was further supported by the suppression

of and TOP-Flash reporter activity in human cells and inhibition of axis duplication as well as

the overexpression phenotypes in Xenopus embryos. In Xenopus, dorso-ventral axis

determination and formation of the dorsal signaling center, Spemann’s organizer, depends on

maternal Wnt/-Catenin signaling (reviewed in (Hikasa and Sokol, 2013)). Interfering with

this early Wnt/-Catenin function e.g. by knock-down or overexpression of -Catenin

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impairs or augments organizer formation and consequently the development of dorsal

structures. Pgam5 morphant embryos were not dorsalized most likely due to the presence of

maternal Pgam5 protein, which is not depleted by morpholino injections. By contrast, early

and global overexpression of Pgam5 led to the loss of dorsal structures reminiscent of the -

Catenin knock-down phenotype (Heasman et al., 2000), which clearly demonstrated the

ability of Pgam5 to suppress endogenous Wnt/-Catenin signaling. Targeted injections of

Pgam5 RNA at the eight-cell stage resulted in enlarged heads consistent with augmented

anterior inhibition of Wnt/-Catenin signaling. However, we also observed smaller heads

with similar frequencies, which appears contradictory. Injections into the dorso-animal

blastomeres of eight-cell stage embryos targets predominantly the anterior ectoderm, but

partially also the marginal zone and thereby the future organizer. Therefore, this phenotype

might be explained by effects of Pgam5 gain-of-function on organizer formation or function,

which have also been reported for dorso-vegetal -Catenin depletion (Heasman et al., 2000).

In addition, pgam5 is expressed in the cranial neural crest. Accordingly, anterior

overexpression might also affect head morphogenesis and thereby indirectly head size.

We have further investigated the molecular mechanism underlying this novel Pgam5 function

and identified Dvl2 as a substrate of Pgam5. Dvl associates with the Frizzled/LRP5/6

receptor complex upon Wnt stimulation and becomes highly phosphorylated in response to

Wnt signaling, which is required for intracellular signal transduction together with Arrb2

(Bryja et al., 2007; Lee et al., 1999; Tauriello et al., 2012; Tolwinski et al., 2003; Wong et al.,

2003). In the context of Wnt/-Catenin signaling, particularly CK1-mediated phosphorylation

of Dvl appears critical (Cong et al., 2004a; Kishida et al., 2001; Klimowski et al., 2006;

Peters et al., 1999). Dvl contributes to inhibition of the -Catenin destruction complex and

thereby to -Catenin stabilization (Cong et al., 2004b; Fiedler et al., 2011; Metcalfe et al.,

2010; Wong et al., 2003; Zeng et al., 2008). Interestingly, an additional nuclear function of

Dvl has been reported by several studies (Barry et al., 2013; Gan et al., 2008; Itoh et al.,

2005) and recently, Wang and colleagues showed that nuclear import of Dvl was

phosphorylation-dependent (Wang et al., 2015). Therefore, dephosphorylation of Dvl by

Pgam5 might inhibit Dvl function on multiple levels and our data support this hypothesis.

We observed stabilization of -Catenin, an increase of dephosphorylated, active -Catenin

and phospho-Dvl2 after Pgam5 depletion in human cells and in Xenopus embryos as well as

decreased nuclear -Catenin levels in Pgam5 overexpressing cells. We have identified Dvl2

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as a substrate of Pgam5 and a phosphatase-deficient mutant of Pgam5 was less effective in

antagonizing Wnt/-Catenin signaling. Thus, the data presented here support the conclusion

that Pgam5 inhibits phosphorylation-dependent Dvl function in Wnt/-Catenin signaling

upstream of -Catenin degradation. However, we cannot rule out that other substrates of

Pgam5 such as the protein kinase ASK1 (Takeda et al., 2009), which was found to promote

-Catenin degradation (Noh et al., 2010), or additional, so far unknown substrates also

contribute to the inhibition of Wnt/-Catenin signaling.

Notably, Pgam5 was still able to inhibit reporter gene activation and axis duplication in

Xenopus by overexpressed -Catenin and moreover, also by the stabilized -Catenin 4ST/A

or S33Y mutants. These observations suggested an additional role of Pgam5 downstream of

-Catenin stabilization. Indeed, we observed less binding between -Catenin and Tcf1 in

cells that overexpressed Pgam5. Although this effect might be explained by failure of these

cells to stabilize -Catenin in response to a Wnt signal, the same was true for stabilized -

Catenin 4ST/A. The latter indicated that Pgam5 modulated the composition or stability of

Tcf1 transcription complexes. Interestingly, we also found that Pgam5 itself co-precipitated

with Tcf1 and -Catenin and thus could directly dephosphorylate components of this

complex.

We observed a marked decrease of phosphorylated Dvl2 in the nuclear fraction of HEK 293T

cells overexpressing Pgam5, indicating Pgam5-mediated regulation of nuclear Dvl2 function.

Moreover, only one, slow migrating band was co-precipitated with Tcf1 indicating that Tcf1

preferentially interacts with phosphorylated Dvl2 and we found that interaction of Dvl2 with

Tcf1 was strongly impaired by Pgam5. Therefore, we conclude that Pgam5-mediated

dephosphorylation of Dvl2 inhibits its function both upstream of -Catenin stabilization and

its downstream nuclear function in the Tcf transcription complex. Again, we cannot exclude

additional targets of Pgam5 since also the nuclear localization, interaction and transcriptional

activity of -Catenin and Tcf/Lef transcription factors is regulated by site-specific

phosphorylation (Fang et al., 2007; Lee et al., 2001; Mahmoudi et al., 2009; Shitashige et al.,

2010; Wang and Jones, 2006; Wu et al., 2008) and thus might be influenced by Pgam5.

Moreover, the interaction of Pgam5 with Tcf1 was attenuated in WNT3A stimulated cells,

indicating that it is dynamically regulated by Wnt signaling. One might speculate that the

regulation of Pgam5 protein-protein interactions and potentially also enzymatic activity could

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be part of the machinery that fine-tunes the cellular response to Wnt signals, which however

needs to be addressed experimentally.

A phosphatase-deficient mutant of Pgam5, PGAM5-H105A, was not able to rescue the

Pgam5 morphant phenotype and was less efficient in inhibiting Wnt/-Catenin signaling,

indicating that the developmental function of Pgam5 depends on its phosphatase activity.

