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Protein associated with SMAD1
(PAWS1/FAM83G) is a substrate for type I bone
morphogenetic protein receptors and
modulates bone morphogenetic protein
signalling
ARTICLE in OPEN BIOLOGY · FEBRUARY 2014
Impact Factor: 5.78 · DOI: 10.1098/rsob.130210 · Source: PubMed
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Research
Cite this article: Vogt J, Dingwell KS,
Herhaus L, Gourlay R, Macartney T, Campbell
D, Smith JC, Sapkota GP. 2014 Protein
associated with SMAD1 (PAWS1/FAM83G) is a
substrate for type I bone morphogenetic
protein receptors and modulates bone
morphogenetic protein signalling. Open Biol. 4:
130210.
http://dx.doi.org/10.1098/rsob.130210
Received: 21 November 2013
Accepted: 30 January 2014
Subject Area:
biochemistry/cellular biology/developmental
biology/genetics/molecular biology
Keywords:
bone morphogenetic protein, SMAD1, FAM83G,
PAWS1, ALK3, BMPR1
Author for correspondence:
Gopal P. Sapkota
e-mail: g.sapkota@dundee.ac.uk
Protein associated with SMAD1
(PAWS1/FAM83G) is a substrate
for type I bone morphogenetic
protein receptors and modulates
bone morphogenetic protein
signalling
Janis Vogt1, Kevin S. Dingwell2, Lina Herhaus1, Robert Gourlay1,
Thomas Macartney1, David Campbell1, James C. Smith2
and Gopal P. Sapkota1
1
MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dow St.,
Dundee DD1 5EH, UK
2
Division of Systems Biology, MRC National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, UK
1. Summary
Bone morphogenetic proteins (BMPs) control multiple cellular processes in
embryos and adult tissues. BMPs signal through the activation of type I BMP
receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical
pathway, this triggers the association of these SMADs with SMAD4 and their
translocation to the nucleus, where they regulate gene expression. BMPs can
also signal independently of SMAD4, but this pathway is poorly understood.
Here, we report the discovery and characterization of PAWS1/FAM83G as a
novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4independent manner, and BMP signalling induces the phosphorylation of
PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP
is essential for activation of the SMAD4-independent BMP target genes
NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD
substrate for type I BMP receptor kinases and as a novel player in the BMP
pathway. We also demonstrate that PAWS1 regulates the expression of several
non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway.
2. Introduction
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsob.130210.
The bone morphogenetic proteins (BMPs) belong to the transforming growth
factor b (TGF-b) family of ligands, and play key roles in development and
tissue homeostasis [1– 5]. BMPs control many cellular processes, including
differentiation, proliferation, survival, migration and morphogenesis in diverse
biological contexts [1], and as a result abnormal BMP signalling is associated
with the pathogenesis of several human diseases, including bone and developmental defects as well as cancer [6 –10]. The actions of BMP ligands on their
target cells are tightly regulated. This is achieved through several processes,
& 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original
author and source are credited.
3. Results
3.1. PAWS1/FAM83G associates with SMAD1
In an effort to uncover novel regulators of the BMP pathway,
we used a proteomic approach to identify partners of
SMAD1. An N-terminally FLAG-tagged SMAD1 fragment
comprising the linker and MH2 (L þ MH2) domains, or an
3.2. PAWS1 forms a complex with SMAD1 independent
of SMAD4
The observation that endogenous PAWS1 and SMAD1 interact with each other prompted us to ask whether they form a
macromolecular complex. To this end, extracts from
2
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empty vector control, were expressed in HEK293 cells
which were then immunoprecipitated with anti-FLAG antibody. The immunoprecipitates (IPs) were incubated with
cleared HeLa extracts, and interacting proteins were resolved
by SDS –PAGE, excised, digested with trypsin and identified
by mass fingerprinting (figure 1a). LEMD3, SMURF2 and
SMAD4, previously reported to be interacting partners of
SMAD1, were identified only in FLAG-SMAD1[L þ MH2]
IPs [28 –30]. We also identified a previously uncharacterized
protein termed FAM83G (figure 1a), which we renamed
protein associated with SMAD1 (PAWS1).
To verify the interaction between PAWS1 and SMAD1 and
to assess the specificity of their interaction, a mammalian
expression construct encoding PAWS1 with a FLAG tag at
the N-terminus (FLAG-PAWS1) was co-expressed in HEK293
cells with constructs encoding R-SMADs with N-terminal
haemagglutinin (HA)-tags. HA-SMAD1 was identified in
FLAG-PAWS1 IPs, whereas HA-SMADs 2 and 3 were not
(figure 1b). The expression of HA-SMAD5 and HA-SMAD8
was too low to assess their interactions with FLAG-PAWS1
(figure 1b). To overcome this, FLAG-PAWS1 was co-expressed
in HEK293 cells with constructs encoding SMADs 1, 5 and 8
containing N-terminal GFP tags (the electronic supplementary
material, figure S1a). GFP-SMAD1, GFP-SMAD5 and GFPSMAD8 were all identified in FLAG-PAWS1 IPs, suggesting
that BMP-SMADs interact with PAWS1. GFP-SMAD4 did
not interact with FLAG-PAWS1 (the electronic supplementary
material, figure S1b).
To map the interaction between PAWS1 and SMAD1, FLAGPAWS1 was co-expressed with N-terminal HA-tagged truncation fragments of SMAD1 in HEK293 cells (figure 1c). As
expected, FLAG-PAWS1 interacted with full-length SMAD1
(figure 1c). Of the SMAD1 fragments, only the HA-MH2
domain of SMAD1 interacted with FLAG-PAWS1, whereas the
HA-MH1 þ linker domain did not interact (figure 1c). The
expression of the HA-MH1 domain or the HA-linker domain
was not detected. We also co-expressed N-terminal FLAGtagged truncation fragments of SMAD1 in HEK293 cells with
HA-PAWS1. HA-PAWS1 was detected in FLAG IPs of WT
SMAD1, the MH1 domain and the MH2 domain, but not the
linker (the electronic supplementary material, figure S1c).
To ask whether endogenous SMAD1 and PAWS1 interact,
SMAD1 IPs from human keratinocyte HaCaT extracts were
subjected to immunoblot analysis with an anti-PAWS1 antibody (figure 1d). Endogenous PAWS1 was detected in
SMAD1 IPs but not in IPs using pre-immune IgG (figure 1d).
SMAD1 IPs also failed to pull down PAWS1 from HaCaT
cells transfected with PAWS1 siRNA, which resulted in
almost complete loss of PAWS1 protein expression (figure 1d).
Similarly, we detected endogenous SMAD1, but not
SMAD2/3, in PAWS1 IPs from HaCaT cell extracts (figure 1e).
Treatment of cells with BMP or TGF-b, to induce phosphorylation of SMAD1 and SMAD2/3, respectively, did not
significantly alter the association of PAWS1 with SMAD1 or
SMAD2/3 in extracts (figure 1e).
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from limiting access of BMPs to their receptors by secreted
molecules such as noggin, to the regulation of the activities
of the downstream pathway components [11 –14].
Upon binding their cognate receptor serine/threonine kinase
pairs, BMP ligands facilitate the phosphorylation and activation
of type I BMP receptors (ALKs 2, 3 and 6) by type II BMP receptors (BMPRII, ActRIIA and ActRIIB). The type I receptors, in
turn, phosphorylate the highly conserved receptor-regulated
SMAD transcription factors (R-SMADs 1, 5 and 8) on two
serine residues at their conserved C-terminal tail SXS motifs.
