JCS ePress online publication date 13 March 2007
1216
Research Article
Endofin acts as a Smad anchor for receptor activation
in BMP signaling
Weibin Shi1, Chenbei Chang2, Shuyi Nie2, Shutao Xie1, Mei Wan1 and Xu Cao1,*
1
Department of Pathology and 2Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 10 January 2007
Journal of Cell Science 120, 1216-1224 Published by The Company of Biologists 2007
doi:10.1242/jcs.03400
Summary
Signaling through receptors of the transforming growth
factor ␤ (TGF␤) superfamily is mediated by cytoplasmic
Smad proteins. It has been demonstrated that Smad anchor
for receptor activation (SARA) facilitates TGF␤ and
activin/nodal signaling by recruiting and presenting
Smad2/3 to the receptor complex. SARA does not bind
Smad1 and hence does not enhance bone morphogenetic
protein (BMP) signaling. Here we report for the first time
that the endosome-associated FYVE-domain protein
endofin acts as a Smad anchor for receptor activation in
BMP signaling. We demonstrate that endofin binds Smad1
preferentially and enhances Smad1 phosphorylation and
nuclear localization upon BMP stimulation. Silencing of
endofin by RNAi resulted in a reduction in BMP-dependent
Key words: BMP, Smad, SARA, Protein phosphatase, Osteoblast
Introduction
Growth factors of the TGF␤ superfamily regulate cell
proliferation, differentiation, migration and apoptosis and are
crucial for the development and maintenance of many different
tissues (Cheifetz, 1999; Francis-West et al., 1999). Bone
morphogenetic proteins (BMPs) in this superfamily induce
bone marrow mesenchymal stem cell differentiation into
osteoblasts and enhance periodontal regeneration in surgically
created defects. BMP ligands, as well as other ligands of the
TGF␤ superfamily, initiate cellular signaling by binding to
type I and type II receptors, both of which are serine/threonine
kinase receptors, thereby inducing the formation of a heterooligomeric receptor complex. The type II receptor then
phosphorylates and activates the type I receptor, which
subsequently transiently associates with and phosphorylates a
subclass of a unique family of intracellular signaling molecules
– the receptor-regulated Smads or R-Smads (Godin et al., 1999;
Ghosh-Choudhury et al., 1994). The R-Smad Smad1
transduces signals from BMPs, whereas the R-Smads Smad2
and Smad3 mediate signals from the TGF␤/activin/nodal
subfamily members. The recruitment of R-Smads to the
receptor complex is the initial step of intracellular TGF␤ signal
transduction and plays a critical role in regulating signal
transduction by the TGF␤ receptor family.
TGF␤ signaling can be enhanced by a protein, Smad anchor
for receptor activation or SARA, which binds
unphosphorylated Smad2 and Smad3 and recruits them to
membranes (Tsukazaki et al., 1998). SARA contains a FYVE
domain adjacent to its Smad-binding domain. The double zincfinger motifs in FYVE domains have been shown to bind
phosphatidylinositol 3-phosphate and anchor FYVEcontaining proteins to cytoplasmic or endosomal membranes.
This enables proteins containing the FYVE domain to recruit
other molecules to membranes for endocytosis or
relocalization (Gaullier et al., 1998; Kutateladze and Overduin,
2001). The FYVE domain in SARA is required for recruitment
of Smad2/3 to the appropriate subcellular locations that
position it adjacent to the activated T␤RI (Tsukazaki et al.,
1998; Wu et al., 2000). Activation of TGF␤ signaling causes
phosphorylation of Smad2, subsequent dissociation from
SARA, and nuclear translocation with the common Smad,
Smad4 (Massague, 1998; Heldin et al., 1997).
Recent data suggest that SARA can also act as a negative
regulator of TGF␤ signaling. In Drosophila, the catalytic
subunit of protein phosphatase 1 (PP1c) has been shown to
bind SARA and negatively regulate signaling through Dpp, a
BMP homologue (Bennett and Alphey, 2002). Expression of a
mutant SARA with a mutation in the PP1c-binding domain
(F678A) resulted in hyperphosphorylation of the type I
receptor and elevated expression of dpp target genes (Bennett
and Alphey, 2002). In our previous studies, we also found that
SARA facilitates dephosphorylation of T␤RI in cell culture
through a Smad7-mediated negative feedback loop. The
inhibitory Smad7 recruits a protein phosphatase holoenzyme,
GADD34-PP1c, to inhibit TGF␤-induced cell cycle arrest and
confer cell resistance to TGF␤ in response to UV irradiation.
SARA facilitates the formation of GADD34-PP1c by
presenting PP1c to this holoenzyme, and thus acts as a
component of Smad7-mediated negative feedback loop of this
signaling pathway (Shi et al., 2004). SARA is therefore a
Smad1 phosphorylation. Moreover, disruption of the
membrane-anchoring FYVE motif by point mutation led
to a reduction of BMP-responsive gene expression in cell
culture and Xenopus ectodermal explants. Furthermore,
we demonstrate that endofin contains a proteinphosphatase-binding motif, which functions to negatively
modulate
BMP
signals
through
receptor
dephosphorylation. Taken together, our results suggest that
endofin plays an important role in both positive and
negative feedback regulation of the BMP signaling
pathway.
