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. Author manuscript; available in PMC: 2016 Jul 11.
Published in final edited form as: Development. 2008 Sep;135(17):2927–2937. doi: 10.1242/dev.020842

Two highly-related regulatory subunits of PP2A exert opposite effects on TGF-β/Activin/Nodal signalling

Julie Batut 1,4,6, Bernhard Schmierer 1,4, Jing Cao 2, Laurel A Raftery 2, Caroline S Hill 1,3, Michael Howell 1,3,5
PMCID: PMC4940033  NIHMSID: NIHMS123247  PMID: 18697906

Summary

We identify Bα (PPP2R2A) and Bδ (PPP2R2D), two highly-related members of the B family of regulatory subunits of the protein phosphatase PP2A, as important modulators of TGF-β/Activin/Nodal signalling, which affect the pathway in opposite ways. Knockdown of Bα in Xenopus embryos or mammalian tissue culture cells suppresses TGF-β/Activin/Nodal-dependent responses, whereas knockdown of Bδ enhances these responses. Moreover, in Drosophila, overexpression of Smad2 rescues a severe wing phenotype caused by overexpression of the single Drosophila PP2A B subunit, Twins. We show that in vertebrates Bα enhances TGF-β/Activin/Nodal signalling by stabilising the basal levels of type I receptor, whereas Bδ negatively modulates these pathways by restricting receptor activity. Thus, these highly-related members of the same subfamily of PP2A regulatory subunits differentially regulate TGF-β/Activin/Nodal signalling to elicit opposing biological outcomes.

Introduction

TGF-β superfamily ligands signal through complexes of type I and type II receptors, both of which are serine/threonine kinases. Binding of a TGF-β superfamily ligand allows the type II receptor kinase to phosphorylate and activate the type I receptor kinase, which in turn phosphorylates receptor-regulated Smads or R-Smads. Phosphorylated R-Smads form complexes with Smad4, which accumulate in the nucleus and directly regulate target gene transcription (ten Dijke and Hill, 2004). The pathway consists of two distinct branches, which are defined by the type I receptors and the R-Smads that are activated. The type I receptors ALK4, ALK5 and ALK7 specifically phosphorylate Smad2 and Smad3, whereas the type I receptors ALK1, ALK2, ALK3 and ALK6 are specific for Smad1, Smad5, and Smad8 (Schmierer and Hill, 2007). Broadly speaking, the Activin, Nodal and TGF-β subfamilies of ligands induce phosphorylation and activation of ALK4/5/7 and thus Smad2/3, whereas ligands of the BMP subfamily activate ALK1/2/3/6 and consequently Smad1/5/8. These two branches are frequently referred to as the TGF-β/Activin/Nodal-branch and the BMP/GDF-branch, respectively (Feng and Derynck, 2005).

Receptor levels are an obvious determinant of a cell's responsiveness to TGF-β superfamily ligands and are extensively regulated. Irrespective of the absence or presence of a signal, endocytosis of receptors by a clathrin-dependent mechanism operates in parallel with a caveolin-dependent mechanism, the former recycling receptors to the membrane, the latter promoting receptor degradation through the proteasomal pathway (Di Guglielmo et al., 2003). A balance between the two is thought to determine the amount of receptors that are present at the plasma membrane and thus competent for signalling. Lysosomal degradation of ALK4 and ALK5 also occurs and is promoted by the protein Dapper2 in zebrafish (Zhang et al., 2004), and degradation of BMP receptors in Xenopus is promoted by a phosphatase, Dullard (Satow et al., 2006).

In addition to receptor levels, the phosphorylation status of both the receptors and the Smads is also tightly controlled. Thus, protein phosphatases have long been postulated to influence TGF-β superfamily signalling, but concrete roles have only recently started to emerge. PP1 is targeted to active receptors by signal-induced feedback to dephosphorylate and inactivate the type I receptor (Shi et al., 2004) in both mammalian cells and Drosophila (Bennett and Alphey, 2002). Downstream of the receptors, a number of different phosphatases have been implicated in the removal of activating phosphates from the R-Smads (Chen et al., 2006; Knockaert et al., 2006; Lin et al., 2006). Finally, PP2A, a regulatory subunit of which can be phosphorylated by ALK5, has been implicated in the TGF-β signalling pathway as a downstream effector (Griswold-Prenner et al., 1998; Petritsch et al., 2000).

Here, we report an entirely novel role for specific regulatory subunits of PP2A in modulating TGF-β/Activin/Nodal signalling at the receptor level. PP2A is a multimeric serine/threonine protein phosphatase consisting of a 36 kD catalytic subunit (PP2AC) and a 65 kD scaffolding subunit (PR65 or A subunit) (Janssens et al., 2005). An additional regulatory subunit, of which four distinct classes exist (B, B′, B″ and B‴), associates with this dimer. The B family (PR55) comprises four highly homologous, mammalian genes (PPP2R2A, PPP2R2B, PPP2R2C and PPP2R2D) with PPP2R2A and PPP2R2D being widely expressed, and PPP2R2B and PPP2R2C expression being restricted to neural tissues (Janssens and Goris, 2001; Strack et al., 1999). To date, only redundant functions of these family members have been described (Adams et al., 2005). Here we show that PPP2R2A (referred to throughout as Bα) and PPP2R2D (referred to throughout as Bδ) have distinct and opposing roles in the regulation of TGF-β/Activin/Nodal signalling.

Materials and Methods

Plasmids and recombinant proteins

Full length mouse Bα, Bδ, B′δ and Xenopus Bδ were PCR-amplified from EST clones, sequence checked and cloned into pEF-Flag and pFTX9 (Howell et al., 2002), for N-terminal Flag tagging. Anti Xenopus Bα and Bδ in situ hybridisation probes corresponding to nucleotides 1988-2176 and 9-194 of the full length mRNA, respectively, were generated by PCR from Xenopus cDNA libraries and cloned into pCR2.1 (Invitrogen). Xbra and gsc probes (Howell et al., 2002), pCS2-GFP, pFTX4K-EGFPSmad2 and the plasmid expressing activated ALK4 (Batut et al., 2007), the plasmid expressing HA-ALK4 (Jullien and Gurdon, 2005), pCMV5-HA-ALK5 and pCMV-HA ALK5ca (Nicolás and Hill, 2003) and EF-LacZ (Bardwell and Treisman, 1994) were as described. HA-Raf1 was a kind gift from Richard Marais. Recombinant full-length human Smad2 was purified from bacterial lysates as a GST fusion by affinity purification followed by thrombin cleavage. Concentration and quality of the recombinant Smad2 were checked by SDS-PAGE and Coomassie staining. In vitro translations using the TnT system (Promega) were performed according to manufacturer's instructions.

Morpholinos, RNA isolation, RT-PCR and q-PCR

Extraction of total mRNA from Xenopus embryos, reverse transcription and q-PCR were performed as described (Batut et al., 2007). RT-PCR was performed as described (Levy and Hill, 2005). For the sequences of the morpholino oligonucleotides (Gene Tools, LLC) and the oligonucleotides used for RT-PCR and q-PCR see Table S1.

Cell culture and transfection of siRNA and plasmids

HeLa TK- and HaCaT cell lines expressing EGFP-Smad2 were generated and cultured as previously described (Nicolas et al., 2004; Schmierer and Hill, 2005). TGF-β (PeproTech) was used at 2 ng/ml. Bafilomycin A1 (Calbiochem) at 10 nM, MG132 and lactacystin (Sigma) at 25 μM. Treatment of extracts with PNGase F was as described (Dorey and Hill, 2006). Cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche), and with siRNAs using Dharmafect II (Dharmacon). Pools of 4 siRNA oligos (SMARTpools, Dharmacon) targeting Bα, Bδ, Dapper-2 or TβR-II were transfected at a final concentration of 75 nM. Experiments were performed 72 hours after the transfection. As controls, a SMARTpool of non-targeting siRNAs were used. Knockdowns using the individual oligonucleotides of a SMARTpool were performed as above at a final concentration of 75 nM. For siRNA sequences see Table S1. Knockdown efficiency was assessed by RT-PCR and/or immunoblotting.