Moreover, when overexpressed either globally or targeted to the dorso-animal blastomeres of

eight-cell stage embryos, this phosphatase-deficient mutant phenocopied Pgam5 loss-of-

function, which pointed towards a dominant-negative activity. Interestingly, PGAM5-H105A

increased the amount of phospho-DVL2 in the cytoplasm even in the absence of a Wnt

stimulus, but decreased nuclear phospho-DVL2. This observation might be explained by

failure to dephosphorylate DVL2, but retained binding and thereby sequestering of phospho-

DVL2 in the cytoplasm. Elevated levels of phosphorylated Dvl2 in the cytoplasm have also

been observed in PGAM5-deficient cells and in Pgam5 morphant embryos, which could

explain the dominant-negative function of PGAM5-H105A. However, we also observed low

residual activity of PGAM5-H105A in repressing axis duplication and activation of the TOP-

Flash reporter. The simultaneously lower amounts of phospho-Dvl in the nucleus would

explain this residual activity.

It is undebated that Dvl function is regulated by post-translational modifications, namely

phosphorylation by multiple kinases. However, one would expect that the activity of

modifying enzymes was counteracted and balanced in this case by phosphatases to allow

dynamic regulation of cellular signaling and function. In this respect the number of putative

candidates involved in Wnt/-Catenin signaling was and still is very limited. With the

identification of the phosphatase Pgam5 as Wnt/-Catenin antagonist in Xenopus

development and human cells, one element was added to the kinase and phosphatase network

that regulates Dvl function in Wnt signaling.

Materials and Methods

Xenopus laevis embryos

Xenopus embryos were generated and cultured according to general protocols and staged

according to the normal table of Nieuwkoop and Faber. All procedures were performed

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according to the German animal use and care law (Tierschutzgesetz) and approved by the

local authorities and committees (animal care and housing approval: I/39/EE006,

Veterinäramt Erlangen; animal experiments approval: 54-2532.2-8/08, German state

administration Bavaria / Regierung von Mittelfranken).

RNA for microinjection was prepared from the respective plasmids (Supplementary Table 1)

using the mMessage mMachine Kit (Ambion, Austin, TX). Embryos were injected as

indicated and cultivated until they reached the desired stage. In situ hybridizations were

carried out as described by Harland (Harland, 1991).

Co-immunopurifications and Quantitative Mass Spectrometry

HEK293T cells were transiently transfected with Arrb2-Flag and Dvl2-GFP. Bait proteins

were affinity-isolated on anti-Flag beads (Sigma-Aldrich, Munich, Germany) or on anti-GFP

beads prepared by coupling an anti-GFP antibody (Acris antibodies, Herford, Germany)

covalently to agarose beads (Direct IP Kit, Thermo Scientific, Germany). Eluates were

digested with trypsin and subsequently with Lys-C in-solution. After addition of protein

digests as quantitative standards the samples desalted on C-18 stage tips (Nest Group, NY,

USA) and analyzed by nanoflow HPLC-MS/MS (2D-NanoLC, Eksigent, CA, USA; Orbitrap

Velos MS, ThermoFisher Scientific, Bremen, Germany, (Vasilj et al., 2012)). Protein

identification was performed with Mascot V2.2 (Matrixscience, London, UK) and

determination of peptide ion signals for label-free quantification was performed with

Progenesis LCMS V2.6 (Nonlinear Dynamics, Newcastle o.T., UK) and relative

quantification was based on the most intense three signals (MI3, (Groessl et al., 2012; Silva

et al., 2006). Isoform specificity of peptides was obtained from the proteomicsdb database

(www.proteomicsdb.org, (Wilhelm et al., 2014)).

Cell Culture and Transfection

HEK293T human embryonic kidney cells (Leibniz Institute Collections of Microorganisms

and Cell Culture, DSMZ, Germany), SW480 colorectal cancer cells and C2C12 cells were

cultured in DMEM supplemented with 10% fetal calf serum (Life Technologies, CA, USA)

at 37 °C in a humidified atmosphere of 10% CO2 and transfected as indicated using Rotifect

(Carl Roth, Germany), Polyethylenimine (Sigma-Aldrich, Germany) or Oligofectamine

(ThermoFisher, USA) according to the manufacturer’s instructions.

Xenopus embryonic fibroblasts (XTC, a kind gift from Ana Losada) were cultured in 67%

DMEM/H2O supplemented with 10% fetal calf serum at 25°C in a humidified atmosphere of

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5% CO2 and transfected as indicated. Information on plasmids used for transfection is

summarized in Supplementary Table 1.

Immunofluorescence imaging and image analysis

For immunofluorescence staining, cells were transfected and stimulated as indicated and

fixed with 4% paraformaldehyde 24h post-transfection. Cells were permeabilized with 0.1%

Triton X-100, blocked with 10% horse serum and endogenous -Catenin was detected using

rat anti--Catenin and anti-rat-Cy3 (Dianova, 1:400) antibodies; nuclei were stained with

DAPI. Immunofluorescence imaging was carried out using a Zeiss Apotome imaging system

(Zeiss, Oberkochen, Germany) or a Leica SP5 confocal laser-scanning microscope (Leica,

Wetzlar, Germany). Quantification of fluorescence intensity was done using the Image J

software package. Briefly, nuclei were identified based on DAPI staining and -Catenin

intensity was measured in individual nuclei of Pgam5-GFP-positive and –negative cells.

Statistical analysis

Statistical differences in phenotype frequencies between two conditions were determined

using the χ2-test. For graded phenotypes such as ventralization or axis duplication, pairwise

comparisons using a two-sample Wilcoxon rank sum test were performed. For quantitative

measurements, Student’s t-tests were applied as indicated. All statistical calculations were

carried out using either built-in functions or user-defined calculations in Microsoft Excel.

Preparation of Embryo Lysates, Co-immunoprecipitation and Western Blotting

Dorsal tissue explants were prepared at stage 11.5, sectioned into anterior, mid and posterior

part and snap-frozen until analysis. Embryos, cells or tissue explants were lysed in 20 mM

HEPES pH 7.4, 150mM NaCl, 0.5% NP-40, 10% Glycerol supplemented with Protease

Inhibitor and Phosphatase Inhibitor Cocktails (Roche, Germany) at 4 °C. Lysates were

cleared at 16,000 x g for 10 min, and the protein concentration was determined using a BCA

Assay, according to the manufacturer’s instructions (Applichem, Germany). For co-

immunoprecipitation, lysates were incubated for 4 h at 4 °C with the appropriate antibody

and protein G beads (Life Technologies, CA, USA). Immunoprecipitates were collected,

washed four times with lysis buffer and eluted with SDS sample buffer. Samples were

separated on polyacrylamide gels, transferred to PVDF membranes and probed with the

indicated antibodies using chemoluminescence or colorimetric detection as described

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previously (Pfister et al., 2012). For a complete list of primary antibodies see Supplementary

Table 2.

Cell Fractionation and Nuclear extracts

Cytoplasmic fractions were obtained by hypotonic lysis and cleared by centrifugation at

16000 x g (20mM Tris-HCl pH 7.5, 1mM EDTA); expression levels of non-cytoplasmic

proteins were determined in Triton X-100 extracts of the fraction insoluble in hypotonic lysis

buffer. Nuclei were prepared and extracted as described (Dignam et al., 1983).