Endofin in BMP signaling
Journal of Cell Science
central regulator of TGF␤ signaling and can act to either
promote or inhibit TGF␤ signals.
The enhancement of TGF␤/activin signaling by another
FYVE-domain protein, Hgs/Hrs, further indicates that other
SARA-like molecules may participate in the regulation of
TGF␤/activin signaling (Miura et al., 2000). By analogy, it
would be anticipated that BMP signal transduction might also
employ SARA-like regulators to help bring Smad1 to the
receptor complexes; however, up to now, no SARA-based
mechanism had been identified for the BMP pathway. In this
study, we present evidence indicating an important role for the
endosome-associated FYVE-domain protein endofin in BMP
signaling (Seet and Hong, 2001; Seet et al., 2004; Seet and
Hong, 2005). We show that endofin binds preferentially to
Smad1 and regulates Smad1 signaling by modulation of its
phosphorylation and nuclear translocation upon BMP
activation. Endofin also has a functional PP1c-binding domain,
which recruits PP1c and facilitates dephosphorylation of the
type I BMP receptor. Our results identify for the first time a
BMP-specific Smad anchor as a receptor activation molecule
that plays a role in transduction and negative feedback
regulation of BMP signals.
Results
Endofin interacts with Smad1 and PP1c
Endofin has been shown to localize to early endosomes, as does
SARA; but it does not bind to Smad2 or influence TGF␤
signals (Seet and Hong, 2001). The relatively high divergence
of the Smad-binding domain suggested that endofin might bind
R-Smads other than the TGF␤-specific Smad-2, such as the
BMP-specific Smad1. To test this possibility directly, we
performed a co-immunoprecipitation assay using the lysate of
COS cells transfected with endofin and Smad1 or Smad2. We
found that Smad1, but not Smad2, was co-precipitated with
endofin (Fig. 1A), suggesting that endofin preferentially binds
to Smad1. To further confirm the interaction in situ, we
performed a protein-fragment complementation assay (PCA)
using yellow fluorescent protein (YFP) as a marker. The
principle of the PCA strategy is that complementary fragments
(F1 and F2) of a reporter protein (such as YFP) will fold into
an active form only when brought together in close proximity
by fusion to two proteins that interact with each other (Remy
et al., 2004; Li et al., 2006). In our experiments, we fused
Smad1 and endofin to N-terminal (a.a. 1-158, YFP1) and Cterminal (a.a. 159-239, YFP2) fragments of YFP, respectively.
When expressed alone, neither YFP1-Smad1 nor endofinYFP2 produced fluorescent signals in the cells. Co-expression
of the vectors encoding YFP1 and YFP2 did not generate
fluorescent signals either (Fig. 1B). When the fusion proteins
were co-transfected into the COS cells, however, we observed
strong yellow fluorescence (Fig. 1B). The intensity of the
fluorescence signal is similar to that observed when Smad1 and
its interacting protein Hoxa1 were co-transfected in a similar
way in parallel (Fig. 1B). These results indicated that Smad1
and endofin interact in transfected cells. To confirm this
interaction, we also examined the localization of tagged Smad1
and endofin in transfected cells by fluorescence
immunocytochemistry. Endofin and Smad1 co-localized in the
cytosol in a punctuate pattern (Fig. 1C), suggesting that the two
proteins interact at intracellular vesicles in the cells. To
eliminate the possibility of artifacts generated by the
1217
overexpression system, we also analyzed endogenous proteins
by using antibodies against Endofin and Smad1. As shown in
the merged panels in Fig. 1C, the endogenous proteins were
also co-localized in the cytosol of the cells. All our data are
consistent with the idea that endofin and Smad1 associate with
each other in the cell.
In addition to the Smad-binding motif, all known SARA
homologs also contain a canonical PP1c-binding motif
(K/R)xVxF or (K/R)VxF (Bennett and Alphey, 2002) and this
motif is involved in negative feedback regulation of Dpp
signaling in Drosophila. We therefore examined whether
endofin can also associate with PP1c. Co-immunoprecipitation
analyses indicated that endofin indeed interacts with PP1c.
Interestingly, our results further showed that overexpression of
PP1c enhanced the interaction of endofin with Smad1 (Fig.
1D). As PP1c can down-regulate BMP signaling resulting in
hypophosphorylation of BMP signal components, our
observation implies that endofin may preferentially bind to
unphosphorylated Smad1. To verify this hypothesis, we
assayed for endogenous interaction of endofin with Smad1,
phosphorylated Smad1 (pSmad1) and PP1c in the presence or
absence of BMP2. We observed that BMP2 stimulated binding
of endofin to PP1c, and the phosphorylated form of Smad1 has
very low affinity for endofin (Fig. 1E). To examine the
specificity of the interaction between endofin and Smad1, the
interaction of SARA and Smad1 was also detected. BMP2 does
not stimulate the binding of SARA with either Smad1 and
phosphorylated Smad1. Our results confirm that endofin, but
not SARA, binds preferentially to the inactive form of Smad1.