IP phosphatase assay

As substrates, endogenous and EGFP-tagged phospho-Smad2 were immunoprecipitated from TGF-β-treated HaCaT EGFPSmad2 cells (Batut et al., 2007; Schmierer and Hill, 2005) with an anti-Smad2/3 antibody in lysis buffer (50 mM Tris-Cl pH7.5, 100 mM NaCl, 10% glycerol, 0.1% NP40, 2 mM DTT, protease inhibitors). HA-Raf-1 was expressed in HeLa TK- cells and immunoprecipitated with anti-HA antibody. Flag-Bα, Flag-Bδ or Flag-B′δ were expressed in HeLa TK- cells. PP2A holocomplexes containing these subunits were purified by Flag pulldown and eluted with Flag peptide. Alternatively, complexes were purified from stable HEK T-Rex cell lines harbouring Flag-Bα or FLAG-Bδ in the tetracycline-inducible pcDNA5/TO construct (Adams et al., 2005) induced with 1 μg/ml tetracycline for 48 h, and treated or not for 1 hour with TGF-β. Phosphatase activity in the eluates was routinely assessed by phosphate release from a synthetic phosphopeptide using a colorimetric assay (Upstate). Phospho-Smad2 or HA-Raf-1 bound to protein G beads were incubated with equal phosphatase activities for 1 hr at 37°C in lysis buffer and analysed for dephosphorylation by immunoblotting.

IP kinase assay

Active, endogenous ALK5 receptor kinase was immunoprecipitated from TGF-β treated HaCaT cells lysed in lysis buffer using anti-ALK5 antibodies that had been chemically crosslinked (Harlow and Lane, 1988) to protein A beads. Kinase activity was assessed by in vitro phosphorylation of 800 ng recombinant human Smad2 protein in the presence of 5 mM ATP, 2 mM MgCl2 and 2 mM MnCl2 at 37°C for 90 minutes. For IP kinase phosphatase assays, purified PP2A holocomplexes containing Flag-Bα, Flag-Bδ or Flag-B′δ were included in the reaction mixture.

Xenopus embryo injections, in situ hybridisation and immunofluorescence

Fertilisation, culture, staging, preparation of synthetic mRNAs and microinjection of Xenopus embryos were performed as described (Howell et al., 2002). Activin A (R&D Systems) was used at 20 ng/ml. Okadaic acid (Calbiochem) was used at 25 nM. In vitro transcribed mRNAs were injected into Xenopus at 500 pg for EGFP-Smad2, 250 pg for HA-ALK4, 100 pg for activated ALK4 and 200-500 pg for Bα and Bδ. GFP mRNA was used as a tracer at 50 pg. Total concentrations of morpholinos were 20 ng. In all cases the injection volume was 4-5 nl. Animal caps were dissected at stage 8-9 and harvested at the indicated stages. Whole-mount in situ hybridisation and immunofluorescence were performed as described (Batut et al., 2007). Antisense probes for in situ hybridisation were labelled with digoxigenin-UTP (Roche).

Genetic Interactions in the Drosophila wing

The following fly strains were used:

  1. w P{GAL4}A9 (Brand and Perrimon, 1993)

  2. w; P{UAS-tws.B}23 (II) (Bajpai et al., 2004)

  3. w; +/CyO; P{UAS-dSmad2.Z}8D3 (III) (Zheng et al., 2003)

  4. y[1] w[1118]; Sp; +/TSTL, CyO, TM6B, Tb[1]

  5. tws[60]/TM6b, Tb Hu (Uemura et al., 1993).

  6. w[67c23] P{w[+mC]=lacW}Smox[G0348]/FM7c (Peter et al., 2002).

Flies were reared on cornmeal-agar-dextrose. Loss of function interactions were tested at 25°. smox/FM7a females were mated to a loss of function tws strain or to y[1] w[c67c23]. Progeny that were smox/Y; tws/+ had no difference in viability or visible phenotypes compared to control smox/Y. Gain of function interaction tests were performed at 22° and 25° in males, which had stronger tws overexpression phenotypes. Wings were mounted in Euparal and imaged digitally using a 4× objective. It was previously reported that larger wings result from A9-GAL4-driven expression of P{UAS-Smox.A} (Marquez et al., 2001). The genotype used here gave only a subtle increase in the average wing dimensions compared to the control genotype. Control matings to produce w P{GAL4}A9/Y; P{UAS-tws.B}23/+ were performed at 23-24°C to permit recovery of mature wings. Tests for genetic interactions between loss-of-function mutations in each gene yielded no phenotypic evidence for a trans-heterozygous interaction (data not shown).

Western blotting, immunoprecipitation, antibodies and confocal microscopy

Whole cell extracts from Xenopus embryos and tissue culture cells, Western blotting and immunoprecipitation procedures were as described (Batut et al., 2007; Howell et al., 1999). Confocal microscopy was performed as described (Batut et al., 2007; Schmierer and Hill, 2005). The following commercial antibodies were used: anti-phospho-Smad2 (S465/S467), anti-phospho-Smad2 (S245/S250/S255), anti-phospho-Raf-1 (S259), anti-phospho-ERK (all Cell Signaling Technology); anti-Smad2/3 (BD Biosciences Pharmingen); anti-ALK5 (v-22, Santa Cruz Biotechnology); anti-TβRII, anti-pan B subunit and anti-PP2A catalytic subunit (Upstate); anti-β-Catenin, anti-Flag, anti-Flag-HRP and anti-Flag beads (Sigma); anti-HA, anti-HA-HRP and anti-GFP (Roche); anti-tubulin (YL1/2, Abcam).

Results

We initially identified the PP2A Bδ subunit as a protein whose overexpression in early Xenopus embryos causes loss of anterior structures. Embryos overexpressing Bδ exhibited a delayed gastrulation as judged by the time of blastopore closure, and showed greatly reduced anterior structures at the tailbud stage (Fig. 1A and Table S2). In contrast, knockdown of Bδ using a specific morpholino resulted in embryos with a shortened axis compared with wild type embryos and slightly larger anterior structures relative to the trunk (Fig. 1A and Table S2). The morphant phenotype was identical when two distinct morpholino oligonucleotides were used (data not shown), and was rescued by co-expression of a mouse Bδ mRNA (Fig. 1B).

Fig. 1. Manipulating the expression of Bα and Bδ in Xenopus embryos produces distinct phenotypes.

Fig. 1

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(A) Xenopus embryos were injected with either control GFP mRNA, Flag-tagged mouse Bδ mRNA (Bδ), a morpholino control (MoC) or a specific morpholino against Xenopus Bα (MoBα) or Bδ (MoBδ) at the one-cell stage, and fixed when control embryos had reached either early gastrula or tailbud (stage 25). Representative embryos are shown, arrows indicate anterior. The anterior regions of embryos are magnified below. Note the lack of head structures in Bδ-injected embryos and MoBα-injected embryos.

(B) Embryos were injected as in A with the indicated mRNAs and morpholinos. The effect of MoBδ or MoBα could be rescued by coinjection with the cognate mRNA (mouse Bδ or Bα). Percentages of embryos showing wild type phenotype when control-injected embryos had reached stage 22 are given. Arrows indicate anterior.

To understand how specific these effects were for this particular B subunit, we investigated the effects of manipulating levels of the highly homologous Bα, which we found also to be expressed in early Xenopus embryos in a similar pattern to Bδ (Fig. S1). Surprisingly, morpholino knockdown of Bα resulted in a very different phenotype to the Bδ knockdown. The embryos exhibited a short anterior-posterior axis, but in this case anterior structures were much reduced (Fig. 1A and Table S2). In fact, the phenotype was similar to that caused by overexpression of Bδ. The effect of Bα knockdown was rescued by overexpression of mouse Bα (Fig. 1B). Embryos overexpressing Bα were phenotypically normal (Fig. 1B), perhaps because Bα levels are not limiting in the early embryo. Consistent with the distinct phenotypes resulting from knockdown of Bα and Bδ, overexpression of Bα could not rescue Bδ morphant embryos and overexpression of Bδ could not rescue Bα morphant embryos (Fig. S2). We thus conclude that these two regulatory B subunits have distinct functions in the early Xenopus embryo.