RT-PCR

Total RNA was extracted from Xenopus embryos of the indicated developmental stages

(High Pure RNA Isolation Kit, Roche, Germany) and reverse transcribed using MMLV

reverse transcriptase (New England Biolabs, USA). Quantitative RT-PCR (qRT-PCR) was

carried out using Brilliant III Sybr Green Master Mix and the Agilent AriaMX system

(Agilent Technologies, USA). Primer sequences are listed in Supplementary Table 3.

Reporter Gene assays

Activation of Wnt/-Catenin activity was analyzed using HEK293T, C2C12 or SW480 cells,

as indicated, transfected with the TOP-Flash or FOP-Flash reporter plasmid (Korinek et al.,

1997) together with constitutively expressed Renilla luciferase (TK Renilla, Promega,

Mannheim, Germany) or -Galactosidase for normalization as indicated. Overexpression or

knock-down was achieved by co-transfection of the respective expression plasmids or

siRNAs. Cells were lysed 24 hours post-transfection (plasmid transfection) or 72h post-

transfection (siRNA transfection) and reporter gene activity was measured in the lysates and

normalized. All samples were measured as technical duplicates and at least three biological

replicates.

Production of recombinant GST-PGAM5

GST-PGAM5 lacks amino acids 13 to 50, thereby removing the mitochondrial membrane

targeting sequence for facilitated purification. GST-PGAM5 was expressed upon IPTG

induction in Rosetta (DE3) bacteria. Bacteria were lysed in Triton X-100 based buffer

containing lysozyme and GST-PGAM5 was precipitated on glutathione beads, washed on

beads and eluted by addition of soluble glutathione.

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Acknowledgements

The authors thank A. Losada, G. Weidinger, K. Takeda, A. Kikuchi, E. Arenas, and R.T.

Moon for providing plasmids and reagents; the Tcf1-Flag construct and the rat anti--Catenin

antibody were kind gifts from K. Mansperger and R. Rupp. We are grateful to J. Ernesti and

T. Hübler for their help with Xenopus experiments and for helpful discussions and support by

Andrej Shevchenko. M.G. was supported by the Max Planck Society (MPG) and by a

research grant from the German Research Foundation (DFG, SH91/1-1 and 1-2) in the

framework of the clinical research unit KFO 249. This work was funded by grants from IZKF

Erlangen to J.B. (D22) and D.B.B. (J58) and a research grant from the German Research

Foundation (DFG) to A.S. (SCHA 965/6-2).

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Figures

Figure 1

PGAM5 physically interacts with Arrb2 and Dvl2

(A) Interactome analysis by quantitative HPLC-MS-MS with Arrb2 as a bait and the

corresponding back-tagging experiments yielded various protein kinases and identified

PGAM5 as a novel binding partner of Dvl2 and Arrb2. Relative abundances are summarized

in the table. Asterisks indicate that proteins were detected in only one biological replicate.

The interaction network with selected preys was illustrated using Cytoscape; the bait proteins

Dvl2 and Arrb2 are colored yellow and orange respectively, kinases are shown in green,

phosphatases in red. The line width indicates the abundance of the respective preys relative to

Dvl2 or Arrb2 as bait.

(B) Co-immunoprecipitation of overexpressed proteins confirms the interaction of Pgam5

with Dvl2 and the Dvl2-Arrb2 complex. (C) Endogenous co-immunoprecipitation of PGAM5

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and ARRB2 with DVL2. Band intensities have been quantified and relative intensities are

given below the respective blots. (D) The binding site of Pgam5 to Dvl2 was mapped to the

region interspacing the PDZ and DEP domain of Dvl2 by co-immunoprecipitation of Pgam5-

HA with a series of truncated Myc-Dvl2 constructs as illustrated in the schematic.

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Figure 2

DVL2 is a substrate of PGAM5

(A) Knock-down of PGAM5 in HEK 293T cells was sufficient to induce

hyperphosphorylation of DVL2. (B) Overexpressed HA-DVL2 yielded two distinct bands on

Western Blots. Co-expression of PGAM5-Flag visibly diminished the slower migrating,

hyperphosphorylated band whereas the phosphatase-deficient mutant PGAM5-H105A-Flag

did not affect electrophoretic mobility of HA-DVL2. (C) Cell lysates were incubated with

recombinant GST (control) or GST-tagged PGAM5 (lacking the N-terminal mitochondrial

targeting sequence) for 30 min. In the presence of recombinant GST-PGAM5, DVL2 was

gradually dephosphorylated. For (B) and (C), intensities have been quantified and the ratio

between the hyperphosphorylated band (a) and the faster migrating band (b) is provided

below the corresponding blots.

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Figure 3

pgam5 is expressed in the anterior neuroectoderm and required for head induction in Xenopus

laevis.

(A) pgam5 RNA was detected by whole mount in situ hybridization in Xenopus embryos at

Nieuwkoop and Faber (NF, (Nieuwkoop and Faber, 1975) stages 6.5 (i), 9 (ii), 11.5 (iii), 13

(iv), 20 (v), 24 (vi) and 35 (vii). Expression was detected in the animal ectoderm at blastula

stages and was more ubiquitous from gastrula to early tadpole stages, although strongest in

the anterior neuroectoderm. At stage 35 pgam5 was detected in the central nervous system

(arrow heads), neural crest (arrows), eye (open arrowhead), otic vesicle and weakly in the

ventral part of somites. Scale bar (500 µm) applies to all images; arch – archenteron, bc –

blastocoel, bp – blastopore, e – ectoderm, fb – forebrain, hb – hindbrain, m- mesoderm, n –

notochord, ne – neuroectoderm, nt – neural tube, oe - oral evagination, sc – spinal chord. (B)

Phenotypes of embryos injected with 0.4 pmol Pgam5 MO1 or Control MO in both

blastomeres at the two-cell stage followed by injection with 100 pg PGAM5 or PGAM5-

H105A RNA (H105A RNA) both dorso-animal blastomeres at the eight-cell stage. The scale

bar (500 µm) applies to all images. The frequencies of the indicated phenotypes from at least

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three independent experiments with the indicated total numbers of embryos are summarized

in the graph (** p-value < 0.01, χ2-test).

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Figure 4

Pgam5 modulates Wnt/-Catenin signaling in Xenopus laevis.