The phosphorylation of R-SMADs triggers their association
with SMAD4 and their subsequent translocation to the nucleus,
where SMAD transcriptional complexes assemble to regulate the
expression of hundreds of target genes [14,15]. The SMAD4dependent transcriptional programme driven by the BMP
ligands is often referred to as ‘canonical’ BMP signalling.
Consistent with the central role that SMAD4 plays in BMP
and TGF-b signalling, the loss of SMAD4 expression is a
common feature in many human cancers [16,17]. However,
many studies have suggested that BMP ligands can also drive
SMAD4-independent and, in some cases, even SMAD1/5/8independent signalling, collectively termed as ‘non-canonical’
BMP signalling [18–23]. For example, in SW480 colorectal
cancer cells, which are SMAD4-deficient, BMPs modulate the
transcription of about 90 genes, including NEDD9, ASNS and
PTEN [18,23], and non-canonical signalling influences a range
of cellular responses, including the suppression of cell proliferation and chemotaxis [19–21,23]. However, the mechanisms by
which BMP activates non-canonical signalling remain elusive.
In the course of a proteomic approach aimed at uncovering
novel regulators of the BMP pathway, we identified FAM83G
(hereafter referred to as protein associated with SMAD 1;
PAWS1) as a SMAD1 interactor. PAWS1 is conserved in
vertebrates but no biochemical roles have yet been reported.
PAWS1 belongs to a family of hypothetical proteins,
FAM83A–H, defined by the presence of a conserved N-terminal
domain of unknown function termed DUF1669, which contains
a putative pseudo-phospholipase D motif [24]. Recently,
FAM83A and B have been reported to be oncogenes and
mediators of resistance to tyrosine kinase inhibitors [25,26].
Mutations in FAM83H have been implicated in amelogenesis
imperfecta, a condition characterized by dental-enamel defects
[27]. However, the precise biochemical roles of the FAM83
family of proteins remain undefined.
Here, we demonstrate that PAWS1 forms a macromolecular complex with SMAD1 that is independent of SMAD4. In
addition, we show that PAWS1 is a novel substrate for
ALK3 and that BMP-induced phosphorylation of PAWS1
regulates the expression of the SMAD4-independent BMP
target genes NEDD9 and ASNS. In the course of our experiments, we show that PAWS1 regulates the BMP pathway
and that it can regulate the expression of several genes
independent of BMP stimulation.
(a)
(c)
FLAG control
FLAG-SMAD1[L + MH2]
MH-1
–
+
250
150
100
75
IPs
MH-1
SMAD1(L+MH2)
25
20
15
10
MH-2
SXS
3
HA-SMAD2
– – + – – – – – + – – –
HA-SMAD3
HA-SMAD5
– – – + – – – – – + – –
– – – – + – – – – – + –
HA-SMAD8
– – – – – + – – – – – +
linker
– – – – – – + + + + + +
– + – – – – – + – – – –
HA-SMAD1 MH-1
– – + – – – – – + – – –
HA-SMAD1 MH-1+linker
HA-SMAD1 linker
– – – + – – – – – + – –
– – – – + – – – – – + –
HA-SMAD1 MH-2
– – – – – + – – – – – +
IB: FLAG
IP: FLAG
IB: HA
IB: FLAG
IB: FLAG
input
IP: FLAG
IB: HA
IB: HA
IB: FLAG
input
IB: HA
IB: GAPDH
(d)
S1
+
–
S1
–
+
input
IgG
siControl +
siPAWS1 –
IgG
IP
input
+
–
–
+
–
+
(e)
–
TGF-b
IP: PAWS1
BMP
–
TGF-b
input
BMP
–
TGF-b BMP
IB: PAWS1
IB: P-SMAD1
IB: SMAD1
IB: P-SMAD2
IB: SMAD2/3
IB: GAPDH
IB: PAWS1
IB: SMAD1
IB: SMAD1/protein G-HRP
(no heavy chain detection)
Figure 1. PAWS1 interacts with SMAD1. (a) Anti-FLAG IPs from HEK293 extracts transfected with vectors either encoding FLAG control or FLAG-tagged SMAD1[L þ
MH2] fragment were incubated with HeLa extracts. Elution was performed with 3X FLAG peptide. Eluted proteins were denatured and resolved by SDS – PAGE and
the gel was Coomassie-stained. Gel pieces (2 mm) covering the entire lane of each sample were excised for identification by mass fingerprinting. The positions of
some of the identified proteins are indicated. (b) HEK293 cells were transfected with the indicated HA-SMAD constructs either alone or together with FLAG-PAWS1
construct. The extracts and anti-FLAG IPs were analysed by immunoblotting using the indicated antibodies. (c) HEK293 cells transfected with constructs encoding
either HA-SMAD1 or indicated HA-SMAD1 truncation mutants either individually or together with construct encoding FLAG-PAWS1. The extracts and anti-FLAG IPs
were analysed by immunoblotting using the indicated antibodies. (d ) HaCaT cells were transfected with a pool of two different siRNAs against either PAWS1
(150 pM each), or with siRNA against FOXO4, for 48 h prior to lysis. Extracts and IPs, using either anti-SMAD1 antibody or pre-immune IgG, were analysed by
immunoblotting using the indicated antibodies. For SMAD1/protein-G-HRP immunoblot, the membrane was first blocked in 5% milk containing 500 ng ml21
protein G, incubated with SMAD1 antibody as primary, and protein-G HRP was used as secondary. This strategy excludes the detection of antibody heavy
chains in IP samples. (e) HaCaT cells were treated with either BMP-2 (25 ng ml21; 1 h), TGF-b (50 pM; 1 h) or left untreated prior to lysis. Extracts and
anti-PAWS1 IPs were analysed by immunoblotting with the indicated antibodies.
untreated HaCaT cells or from cells treated with BMP or
TGF-b were separated into 32 fractions by size-exclusion
chromatography (figure 2). Under basal unstimulated conditions, SMAD1 and SMAD2/3 were mostly detected in
fractions corresponding to their predicted molecular weights
(approx. 50 –55 kDa), indicating that they exist predominantly
as monomers (fractions X –Z; figure 2a). BMP stimulation,
which causes an increase in phosphorylation of SMAD1 over
basal levels, caused a portion of SMAD1 and phosphoSMAD1 to elute in slightly higher-molecular-weight fractions
(fractions V –W as well as X –Z; figure 2b). TGF-b stimulation,
which induces phosphorylation of SMAD2 and SMAD3,
caused a more dramatic change in the elution profile of
phospho-SMADs 2 and 3, which were now detected in fractions corresponding to much higher molecular weights
(fractions T –Y; figure 2c).
Consistent with the idea that activated R-SMADs form a
complex with SMAD4 [15], BMP-induced phospho-SMAD1
(figure 2b) and, in particular, TGF-b-induced phospho-SMADs
2 and 3 (figure 2c) eluted in fractions that overlapped with
those containing SMAD4 (fractions T–X; figure 2b,c).
Surprisingly, the elution profile of SMAD4 itself was unchanged
by BMP or TGF-b stimulation, suggesting that SMAD4 exists
in an oligomeric state with itself or with other proteins prior to
formation of active R-SMAD/SMAD4 complexes (figure 2a–c).