Both Bα and Bδ affect Activin/Nodal-dependent processes

The phenotype of Bα morphant and Bδ overexpressing embryos was similar to the phenotypes of various Nodal signalling mutants in fish (Schier, 2001), and also to phenotypes of fish and frog embryos in which Nodal signalling had been inhibited by the pharmacological type I receptor inhibitor, SB-431542 (Batut et al., 2007; Ho et al., 2006; Sun et al., 2006). Conversely, the phenotype of Bδ morphant embryos was consistent with increased Activin/Nodal and/or Wnt signalling (Tada et al., 2002; Whitman, 2001). To investigate this in more detail, we examined the effect of manipulating the levels of Bα and Bδ on the expression of the Activin/Nodal target genes gsc and Xbra (Howell et al., 2002). Both in situ hybridisation and quantitative PCR revealed that either overexpression of Bδ or knockdown of Bα greatly inhibited the expression of gsc and Xbra in early gastrula embryos (Fig. 2). In contrast, knockdown of Bδ increased the expression of these genes (Fig. 2). We also analysed β-catenin localisation as a readout for Wnt activity, but found no evidence for enhanced Wnt activity in Bδ morphant embryos or reduced Wnt activity in Bα morphant and Bδ-overexpressing embryos (see Fig. 5A). Thus, the observed phenotypes are most likely due to a modulation in the intensity of Nodal signalling, with knockdown of Bδ promoting Nodal signalling and knockdown of Bα or overexpression of Bδ, inhibiting Nodal signalling.

Fig. 2. Manipulating the expression of Bα and Bδ in Xenopus embryos has opposing effects on Activin/Nodal target gene expression.

Fig. 2

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(A) In situ hybridisation of gastrula-stage embryos injected with either a morpholino control (MoC) or a specific morpholino against Xenopus Bα (MoBα) or Bδ (MoBδ) or with Flag-tagged mouse Bδ mRNA (Bδ) at the one-cell stage. The probes used were against gsc or Xbra. Staining was visualised with BM purple. Note the increased staining for both Xbra and Gsc in Bδ morphants, and decreased staining in Bα morphants and embryos overexpressing Bδ.

(B) Analysis of gene expression by q-PCR. Total RNA was isolated from stage 10.5 embryos that had been injected at the one-cell stage with either morpholino control, or morpholinos against Bα or Bδ. Expression levels are normalised to ornithine decarboxylase (ODC).

Fig. 5. Bα and Bδ exert differential effects on the level of phosphorylated Smad2.

Fig. 5

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(A) Embryos were injected at the one-cell stage with morpholinos (Mo) against Bα or Bδ, or Bδ mRNA as indicated. Embryos were harvested at stage 10, fixed, dissected through the lip and analysed by immunofluorescence using anti-Smad2 and anti-β-Catenin antibodies. The nuclei were visualised with DAPI. Panels a and b show an area from the ventral vegetal region and panels c-e show an area from the dorsal vegetal region.

(B) Embryos remained uninjected (ui) or were injected with two doses of mouse Bα mRNA or mouse Bδ mRNA, cultured until control embryos reached stage 9 and analysed by immunoblotting with anti-phospho-Smad2, Smad2/3, or phospho-ERK (pERK) antibodies.

(C) Embryos were injected with distinct morpholinos (labelled 1 or 2) targeting Bδ, or Bα respectively, or with a control morpholino and analysed as in (B).

(D) Animal caps from stage 8 embryos were incubated with or without okadaic acid (OA, 25 nM) for 1 hr, treated with or without Activin for 20 min and processed for immunoblotting.

(E) HeLa EGFP-Smad2 cells were transfected with either an siRNA SMARTpool control or a human Bα- or Bδ-specific SMARTpool. Cells were incubated with TGF-β for the times indicated, fixed and visualised by confocal microscopy.

(F) HeLa EGFP-Smad2 cells were transfected as in E and incubated with TGF-β for the times indicated. Samples were analysed by Western blotting with anti-phospho-Smad2, anti-Smad2/3 and anti-pan B subunit antibodies.

To corroborate our data from whole embryos we investigated the effects of Bα and Bδ on Activin-dependent animal cap elongation, which is a functional readout for Activin/Nodal activity (Smith, 1993). Animal cap explants cultured in buffer alone heal into balls of ciliated epidermis, whilst those treated with Activin elongate and differentiate to form mesoderm. Activin-induced elongation of animal cap explants was completely abrogated by overexpression of Bδ, but not Bα (Fig. 3A). Conversely, morpholino knockdown of Bδ enhanced cap elongation, whereas knockdown of Bα completely abolished elongation (Fig. 3B). Importantly, the effects of Bα knockdown could be rescued by overexpression of an EGFP-tagged version of Smad2, which is the intracellular mediator of the Activin/Nodal pathway (Fig. 3C). As a control, overexpression of EGFPSmad2 had no effect on Activin-induced animal cap elongation by itself (Fig. 3C).

Fig. 3. Bα and Bδ act on the Activin/Nodal signalling pathway in Xenopus.

Fig. 3

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(A-C) One-cell embryos were injected with the indicated mRNAs (Bα, Bδ or EGFP-Smad2) and morpholinos (MoC, MoBα or MoBδ). When control embryos had reached stage 8, the animal pole was excised and incubated with or without Activin for 16 hrs, and visualised for the degree of elongation.

Altogether, these results indicate that altering the expression levels of Bα or Bδ in the early embryo modulates the Activin/Nodal signalling pathway. Importantly, Bα and Bδ affect the strength of Activin/Nodal signalling in Xenopus in opposite directions, with Bα normally acting positively, and Bδ acting negatively.

Wing phenotypes caused by overexpression of the Drosophila B subunit Twins can be rescued by overexpression of Smad2 and vice versa

We next extended our analysis of the role of the B family of PP2A regulatory subunits in Activin/Nodal signalling by analysing the genetic interaction between twins and smox in Drosophila. Twins is the only Drosophila B family member (Mayer-Jaekel et al., 1993), and Smox is Drosophila Smad2 (Henderson and Andrew, 1998), which transduces signals from the Activin type I receptor Baboon (Brummel et al., 1999; Parker et al., 2006; Serpe and O'Connor, 2006). Overexpression tests yielded a genetic interaction between smox and twins. Overexpression of Twins alone throughout the developing wing primordium with the UAS-Gal4 binary expression system (Brand and Perrimon, 1993) yielded very small wings with minimal venation in males, which had reduced survival (Fig. 4B); control wings that lacked the A9-Gal4 expression driver were wild type in phenotype (Fig. 4A). Overexpression of Smox alone yielded wings with abnormal venation (n>40, Fig. 4C). When the two transgenes were co-expressed, however, 24 out of 30 wings were completely suppressed to wild type shape and venation; 6 wings had partially suppressed phenotypes (Fig. S3). Co-expression of the Baboon A isoform with Twins also suppressed, but not as well as Smox (Fig. S3). In summary, the effects of Twins overexpression on wing size and vein structure was strongly or completely suppressed by increased levels of Smox/dSmad2 in 93% of cases, suggesting that Twins antagonises Activin/Smad2 signalling in the wing primordium. In Drosophila therefore, the only B subunit Twins acts similarly to Bδ in Xenopus.

Fig. 4. Overexpression of Drosophila Smad2 (Smox) can rescue the effects of overexpression of Drosophila B subunit, Twins in the wing.

Fig. 4

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(A) Phenotypically wild type wing from +/Y; UAS-tws23; UAS-Smox8D3 male.