Embryos were injected with 0.4 pmol Pgam5 MO1 or Control MO in both blastomeres at the

two-cell stage. (A) The Pgam5 MO1 phenotypes are rescued by co-injection of either 100 pg

MO-insensitve Xenopus pgam5-RNA or 10 pg dkk1-gfp DNA as indicated. Scale bar (500

µm) applies to all images. The frequencies of the indicated phenotypes from at least three

independent experiments with the indicated total numbers of embryos are summarized in the

graph (** p-value < 0.01, χ2-test). (B) Pgam5 depletion resulted in overall elevated levels of

active dephosphorylated -Catenin (ABC) and hyperphosphorylation of Dvl2 in NF stage

11.5 embryos. Band intensities of ABC, -Catenin (BC), hyperphosphorylated and

dephosphorylated Dvl2 (a and b respectively) have been quantified and the intensity ratios for

ABC/BC and Dvl2 a/b are given below the respective blots.

Pgam5 knock-down resulted in upregulation of endogenous Wnt/-Catenin target genes: (C)

The graphs show relative expression of the indicated genes from four independent

experiments (average ± SD). Statistically significant deviations are indicated by asterisks (**

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p-value < 0.01, * p-value < 0.05, (*) p-value < 0.1, t-test for the hypothesis of the mean). (E)

Knock-down of PGAM5 also strongly enhanced responsiveness of HEK 293T cells to

WNT3A stimulation as determined by TOP-Flash reporter gene assays.

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Figure 5

Pgam5 is required for anterior inhibition of Wnt/-Catenin signaling and ectodermal anterior-

posterior patterning

(A) The prospective neural plate was explanted from embryos injected with 0.4 pmol Pgam5

MO1 or Control MO at NF stage 11.5 and dissected into anterior, mid and posterior third as

indicated in the schematic. Active dephosphorylated -Catenin (ABC) was detected in an

anterior to posterior gradient in lysates from control dorsal explants. The blots show one

representative experiment; average ratios ± SD of ABC/-Catenin from four independent

experiments are summarized in the graph (* p-value < 0.05, separate variances t-test). (B)

Embryos were injected into one dorsal blastomere at the four-cell stage with 0.4 pmol Pgam5

MO1 or Control MO, pCS2+ lacZ as lineage tracer and PGAM5 RNA or PGAM5-H105A

RNA as indicated. Embryos were stained for -Galactosidase to identify the injected side

(asterisk) and analyzed for the expression of krox20 and otx2 at NF stage 21 by in situ

hybridization. Images show representative embryos; the corresponding pattern is

schematically shown in the upper right corner. The graph summarizes the results from five

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independent experiments (** p-value < 0.01, * p-value < 0.05, n.s. not significant, Wilcoxon-

Rank-Sum test).

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Figure 6

PGAM5 overexpression inhibits Wnt/-Catenin signaling in Xenopus embryos and human

cells

(A) Secondary body axes were induced in Xenopus laevis embryos by injection of 5 pg wnt8

RNA and 100 pg PGAM5 or PGAM5-H105A RNA as indicated into both ventral blastomeres

at the four-cell stage. Images show representative examples of embryos; filled arrowheads

indicate primary axes, open arrowheads indicate secondary axes. The graph shows the

frequency of complete and partial axis duplication from five independent experiments. (B)

Phenotypes of embryos injected with 200 pg egfp, PGAM5 RNA or PGAM5-H105A RNA

(H105A RNA) in both blastomeres at the two-cell stage. Arrowheads indicate the dorsal fin

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and somites. The frequencies of the indicated phenotypes from at least three independent

experiments are summarized in the graph. For (A) and (B) statistical significance of

deviations was calculated using the Wilcoxon Rank Sum test; ** p-value < 0.01, * p-value <

0.05.

(C) Pgam5 overexpression reduced nuclear -Catenin levels in Xenopus embryonic

fibroblasts (XTC cells). Cells were transfected as indicated, stimulated with WNT3A

conditioned medium for 1h and immunostained for endogenous -Catenin. The scale bar (50

µm) applies to all images. The graph summarizes nuclear -Catenin intensity of at least 20

GFP-positive and GFP-negative cells (** p-value < 0.01, separate variance t-test).

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Figure 7

PGAM5 interferes with Wnt/-Catenin signaling downstream of -Catenin stabilization

(A) PGAM5, but not PGAM5-H105A antagonized TOP-Flash activation by LRP6, DVL2

and stabilized -Catenin. (B) Xenopus Pgam5 blocked stimulation of the TOP-Flash reporter

by WNT3A in the presence of exogenous Tcf1. The graphs in (A) and (B) show normalized

average Luciferase activity ± SD from at least three independent experiments (** p-value <

0.01, * p-value < 0.05, n.s. not significant, separate variances t-test). (C) WNT3A stimulation

enhanced interaction of Tcf1-Flag with endogenous -Catenin in HEK 293T cells, which was

attenuated by co-expression of Pgam5-HA. (D) Tcf1-Flag co-precipitated with stabilized -

Catenin 4ST/A-Myc irrespective of WNT3A stimulation. Co-expression of Pgam5-HA

strongly reduced interaction between Tcf1-Flag and -Catenin 4ST/A-Myc.

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Figure 8

PGAM5-mediated dephosphorylation inhibits nuclear function of DVL2

(A) Endogenous -Catenin and Dvl2 were detected in the cytoplasmic and nuclear fractions

fromWNT3A stimulated HEK293T cells transfected as indicated. GAPDH, LAMIN-B and

pan-Cadherin were used as markers for cytoplasm, nuclei and membrane fractions

respectively. The ratio between the hyperphosphorylated band (a) and the faster migrating

band (b) of DVL2 in nuclear and cytoplasmic fractions are plotted in the graph (average ± SD

from at least three independent experiments; * p-value < 0.05, separate variances t-test). (B)

In whole-cell lysates, Tcf1-Flag co-precipitated with overexpressed Myc-Dvl2 and

endogenous -Catenin irrespective of WNT3A stimulation. Co-expression of Pgam5-HA

strongly reduced interaction between Tcf1-Flag, Myc-Dvl2 and endogenous -Catenin;

WNT3A stimulation moderately enhanced interaction of Tcf1 with Dvl2, but not -Catenin

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in the presence of exogenous Pgam5. (C) In the inverse co-immunoprecipitation experiment

Myc-Dvl2 was immunoprecipitated. The WNT3A-induced interaction of Tcf1-Flag with

Myc-Dvl2 was blocked by co-expression of Pgam5.

sp|Q6GL33|PGAM5_XENTR/1-278XB-GENE-17344731|PGAM5.L/1-279sp|Q5FWM4|PGAM5.S_XENLA/1-275sp|Q8BX10|PGAM5_MOUSE/1-288sp|Q96HS1|PGAM5_HUMAN/1-289sp|Q502L2|PGAM5_DANRE/1-289

