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First published online August 12, 2008
doi: 10.1242/10.1242/dev.020842
,
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.
Authors for correspondence (e-mails:
caroline.hill{at}cancer.org.uk;
michael.howell{at}cancer.org.uk)
Accepted 1 July 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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(PPP2R2A) and B
(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 that
affect the pathway in opposite ways. Knockdown of B in Xenopus
embryos or mammalian tissue culture cells suppresses
TGF-β/Activin/Nodal-dependent responses, whereas knockdown of B
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,
B enhances TGF-β/Activin/Nodal signalling by stabilising the basal
levels of type I receptor, whereas B 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.
Key words: PP2A regulatory B subunits, TGF-β/Activin/Nodal signalling, Xenopus, Drosophila
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). 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 the responsiveness of a cell
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
kDa catalytic subunit (PP2AC) and a 65 kDa 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
B) and PPP2R2D (referred to throughout as B) have distinct and
opposing roles in the regulation of TGF-β/Activin/Nodal signalling.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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, B, B' and Xenopus
B 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 B
and B 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). Details of the sequences of the morpholino oligonucleotides
(Gene Tools) and the oligonucleotides used for RT-PCR and q-PCR can be
provided on request.
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 four
siRNA oligos (SMARTpools, Dharmacon) targeting B, B,
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. siRNA sequences can be provided on request.
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 (pH 7.5), 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-B, Flag-B or Flag-B' was 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-B or FLAG-B in
the tetracycline-inducible pcDNA5/TO construct
(Adams et al., 2005) induced
with 1 µg/ml tetracycline for 48 hours, 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 hour 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-B, Flag-B or Flag-B' 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 B and B. 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);
and (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°C. 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 with control smox/Y. Gain-of-function interaction
tests were performed at 22°C and 25°C in males, which had stronger
tws overexpression phenotypes. Wings were mounted in Euparal and
imaged digitally using a 4x objective. It has previously been reported
that larger wings result from A9-GAL4-driven expression of
P{UAS-Smox.A} (Marquez et al.,
2001). The genotype used here produced only a subtle increase in
the average wing dimensions compared with 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.
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).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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subunit as a protein whose
overexpression in early Xenopus embryos causes loss of anterior
structures. Embryos overexpressing B exhibited a delayed gastrulation,
as judged by the time of blastopore closure, and showed greatly reduced
anterior structures at the tailbud stage
(Fig. 1A; see Table S1 in the
supplementary material). By contrast, knockdown of B 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; see Table S1 in the
supplementary material). The morphant phenotype was identical when two
distinct morpholino oligonucleotides were used (data not shown), and was
rescued by co-expression of a mouse B mRNA
(Fig. 1B).
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, which we found also to be expressed in early
Xenopus embryos in a similar pattern to B (see Fig. S1 in the
supplementary material). Surprisingly, morpholino knockdown of B
resulted in a very different phenotype to the B knockdown. The embryos
exhibited a short anterior-posterior axis, but in this case anterior
structures were much reduced (Fig.
1A; see Table S1 in the supplementary material). In fact, the
phenotype was similar to that caused by overexpression of B. The effect
of B knockdown was rescued by overexpression of mouse B
(Fig. 1B). Embryos
overexpressing B were phenotypically normal
(Fig. 1B), perhaps because
B levels are not limiting in the early embryo. Consistent with the
distinct phenotypes resulting from knockdown of B and B,
overexpression of B could not rescue B morphant embryos and
overexpression of B could not rescue B morphant embryos (see
Fig. S2 in the supplementary material). We thus conclude that these two
regulatory B subunits have distinct functions in the early Xenopus
embryo.
Both B and B affect Activin/Nodal-dependent processes
The phenotype of B morphant and B-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 B 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 B and B 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
B or knockdown of B greatly inhibited the expression of
gsc and Xbra in early gastrula embryos
(Fig. 2). By contrast,
knockdown of B 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 B morphant embryos or reduced Wnt
activity in B morphant and B-overexpressing embryos (see
Fig. 5A). Thus, the observed
phenotypes are most probably due to a modulation in the intensity of Nodal
signalling, with knockdown of B promoting Nodal signalling and
knockdown of B or overexpression of B inhibiting Nodal
signalling.