(B) Small, blistered wing from A9-GAL4; UAS-tws23; + male. All wings from these males are smaller than wild type; approximately 80% were cupped and blistered with little or no evidence of veins.

(C) Wing from A9-GAL4; +; UAS-Smox8D3 male. In this genotype, wing veins formed a delta at the margin, and additional wing vein material was often observed (arrowheads).

(D) Phenotypically normal wing from A9-GAL4; UAS-tws23; UAS-Smox8D3 male. All veins terminated normally at the margin (arrows).

Bα and Bδ exert opposite effects on the levels of active Smad2

We next analysed in detail at what point in the TGF-β/Activin/Nodal pathway these phosphatase subunits acted. TGF-β/Nodal/Activin stimulation leads to C-terminal phosphorylation of Smad2 (and Smad3), which then accumulate in the nucleus (Massagué et al., 2005). At early gastrula stages endogenous Nodal signalling in Xenopus embryos is stronger dorsally (Lee et al., 2001) as seen by nuclear localisation of Smad2 on the dorsal, but not on the ventral side (Fig. 5A panels a and c). Knockdown of Bα or overexpression of Bδ suppressed this Smad2 nuclear accumulation on the dorsal side, whilst knockdown of Bδ permitted Smad2 nuclear accumulation even on the ventral side (Fig. 5A, top panels). The effects were specific to Nodal signalling as levels of nuclear β-catenin, which reflect active Wnt signalling, were not similarly affected (Fig. 5A, middle panels). In animal cap explants expressing a constitutively active version of the Activin/Nodal type I receptor ALK4 to mimick Nodal signalling (Wieser et al., 1995), morpholino knockdown of Bα prevented nuclear accumulation of EGFP-Smad2 (Fig. S4A). Moreover, Activin-induced nuclear accumulation of EGFP-Smad2 in animal cap explants was also abrogated by overexpression of Bδ, but not Bα (Fig. S4B). Taken together, these results indicate that Bα and Bδ act on Activin/Nodal signalling downstream of the ligands.

TGF-βActivin/Nodal-induced nuclear accumulation of Smad2 is driven by Smad2 C-terminal phosphorylation (Massagué et al., 2005). In accordance with the observed changes in Smad2 nuclear accumulation, we found that overexpression of Bδ caused a decrease in Smad2 phosphorylation in whole embryos in response to endogenous Nodal signalling, whereas overexpression of Bα had no effect (Fig. 5B). The same was true in animal caps in response to exogenous Activin (Fig. S5 and data not shown). In knockdown experiments, Smad2 phosphorylation was increased by Bδ knockdown and decreased by Bα knockdown (Fig. 5C). The effect of Bδ knockdown could be mimicked by a 1-hour treatment of animal caps with the PP2A catalytic subunit inhibitor, okadaic acid (Fig. 5D), suggesting that Bδ's action on TGF-β/Activin/Nodal signalling requires the phosphatase activity of PP2A (see also Discussion). Previous work has suggested that B family members have cell type specific effects on the ERK MAPK pathway (Adams et al., 2005; Strack, 2002; Van Kanegan et al., 2005). However in Xenopus embryos, knockdown of Bδ or Bα or their overexpression had no effect on ERK phosphorylation in response to endogenous receptor tyrosine kinase signalling (Fig. 5B and C).

The observed effects on Smad2 phosphorylation and nuclear accumulation are not confined to Xenopus embryos but are conserved in mammalian systems. We used siRNAs to knock down Bα and Bδ in a HeLa cell line stably expressing EGFP-Smad2. As in Xenopus, knockdown of Bα decreased the nuclear accumulation of Smad2 in response to TGF-β in these cells (Fig. 5E) and inhibited Smad2 phosphorylation (Fig. 5F). In contrast, knockdown of Bδ enhanced Smad2 nuclear accumulation relative to control cells (Fig. 5E) and increased Smad2 phosphorylation (Fig. 5F). These effects were observable in a number of different human and mouse cell lines (data not shown) and were specific, as several different individual siRNA oligonucleotides which were proven to specifically knock down Bα or Bδ (Fig. S6A, B), elicited the same effect (Fig. S6B, C). Consistent with the decrease in TGF-β-induced Smad2 phosphorylation caused by Bα knockdown, TGF-β was also less effective at mediating growth arrest in these conditions (Fig. S7). Overexpression of either Bα or Bδ had little effect on the level of phosphorylated Smad2 in tissue culture cells (data not shown), perhaps because their levels are not limiting or because free subunits which are not incorporated into holocomplexes, are unstable (Strack et al., 2002).

In conclusion, Bα and Bδ modulate the level of active phosphorylated Smad2 and hence its nuclear accumulation in both Xenopus embryos and tissue culture cells, with Bα normally promoting Smad2 phosphorylation and Bδ inhibiting Smad2 phosphorylation.

Bα- and Bδ-containing PP2A holocomplexes do not dephosphorylate pSmad2

The simplest hypothesis to explain the effects on Smad2 phosphorylation was that Bδ acted directly on the C-terminal phosphates of activated Smad2 and that Bα negated this action. We therefore immunopurified heterotrimeric active PP2A complexes containing either Flag-tagged Bα or Bδ or a distinct regulatory subunit, B′δ (Janssens and Goris, 2001) together with the catalytic and scaffolding subunits (Fig. 6A-C and data not shown). These complexes were all active since they dephosphorylated a control phospho-peptide (Fig. 6C) and the PP2A complexes containing either Bα or Bδ, but not complexes containing B′δ, efficiently dephosphorylated an immunopurified Raf substrate (Fig. 6D), as previously reported (Adams et al., 2005). However, we could not detect any dephosphorylation of an immunopurified, C-terminally phosphorylated Smad2 substrate by any of the phosphatase complexes (Fig. 6D). This was also true for PP2A holocomplexes purified from TGF-β-induced cells (Fig. 6E and F). Furthermore, we could not detect significant dephosphorylation of a number of phosphorylated residues within the Smad2 linker region (Kretzschmar et al., 1999) (Fig. 6G). Thus, neither Bα nor Bδ seem to act directly on phosphorylated Smad2.

Fig. 6. Bα and Bδ do not act directly on phosphorylated Smad2.

Fig. 6

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(A) Outline of the experimental procedure to isolate Bα- and Bδ-containing active PP2A holocomplexes and to perform phosphatase assays.

(B) Silver-stained gel showing the composition of complexes isolated by Flag pulldown from HeLa cells transfected with the indicated Flag-tagged B subunits. The components of the complex are indicated including the catalytic subunit (PP2AC) and the structural subunit (PP2AA). *indicates that the Flag-Bδ overlies PP2AA.

(C) Western blot analysis of immunopurified complexes showing the presence of appropriate B or B′δ (PPP2R5D) subunit (Flag blot) and co-purified catalytic subunit (anti-PP2AC blot) for each complex. Phosphatase activity was assessed by a colorimetric assay using a phospho-peptide as substrate (bars).

(D) PP2A complexes as in (C) were incubated with phospho-Smad2 immunopurified from TGF-β-induced HaCaT EGFP-Smad2 cells. The reactions were then analysed by immunoblotting with anti-pSmad2 and anti-Smad2/3 antibodies. All PP2A complexes tested failed to dephosphorylate phospho-Smad2. Bα- and Bδ-containing complexes dephosphorylated pS259 of immunoprecipitated HA-tagged Raf-1 (lower panels)

(E) TGF-β treatment prior to immunopurification of the PP2A complexes does not affect the amount of co-purified catalytic subunit, nor the activity of the complexes in the colorimetric assay.

(F) As in (D), but PP2A complexes were purified from untreated (-) or TGF-β-induced cells (+), as shown in (E).