111111

MYLRKAL--IAGGSAAAAAAAILGAAAVGKSKG--------GPDLDILSVVPA--------ATGQWDRNWDRREPIMYLRRAL--IAGGSAAAAAAAILGAVAAGKSKG--------GSDLDIVSVPPP-------LATGQWDRNWDRREPIMYLRRAL--IAGGSAA---AAILGVVAAGKSKG--------GSDSEILSVAPP--------ATGQWDRNWDRREPIMAFRQALQLAACGLAGGSAAVLFSAVAVGKPRG--------GGDADTRATEPPAWTG-ARAGRGVWDTNWDRREPLMAFRQALQLAACGLAGGSAAVLFSAVAVGKPRA--------GGDAEPRPAEPPAWAGGARPGPGVWDPNWDRREPLMSFRRALS-LACGFAGGSAVLVCAAVVADKNGYFGEGRRVTETLAAVNAAHPPAW-----PTANGWDYNWDKREPS

mitochondrial targeting

sp|Q6GL33|PGAM5_XENTR/1-278XB-GENE-17344731|PGAM5.L/1-279sp|Q5FWM4|PGAM5.S_XENLA/1-275sp|Q8BX10|PGAM5_MOUSE/1-288sp|Q96HS1|PGAM5_HUMAN/1-289sp|Q502L2|PGAM5_DANRE/1-289

596056686971

SMVNLSKINGETGEEELQLHLNKHKPKATRHIFLIRHSQYKQDGKTDFDRVLTPLGREQADLTGKRLSSLGFKYNHSMFNLSKINGETGEEELQLHRNKHKPKATRHIFLIRHSQYKLDGKSDFDRVLTPLGRQQADLTGQRLASLGYKYTHSMVNLSKINAETGEEELQLHLNKHKPKATRHIFLIRHSQYKLDGKTDFDRVLTPLGREQADLTGQRLASLGHKYNHSLINLKKRNVESGEDELTSRLDHYKAKATRHIFLIRHSQYHVDGSLEKDRTLTPLGREQAELTGLRLASLGLKFNKSLINVRKRNVESGEEELASKLDHYKAKATRHIFLIRHSQYHVDGSLEKDRTLTPLGREQAELTGLRLASLGLKFNKSMVNGKRKES-TG-ENGSQDAENNKPRATRHIFLIRHSQYNLKGDGDKERFLTPLGREQAEFTGQRLASFGLKYDT

sp|Q6GL33|PGAM5_XENTR/1-278XB-GENE-17344731|PGAM5.L/1-279sp|Q5FWM4|PGAM5.S_XENLA/1-275sp|Q8BX10|PGAM5_MOUSE/1-288sp|Q96HS1|PGAM5_HUMAN/1-289sp|Q502L2|PGAM5_DANRE/1-289

135136132144145145

IVYSTMTRAKETTEIISKYLPDVKKSSSDLLREGAPIRPEPQVCHWKPDF-VYYEDGSRIEAAFRHFIHRADPKQEIVYSTMTRAKETTEIISKYLPDVKKSSSDLLREGAPIRPEPQVCHWKPDF-VYYEDGPRIEAAFRQFIHRADPKQEIVYSTMTRAKETTEIISKYLPDVNKSSSDLLREGAPIRPEPQVCHWKPDF-VYYEDGPRIEAAFRHFIHRADPKQEIVHSSMTRAVETTDIISKHLPGVSRVSTDLLREGAPIEPDPPVSHWKPEAVQYYEDGARIEAAFRNYIHRADARQEIVHSSMTRAIETTDIISRHLPGVCKVSTDLLREGAPIEPDPPVSHWKPEAVQYYEDGARIEAAFRNYIHRADARQELIHSSMTRATETANIISKYLPGVELVSCDLLREGAPIEPVPPVTHWKPEAVQYHEDGARIEAAFRRYIHRADAKQK

sp|Q502L2|PGAM5_DANRE/1-289

sp|Q6GL33|PGAM5_XENTR/1-278XB-GENE-17344731|PGAM5.L/1-279sp|Q5FWM4|PGAM5.S_XENLA/1-275sp|Q8BX10|PGAM5_MOUSE/1-288sp|Q96HS1|PGAM5_HUMAN/1-289

210211207220221221

ADSYEILICHANVIRYIVCRALQLPPEAWLRMFLNNGSISYLVIRPNGNVSLRMLGDSGFMPPEKISRTEDSYEILICHANVIRYIVCRALQFPPEAWLRISLNNGSITSLVIRPNGNVSLRMLGDSGFMPPEKISRTEDSYEILICHANVIRYVVCRALQFPPEAWLRISLNNGSITYLVIRPNGNVSIRMLGDSGFMPAEKISRTEDSYEIFICHANVIRYIVCRALQFPPEGWLRLSLNNGSITHLVIRPNGRVALRTLGDTGFMPPDKITRSEDSYEIFICHANVIRYIVCRALQFPPEGWLRLSLNNGSITHLVIRPNGRVALRTLGDTGFMPPDKITRSEDSYEIIVCHANVIRYFVCRALQFPPEGWLRLGLNNGSITWLTVRPSGRVSLRALGDSGFMPPDKLTRT

TM helix (pred.)

*

Supplementary Figure 1

Alignment of Xenopus tropicalis Pgam5, Xenopus laevis Pgam5.S and Pgam5.L to the zebrafish, human and mouse orthologs (Uniprot Accession Numbers X. tropicalis Pgam5 Q6GL33, X. laevis Pgam5.S Q5FWM4, X. laevis Pgam5 was translated from XB-GENE-17344731, D. rerio Pgam5 Q502L2, H. sapiens PGAM5 Q96HS1, M. musculus Pgam5 Q8BX10). Identical amino acids are shaded grey. The boxes mark functional features: blue – mitochondrial targeting sequence (Lo and Hannink, 2008), black: predicted transmembrane helix, green – PGAM domain. Histidine 105 (human numbering) in the catalytic center is labeled with an asterisk.

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GS

T-P

GA

M5Δ

HA (DVL2)

GS

T

CIP

C

D

A

HA

100 kDa

37 kDa

Myc

Pgam5-HA+ + + + ++ + +

IP a

nti-M

yc

+

1

HA

Myc

37 kDa

100 kDa

2 3 4 5 6 7 8

Inpu

t

−B

7

2 DIX PDZ

DIX PDZ DEP1

DIX PDZ3

DIX PDZ DEP4

PDZ DEP5

DEP6

PDZ DEP8

Myc-

Myc-

Myc-

Myc-

Myc-

Myc-

Myc-

Myc-

α-TUBULIN

PGAM5

DVL2

+WNT3A

−

100

35

35

PG

AM

5

PG

AM

5

−

0.3 0.3 1.1 0.8

ab

intensity ratio (a/b)