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and B 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,
whereas those treated with Activin elongate and differentiate to form
mesoderm. Activin-induced elongation of animal cap explants was completely
abrogated by overexpression of B, but not of B
(Fig. 3A). Conversely,
morpholino knockdown of B enhanced cap elongation, whereas knockdown of
B completely abolished elongation
(Fig. 3B). Importantly, the
effects of B 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 EGFP-Smad2 had no effect on
Activin-induced animal cap elongation by itself
(Fig. 3C).
Altogether, these results indicate that altering the expression levels of
B or B in the early embryo modulates the Activin/Nodal
signalling pathway. Importantly, B and B affect the strength of
Activin/Nodal signalling in Xenopus in opposite directions, with
B normally acting positively and B 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, whereas no trans-heterozygous interaction was evident with
loss-of-function mutations (see Materials and methods). 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; six wings
had partially suppressed phenotypes (see Fig. S3 in the supplementary
material). Co-expression of the Baboon A isoform with Twins also suppressed,
but not as well as Smox (see Fig. S3 in the supplementary material). In
summary, the effects of Twins overexpression on wing size and vein structure
were 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 B in Xenopus.
B and B 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. TGFβ/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, compare parts a and
c). Knockdown of B or overexpression of B suppressed this Smad2
nuclear accumulation on the dorsal side, whereas knockdown of B
permitted Smad2 nuclear accumulation even on the ventral side
(Fig. 5A, top row). 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 row). In
animal cap explants expressing a constitutively active version of the
Activin/Nodal type I receptor ALK4 to mimic Nodal signalling
(Wieser et al., 1995),
overexpression of B prevented nuclear accumulation of EGFP-Smad2 (see
Fig. S4A in the supplementary material). Taken together, these results
indicate that B and B act on Activin/Nodal signalling downstream
of the ligands.
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). In accordance with the observed changes in Smad2 nuclear
accumulation, we found that overexpression of B caused a decrease in
Smad2 phosphorylation in whole embryos in response to endogenous Nodal
signalling, whereas overexpression of B had no effect
(Fig. 5B). The same was true in
animal caps in response to exogenous Activin (see Fig. S5 in the supplementary
material; data not shown). In knockdown experiments, Smad2 phosphorylation was
increased by B knockdown and decreased by B knockdown
(Fig. 5C). The effect of
B knockdown could be mimicked by a 1-hour treatment of animal caps with
the PP2A catalytic subunit inhibitor, okadaic acid
(Fig. 5D), suggesting that the
action of B 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 B
or B or their overexpression had no effect on ERK phosphorylation in
response to endogenous receptor tyrosine kinase signalling
(Fig. 5B,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 B and B in a HeLa cell
line stably expressing EGFP-Smad2. As in Xenopus, knockdown of
B decreased the nuclear accumulation of Smad2 in response to TGF-β
in these cells (Fig. 5E) and
inhibited Smad2 phosphorylation (Fig.
5F). By contrast, knockdown of B 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 B
or B (see Fig. S6A,B in the supplementary material), elicited the same
effect (see Fig. S6B,C in the supplementary material). Consistent with the
decrease in TGF-β-induced Smad2 phosphorylation caused by B
knockdown, TGF-β was also less effective at mediating growth arrest in
these conditions (see Fig. S7 in the supplementary material). Overexpression
of either B or B 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 that are not
incorporated into holocomplexes, are unstable
(Strack et al., 2002).
In conclusion, B and B modulate the level of active
phosphorylated Smad2 and hence its nuclear accumulation in both
Xenopus embryos and tissue culture cells, with B normally
promoting Smad2 phosphorylation and B inhibiting Smad2
phosphorylation.
B- and B-containing PP2A holocomplexes do not dephosphorylate pSmad2
The simplest hypothesis to explain the effects on Smad2 phosphorylation was
that B acted directly on the C-terminal phosphates of activated Smad2
and that B negated this action. We therefore immunopurified
heterotrimeric active PP2A complexes containing either Flag-tagged B or
B or a distinct regulatory subunit, B'
(Janssens and Goris, 2001),
together with the catalytic and scaffolding subunits
(Fig. 6A-C; data not shown).
These complexes were all active as they dephosphorylated a control
phospho-peptide (Fig. 6C); the
PP2A complexes containing either B or B, but not complexes
containing B', 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,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 B nor B seems to act directly on phosphorylated
Smad2.
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and B do not affect receptor kinase activity in vitro and B 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 B,
B, B'). Thus, in vitro, neither phosphatase subunit
affected receptor kinase activity.