(G) Phosphorylated serines 245, 250 and 255 of Smad2 are not substrates for immunopurified Bα and Bδ complexes. Phosphatase complexes were immunopurified from either control cells (C) or cells expressing Flag-tagged Bα or Bδ as indicated, and incubated with either a Smad2/3 immunoprecipitate from TGF-β-induced HaCaT cells (upper panels) or, as a control, an immunopurified phosphorylated Raf substrate from HeLa cells expressing HA-Raf (lower panel). Samples were analysed by Western blotting using antibodies recognising Smad2 phosphorylated at residues S245, S250, S255, as well as anti-Smad2/3, anti-phospho Raf and anti-HA as indicated.

Bα and Bδ do not affect receptor kinase activity in vitro

Since Bα and Bδ do not act on Smad2 directly, but do affect Smad2 phosphorylation in vivo we asked whether they could regulate the activity of the TGF-β receptor complex. The TGF-β receptor complex comprises two type II receptors (TβR-II) and two type I receptors (ALK5). ALK5 activity requires its phosphorylation by the constitutively active type II receptor, and thus we reasoned that dephosphorylation of ALK5 by a PP2A complex in vitro would reduce ALK5 activity. In an in vitro kinase assay (see Fig. 7A for the experimental scheme) immunopurified active receptor complexes from TGF-β-induced cells phosphorylated recombinant Smad2 at its C-terminus (Fig. 7B, lane C). However, incubation of receptor and substrate with either phosphatase complex had no significant effect on the ability of receptors to phosphorylate Smad2 (Fig. 7B, lanes Bα, Bδ, B′δ). Thus in vitro, neither phosphatase subunit affected receptor kinase activity.

Fig. 7. Bα regulates the basal level of the type I receptor and Bδ regulates its activity.

Fig. 7

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(A) Outline of the experimental procedure to isolate Bα- and Bδ-containing active PP2A holocomplexes and to assay their ability to affect the kinase activity of ALK5.

(B) The presence of neither PP2A complex affects the kinase activity of ALK5 in vitro. Endogenous ALK5 complexes immunopurified from untreated or TGF-β-treated HaCaT cells were incubated with recombinant Smad2 substrate in the absence or presence of B-subunit-specific PP2A complexes purified as in Figure 6. C-terminal Smad2 phosphorylation was detected by immunoblotting. The activity of the PP2A complexes was confirmed by their ability to dephosphorylate pS259 of Raf-1 (lower panel).

(C) Knockdown of Bδ promotes ALK4 clustering. Animal caps from embryos expressing either HA-ALK4 mRNA alone (upper panels) or in combination with morpholino against Bδ (MoBδ, middle panels) were incubated for 1 hr in the presence or absence of Activin and stained with anti-HA antibody. HA-ALK4 clusters in response to Activin and in untreated embryos injected with MoBδ. Okadaic acid (OA) treatment (lower panels) also induces HA-ALK4 clustering and thus mimics Bδ knockdown.

(D) Bα knockdown strongly decreases basal protein levels of ALK5. HaCaT cells were transfected with siRNAs and treated with TGF-β as indicated. Extracts were immunoblotted with antibodies against ALK5, phospho-Smad2, pan B-subunits and Smad2/3.

(E) Bα knockdown has no effect on TβR-II levels. HaCaT cells were transfected with the indicated siRNAs. Extracts were immunoblotted with antibodies against TβR-II, pan B-subunits and Smad2/3. Prior to electrophoresis, extracts were treated ± PNGase F to remove N-linked sugars from TβR-II and visualise it more clearly.

(F) Bα knockdown or Bδ overexpression decreases protein levels of HA-ALK4. Xenopus embryos were injected at the one-cell stage with HA-ALK4 and GFP mRNAs as well as with morpholinos or Bδ mRNA as indicated, cultured until uninjected embryos (Ui) had reached stage 9 and analysed by immunoblotting.

(G) Model of the modulation of TGF-β/Activin/Nodal signalling by Bα and Bδ. Bα normally stabilises the type I receptors ALK4 and ALK5, and Bα knockdown promotes their basal degradation. Bδ normally restricts ligand-dependent activation of ALK4 and ALK5, and Bδ knockdown facilitates such activation. When overexpressed, Bδ additionally inihibits endogenous Bα by replacing it in the PP2A holoenzyme due to its higher affinity for the catalytic subunit.

Knockdown of Bδ enhances ALK4 activity

We therefore investigated whether Bα or Bδ affected receptor activity in vivo. We have recently shown that ALK4 receptor clustering provides a convenient readout of receptor activity in vivo (Batut et al., 2007) as it is induced in response to ligand and requires ALK4 kinase activity (Fig. S8). In untreated animal caps, which exhibit very low levels of Activin/Nodal signalling, morpholino knockdown of Bδ, or inhibition of PP2A catalytic activity by okadaic acid, was sufficient to induce ALK4 clustering (Fig. 7C, compare untreated animal caps). Thus a reduction of Bδ activity lowers the threshold of ligand required for ALK4 signalling, indicating that Bδ normally restricts receptor activity and might act to suppress signalling at very low ligand concentrations.

Knockdown of Bα promotes degradation of ALK4 and ALK5

In the course of investigating whether knockdown of Bα would inhibit Activin/Nodal-induced ALK4 clustering, we noticed that levels of HA-ALK4 were extremely low when Bα was depleted (Fig. 7D). Moreover, overexpression of Bδ, which has the same functional effects as Bα knockdown, similarly reduced HA-ALK4 levels (Fig. 7D).

These data were corroborated in HaCaT cells, where knockdown of Bα led to a strong decrease in levels of endogenous ALK5 protein (Fig. 7E). This effect was specific as knockdown of Bα or Bδ did not affect endogenous TβR-II levels (Fig. 7F). We conclude that the effect on receptor levels is post-transcriptional in both model systems. In tissue culture cells, knockdown of Bα had no effect on endogenous ALK5 mRNA levels (Fig. S9A) and also substantially reduced protein levels of exogenously expressed HA-tagged ALK5 (Fig. S9B). Similarly in Xenopus embryos, the effects of Bα knockdown or Bδ overexpression on HA-ALK4 are not at the level of transcription since the receptor is expressed from an injected synthetic mRNA. In principle, Bα could affect translation of the type I receptors, or their stability. We favour the latter possibility since the mRNAs used in both systems have no 3′ or 5′ UTRs, which are usually required for translational regulation. Furthermore, we could detect a weak interaction between HA-ALK4 and Flag-tagged Bα in a co-immunoprecipitation (Fig. S10) as was reported previously for ALK5 (Griswold-Prenner et al., 1998), suggesting that Bα might affect ALK4 and ALK5 at the protein level.

To identify the pathway of degradation, we asked whether inhibitors of the lysosome (bafilomycin A1) or the proteasome (lactacystin or MG132) could rescue the effects of Bα knockdown in tissure culture cells. We found that in HeLa cells overexpressing ALK5, the effects of Bα knockdown was rescued by bafilomycin A1, but not MG132 (Fig. S11A). Similarly in HaCaT cells, a partial rescue of endogenous ALK5 levels was observed with bafilomycin A treatment, but not by treatment with lactacystin or MG132 (Fig. S11B).

Taken together, these data suggest that knockdown of Bα (and in Xenopus, also overexpression of Bδ) promotes degradation of the type I receptors ALK4 and ALK5 via a lysosomal pathway. We speculated that PP2A might regulate Dapper2, which is known to promote lysosomal degradation of ALK4 and ALK5 (Zhang et al., 2004). However, knockdown of Dapper2 did not ameliorate the decrease in ALK5 levels resulting from Bα knock-down, even though Dapper2 knockdown alone clearly raised levels of ALK5 (Fig. S11C). Thus, it is more likely that Dapper2 regulates PP2A or that the two act independently.

Discussion

It has long been speculated that the individual, yet highly homologous, members of each subfamily of PP2A regulatory subunits would have unique and specific roles in addition to their established redundant activities. We have demonstrated here for the first time such non-redundant functions of two members of the B subfamily of PP2A regulatory subunits. We have shown that Bα and Bδ perform important functions in modulating the intensity of TGF-β/Activin/Nodal signalling in different species by stabilising basal levels of the ALK4 and ALK5 receptors (Bα) and by restricting receptor activity (Bδ) (Fig. 7G). Perturbation of these mechanisms has dramatic functional consequences in vivo as demonstrated by altered gene expression and serious developmental defects.