37 kDa

100 kDa

50 kDa

Arrb2-FlagMyc-Dvl2Pgam5-HA++

++

+

+

HA

Flag

Myc

InputIP anti-Flag

++++

+

+

Supplementary Figure 2

(A) Co-immunoprecipitation of overexpressed proteins shows a weak interaction of Pgam5 with Arrb2, which is slightly increased by co-expression of Dvl2. (B) Western blot corresponding to Fig. 1D. The binding site of Pgam5 to Dvl2 was mapped to the region interspacing the PDZ and DEP domain of Dvl2 by co-immunoprecipitation of Pgam5-HA with a series of truncated Dvl2-Myc constructs (1-8) as illustrated.(C) Overexpression of PGAM5 partially reverted Wnt3a induced electrophoretic mobility shifts of endogenous DVL2 in HEK 293T cells, indicating that PGAM5 dephosphorylates DVL2. (D) HA-DVL2 was immunoprecipitated, washed with high stringency to remove bound proteins and incubated with recombinant GST, GST-PGAM5Δ (lacking the N-terminal mitochondrial targeting sequence) or Calf Intestinal Alkaline Phosphatase (CIP) for 30 min at 37°C. The sample incubated with GST showed two bands. After incubation with GST-PGAM5Δ or CIP only one band with higher electrophoretic mobility remained, which confirmed that DVL2 is directly dephosphorylated by PGAM5.

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C

pgam5.S

odc

NF stage 4 9 10 10.5 11 12 17 20 24

B

pgam

5 se

nse

NF st.6.5

NF st.24NF st.35

NF st.9 NF st.11.5 NF st.13

NF st.20NF st.18 NF st.24

NF st.4

NF st.4

animal-vegetalsection

NF st.6.5 NF st.18

dorsal view lateral view sagittal section

n arch.nt

nt

lateral view

pgam

5 an

tisen

se

D

NF stage11.5 204

pgam5.S

pgam5.L

odc

A

Supplementary Figure 3

(A) Transcripts of pgam5.S and pgam5.L homeologs were amplified by RT-PCR from whole embryo of the indica-ted stages; ornithine decarboxylase (odc) was used as house-keeping gene. The arrows indicate the expected fragment size. In contrast to pgam5.S, transcripts of pgam5.L were barely detectable and only in stage 4 embryos. (B) pgam5.S transcripts were present maternally (NF stage 4) and throughout early embryonic development (NF stages 9-24).(C) Supplementary in situ hybridization data. Maternal pgam5.S transcripts were detected predominantly in the animal blastomeres at NF stage 4 (8-cell stage) and at early blastula stage 6.5. At stage 18/19 pgam5 was broadly expressed in the dorsal ectoderm and the neural tube (nt). A sagittal section showed that expression was strongest in the anterior neural tube and gradually decreased towards posterior (arrowheads indicate neural tube (nt), n – notochord, arch – archenteron). The archenteron cavity appears stained due to trapped dye. (D) Sense controls corresponding to images in (A) and in Figure 3A.

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A

Pgam5

Gapdh

5MM

Con

trol M

O

Pga

m5

MO

1

Pga

m5

MO

2

37 kDa

37 kDa

C

short a/p axislarger headnormalsmall headno head

0

100

80

60

40

20ph

enot

ype

frequ

ency

(%)

Pgam5 MO2pgam5 RNA

# embryos 138 35

+++

**Pgam5 MO2

Pgam5 MO2 + pgam5 RNA

D

B

5‘ UTR CDS

gctttctccgtctgagaacgtgtgtgagatccataagctgccaATGTATCTTCGTAGGGCTCTGAT ||||||||||||||||||||||||| |||||||||||||||||||||||||

Pgam5 MO2 agaggcagactcttgcacacactct attcgacggttacatagaagcatcc

Pgam5 MO1

Pgam5 5MM MO agaggcacagtcttccacagagtct

pgam5.S

5‘ UTR CDS

ccgcttcatccgcctgaacctgtgtgtgtgagcgagagattcataagctgacgATGTATCTTCGTAGGGCTCTGATgat | |||| |||| ||||||| ||

||||||| | |||||||||||||||

Pgam5 MO2 agaggcagactcttgcacacactct attcgacggttacatagaagcatcc

Pgam5 MO1

pgam5.L

Supplementary Figure 4

(A) Translation-blocking antisense morpholino oligonucleotides were used to knock-down Pgam5 in Xenopus laevis embryos. Pgam5 MO1 and Pgam5 MO2 target two non-overlapping sites in pgam5.S transcripts at and close to the translation start respectively. The alignment shows the binding sites of MO1 and MO2 to the pgam5.S mRNA and base exchanges in the Pgam5 5-mismatch Control MO. (B) Alignment of Pgam5 MO1 and Pgam5 MO2 to pgam5.L. Pgam5 MO1 aligns with only two mismatches also to pgam5.L and therefore MO1 can be expected to suppress also translation from pgam5.L. The sequence in pgam5.L corresponding to the Pgam5 MO2 binding site contains seven nucleotide exchanges, therefore Pgam5 MO2 is not expected to affect pgam5.L translation. (C) Injection of either Pgam5 MO1 or Pgam5 MO2 into both blastomeres at the two-cell stage resulted in knock-down of Pgam5 at stage 11.5.

(D) Representative phenotypes of embryos injected with 0.4 pmol Pgam5 MO2 or co-injected with MO-insensitive pgam5 RNA as indicated. Co-injection of pgam5 RNA rescues the phenotype of Pgam5 morphant embryos. Frequencies of phenotypes from at least three independent experiments are summarized in the graph; the total numbers of embryos are provided below each column. The χ2-test was used for statistical analysis (** p-value < 0.01).

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A Control MO Pgam5 MO1 Pgam5 MO2 Staurosporine

100

80

60

40

20

0

Pgam5 M

O1

aver

age

AO

inte

nsity

Pgam5 M

O2

Contro

l MO

# embryos 67 63 63

120

Contro

l

Stauros

porin

35 31

**

Pgam5 MO1Control MO

Pgam5 MO1 Staurosporine

100

80

60

40

20

Pgam5 M

O1

phen

otyp

e fre

quen

cies

(%)

Contro

l MO

# embryos 55 57 570

Contro

l

Stauros

porin46

**n.s.

equal, high

equal, low

injected side increased

Control MO

C 100

80

60

40

20

Pgam5 MO1

surv

ival

(%)

Contro

l MO

# embryos 419 154 1880

84

**

n.s.

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Supplementary Figure 5

Knock-down of Pgam5 did not cause increased cell death.