In extracts from untreated, BMP-treated and TGF-b-treated
cells, PAWS1 (whose predicted molecular weight is 91 kDa)
eluted in fractions corresponding to greater than 670 kDa (predominantly fractions O and P; figure 2a–c). A portion of
phospho-SMAD1 was also detected in these fractions, irrespective of whether cells had been untreated or treated with BMP
or TGF-b. The presence of total SMAD1 was confirmed by
immunoblotting SMAD1 IPs from these fractions (the electronic supplementary material, figure S1b). PAWS1 elution
did not overlap with that of SMAD4- or of TGF-b-induced
phospho-SMADs 2 or 3 (figure 2a,c). This is consistent with
our observations that PAWS1 does not interact with SMAD2
or 3 (figure 1b,e) or SMAD4 (the electronic supplementary
Open Biol. 4: 130210
HA-SMAD1
– – – – – – + + + + + +
– + – – – – – + – – – –
FLAG-PAWS1
SXS
FLAG-PAWS1
HA-SMAD1
37
(b)
MH-2
linker
LEMD3
PAWS1
SMAD4
50
linker
MH-1
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+
–
670 kDa
158 kDa 44 kDa
eluted fractions
IB: PAWS1
IB: PAWS1
IB: P-SMAD1
IB: P-SMAD1
IB: P-SMAD2
IB: P-SMAD3
IB: SMAD1
IB: P-SMAD2
IB: P-SMAD3
IB: SMAD2/3
IB: SMAD2/3
IB: SMAD4
IB: SMAD4
670 kDa
158 kDa 44 kDa
L M N O P Q R S T U V WX Y Z
eluted fractions
IB: PAWS1
TGF-b stimulated
IB: P-SMAD1
IB: SMAD1
IB: P-SMAD2
IB: P-SMAD3
IB: SMAD2/3
IB: SMAD4
Figure 2. Size-exclusion chromatography. (a) Unstimulated HaCaT cell extracts were fractionated by gel filtration chromatography on a Superose 10/300 GL column
(GE Healthcare). Five microlitres of each recovered fraction was resolved by SDS –PAGE and subsequently analysed by immunoblotting using the indicating antibodies. (b) Same as (a) except the cells were treated with BMP-2 (25 ng ml21) for 1 h prior to lysis. (c) Same as (a) except the cells were treated with TGF-b
(50 pM) for 1 h prior to lysis.
material, figure S1b). Together, these findings suggest that a
portion of SMAD1 forms a macromolecular complex with
PAWS1 that is independent of SMAD4.
3.3. PAWS1 does not affect the extent or kinetics of
bone morphogenetic protein-induced SMAD1
phosphorylation
PAWS1 is expressed in many mouse tissues and in many
human cell lines, although not in PC3 prostate cancer cells
(the electronic supplementary material, figure S2a,b). To investigate the role of PAWS1 in BMP signalling, we therefore made
use of PC3 and HaCaT cells. Because PC3 cells lack endogenous PAWS1, we stably reintroduced, by retroviral infection,
either a control vector (PC3-control) or a vector encoding
human PAWS1 (PC3-PAWS1). PC3-control cells did not
express PAWS1, and PC3-PAWS1 cells expressed PAWS1 at
levels comparable to those seen in HaCaT cells (figure 3a).
To explore the effect of PAWS1 on BMP-induced phosphorylation of SMAD1, PC3-control and PC3-PAWS1 cells
were treated with BMP and assayed at intervals thereafter
(figure 3b). In both cell types, BMP induced SMAD1
phosphorylation within 15 min, the levels reaching a maximum by 1 h and falling thereafter (figure 3b). PAWS1 had
no detectable effect on the kinetics or extent of BMP-induced
SMAD1 phosphorylation, or on the levels of SMAD1 protein
(figure 3b). To ask whether PAWS1 affects cellular sensitivity
to BMP signals, PC3-control and PC3-PAWS1 cells were
treated with increasing amounts of BMP, and SMAD1 phosphorylation was monitored by immunoblotting. There was
no significant difference in the levels of phospho-SMAD1 in
the two cell types (figure 3c).
To confirm that PAWS1 does not affect BMP-induced phosphorylation of SMAD1, a loss-of-function study was performed.
HaCaT cells were transfected with siRNA oligonucleotides targeting PAWS1 or (as a control) FOXO4, and treated with BMP.
In cells transfected with PAWS1 siRNA, PAWS1 protein
expression was depleted by approximately 90% compared
with control. PAWS1 depletion did not significantly alter the
levels of phospho-SMAD1 induced by BMP (figure 3d).
Treatment of cells with BMP causes the nuclear translocation
of phospho-SMAD1 [15]. To examine the subcellular localization
of PAWS1, control- or ligand-stimulated HaCaT cells were separated into nuclear and cytosolic fractions. Under basal- or TGF-bstimulated conditions, PAWS1 was detected predominantly in
4
Open Biol. 4: 130210
unstimulated
L M N O P Q R S T U V WX Y Z
eluted fractions
IB: SMAD1
(c)
158 kDa 44 kDa
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L M N O P Q R S T U V WX Y Z
670 kDa
(b)
BMP stimulated
(a)
PC3 PAWS1
PC3 control
HaCaT
(d)
siPAWS1
–
–
+
+
siControl
+
+
–
–
BMP
–
+
–
+
5
IB: PAWS1
IB: PAWS1
IB: SMAD1
IB: P-SMAD1
IB: GAPDH
IB: SMAD1
IB: GAPDH
HaCaT cells
0 15 30 60 240 360 0 15 30 60 240 360 BMP (min)
+ + + + + + – – – – – – PC3 control
– – – – – – + + + + + + PC3 PAWS1
IB: PAWS1
(e)
IB: P-SMAD1
IB: SMAD1
Cytosolic/Nuclear
C N C N C N
WCL
– – T T B B
BMP/TGF-b
*
IB: PAWS1
IB: GAPDH
IB: P-SMAD1
(c)
0
+
–
5
+
–
10
+
–
25
+
–
50
+
–
0
–
+
5
–
+
10
–
+
25
–
+
50 BMP (ng ml–1)
– PC3 PAWS1
+ PC3 control
IB: PAWS1
IB: SMAD1
IB: P-SMAD2
IB: SMAD2/3
IB: P-SMAD1
IB: GAPDH
IB: SMAD1
IB: lamin A/C
IB: GAPDH
* PAWS1 siRNA transfected
Figure 3. Effect of PAWS1 on BMP-induced SMAD1 phosphorylation. (a) Extracts (20 mg protein) from either HEK293, HaCaT, PC3-control (PC3 cells stably integrated
with a control vector), or PC3-PAWS1 cells (PC3 cells stably integrated with a vector encoding wild-type PAWS1) were resolved by SDS – PAGE and analysed by
immunoblotting using the indicated antibodies. (b) PC3-control and PC3-PAWS1 cells were treated with BMP-2 (25 ng ml21) for the indicated time (min) prior to
lysis. Extracts (20 mg protein) were resolved by SDS – PAGE and analysed by immunoblotting using the indicated antibodies. (c) PC3-control and PC3-PAWS1 cells
were treated with the indicated concentrations of BMP-2 for 1 h prior to lysis. Extracts (20 mg protein) were resolved by SDS – PAGE and analysed by immunoblotting using the indicated antibodies. (d ) PAWS1-depleted HaCaT cells (siPAWS1) and HaCaT cells expressing FOXO4 siRNA (siControl) were treated either with or
without BMP-2 (25 ng ml21) for 1 h prior to lysis. Extracts (20 mg protein) were resolved by SDS – PAGE and immunoblotted with the indicated antibodies.
(e) Extracts from HaCaT cells treated with either BMP-2 (25 ng ml21) or TGF-b (50 pM) for 1 h, or left untreated were separated into cytosolic and nuclear fractions.