We do not yet know the exact mechanism whereby Bα and Bδ regulate receptor levels and activity, respectively. Nevertheless it is clear from our knockdown experiments that each subunit affects a separate and distinct aspect of receptor biology, ruling out the possibility that Bα and Bδ merely compete with each other for catalytic and scaffolding subunits, so that knockdown of one B subunit increases the levels of complexes containing the other B subunit. The results of our loss of function experiments also exclude the possibility that Bα and Bδ have opposing activities on a single common substrate. However, it is likely that subunit competition within the PP2A holoenzyme does explain why Bδ overexpression mimicks Bα knockdown (both manipulations causing ALK4 destabilisation in Xenopus embryos). Bδ has a higher affinity for the catalytic subunit than Bα (Fig. 6C and E). Overexpressed Bδ is thus more likely to compete out endogenous Bα than vice versa, and Bδ overexpression would mimick Bα knockdown, whereas Bα overexpression would not necessarily have an effect, as we observe.

We have shown that the PP2A catalytic inhibitor, okadaic acid, can mimick the effects of Bδ knockdown suggesting that Bδ functions as part of a PP2A holoenzyme complex. However, as okadaic acid is not absolutely specific for PP2A, a mechanism independent of the PP2A catalytic subunit cannot be definitively ruled out. We have shown that knockdown of Bδ promotes receptor clustering at low endogenous ligand concentrations, suggesting that Bδ normally inhibits receptor clustering and thus receptor activation at sub-threshold levels of ligand. Whether it does this by removing (in the context of a PP2A holoenzyme complex) an activating phosphate from serine/threonine residues in the receptors themselves, or via dephosphorylation of another component remains to be investigated.

In contrast to the role ascribed to Bδ, our data demonstrate that Bα regulates the levels of the type I receptors ALK4 and ALK5, most likely by stabilising the receptors and preventing their degradation via the lysosomal pathway. The involvement of phosphatases in protein turnover is not unprecedented. The protein phosphatase Dullard has recently been reported to promote the degradation of BMP type II receptors in Xenopus, thus repressing BMP-dependent phosphorylation of the BMP type I receptor (Satow et al., 2006). More specifically to PP2A, the cycling of the protein Period in Drosophila is dependent upon the activity of Twins and loss of PP2A activity reduces Period expression (Sathyanarayanan et al., 2004). In this case, Twins acts in the context of a PP2A holoenzyme to dephosphorylate Period directly. Moreover, Twins also affects the levels of Armadillo, the Drosophila β-Catenin homologue, since it is required for stabilisation of Armadillo in response to Wingless signalling (Bajpai et al., 2004). Consistent with our demonstration that Drosophila Twins acts like vertebrate Bδ, we have observed a reduction in levels of nuclear β-Catenin in Bδ morphant embryos (Fig. 5A). Importantly however, this effect of Bδ on the Wnt signalling pathway cannot explain the phenotypes we observe in Bδ morphant embryos. In our present study it is not yet clear whether Bα stabilises ALK4 and ALK5 by acting to dephosphorylate the receptors themselves, or whether it acts indirectly. Consistent with a previous report (Griswold-Prenner et al., 1998) we could detect a weak interaction between Bα and ALK4, suggesting that it may act on the receptor directly, or possibly on another component of the receptor complex. We find that the effect of Bα on type I receptor levels occurs in the absence of signalling, indicating that Bα is required for regulating the Basal levels of receptor and not responsible for downregulating receptor levels after signal transduction.

In summary, we conclude that Bα- and Bδ-containing phosphatase complexes have distinct substrates whose net effects on the TGF-β/Activin/Nodal pathway are opposite. This strongly suggests that the ratio of Bα to Bδ in a particular cell will influence the threshold response to TGF-β/Activin/Nodal ligands, which will in turn determine the levels of target gene transcription and thus developmental programmes.

Supplementary Material

Supplementary Figure 1. In situ hybridisation of endogenous Xenopus Bα and Bδ mRNA. Embryos were collected at stage 8, stage 10 and tailbud stage and hybridised with an antisense probe specific for Xenopus Bα mRNA or Xenopus Bδ mRNA. Whole embryos and saggital sections are shown. At Stage 10 the sections were through the blastopore lip. The position of the dorsal blastopore lip is indicated by asterisks. At Stage 8 and 10, both B subunits are predominantly expressed in the animal cap and more weakly in the marginal zone. Note that Bδ mRNA is particularly well expressed in the ectodermal inner layer of stage 10 embryos (arrow). At tailbud stages Bα is strongly expressed in the branchial arches, whilst Bδ is much more weakly expressed.

Supplementary Figure 2. Bα and Bδ mRNA expression specifically rescues the phenotype induced by the cognate morpholinos. One-cell embryos were injected with control morpholinos (MoC), morpholinos against Bα (MoBα), or morpholinos against Bδ (MoBδ), either alone (-) or in combination with Bα mRNA (Bα) or Bδ mRNA (Bδ). Embryos were fixed when control embryos had reached stage 10.25. The blastopore lip is absent in MoBα-injected embryos (d). This phenotype can be rescued by the co-expression of Bα (e), but not Bδ mRNA (f). MoBδ injected embryos exhibit a slightly more advanced blastopore lip (compare g and a), which is restored to its expected size by co-expression of Bδ mRNA (i), but not Bα mRNA (h).

Supplementary Figure 3. Suppression of tws overexpression by smox or baboA. Males with A9-Gal4 driven overexpression survived poorly, so experiments were performed at several temperatures. At higher temperature (25°), expression levels from the Gal4-UAS system are higher, so that phenotypes are stronger than at lower temperature (22°) (Brand and Perrimon, 1993). Phenotypes of wings from experimental genotypes were compared to phenotype of wings from A9-Gal4/+; UAS-tws23/+ and grouped into classes. At both 25° (n=14) and 22° (n=16), the majority of wings from A9-Gal4/+; UAS-tws23/+; UAS-smox8D3/+ were wild type in phenotype. Fewer A9-Gal4/+; UAS-tws23/+; UAS-baboA/+ males survived (n=8), and most wings had an intermediate phenotype. Phenotype classes: Complete suppression: wild type shape and venation. Strong suppression: slight loss of tissue at posterior margin (Bajpai et al., 2004) or small changes in vein thickness. Moderate suppression: normal wing size with longitudinal fold down middle or decreased wing size with thick or split veins. Weak or no suppression: small wings with no apparent veins. For graphical depiction, control genotypes were set arbitrarily at 16 wings.

Supplementary Figure 4. Knockdown of Bα or overexpression of Bδ inhibits nuclear accumulation of Smad2 in animal cap explants. A. One-cell embryos were injected with EGFP-Smad2 mRNA and mRNA expressing HA-tagged constitutively active ALK4 (HA-ALK4ca) together with MoC or MoBα as indicated. At stage 8, animal caps were dissected and visualised by confocal microscopy. Two separate caps are shown for each condition. B. One-cell embryos were injected with EGFP-Smad2 mRNA alone or together with Bα or Bδ mRNA as indicated. At stage 8, animal caps were dissected, incubated with or without Activin for 20 min and visualised by confocal microscopy.

Supplementary Figure 5. Overexpression of Bδ in animal caps blocks Smad2 phosphorylation. Embryos were injected at the one-cell stage with EGFP-Smad2 and/or Flag-tagged mouse Bδ mRNA as indicated and cultivated until control embryos reached stage 8. Animal caps were dissected, treated with Activin as indicated and processed for Western blot analysis.