(A) Embryos were injected with 0.4 pmol Control MO, Pgam5 MO1 or Pgam5 MO2 into both blastomeres at the two-cell stage. At NF stage 12.5 embryos were stained for dead cells with 1 µg/ml Acridine Orange in 0.1x MBSH for 30 min, washed three times with 0.1x MBSH and imaged using a Leica M165FC stereomicroscope equipped with epifluorescence optics and a Jenoptik ProgRes C14 camera. As positive control, embryos were treated at NF stage 11.5 for 2h with 1µM staursporine or buffer only, stained with Acridine Orange and imaged as described above. Image acquisition parameters were kept constant for all images. Mean fluorescence intensities were measured for each embryo using the Image J software package. The images show representative embryos injected with Control MO, Pgam5 MO1 or Pgam5 MO2 as well as staurosporine-treated embryos. The average fluorescence intensities ± SD are plotted in the graph; values did not differ significantly between Control MO, MO1 and MO2 (separate variances t-test, p-values > 0.7), staurosporine treatment significantly increased Acridine Orange fluorescence intensity compared to control embryos (** p-value < 0.01). The total numbers of analyzed embryos from four independent experiments are given below each column.

(B) Embryos were injected with 0.4 pmol Control MO or Pgam5 MO1 and co-injected with 100 pg pCS2 LacZ into one blastomere at the four-cell stage. As positive control, embryos were treated with 1µM staurosporine for 1h at NF stage 19. At NF stage 20 embryos were fixed and stained for β-Galactodidase. Cleaved Caspase 3 (Cleaved Casp3) was detected by whole-mount immunostaining and colorimetric Alkaline Phosphatase staining (NBT/BCIP). For MO-injected embryos Cleaved Casp3-positive cells on injected and uninjected sides were compared and categorized into equal (low/high) or increased numbers in the injected side. Staurosporine-treated embryos were compared with control embryos and categorized into low or high number of Cleaved Casp3-positive cells. The graph summarizes the results from four independent experiments (** p-value < 0.01 χ2-test). The total numbers of embryos are provided below each column.

(C) Embryos were injected with 1.6 pmol Control MO or 0.4 pmol, 0.8 pmol or 1.6 pmol Pgam5 MO1 and cultured till NF stage 40. The graph shows the average survival rates from at least three independent experiments. The total numbers of embryos are provided below each column. Injection of 0.4 pmol MO1 did not significantly affect survival, whereas survival rates dropped significantly after injection of higher doses of MO1 (separate variances t-test, * p-value < 0.05).

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si P

GA

M5

β-Catenin

si g

fp

AXIN2

α-TUBULIN

PGAM5

hypo

toni

cTX

-100

α-TUBULIN

100

55

35

100

55

HEK293TA

++

+ +

rela

tive

Luc

activ

ity

(AU

)

40

300

+si gfp

si PGAM5WNT3A

250

0

20

+

C2C12

B

Supplementary Figure 6

(A) Knock-down of PGAM5 by siRNA transfection strongly enhanced responsiveness of murine C2C12 cells to WNT3A stimulation as determined by TOP-Flash reporter gene assays normalized to β-Galactosidase. (B) PGAM5 knock-down in HEK293T cells resulted in stabilization of cytoplasmic β-Catenin and upregulation of the feedback target AXIN2.

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DC

β-Catenin

α-TUBULIN

PGAM5

100

55

35

100

55

70

7070

130

130

LRP6

HA (DVL2)

100

80

60

40

20

0

% a

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dupl

icat

ion

**

184# embryos 168 189

Pgam5-HA + +β-Catenin

β-Catenin

β-Catenin+ Pgam5

B

++

rela

tive

Luc

activ

ity

(AU

)

0.5

1.5

+PGAM5-FlagPGAM5-H105A-Flag

Flag-AXIN

1.0

0

SW480

β-Catenin 4ST/A-Myc

+caLRP6 β-Catenin

*

*

rela

tive

Luc

activ

ity

(AU

)

600

400

0

200

Pgam5-GFP + +

*

Dvl2+

HEK 293T

*

Pgam5-GFP

LRP6/Dvl2/β-catenin

1005050

1005050

100

50

Supplementary Figure 7

(A) Expression of overexpressed proteins corresponding to Figure 7A was confirmed by Western Blot. Filled arrowheads indicate endogenous proteins, open arrowheads indicate overexpressed proteins. (B) Xenopus Pgam5 antagonized TOP-Flash activation by constitutively-active LRP6, Dvl2, β-Catenin or stabilized β-Catenin 4ST/A-Myc. The graph shows average ± SD from at least three independent experiments. Statistical significance of deviations was calculated using the t-test for the hypothesis of the mean (* p-value < 0.05). Expression of overexpressed proteins was confirmed by Western Blot; corresponding blots are provided below the respective columns. (C) Overexpression of PGAM5 in SW480 colon carcinoma cells suppressed constitutive Wnt/β-Catenin activity; the phosphatase-inactive mutant PGAM5-H105A had no effect. Overexpression of AXIN downregulated TOP-Flash activity comparable to PGAM5. TOP-Flash values were normalized to β-Galactosidase. (D) Secondary body axes were induced in Xenopus laevis embryos by injection of 400 pg β-catenin RNA and 100 pg pgam5 RNA was co-injected as indicated into both ventral blastomeres at the four-cell stage. The graph shows the frequency of axis duplication from four independent experiments; statistical significance of deviations was calculated using the χ2-test; ** p-value < 0.01. Images show representative examples of embryos; arrows indicate neural tubes in the primary and – if present – in the secondary axis.