Fractions and whole cell lysates (WCLs) were resolved by SDS – PAGE and analysed by immunoblotting using the indicated antibodies. GAPDH and lamin A/C were
used as cytosolic and nuclear controls, respectively.
the cytosolic fractions. However, upon BMP stimulation, a
small portion of PAWS1 was detected in the nuclear fraction.
As expected, phospho-SMAD1 and phospho-SMAD2 were
detected in the nuclear fractions upon BMP and TGF-b stimulations respectively (figure 3e). Lamin A/C and GAPDH used
as controls were detected in the nuclear fraction and cytosolic
fraction respectively (figure 3e).
Our attempts to explore the intracellular localization of
PAWS1 by immunofluorescence were unsuccessful: neither of
our antibodies proved suitable for endogenous immunostaining.
3.4. PAWS1 is phosphorylated by type I bone
morphogenetic protein receptor in vitro and in vivo
The observations that SMAD1 and PAWS1 interact in a complex, and that a portion of PAWS1 translocates to the nucleus
upon BMP treatment, prompted us to ask whether BMP signalling causes a post-translational modification to occur within
PAWS1. We therefore generated HEK293 cells carrying a
single copy of GFP-PAWS1 and used mass spectrometry to
analyse phospho-modification of material immunoprecipitated
from control cells or cells treated with BMP. BMP-treated cells,
but not controls, proved to contain a triphosphopeptide corresponding to residues 608–623 (RPSVASSVSEEYFEVR) of
human PAWS1. Our mass spectrometric analysis established
Ser610 as one of the phosphosites, but was unable to establish
the two remaining phosphoresidues within the peptide.
We note that this PAWS1 peptide is highly conserved
among vertebrate PAWS1 orthologues (figure 4a), and that
the SSVS motif, corresponding to residues 613–616 of
PAWS1, is identical to the SSXS motif at the C-termini of RSMADs (figure 4a). The second and third serine residues
within the SSXS motif of SMADs 1, 5 and 8 are phosphorylated
Open Biol. 4: 130210
(b)
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HEK293
(a)
(c) 3500
BMP
32P
2500
acetonitrile %
90
80
70
60
50
40
30
20
10
0
961.4382
2000
1500
P2
32P
1000
P3
500
0
0
(d)
20
40
60
6
80 100 120 140 160 180 200 220
time (min)
1000
(cpm)
32P
500
100
autorad image 75
–
+
–
+
–
–
+
+
P2
200
100
32P
+
–
–
+
(cpm)
(e) 300
PAWS1 (523-end)
Smad1
Smad2
BMPR1A (ALK3)
kDa
32P
R P S V A S S V S E E Y F E V R
amino acid
0
(f)
R P S V A S S V S E E Y F E V R
amino acid
PAWS1(523-end)
PAWS1(523-end)-S610A
50
PAWS1(523-end)-S613A/S614A/S616A
100
Coomassie- 75
stained gel
50
ALK3
kDa 75
32P autorad image
50
– + –
– – +
– – –
– – –
– – + – –
– – – + –
+ – – – +
– + + + +
75
Coomassie-stained gel
50
Figure 4. Phosphorylation of PAWS1 by BMPR1A (ALK3). (a) GFP IPs from HEK293 cells stably expressing GFP-PAWS1 treated either with or without BMP-2
(25 ng ml21) were resolved by SDS – PAGE. The gel was Coomassie-stained, and bands representing GFP-PAWS1 were excised, digested with trypsin and phosphopeptides identified by mass spectrometry. The sequence alignment of the PAWS1 triphosphopeptide identified upon BMP treatment compared with other
vertebrates is shown. Also shown for comparison is the sequence alignment of the SSVS motif in different R-SMADs. h, human; m, mouse; x, Xenopus laevis.
(b) Kinase assays were set up with BMPR1A (ALK3) using GST-SMAD1, GST-SMAD2 and GST-PAWS1 (523-end) as substrates using g32P-ATP as described in
the methods. Samples were resolved by SDS – PAGE, the gel was Coomassie-stained and radioactivity was analysed by autoradiography. (c) GST-PAWS1(523end) phosphorylated by BMPR1A in B was excised, digested with trypsin and resolved by HPLC on a C18 column using an increasing acetonitrile gradient as indicated. Three peaks (P1 –3) of 32P radioactivity release were observed. Analysis of peak P1 by LC – MS – MS revealed the phosphopeptide RPSVASSVSEEYFEVR, with an
observed m/z of 961.4382[2þ]. Similarly, peak P2 revealed the diphosphopeptide RPSVASSVSEEYFEVR, with observed m/z of 1001.42 [2þ]. (d ) Solid-phase sequencing of peak P1 showed the 32P radioactivity after the third cycle of Edman degradation. (e) Solid-phase sequencing of peak P2 revealed the release of 32P
radioactivity after the seventh and ninth cycles of Edman degradation. Amino acid sequences in (d,e) were deduced from LC – MS –MS analysis. ( f ) As in (b)
except that BMPR1A (ALK3) was incubated in a kinase assay with GST-PAWS1(523-end), GST-PAWS1(523-end)S610A or GST-PAWS1(523-end)S613A/S614A/
S616A used as substrates.
by type I BMP receptor kinases, causing their translocation to
the nucleus [15]. We therefore reasoned that PAWS1 might
be a novel target for BMP type I receptor kinases. To date,
no non-SMAD substrates for BMP type I receptor kinases
(ALKs 2, 3 and 6) have been reported. To test this idea, we
established an in vitro kinase assay using a GST-PAWS1(523–
823) fragment as a substrate for BMPR1A (ALK3). PAWS1,
like SMAD1, was phosphorylated in vitro by ALK3, whereas
SMAD2, used as a negative control, was not (figure 4b). Activated versions of the type I BMP receptors ALK2 and ALK6
also phosphorylated PAWS1 in vitro (the electronic supplementary material, figure S3), and this phosphorylation was
inhibited by LDN193189, a potent inhibitor of type I BMP
receptor kinases [8,31] (the electronic supplementary material,
figure S3).
We sought to map the in vitro ALK3 phosphorylation sites
within PAWS1 by a combination of mass spectrometry and
solid-phase Edman sequencing. 32P-labelled GST-PAWS1
phosphorylated by ALK3 was digested with trypsin, and the
resulting peptides were separated by reverse-phase chromatography on a C18 column. Three 32P-labelled peaks, one major
(P1) and two minor (P2 and P3), eluted at 26%, 25% and
24% acetonitrile, respectively (figure 4c). The molecular mass
of P1 determined by mass spectrometry (961.4382 Da) corresponded to that of a tryptic phosphopeptide comprising
residues 608–623 with a single phosphorylation modification.
32
P radioactivity was released after the third cycle of Edman
degradation (figure 4d), confirming that phosphorylation of
PAWS1 occurs at Ser610. For P2, 32P radioactivity was released
after the seventh and the ninth cycles of Edman degradation,
Open Biol. 4: 130210
P1
0
(b)
rsob.royalsocietypublishing.org
PAWS1
P1
3000
percentage acetonitrile
–
radioactivity (cpm)
(a)
To investigate the significance of BMP-induced phosphorylation of PAWS1 in vivo, we raised a phospho-specific
antibody recognizing PAWS1 phosphorylated at Ser610
(PAWS1-S610P; figure 5a). We also generated PC3 cells
stably integrated with a PAWS1(S610A) mutant. In PC3PAWS1 cells, treatment with BMP, but not TGF-b, resulted
in the phosphorylation of PAWS1 at Ser610, as detected by
our phospho-specific antibody. By contrast, the PAWS1S610P antibody did not detect a product in PC3-control
cells or in PC3-PAWS1(S610A) cells, confirming the specificity of this reagent (figure 5a). The introduction of
wild-type or the S610A mutant version of PAWS1 in PC3
cells did not significantly alter the levels of BMP-induced
phospho-SMAD1 (figure 5a).