Supplementary Figure 6. A. Specificity of siRNA-induced knockdown of Bα and Bδ in HeLa EGFPSmad2 cells as assayed by RT-PCR. HeLa EGFPSmad2 cells were transfected with different siRNAs against Bα or Bδ, RNA was extracted and subjected to RT-PCR analysis. Levels of Bα and Bδ mRNA are given relative to a GAPDH control. B. Specificity of siRNA-induced knockdown of Bα and Bδ in HeLa EGFPSmad2 cells assayed by Western blotting. HeLa EGFPSmad2 cells were transfected with siRNA SMARTpools and individual siRNAs against Bα or Bδ, induced with TGF-β for the times indicated and analysed by Western blotting with antibodies against phosphorylated Smad2 (pSmad2), pan B subunits, and Smad2/3 as a loading control. C. Specificity of siRNA-induced knockdown of Bα and Bδ in the HeLa EGFPSmad2 cell line as assayed by EGFPSmad2 localisation. HeLa EGFPSmad2 cells were transfected with siRNA SMARTpools and individual siRNAs against Bα or Bδ as indicated, induced with TGF-β for the times indicated and fixed for confocal microscopy. D. Specificity of the pan B antibody. Flag-tagged mouse Bδ, Xenopus Bδ (XL-Bδ) and mouse Bα were transcribed and translated in vitro and analysed by immunoblotting with anti-pan B and anti-Flag antibodies. The anti-pan B antibody recognises mouse Bα and Bδ equally well, but fails to detect Xenopus Bδ. As endogenous Bα and Bδ comigrate on SDS-PAGE and Bα is much more abundant than Bδ (Strack et al., 1999), we could easily monitor knockdown of Bα using this antibody, but could not detect knockdown of Bδ directly on the protein level. Bδ knockdown was thus monitored at the RNA level (A).

Supplementary Figure 7. TGF-β-dependent growth arrest is inhibited by knockdown of Bα. HaCaT cells were treated with siRNAs as indicated. After 48 hours, cells were serum starved, and after 24 hours of starvation shifted into 10% serum in the absence or the presence of different amounts of TGF-β (100 pg/ml and 400 pg/ml). After a further 24 hours, the percentage of cells in G1 was determined by FACS analysis and normalised to the percentage of cells in G1 under control conditions (no TGF-β).

Supplementary Figure 8. Clustering of ALK4 receptor is dependent on ALK4 kinase activity. Embryos were injected at the one-cell stage with HA-ALK4 and EGFP-Smad2 mRNA and cultured until control embryos reached Stage 8. Animal caps were dissected and either left untreated or induced with Activin for 1h in the absence or presence of 10 μM SB-431542 to inhibit ALK4 kinase activity. EGFP-Smad2 was visualised directly, and HA-ALK4 was detected by immunostaining using anti-HA antibodies. Clustering of HA-ALK4 upon Activin induction (arrows) is inhibited by treatment with SB-431542.

Supplementary Figure 9. A. ALK5 mRNA levels are not affected by knockdown of either Bα or Bδ. HeLa EGFP-Smad2 cells were transfected with siRNAs as indicated. C, control siRNA. Total RNA was prepared 72 hours later and reverse transcription with (RT) or without (no RT) reverse transcriptase was performed. cDNA fragments were then amplified. Grb-2 was used as a control.

B. Protein levels of exogenous HA-tagged ALK5 are strongly reduced in Bα knockdown cells. HeLa EGFP-Smad2 cells were transfected with Bα- or Control (C) siRNA. After 48 hours, cells were transfected with a constitutively active version of ALK5 (HA-ALK5ca), and at the same time treated with the ALK5 receptor inhibitor SB-431542. After a further 24 hours, SB-431542 was washed out to reactivate the receptor and to induce Smad2 phosphorylation. Protein extracts were immunoblotted using antibodies against HA, phospho-Smad2, pan-B subunits, and Smad2 (as a loading control). Protein levels of HA-ALK5ca are strongly reduced in Bα knockdown conditions. Correspondingly, TGF-β-independent Smad2 phosphorylation by HA-ALK5ca is attenuated under Bα knockdown conditions. A similar down-regulation of protein levels was seen for wildtype HA-ALK5 (data not shown).

C. Downregulation of HA-ALK5ca in Bα-knockdown cells is not due to impaired transfection efficiency or lower transcription of transgenes. β-Galactosidase activity from a co-transfected lacZ-plasmid is unaffected by Bα-knockdown.

Supplementary Figure 10. Bα interacts with ALK4 in Xenopus embryos. HA-ALK4 was immunoprecipitated from stage 9 embryos injected with HA-ALK4 and Bα or Bδ as indicated. Immunoprecipitates (IP) and inputs were immunoblotted with the indicated antibodies. An immunoprecipitation control using beads alone is also shown.

Supplementary Figure 11. ALK5 degradation triggered by knockdown of Bα is partially rescued by Bafilomycin A1 treatment, but does not occur via a Dapper2-dependent mechanism. A. HeLa TK- cells were transfected with control siRNA or an siRNA against Bα. After 48 hours, they were transfected with a plasmid expressing HA-ALK5. After a further 24 hours, cells were treated or not with 10 nM Bafilomycin A1 for 6 hours. Whole cell extracts were analysed by immunoblotting using antibodies against HA, pan B subunits and Smad2. B. HaCaT cells were transfected with control siRNA or an siRNA against Bα. After 72 hours cells were treated or not with 10 nM Bafilomycin A1, 25 μM MG132 or 25 μM Lactacystin for 6 hours. Whole cell extracts were analysed by immunoblotting using antibodies against ALK5, pan B subunits and Smad2/3. C. Knockdown of Dapper2 in HaCaT cells increases protein levels of endogenous ALK5 (compare lanes 4-6 with lanes 1-3 in the upper panel), however fails to prevent the ALK5 degradation triggered by Bα knockdown (lane 5). Knockdown and loading controls are shown (lower two panels).

Acknowledgments

We thank L. S. Shashidhara, C. Doe, K. Irvine, H. Jaeckle, M.B. O'Connor, U. Schaefer, B. Pellock, and the Bloomington Stock Center for fly strains, R. Marais for the HA-Raf1 construct and B. Wadzinski for the Tet inducible HEK cell lines. We are grateful to Sally Leevers, Peter Parker and members of the Hill lab for helpful discussions and comments on the manuscript. The work was funded by Cancer Research UK, the NIH (grant GM60501 to LAR), by an Erwin Schrödinger Fellowship of the Austrian Science Foundation (J2397-B12) and an EU Marie Curie Fellowship (515294) to BS.

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Associated Data

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Supplementary Materials

Supplementary Figure 1. In situ hybridisation of endogenous Xenopus Bα and Bδ mRNA. Embryos were collected at stage 8, stage 10 and tailbud stage and hybridised with an antisense probe specific for Xenopus Bα mRNA or Xenopus Bδ mRNA. Whole embryos and saggital sections are shown. At Stage 10 the sections were through the blastopore lip. The position of the dorsal blastopore lip is indicated by asterisks. At Stage 8 and 10, both B subunits are predominantly expressed in the animal cap and more weakly in the marginal zone. Note that Bδ mRNA is particularly well expressed in the ectodermal inner layer of stage 10 embryos (arrow). At tailbud stages Bα is strongly expressed in the branchial arches, whilst Bδ is much more weakly expressed.

Supplementary Figure 2. Bα and Bδ mRNA expression specifically rescues the phenotype induced by the cognate morpholinos. One-cell embryos were injected with control morpholinos (MoC), morpholinos against Bα (MoBα), or morpholinos against Bδ (MoBδ), either alone (-) or in combination with Bα mRNA (Bα) or Bδ mRNA (Bδ). Embryos were fixed when control embryos had reached stage 10.25. The blastopore lip is absent in MoBα-injected embryos (d). This phenotype can be rescued by the co-expression of Bα (e), but not Bδ mRNA (f). MoBδ injected embryos exhibit a slightly more advanced blastopore lip (compare g and a), which is restored to its expected size by co-expression of Bδ mRNA (i), but not Bα mRNA (h).