Knockdown of B enhances ALK4 activity
We therefore investigated whether B or B affected receptor
activity in vivo. We have recently shown that in Xenopus 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 (see Fig. S8
in the supplementary material). In untreated animal caps, which exhibit very
low levels of Activin/Nodal signalling, morpholino knockdown of B 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 B
activity lowers the threshold of ligand required for ALK4 signalling,
indicating that B normally restricts receptor activity and might act to
suppress signalling at very low ligand concentrations.
Knockdown of B promotes degradation of ALK4 and ALK5
In the course of investigating whether knockdown of B would inhibit
Activin/Nodal-induced ALK4 clustering, we noticed that levels of HA-ALK4 were
extremely low when B was depleted
(Fig. 7D). Moreover,
overexpression of B, which has the same functional effects as B
knockdown, similarly reduced HA-ALK4 levels
(Fig. 7D).
These data were corroborated in HaCaT cells, where knockdown of B
led to a strong decrease in levels of endogenous ALK5 protein
(Fig. 7E). This effect was
specific, as knockdown of B or B 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 B had no effect on
endogenous ALK5 mRNA levels (see Fig. S9A in the supplementary material) and
also substantially reduced protein levels of exogenously expressed HA-tagged
ALK5 (see Fig. S9B in the supplementary material). Similarly in
Xenopus embryos, the effects of B knockdown or B
overexpression on HA-ALK4 are not at the level of transcription as the
receptor is expressed from an injected synthetic mRNA. In principle, B
could affect translation of the type I receptors, or their stability. We
favour the latter possibility as 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 B in a co-immunoprecipitation (see Fig. S10 in the
supplementary material) as has been reported previously for ALK5
(Griswold-Prenner et al.,
1998), suggesting that B 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 B knockdown in tissue culture cells. We found
that in HeLa cells overexpressing ALK5, the effects of B knockdown were
rescued by bafilomycin A1, but not MG132 (see Fig. S11A in the supplementary
material). 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 (see Fig. S11B in the supplementary material).
Taken together, these data suggest that knockdown of B (and in
Xenopus, also overexpression of B) 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 B knock-down, even though
Dapper2 knockdown alone clearly raised levels of ALK5 (see Fig. S11C in the
supplementary material). Thus, it is more likely that Dapper2 regulates PP2A
or that the two act independently.
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
and B 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 (B) and by restricting
receptor activity (B) (Fig.
7G). Perturbation of these mechanisms has dramatic functional
consequences in vivo, as demonstrated by altered gene expression and serious
developmental defects.
|
and B
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 B
and B 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 B and B have
opposing activities on a single common substrate. However, it is likely that
subunit competition within the PP2A holoenzyme does explain why B
overexpression mimics B knockdown (both manipulations causing ALK4
destabilisation in Xenopus embryos). B has a higher affinity
for the catalytic subunit than B
(Fig. 6C,E). Overexpressed
B is thus more likely to compete out endogenous B than vice
versa, and B overexpression would mimic B knockdown, whereas
B overexpression would not necessarily have an effect, as we
observe.
|
knockdown, suggesting that B 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 B
promotes receptor clustering at low endogenous ligand concentrations,
suggesting that B 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 B, our data demonstrate that
B regulates the levels of the type I receptors ALK4 and ALK5, most
probably 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, as
it is required for stabilisation of Armadillo in response to Wingless
signalling (Bajpai et al.,
2004). Consistent with our demonstration that Drosophila
Twins acts in the same way as vertebrate B, we have observed a
reduction in levels of nuclear β-Catenin in B morphant embryos
(Fig. 5A). Importantly,
however, this effect of B on the Wnt signalling pathway cannot explain
the phenotypes we observe in B morphant embryos. In our present study
it is not yet clear whether B 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 B 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 B
on type I receptor levels occurs in the absence of signalling, indicating that
B is required for regulating the basal levels of receptor and not
responsible for downregulating receptor levels after signal transduction.
|
- and B-containing phosphatase
complexes have distinct substrates, the net effects of which on the
TGF-β/Activin/Nodal pathway are opposite. This strongly suggests that the
ratio of B to B 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 material for this article is available at
http://dev.biologists.org/cgi/content/full/135/17/2927/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Université de Toulouse, Centre de Biologie du
Développement, CBD, UMR 5547, IFR 109, Bat 4R3, 118 route de Narbonne,
31062 Toulouse Cedex 9, France
Present address: High Throughput Screening Laboratory, Cancer Research UK
London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
|
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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).