We next asked whether BMP induces the phosphorylation
of endogenous PAWS1 at Ser610 in HaCaT cells. Treatment of
HaCaT cells with BMP indeed caused phosphorylation of
PAWS1 at Ser610, and this was inhibited by LDN-193189
(figure 5b). The time-course of BMP-induced PAWS1 phosphorylation mirrored that of the phosphorylation of the tail
of SMAD1 (the electronic supplementary material, figure
S5). Interestingly, phosphorylation of Ser610 of PAWS1
does not affect its ability to interact with SMAD1 (figure 5c).
BMP signalling regulates target gene expression in SMAD4dependent and -independent manners [23,32]. For example,
BMP induces ID1 and SnoN in an SMAD4-dependent manner
[32], whereas genes such as NEDD9 and ASNS can be activated
in cells lacking SMAD4 (figure 5d,e and the electronic supplementary material, figure S6b; [23]). Because PAWS1 forms a
complex with SMAD1 independent from SMAD4, we reasoned
that induction of SMAD4-independent BMP target genes might
occur through PAWS1. Consistent with this suggestion, BMP
induced NEDD9 and ASNS expression in PC3-PAWS1 cells,
3.6. PAWS1 regulates the expression of non-bone
morphogenetic protein target genes
To explore further the ability of PAWS1 to regulate gene
expression, we asked whether the introduction of PAWS1
into PC3 cells regulates the expression of 155 known TGFb/BMP target genes (figure 6a and the electronic supplementary material, figure S7a,b). Expression of 20 genes proved to
be changed by more than twofold upon restoration of PAWS1
(figure S7a and the electronic supplementary material, S5a,b).
Among these, we confirmed by RT-PCR that expression of
FST, TGFBI and TGFBR2 was augmented, whereas
expression of TSC22D was diminished (figure 6b and the
electronic supplementary material, figure S7c).
To ensure that these changes in gene expression were
directly linked to PAWS1, we depleted PAWS1 in HaCaT cells
by RNAi and confirmed that expression of both FST and
TGFBI were reduced (figure 6c). However, we also found that
BMP treatment of PC3-control or PC3-PAWS1 cells did not
alter expression of FST, TGFBI, TGFBR2 or TSC22D (figure 6c
and the electronic supplementary material, S6c). Similarly,
BMP treatment did not affect the expression of FST and
TGFBI in control HaCaT cells or those expressing PAWS1
siRNA (figure 6c). These results suggest that PAWS1 also regulates gene expression in a manner that is independent of BMP
treatment. This is discussed below. We also tested the effect of
PAWS1 on the expression of a canonical TGF-b and BMP
target gene, SnoN. The expression of SnoN induced by BMP
or TGF-b was identical in both PC3-control and PC3-PAWS1
cells, implying that PAWS1 had no effect on the expression of
SnoN (figure 6d).
4. Discussion
Our experiments show that PAWS1 forms a complex with
SMAD1 in a SMAD4-independent manner; that it is a
target of type I BMP receptor kinases (and is the first such
non-SMAD target to be identified); and that it is a novel
player in the BMP signal transduction pathway. Of particular
significance, PAWS1 regulates the expression of some
SMAD4-independent BMP target genes as well as some
BMP-independent genes.
4.1. A non-canonical PAWS1–SMAD1 complex
PAWS1 interacts with SMAD1 but not with SMAD2/3.
Although the linker domain of the R-SMADs is the least conserved region, it is the SMAD1-MH2 domain that mediates
the interaction with PAWS1 and presumably provides the
observed specificity. Interaction of the R-SMADs with
SMAD4 to form an active complex [15] occurs following the
7
Open Biol. 4: 130210
3.5. Phosphorylation of PAWS1 at Ser610 regulates the
expression of bone morphogenetic proteindependent SMAD4-independent target genes
but not in PC3-control cells and not in PC3-PAWS1(S610A)
cells, further suggesting that phosphorylation of PAWS1 at
Ser610 is necessary for BMP-induced activation of these genes
(figure 5f ). Expression of BMPR2 was unaffected by the presence of wild-type PAWS1 or the S610A mutant in PC3 cells
(the electronic supplementary material, figure S6a). BMPinduced expression of the SMAD4-dependent target gene ID1
was not affected significantly by restoration of wild-type
PAWS1 expression in PC3 cells (the electronic supplementary
material, figure S7c).
rsob.royalsocietypublishing.org
consistent with phosphorylation at Ser614 and Ser616 of
PAWS1 (figure 4e). There was not enough material for analysis
of the phosphorylation sites within peak P3.
These results indicate that ALK3 phosphorylates PAWS1
predominantly at Ser610 but can also phosphorylate at
Ser614 and Ser616 in vitro. Consistent with this conclusion,
mutation of Ser610 to Ala almost completely abolished phosphorylation of PAWS1 by ALK3 in vitro (figure 4f ), and the
major radioactive peak corresponding to Ser610 (P1 in figure
4c) was lost when the tryptic fragments of PAWS1(S610A)
phosphorylated by ALK3 were subjected to reverse-phase
HPLC as above (the electronic supplementary material,
figure S4). Peak P2, corresponding to Ser614/Ser616 phosphorylation on PAWS1, was unaffected (the electronic
supplementary material, figure S4), and indeed, this dual
Ser614/Ser616 phosphorylation was confirmed by Edman
degradation and mass spectrometry (the electronic supplementary material, figure S4). Mutation of Ser613, Ser614 and Ser616
to Ala resulted in a significant but not complete inhibition of
phosphorylation of PAWS1 by ALK3 in vitro (figure 4f ). It is
Ser614 and Ser616 that correspond to the sites in the SMAD1
SSVS motif that are phosphorylated by ALK3, so it was surprising that PAWS1 is phosphorylated predominantly at
Ser610; this is discussed below.