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Supplementary Methods

Supplementary Table 1

Plasmids and antisense Oligonucleotides

Plasmid / Morpholino / siRNA Reference

Pgam5 MO1 5’-cctacgaagatacattggcagctta-3’ this study

Pgam5 MO2 5’-tctcacacacgttctcagacggaga-3’ this study

Pgam5 5MM MO 5’-tctgagacaccttctgacacggaga-3’ this study

si PGAM5 5’-cgcccgugucucauuggaa -3’ this study

si gfp 5´-gcuaccuguuccauggcca -3` this study

pCRII Pgam5 this study

pCS2+ Pgam5-HA this study

pCS2+ Pgam5-GFP this study

pCS2+ PGAM5-Flag this study

pCS2+ PGAM5-H105A-Flag this study

pcDNA3.1 PGAM5-Flag Takeda et al., 2009

pcDNA3.1 PGAM5-H105A-Flag Takeda et al., 2009

pGEX4T3 PGAM5Δ this study

pN1 PGAM5 this study

pCS2+ DKK1-GFP Kagermeier-Schenk et al., 2011

pcDNA3.1 Arrb2-Flag Seitz et al., 2014

pCS2+ myc-Dvl2 Seitz et al., 2014

pcDNA3.1 HA-DVL2 this study

pcDNA3.1 Flag-AXIN Kishida et al., 1999

pCS2+ LRP6 ΔE1-E4 Mao et al., 2001

pCS2+ β-Catenin-myc Yost et al., 1996

pCS2+ β-Catenin 4ST/A-myc Yost et al., 1996

pcDNA3.1 beta-Catenin S33Y this study

pCS2+ Tcf1-Flag Ralph Rupp, Munich

pCS2+ LacZ Schille et al., 2016

pGL3 Super 8x TOPFlash Korinek et al., 1997

pGL TK Renilla Promega GmbH, Mannheim,

Germany

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Supplementary Table 2

Antibodies and reagents

Antibody / Reagent Dilution Source / Reference

Rabbit anti-PGAM5 1:1000 Abcam plc, Cambridge, UK; # ab126534

Mouse anti-PGAM5 1:1000 Atlas Antibodies AB, Stockholm, Sweden #AMAb90803

Rabbit anti-DVL2 1:1000 Proteintech Europe Inc, Manchester, UK #12037-1-AP

Mouse anti-Dvl2 1:500 bio-techne, Minneapolis, USA; #MAB4216

Rabbit anti-ARRB2 1:1000 Proteintech Europe, Manchester, UK #10171-1-AP

Mouse anti-Arrb2 1:1000 Abcam plc, Cambridge, UK; #ab54790

Rabbit anti-β-Catenin 1:1000 Santa Cruz Biotechnology Inc., Dallas, TX, USA #sc-7199

Rat anti-β-Catenin 1:1000 Ralph Rupp, Ludwig-Maximilians-University Munich, Germany

Rabbit anti-GFP 1:1000 Cell Signaling Technology Inc., Danvers, MA, USA #2956

Rabbit anti-GFP 1:2000 Acris Antibodies GmbH, Herford, Germany; #SP3005P

Mouse anti-GAPDH 1:2000 Proteintech Europe Inc, Manchester, UK; #60004-1-Ig

Rabbit anti-TIMM44 1:500 Proteintech Europe Inc, Manchester, UK #13859-1-AP

Mouse anti-Lamin B2 1:500 Santa Cruz Biotechnology Inc., Dallas, TX, USA #sc-56147

Rabbit anti-panCadherin

1:1000 Cell Signaling Technology Inc., Danvers, MA, USA #4068

Mouse anti-dephospho-β-Catenin

1:500 Merck Millipore, Darmstadt, Germany; #05-665

Mouse anti-Axin2 (C/G7)

1:50 Lustig et al., 2002

Rat anti-α-Tubulin 1:1000 AbD Serotec; #MCA77G

Mouse anti-β-Actin 1:1000 Sigma-Aldrich Chemie GmbH, Munich, Germany; A5441

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Rabbit anti-HA 1:1000 Sigma-Aldrich Chemie GmbH, Munich, Germany; H6908

Mouse anti-GST 1:1000 Sigma-Aldrich Chemie GmbH, Munich, Germany; G1160

Mouse anti-Flag immobilized

assay dependent

Sigma-Aldrich Chemie GmbH, Munich, Germany; #A2220

Dynabeads Protein G assay dependent

ThermoFisher Germany, Darmstadt, Germany; #10004D

Staurosporine 1µM Cell Signaling Technology Inc., Danvers, MA, USA #9953

Acridine Orange 1µg/ml Applichem GmbH, Darmstadt, Germany; #A1398

Rabbit anti-Cleaved Caspase 3

1:500 Cell Signaling Technology Inc., Danvers, MA, USA; #9661

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Supplementary Table 3

Primers for qRT-PCR and RT-PCR

Primer Name Sequence Reference

qRT-PCR

odc fwd cattgcagagcctgggagata Schambony and Wedlich, 2007

odc rev tccactttgctcattcaccataac Schambony and Wedlich, 2007

xnr3 fwd cgagtgcaagaaggtggaca Bryja et al., 2007

xnr3 rev atcttcatggggacacagga Bryja et al., 2007

msgn1 fwd ctggacaaggtctggaggagat this study

msgn1 rev tttatgcccatgatccttcttg this study

hoxd1 fwd ggacaaatttcaccaccaaaca this study

hoxd1 rev ggtatcgttgagctgcagagag this study

dkk1 fwd gatgcctacccgctctacagtt this study

dkk1 rev ggacacaaattccgttgctaca this study

RT-PCR

odc_fwd gatgggctggatcgtatcgt Schille et al., 2016

odc_rev tggcagcagtacagacagca Schille et al., 2016

pgam5.S_fwd1 atcccaagcaggaagaagacag this study

pgam5.S_rev1 agcccaagaaataaggcacaga this study

pgam5.S_fwd2 atctcagtgcagcggaaactc this study

pgam5.S_rev2 tgctctcgtcattgtggagtaaa this study

pgam5.L_fwd1 gacgtcagtgtggtggaaactc this study

pgam5.L_rev1 actgtgagtgtcggatcaggaa this study

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Supplementary References

Bryja, V., Gradl, D., Schambony, A., Arenas, E. and Schulte, G. (2007). Beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo. Proc Natl Acad Sci USA 104, 6690–6695.

Kagermeier-Schenk, B., Wehner, D., Ozhan-Kizil, G., Yamamoto, H., Li, J., Kirchner, K., Hoffmann, C., Stern, P., Kikuchi, A., Schambony, A., et al. (2011). Waif1/5T4 inhibits Wnt/β-catenin signaling and activates noncanonical Wnt pathways by modifying LRP6 subcellular localization. Dev Cell 21, 1129–1143.

Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M. and Kikuchi, A. (1999). DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability. Mol Cell Biol 19, 4414–4422.

Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997). Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science 275, 1784–1787.

Lustig, B., Jerchow, B., Sachs, M., Weiler, S., Pietsch, T., Karsten, U., van de Wetering, M., Clevers, H., Schlag, P. M., Birchmeier, W., et al. (2002). Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol Cell Biol 22, 1184–1193.

Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L., et al. (2001). Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol Cell 7, 801–809.

Schambony, A. and Wedlich, D. (2007). Wnt-5A/Ror2 regulate expression of XPAPC through an alternative noncanonical signaling pathway. Dev Cell 12, 779–792.

Schille, C., Bayerlová, M., Bleckmann, A. and Schambony, A. (2016). Ror2 signaling is required for local upregulation of GFD6 and activation of BMP signaling at the neural plate border. Development 143, 3182–3194.

Seitz, K., Dürsch, V., Harnoš, J., Bryja, V., Gentzel, M. and Schambony, A. (2014). β-Arrestin interacts with the beta/gamma subunits of trimeric G-proteins and dishevelled in the Wnt/Ca(2+) pathway in xenopus gastrulation. PLoS ONE 9, e87132.

Takeda, K., Komuro, Y., Hayakawa, T., Oguchi, H., Ishida, Y., Murakami, S., Noguchi, T., Kinoshita, H., Sekine, Y., Iemura, S.-I., et al. (2009). Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1. Proc Natl Acad Sci USA 106, 12301–12305.

Yost, C., Torres, M., Miller, J. R., Huang, E., Kimelman, D. and Moon, R. T. (1996). The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 10, 1443–1454.

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