Supplementary Figure 3. Suppression of tws overexpression by smox or baboA. Males with A9-Gal4 driven overexpression survived poorly, so experiments were performed at several temperatures. At higher temperature (25°), expression levels from the Gal4-UAS system are higher, so that phenotypes are stronger than at lower temperature (22°) (Brand and Perrimon, 1993). Phenotypes of wings from experimental genotypes were compared to phenotype of wings from A9-Gal4/+; UAS-tws23/+ and grouped into classes. At both 25° (n=14) and 22° (n=16), the majority of wings from A9-Gal4/+; UAS-tws23/+; UAS-smox8D3/+ were wild type in phenotype. Fewer A9-Gal4/+; UAS-tws23/+; UAS-baboA/+ males survived (n=8), and most wings had an intermediate phenotype. Phenotype classes: Complete suppression: wild type shape and venation. Strong suppression: slight loss of tissue at posterior margin (Bajpai et al., 2004) or small changes in vein thickness. Moderate suppression: normal wing size with longitudinal fold down middle or decreased wing size with thick or split veins. Weak or no suppression: small wings with no apparent veins. For graphical depiction, control genotypes were set arbitrarily at 16 wings.

Supplementary Figure 4. Knockdown of Bα or overexpression of Bδ inhibits nuclear accumulation of Smad2 in animal cap explants. A. One-cell embryos were injected with EGFP-Smad2 mRNA and mRNA expressing HA-tagged constitutively active ALK4 (HA-ALK4ca) together with MoC or MoBα as indicated. At stage 8, animal caps were dissected and visualised by confocal microscopy. Two separate caps are shown for each condition. B. One-cell embryos were injected with EGFP-Smad2 mRNA alone or together with Bα or Bδ mRNA as indicated. At stage 8, animal caps were dissected, incubated with or without Activin for 20 min and visualised by confocal microscopy.

Supplementary Figure 5. Overexpression of Bδ in animal caps blocks Smad2 phosphorylation. Embryos were injected at the one-cell stage with EGFP-Smad2 and/or Flag-tagged mouse Bδ mRNA as indicated and cultivated until control embryos reached stage 8. Animal caps were dissected, treated with Activin as indicated and processed for Western blot analysis.

Supplementary Figure 6. A. Specificity of siRNA-induced knockdown of Bα and Bδ in HeLa EGFPSmad2 cells as assayed by RT-PCR. HeLa EGFPSmad2 cells were transfected with different siRNAs against Bα or Bδ, RNA was extracted and subjected to RT-PCR analysis. Levels of Bα and Bδ mRNA are given relative to a GAPDH control. B. Specificity of siRNA-induced knockdown of Bα and Bδ in HeLa EGFPSmad2 cells assayed by Western blotting. HeLa EGFPSmad2 cells were transfected with siRNA SMARTpools and individual siRNAs against Bα or Bδ, induced with TGF-β for the times indicated and analysed by Western blotting with antibodies against phosphorylated Smad2 (pSmad2), pan B subunits, and Smad2/3 as a loading control. C. Specificity of siRNA-induced knockdown of Bα and Bδ in the HeLa EGFPSmad2 cell line as assayed by EGFPSmad2 localisation. HeLa EGFPSmad2 cells were transfected with siRNA SMARTpools and individual siRNAs against Bα or Bδ as indicated, induced with TGF-β for the times indicated and fixed for confocal microscopy. D. Specificity of the pan B antibody. Flag-tagged mouse Bδ, Xenopus Bδ (XL-Bδ) and mouse Bα were transcribed and translated in vitro and analysed by immunoblotting with anti-pan B and anti-Flag antibodies. The anti-pan B antibody recognises mouse Bα and Bδ equally well, but fails to detect Xenopus Bδ. As endogenous Bα and Bδ comigrate on SDS-PAGE and Bα is much more abundant than Bδ (Strack et al., 1999), we could easily monitor knockdown of Bα using this antibody, but could not detect knockdown of Bδ directly on the protein level. Bδ knockdown was thus monitored at the RNA level (A).

Supplementary Figure 7. TGF-β-dependent growth arrest is inhibited by knockdown of Bα. HaCaT cells were treated with siRNAs as indicated. After 48 hours, cells were serum starved, and after 24 hours of starvation shifted into 10% serum in the absence or the presence of different amounts of TGF-β (100 pg/ml and 400 pg/ml). After a further 24 hours, the percentage of cells in G1 was determined by FACS analysis and normalised to the percentage of cells in G1 under control conditions (no TGF-β).

Supplementary Figure 8. Clustering of ALK4 receptor is dependent on ALK4 kinase activity. Embryos were injected at the one-cell stage with HA-ALK4 and EGFP-Smad2 mRNA and cultured until control embryos reached Stage 8. Animal caps were dissected and either left untreated or induced with Activin for 1h in the absence or presence of 10 μM SB-431542 to inhibit ALK4 kinase activity. EGFP-Smad2 was visualised directly, and HA-ALK4 was detected by immunostaining using anti-HA antibodies. Clustering of HA-ALK4 upon Activin induction (arrows) is inhibited by treatment with SB-431542.

Supplementary Figure 9. A. ALK5 mRNA levels are not affected by knockdown of either Bα or Bδ. HeLa EGFP-Smad2 cells were transfected with siRNAs as indicated. C, control siRNA. Total RNA was prepared 72 hours later and reverse transcription with (RT) or without (no RT) reverse transcriptase was performed. cDNA fragments were then amplified. Grb-2 was used as a control.

B. Protein levels of exogenous HA-tagged ALK5 are strongly reduced in Bα knockdown cells. HeLa EGFP-Smad2 cells were transfected with Bα- or Control (C) siRNA. After 48 hours, cells were transfected with a constitutively active version of ALK5 (HA-ALK5ca), and at the same time treated with the ALK5 receptor inhibitor SB-431542. After a further 24 hours, SB-431542 was washed out to reactivate the receptor and to induce Smad2 phosphorylation. Protein extracts were immunoblotted using antibodies against HA, phospho-Smad2, pan-B subunits, and Smad2 (as a loading control). Protein levels of HA-ALK5ca are strongly reduced in Bα knockdown conditions. Correspondingly, TGF-β-independent Smad2 phosphorylation by HA-ALK5ca is attenuated under Bα knockdown conditions. A similar down-regulation of protein levels was seen for wildtype HA-ALK5 (data not shown).

C. Downregulation of HA-ALK5ca in Bα-knockdown cells is not due to impaired transfection efficiency or lower transcription of transgenes. β-Galactosidase activity from a co-transfected lacZ-plasmid is unaffected by Bα-knockdown.

Supplementary Figure 10. Bα interacts with ALK4 in Xenopus embryos. HA-ALK4 was immunoprecipitated from stage 9 embryos injected with HA-ALK4 and Bα or Bδ as indicated. Immunoprecipitates (IP) and inputs were immunoblotted with the indicated antibodies. An immunoprecipitation control using beads alone is also shown.

Supplementary Figure 11. ALK5 degradation triggered by knockdown of Bα is partially rescued by Bafilomycin A1 treatment, but does not occur via a Dapper2-dependent mechanism. A. HeLa TK- cells were transfected with control siRNA or an siRNA against Bα. After 48 hours, they were transfected with a plasmid expressing HA-ALK5. After a further 24 hours, cells were treated or not with 10 nM Bafilomycin A1 for 6 hours. Whole cell extracts were analysed by immunoblotting using antibodies against HA, pan B subunits and Smad2. B. HaCaT cells were transfected with control siRNA or an siRNA against Bα. After 72 hours cells were treated or not with 10 nM Bafilomycin A1, 25 μM MG132 or 25 μM Lactacystin for 6 hours. Whole cell extracts were analysed by immunoblotting using antibodies against ALK5, pan B subunits and Smad2/3. C. Knockdown of Dapper2 in HaCaT cells increases protein levels of endogenous ALK5 (compare lanes 4-6 with lanes 1-3 in the upper panel), however fails to prevent the ALK5 degradation triggered by Bα knockdown (lane 5). Knockdown and loading controls are shown (lower two panels).

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