(a)
(b)
T
B
–
T
B
–
T
B
TGFb/BMP
–
–
–
–
–
–
+
+
+
PC3-PAWS1(S610A)
+
+
+
–
–
–
–
–
–
PC3-PAWS1
–
–
–
+
+
+
–
–
–
PC3-control
BMP
–
+
+
LDN-193189
–
–
+
8
IP: PAWS1;
IB: P-PAWS1 S610
IB: PAWS1
IP: PAWS1;
IB: P-SMAD1
IB: P-PAWS1 S610
IB: PAWS1
IB: SMAD1
IB: P-SMAD1
IB: GAPDH
(c)
PC3
BxPC3
SW480
HaCaT
IB: GAPDH
(d)
IB: PAWS1
IP: SMAD1
PC3-PAWS1
+
–
–
PC3-control
–
+
–
PC3-PAWS1(S610A)
–
–
+
IgG
input
+
–
–
IB: SMAD1
–
+
–
IB: SMAD4
–
–
+
IB: GAPDH
IB: PAWS1
IB: SMAD1
IB: GAPDH
(e)
ASNS
NEDD9
SW480
2.5
p < 0.0001
p < 0.0001
2.5
2.0
fold change
(f)
ASNS
PC3 control
PC3 PAWS1
PC3 PAWS1 S610A
2.0
1.5
1.5
1.0
1.0
0.5
0.5
NEDD9
p = 0.0293
–
BMP 2
treatment
BMP 4/7
3
PC3 control
PC3 PAWS1
PC3 PAWS1 S610A
2
1
0
0
0
4
p = 0.0135
PC3
PC3
PC3
control PAWS1 PAWS1
S610A
PC3
PC3
PC3
control PAWS1 PAWS1
S610A
Figure 5. The role of PAWS1 in the BMP pathway. (a) PC3-control, PC3-PAWS1 and PC3-PAWS1(S610A) cells were treated with either BMP-2 (25 ng ml21), TGF-b
(50 pmol) or left untreated for 1 h prior to lysis. PAWS1 was immunoprecipitated from cell extracts (1 mg protein) using anti-PAWS1 antibody. Anti-PAWS1 IPs and
extract inputs (20 mg protein) were resolved by SDS – PAGE and immunoblotted with the indicated antibodies. (b) HaCaT cells were either left unstimulated or
stimulated with BMP-2 (25 ng ml21) or BMP-2 and LDN193189 (100 nM) for 1 h prior to lysis. PAWS1 was immunoprecipitated from cell extracts (1 mg protein)
using anti-PAWS1 antibody. Anti-PAWS1 IPs and extract inputs (20 mg protein) were resolved by SDS – PAGE and immunoblotted with the indicated antibodies. (c)
PC3-control, PC3-PAWS1 and PC3-PAWS1(S610A) cells were lysed and SMAD1 immunoprecipitated from extracts (1 mg protein) using anti-SMAD1 antibody. IP using
pre-immune IgG was used as control from PC3-PAWS1 cell extracts (1 mg protein). SMAD1 IPs, IgG IP and extract inputs (20 mg protein) were resolved by SDS –
PAGE and immunoblotted with the indicated antibodies. (d ) Extract inputs (20 mg protein) from HaCaT, SW480, BxPC3 and PC3 cells were resolved by SDS– PAGE
and analysed by immunoblotting using the indicated antibodies. (e) SW480 cells were either treated with BMP-2 (25 ng ml21) and BMP-2/7 (10 ng ml21 each) or
left untreated for 6 h prior to RNA isolation. The relative expression of the indicated genes was analysed by qRT-PCR as described in the methods. The results show
the fold change in gene expression relative to unstimulated controls. Data are represented as mean of three biological replicates and error bars indicate standard
deviation (n ¼ 3). ( f ) PC3-control, PC3-PAWS1 and PC3-PAWS1(S610A) cells were either treated with BMP-2 (25 ng ml21) or left untreated for 6 h prior to RNA
isolation. The relative expression of the indicated genes was analysed by qRT-PCR as described in §5. The results show the fold change in gene expression relative to
unstimulated controls. Data are represented as mean of three biological replicates and error bars indicate standard deviation (n ¼ 3).
ligand-induced phosphorylation of R-SMADs at the SXS
motif within the MH2 domain. Further work is required to
understand the nature of the interaction between PAWS1
and the MH2 domain of SMAD1.
Most SMAD1 in our cells forms a ‘canonical’ complex
with SMAD4 upon BMP treatment. However, in both controland BMP-treated extracts, a subfraction of SMAD1 associates
with PAWS1 in a high-molecular-weight complex that does
not include SMAD4. We do not know the identity of the
other proteins in this complex, but our results suggest that
it plays a hitherto unrecognized role in BMP signalling. The
interaction between PAWS1 and SMAD1 is not affected by
treatment of cells with BMP or TGF-b, suggesting that the
association is constitutive. BMP treatment of cells causes
Open Biol. 4: 130210
IB: SMAD1
rsob.royalsocietypublishing.org
–
(a)
(c)
FST
change PAWS1/control
log fold change
FST
0.4
TGFBR2
fold change
0.6
fold change
0.8
p < 0.0001
2
1
30
20
10
0
0.2
0
siPAWS1
0
p = 0.0284
siControl
siPAWS1
siControl
ID1
–0.2
PAWS1
–0.4
30
–0.8
TSC22D
genes below threshold
genes above threshold
control
p < 0.0001
BMP
20
HaCaT cells
10
0
siPAWS1
(b)
control
(d)
BMP
TGFBI
FST
4
40
30
2
20
1
10
0
PC3PAWS1
PC3 cells
p < 0.0001
3
2
p < 0.01
unstimulated
BMP
TGF-b
p < 0.0213
1
0
PC3control
SnoN
p < 0.0001
p < 0.0001
p < 0.0001
fold change
fold change
3
siControl
PC3control
PC3PAWS1
0
PC3-control
PC3-PAWS1
PC3 cells
Figure 6. PAWS1 impacts the expression of multiple genes in the TGF/BMP pathways independent of BMP treatment. (a) Scatter plots of log fold change in expression
in PC3-PAWS1 over PC3-control cells of 150 TGF-b/BMP pathway components and target genes analysed by qPCR macroarray. Each dot represents the expression of a
single gene. (b) PC3-PAWS1 and PC3-control cells were treated either with or without BMP-2 (25 ng ml21) for 6 h prior to lysis and the expression of FST and TGFBI was
analysed by qRT-PCR as described in the methods. The results show the fold change in gene expression relative to the levels observed for unstimulated PC3-control cells.
Data are represented as mean of three biological repeats and error bars indicate standard deviation (n ¼ 3). (c) PAWS1-depleted HaCaT cells (siPAWS1) or HaCaT cells
expressing FOXO4 siRNA (siControl) were treated with or without BMP-2 (25 ng ml21) for 6 h prior to lysis and the expression of FST, TGFBI and PAWS1 was analysed by
qRT-PCR. The results show the fold change in gene expression relative to the levels observed for unstimulated siPAWS1 HaCaT cells. Data are represented as mean of
three biological repeats and error bars indicate standard deviation (n ¼ 3). (d). PC3-control and PC3-PAWS1 cells were treated with control, BMP-2 (25 ng ml21) or
TGFb (50 pM) for 6 h prior to lysis, and the expression of SnoN was analysed by qRT-PCR. The results show the fold change in SnoN expression relative to the levels
observed for control-stimulated PC3-control cells. Data are represented as mean of three biological repeats and error bars indicate standard deviation (n ¼ 3).
some PAWS1 to translocate to the nucleus. This nuclear
accumulation of PAWS1 may occur through interaction
with SMAD1: BMP can induce the nuclear localization of
phosphorylated SMAD1 even in the absence of SMAD4 [33].
4.2. PAWS1: the first non-SMAD type I bone
morphogenetic protein receptor substrate
Immunoprecipitation of PAWS1 from BMP-treated cell extracts
allowed the identification of a triphosphopeptide that includes
an SSVS motif that is present in SMAD1 and which, in that
molecule, is phosphorylated by the type I BMP receptor
kinase. No non-SMAD substrates for type I BMP receptor
kinases have previously been reported, so it is significant that
BMPR1A (ALK3) phosphorylated PAWS1 at Ser610, Ser614
and Ser616 in vitro. Comparison with the SSVS motif of
SMAD1 would predict that the major phosphorylation site of
PAWS1 would be Ser614 and Ser616, so it was surprising
that Ser610 was the major PAWS1 phosphorylation site. Nevertheless, we go on to show that PAWS1 is also phosphorylated
at Ser610 in response to BMP in vivo, and that Ser610 is necessary for the activation of SMAD4-independent BMP target
genes such as NEDD9 and ASNS (see below).
The implication that type I BMP and TGF-b receptor
kinases (ALKs 1– 7) have substrates other than SMADs is
consistent with knockout studies in mice, where the loss of
ALKs 2, 3 or 6 result in phenotypes that cannot fully be
explained simply by the failure to activate SMADs 1, 5 or 8
[34 –38]. There are likely to be many more non-SMAD
substrates for type I BMP and TGF-b receptor kinases.
