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Development 135 (17): 2927-2937

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

Julie Batut1,*,{dagger}, Bernhard Schmierer1,*, Jing Cao2, Laurel A. Raftery2, Caroline S. Hill1,§, and Michael Howell1,{ddagger},§

1 Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
2 Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Bldg. 149 13th Street, Charlestown, MA 02129, USA.


Figure 1
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  		Fig. 1. Manipulating the expression of B{alpha} and B{delta} in Xenopus embryos produces distinct phenotypes. (A) Xenopus embryos were injected with either control GFP mRNA, Flag-tagged mouse B{delta} mRNA (B{delta}), a morpholino control (MoC) or a specific morpholino against Xenopus B{alpha} (MoB{alpha}) or B{delta} (MoB{delta}) at the one-cell stage, and fixed when control embryos had reached either early gastrula or tailbud (stage 25). Representative embryos are shown, arrowheads indicate anterior. The anterior regions of embryos are magnified below. Head structures are lacking in B{delta}-injected embryos and MoB{alpha}-injected embryos. (B) Embryos were injected as in A with the indicated mRNAs and morpholinos. The effect of MoB{delta} or MoB{alpha} could be rescued by co-injection with the cognate mRNA (mouse B{delta} or B{alpha}). Percentages of embryos showing wild-type phenotype when control-injected embryos had reached stage 22 are given. Arrowheads indicate anterior.

 

Figure 2
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  		Fig. 2. Manipulating the expression of B{alpha} and B{delta} in Xenopus embryos has opposing effects on Activin/Nodal target gene expression. (A) In situ hybridisation of gastrula-stage embryos injected with either a morpholino control (MoC) or a specific morpholino against Xenopus B{alpha} (MoB{alpha}) or B{delta} (MoB{delta}), or with Flag-tagged mouse B{delta} mRNA (B{delta}) at the one-cell stage. The probes used were against gsc or Xbra. Staining was visualised with BM purple. There is increased staining for both Xbra and Gsc in B{delta} morphants, and decreased staining in B{alpha} morphants and embryos overexpressing B{delta}. (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 B{alpha} or B{delta}. Expression levels are normalised to ornithine decarboxylase (ODC).

 

Figure 3
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  		Fig. 3. B{alpha} and B{delta} act on the Activin/Nodal signalling pathway in Xenopus. (A-C) One-cell embryos were injected with the indicated mRNAs (B{alpha}, B{delta} or EGFP-Smad2) and morpholinos (MoC, MoB{alpha} or MoB{delta}). (A) Overexpression of B{delta}, but not B{alpha}, inhibits elongation of animal caps in response to Activin. (B) Knockdown of B{alpha} inhibits Activin-induced animal cap elongation, while knockdown of B{delta} promotes it. (C) EGFP-Smad2 expression rescues the effect of knocking down B{alpha} on Activin-induced animal cap elongation. When control embryos had reached stage 8, the animal pole was excised and incubated with or without Activin for 16 hours, and visualised for the degree of elongation.

 

Figure 4
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  		Fig. 4. Overexpression of Drosophila Smad2 (Smox) can rescue the effects of overexpression of the Drosophila B subunit Twins in the wing. (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; ~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 (arrows). (D) Phenotypically normal wing from A9-GAL4; UAS-tws23; UAS-Smox8D3 male. All veins terminated normally at the margin (arrows).

 

Figure 5
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  		Fig. 5. B{alpha} and B{delta} exert differential effects on the level of phosphorylated Smad2. (A) Embryos were injected at the one-cell stage with morpholinos (Mo) against B{alpha} or B{delta}, or B{delta} 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. Parts a and b show an area from the ventral vegetal region and parts c-e show an area from the dorsal vegetal region. (B) Embryos remained uninjected (ui) or were injected with two doses of mouse B{alpha} mRNA or mouse B{delta} 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 B{delta} or B{alpha}, 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 hour, treated with or without Activin for 20 minutes and processed for immunoblotting. (E) HeLa EGFP-Smad2 cells were transfected with either an siRNA SMARTpool control or a human B{alpha}- or B{delta}-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.

 

Figure 6
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  		Fig. 6. B{alpha} and B{delta} do not act directly on phosphorylated Smad2. (A) Outline of the experimental procedure to isolate B{alpha}- and B{delta}-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). Asterisk indicates that the Flag-B{delta} overlies PP2AA. (C) Western blot analysis of immunopurified complexes showing the presence of appropriate B or B'{delta} (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-phospho-Smad2 and anti-Smad2/3 antibodies. All PP2A complexes tested failed to dephosphorylate phospho-Smad2. B{alpha}- and B{delta}-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 B{alpha} and B{delta} complexes. Phosphatase complexes were immunopurified from either control cells (C) or cells expressing Flag-tagged B{alpha} or B{delta} 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.

 

Figure 7
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  		Fig. 7. B{alpha} regulates the basal level of the type I receptor and B{delta} regulates its activity. (A) Outline of the experimental procedure to isolate B{alpha}- and B{delta}-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 Fig. 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 B{delta} promotes ALK4 clustering. Animal caps from embryos expressing either HA-ALK4 mRNA alone (top row) or in combination with morpholino against B{delta} (MoB{delta}, middle row) were incubated for 1 hour 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 MoB{delta}. Okadaic acid (OA) treatment (bottom row) also induces HA-ALK4 clustering and thus mimics B{delta} knockdown. (D) B{alpha} 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) B{alpha} 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 with or without PNGase F to remove N-linked sugars from TβR-II and visualise it more clearly. (F) B{alpha} knockdown or B{delta} 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 B{delta} mRNA as indicated, cultured until uninjected embryos had reached stage 9 and analysed by immunoblotting. (G) Model of the modulation of TGF-β/Activin/Nodal signalling by B{alpha} and B{delta}. B{alpha} normally stabilises the type I receptors ALK4 and ALK5, and B{alpha} knockdown promotes their basal degradation. B{delta} normally restricts ligand-dependent activation of ALK4 and ALK5, and B{delta} knockdown facilitates such activation. When overexpressed, B{delta} additionally inhibits endogenous B{alpha} by replacing it in the PP2A holoenzyme owing to its higher affinity for the catalytic subunit (not shown).

 

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