4.3. PAWS1 and the bone morphogenetic protein
signalling pathway
The absence of SMAD4 in the complex that contains PAWS1
and SMAD1 suggests that PAWS1 may play a unique function
in the BMP signalling pathway. Consistent with this notion,
PAWS1 does not influence BMP-induced phosphorylation of
SMAD1 or the expression of SMAD4-dependent BMP target
genes such as ID1 and SnoN. However, the activation of
NEDD9 and ASNS in response to BMPs was lost in PC3 cells
lacking PAWS1 and was restored upon the reintroduction of
wild-type PAWS1 but not the PAWS1(S610A) mutant. We
note that NEDD9 has been implicated in cellular migration as
well as in the invasion and metastasis of cancer cells [39,40],
and that unregulated ASNS expression has also been linked
Open Biol. 4: 130210
–1.0
fold change
–0.6
rsob.royalsocietypublishing.org
TGFBI
1.0
9
TGFBI
40
3
with cancer [41]. It will be interesting to discover whether the
expression of PAWS1 itself is misregulated in cancer.
5. Material and methods
5.1. General
A PAWS1 antibody was generated by injecting GST-PAWS1
(amino acids 715–815) into sheep. The P-PAWS1 S610 antibody
was generated by injecting peptide GPGPRRPS*VAS (* denotes
phospho-Ser) into rabbit. The antibodies were subsequently affinity purified. Anti-FLAG-M2-horseradish peroxidase (HRP)
antibody was from Sigma. HA-HRP antibody was from Roche.
Antibodies recognizing phospho-SMAD1/5/8, phosphoSMAD2, phospho-SMAD3, SMAD2/3, GAPDH and lamin A/
C were from Cell Signalling Technology. HRP-conjugated secondary antibodies and light-chain-specific HRP-conjugated
antibodies were from Jackson Laboratories. BMP-2 and BMP-4/
7 were from R&D Systems. The nuclear cytoplasmic extraction
kit was from Thermo Scientific. The first strand cDNA synthesis
kit was from Invitrogen. 2X SYBR green master mix was from
BioRad. pBABE-Puro, pCMV-Gag-Pol and pCMV-VSVG constructs were from Cell Biolabs. All plasmids for mammalian cell
expression were cloned into pCMV5, pBABE-puro or pcDNAFRT-TO vectors with N-terminal FLAG, HA or GFP tags as indicated. For bacterial expression of proteins, SMAD1, SMAD2 and
PAWS1 (523-end; other mutants) were cloned into pGEX6T vectors. All DNA constructs were verified by DNA sequencing (by
the DNA Sequencing Service at University of Dundee; www.
dnaseq.co.uk). Bacterial protein expression in BL21 cells and
purification were performed as described previously [31].
5.2. Cell culture, transfection and lysis
Unless stated otherwise, prostate cancer-derived PC3 cells,
human embryonic kidney HEK293 cells, HeLa cells, SW480
5.3. Generation of PC3 cells stably expressing wild-type
PAWS1 or PAWS1-S610A mutant
Retroviral pBABE-puromycin control vector (1 mg each) or
vectors encoding PAWS1 and PAWS1-S610A mutant were
co-expressed with CMV-Gag/Pol (0.9 mg) and CMV-VSVG
(0.1 mg) constructs in HEK293T cells. Retroviruses were collected 48 h post-transfection from the culture medium by
filtration through 0.45 mm filters into sterile Falcon tubes as
described previously [44]. PC3 cells, plated at approximately
40% confluence 24 h previously, were infected by transferring filtered retroviruses directly onto the cells together with
8 mg ml21 polybrene. Twenty-four hours post-infection, cells
were cultured in the presence of medium containing puromycin
(2 mg ml21) for selection of infected cells.
5.4. Immunoprecipitation
Snap frozen cell extracts were allowed to thaw on ice and centrifuged at 14 000 rpm for 10 min at 48C. Protein concentration
was determined spectrophotometrically. Extracts (1 mg unless
stated otherwise) were then subjected to immunoprecipitation
using 10 ml packed beads (GFP-trap, anti-FLAG-M2 or specific
antibody covalently bound to protein G sepharose beads or
magnetic Dyna-beads (1 mg antibody per 5 ml packed beads))
in a rotating platform for 2 h at 48C. IPs were then washed
twice in lysis buffer with 0.5 M NaCl, and twice in buffer A
(50 mM Tris–HCl pH 7.5, 0.1 mM EGTA, 0.1% b-mercaptoethanol) at 48C. Samples were reduced in SDS–sample
buffer (250 mM Tris–HCl pH 6.8, 10% SDS, 50% glycerol,
0.1% bromophenol blue; 0.1% b-mercaptoethanol), boiled for
5 min and resolved by SDS–PAGE.
5.5. Mass spectrometry
FLAG control or a FLAG-SMAD1(L þ MH2) fragment
expressed in HEK293 cells was isolated from cleared extracts
(50 mg protein) by immunoprecipitation with anti-FLAG-M2
antibody coupled to agarose beads. The IPs were washed and
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Our analysis of 155 known TGF-b/BMP target genes indicates that several of these are differentially expressed upon
reintroduction of PAWS1 in PC3 cells, and that this occurs
in a ligand-independent fashion. These observations were
confirmed for the genes FST and TGFBI in PC3 cells following introduction of PAWS1 and in HaCaT cells following
depletion of PAWS1 by RNAi. PAWS1 therefore regulates
gene expression independent from BMP signalling as well
as in a ligand-dependent manner, and a global transcriptomic
analysis of genes affected by PAWS1 may yield clues to
possible biochemical roles beyond the BMP pathway.
To complement such an analysis, it will be necessary to
understand more about PAWS1 as a protein. Sequence
analysis offers few functional clues beyond the presence of a
putative pseudo-phospholipase D (PLD) active site motif, but
we note that PLD activity was not detected in the related proteins FAM83A and B [25,26]. It is, nevertheless, possible that
PAWS1 interacts with phospholipids and/or other PLDs, or
that it acts as a scaffolding protein to control signalling pathways downstream of PLDs. Uncovering the precise functional
roles of PAWS1 will enable us to ask how BMP signalling
and SMAD1 impact on the biochemical properties of PAWS1.
10
rsob.royalsocietypublishing.org
4.4. PAWS1: beyond the bone morphogenetic protein
signalling pathway
cells, BxPC3 cells and human keratinocyte HaCaT cells were
propagated in DMEM supplemented with 1% penicillin/streptomycin, 2 mM L-glutamine (Gibco) and 10% FBS (Hyclone).
Cells were kept at 378C in a humidified incubator with 5%
CO2. pcDNA-FRT-TO plasmids encoding GFP- or GFPtagged PAWS1 were used to generate stable tetracycline-inducible FlpIN-TRex (Invitrogen) HEK293 and U2OS cell lines
following the manufacturer’s protocol. The cells were grown
in medium that additionally contained 100 mg ml21 hygromycin and 15 mg ml21 blasticidin as described previously [42].
For overexpression of pCMV5 plasmids encoding FLAG- or
HA-tagged proteins, HEK293 cells were transfected using
PEI as described previously [43]. The siRNA oligonucleotides
(300 pmole total/10-cm diameter dish) were transfected into
HaCaT cells using Transfectin (BioRad). Cells were harvested
48 h post-transfection. For protein analysis, cells were scraped
directly with cell lysis buffer (50 mM Tris–HCl pH 7.5, 1 mM
EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM activated sodium
orthovanadate, 50 mM sodium fluoride, 5mM sodium pyrophosphate, 0.27 M sucrose, 5 mM b-glycerophosphate, 0.1%
b-mercaptoethanol and one tablet of protease inhibitor cocktail
(per 25 ml)) and snap frozen in liquid nitrogen.
5.6. Gel filtration chromatography
5.7. Immunoblotting
Cell extracts were cleared by centrifuging at 14 000 rpm for
10 min at 48C. Extracts (20 mg) or IPs (30% of total unless
stated otherwise) were reduced in SDS sample buffer and
boiled for 5 min, resolved using SDS– PAGE and transferred onto PVDF membranes. Membranes were blocked
with 5% non-fat dry milk powder in TBST (50 mM
Tris, 150 mM NaCl, 0.2% Tween-20) incubated overnight
at 48C with primary antibody, followed by incubation
with an HRP-conjugated secondary antibody (1 : 10 000).
Antigen –antibody complexes were detected with enhanced
chemiluminescence reagents.
5.8. Quantitative PCR
Real-time quantitative reverse transcription PCR (qRT-PCR)
was carried out using 1 mg of isolated RNA and the SuperScript cDNA kit (Invitrogen) according to the manufacturer’s
protocol. qRT-PCRs were performed in triplicate (10 ml)
according to the manufacturer’s protocol, including forward
and reverse primers (0.5 mM each), 50% SYBR green master
mix (BioRad) and a cDNA equivalent of 1 ng ml21 RNA in a
CFX 384 real-time system qRT-PCR machine (BioRad). The
data were normalized to the geometrical mean of two housekeeping genes (GAPDH and HPRT1) and analysed by the
Pfaffl method [45].
5.9. RNAi and qRT-PCR primers
Primers were designed using PERLPRIMER and purchased from
Invitrogen. Primers (50 –30 ): PAWS1 forward: CACAGAAGG
TGATAGCTGTG; reverse: ACTTGACGTTACTCTCATCCA;
FOXO4 forward: TTGGAGAACCTGGAGTGTGACA; reverse:
AAGCTTCCAGGCATGACTCAG; ID1 forward: AGGCTGGATGCAGTTAAGGG; reverse: GGTCCTTTTCACCAGCAA
GCT; GAPDH forward: TGCACCACCAACTGCTTAGC;
reverse: GGCATGGACTGTGGTCATGAG; HPRTI forward:
TGACACTGGCAAAACAATGCA; reverse: GGTCCTTTTCA
CCAGCAAGCT; NEDD9 forward: GCTCTATCAAGTGCCA
AACCC; reverse: GGTTCCCCCAATGCTTCTCT; ASNS forward: AACTGCTGCTTTGGATTTCAC; reverse: GCTGTTGC
ATCTTCTTATGGT; BMPR2 forward: TGGAACATACCGTTT
5.10. Statistical analysis
Data are presented as the mean + s.d. The statistical significance of differences between experimental groups was
assessed using the two-way analysis of variance test with
Bonferroni post-tests. Differences in means were considered
significant if p , 0.05.
5.11. Analysis of 32P-labelled phosphorylation sites
For kinase assays, 20 ml reactions were set up consisting of 150 ng
of kinase (GST-ALK3; GST-ALK2 or GST-ALK6; all from Carna
Biosciences) and 2 mg substrate protein (GST-SMAD1, GSTSMAD2, GST-PAWS1 (523-end) or other mutants of PAWS1 as
indicated) in a buffer containing 50 mM Tris–HCl pH 7.5, 0.1%
2-mercaptoethanol, 0.1 mM EGTA, 10 mM MgCl2, 0.5 mM
microcystein-LR and 0.1 mM g32P-ATP (500 cpm pmole21 for
routine autorad analysis; 10000 cpm pmole21 for mapping phosphoresidues). Assays were performed at 308C for 30 min and
stopped by adding 1 SDS sample buffer and heating to
958C for 5 min. The samples were resolved by SDS–PAGE, the
gels stained with Coomassie blue and dried. Radioactivity was
analysed by autoradiography. Identification of the phosphoresidues within PAWS1 was performed as described [46] except that
mass spectrometric analysis of phosphopeptides was performed
as above for fingerprinting with the addition of multi-stage
activation during the MS2 analysis.
5.12. Cellular fractionation
Nuclear/cytosolic fractionation was performed using the
nuclear and cytosolic extraction kit from Thermo Scientific
according to the manufacturer’s instructions. Proteins were
denatured by boiling for 5 min in SDS sample buffer prior
to SDS– PAGE.
Acknowledgements. We thank Joby Varghese, Patrick Pedrioli and
Matthias Trost for help with mass spectrometry. We thank Kirsten
McLeod and Janis Stark for help with tissue culture, the staff at the
Sequencing Service (School of Life Sciences, University of Dundee,
Scotland) for DNA sequencing and the protein production teams at
the Division of Signal Transduction Therapy (DSTT; University of
Dundee) coordinated by Hilary McLauchlan and James Hastie for
the expression and purification of proteins and antibodies. We
thank the UK Medical Research Council, and the pharmaceutical
companies supporting the DSTT (AstraZeneca, BoehringerIngelheim, GlaxoSmithKline, Merck-Serono, Pfizer and Janssen) for
11
Open Biol. 4: 130210
Extracts from HaCaT cells treated with or without the indicated ligands were cleared by centrifugation and further
cleared through Spin-X tubes. Protein extract (1 mg) was
subjected to separation through a Superose 6 10/300 GL
column (GE Healthcare), which was washed and equilibrated
with buffer containing 50 mM Tris–HCl 7.5, 150 mM NaCl,
0.03% Brij-35. Thirty-two fractions were collected, and they
were processed as described previously [43].
CTGCT; reverse: GAATGAGGTGGACTGAGTGG; TGFBI forward: ATCACCAACAACATCCAGCA; reverse: CCGTTACCT
TCAAGCATCGT; FST forward: GATCTTGCAACTCCATTTC
GG; reverse: GGCTATGTCAACACTGAACAC; TGFBR2 forward: GCTGTATGGAGAAAGAATGACGA; reverse: ACAG
GAACACATGAAGAAAGTC; TSC22D1 forward: CTATCAG
TGGTGACAGTGGG; reverse: TTCACTAGATCCATAGCTTG
CTC; SnoN forward: GAGGCTGAATATGCAGGACAG;
reverse: CTATCGGCCTCAGCATGG.
siRNA against PAWS1 were purchased from Qiagen and
targeted to the following sequences: siRNA PAWS1-1:
AAGATGATGACGACTACGTAA (catalogue no. SI03683897).
siRNA
PAWS1-2:
CCGGGCTAGCGTCTACATGCA
(catalogue no. SI03683904).
siRNA against FOXO4 was purchased from Eurofins and
targeted to the following sequence: siRNA FOXO4: CCCGAC
CAGAGAUCGCUAA.
rsob.royalsocietypublishing.org
incubated with cleared HeLa extracts (100 mg) at 48C for 4 h.
After washing, FLAG-proteins and any interacting partners
were eluted using 3X FLAG peptide following the manufacturer’s protocol. Eluted proteins were reduced in sample
buffer and resolved by SDS–PAGE. Coomassie-stained
bands were excised and tryptically digested. Mass spectrometric analysis of the resulting peptides was performed by
LC–MS– MS as described previously [43].
constructs. G.P.S. conceived and designed the project. J.V., K.S.D.,
J.C.S. and G.P.S. contributed to writing the manuscript.
Funding statement. J.C.S. and K.D. were supported by the Medical
Research Council ( programme no. U117597140).
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