To further determine whether the putative Smad- and PP1binding domains (SBD and PBD respectively) mediate the
interaction of endofin with Smad1 and PP1c, we performed coimmunoprecipitation experiments with mutant endofin. Three
mutants were generated that contain point mutations in FYVE
and PBD domains or deletion of the SBD motif (Fig. 2A). We
found that disruption of the SBD or mutation of FYVE domain
resulted in a diminishment of the Smad1-endofin interaction
(Fig. 2B), while mutation of the PBD almost abolished the
interaction between PP1c and endofin (Fig. 2C). The results
demonstrate that SBD and PBD mediate the binding of endofin
to Smad1 and PP1c respectively.
Endofin facilitates dephosphorylation of the BMP type I
receptor by PP1
Expression of a PP1c-binding mutant of SARA has been
shown to result in hyperphosphorylation of the type I receptor
and elevation of expression of a target of TGF-␤ signaling
(Bennett and Alphey, 2002). To assess whether endofin has a
similar activity, we performed an in vivo phosphorylation
assay. As shown in Fig. 3A, overexpression of endofin reduced
phosphorylation of the type I BMP receptor ALK3 in response
to BMP2, whereas the mutant endofin with impaired PBD
enhanced ALK3 phosphorylation. Phosphatase inhibitor 1, an
inhibitor for PP1c, also increased ALK3 phosphorylation. The
results suggest that endofin can regulate ALK3 activation
through its interacting phosphatase PP1c.
In our previous study of SARA, we found that
dephosphorylation of the type I TGF␤ receptor ALK5 is
mediated by a protein phosphatase regulatory subunit
GADD34, which binds to ALK5 through a bridging
mechanism involving an inhibitory Smad, Smad7 (Shi et al.,
Journal of Cell Science 120 (7)
Journal of Cell Science
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Fig. 1. Endofin interacts with Smad1. (A) Co-immunoprecipitation (co-IP) of endofin with Smads. Flag-tagged Smad1 or Smad2 was cotransfected with HA-tagged endofin into COS1 cells. The lysate was subjected to co-IP with anti-HA antibody and probed with anti-Flag
antibody, or co-IP with anti-Flag and probed with anti-HA. (B) Confirmation of the interaction between Smad1 and endofin by proteinfragment complementation assays (PCA). The complementary fragments (YFP1 and YFP2) of YFP were fused with Smad1 and endofin,
respectively, to create YFP1-Smad1 (a.a. 1-158 of YFP) and endofin-YFP2 (a.a. 159-239 of YFP), and then transfected into COS1 cells. The
interaction between endofin and Smad1 is indicated here as yellow fluorescence. Transfection of pcDNA3, co-transfection of pcDNA3-YFP1
and pcDNA3-YFP2 and co-transfection of YFP1-Smad1 and Hoxa1-YFP2 serve as negative and positive controls, respectively (Li et al., 2006).
Bar, 10 ␮m. (C) Colocalization of Smad1 and endofin. Upper panel: C2C12 cells were transfected with HA-Endofin and Flag-Smad1. After
transfection for 36 hours, cells were processed for immunofluorescence using antibodies against HA (rabbit polyclonal) and Flag (mouse
monoclonal). Lower panel: Endogenous protein co-localization. Naive C2C12 cells were subjected to immunofluorescent microscopy using
rabbit polyclonal anti-endofin and mouse monoclonal anti-Smad1. Nuclei were counterstained with Hoechst 33342. Representative images are
shown. (D) Endofin interacts with both Smad1 and PP1c. COS1 cells were transfected with Flag-Smad1, HA-tagged endofin and Myc-tagged
PP1c. Cells were treated with rhBMP2 (200 ng/ml) for 2 hours before lysis. The lysate was subject to co-immunoprecipitation with antibody
against HA, the presence of Smad1 and PP1c in the precipitate was detected with anti-Flag and anti-Myc antibodies, respectively. (E) Co-IP of
endogenous endofin, Smad1 and PP1c. C2C12 cells were cultured until subconfluent, with or without treatment of rhBMP2 (200 ng/ml, 2
hours), cells were lysed, and the lysate was subjected to immunoprecipitation with antibody against either endofin or SARA. The presence of
Smad1 and PP1c in the precipitate was detected by anti-Smad1 and anti-PP1c antibodies. Bar, 5 ␮m.
2004). To see whether a similar mechanism applies to endofin,
we tested the interaction of the BMP type I receptors ALK3
and ALK6 with GADD34, as well as the potential requirement
for inhibitory Smads Smad6 and Smad7. We found that both
ALK3 and ALK6 readily interact with GADD34 without any
auxiliary factors (Fig. 3B), and sequential co-
immunoprecipitation further clarified that although ALK3 and
ALK6 bind inhibitory Smads, they do not form a ternary
complex with GADD34 and Smad6 or Smad7 (Fig. 3C). Our
data indicate that unlike ALK5, ALK3 and ALK6 directly
associate with the regulatory subunit of protein phosphatase 1
in the absence of inhibitory Smads.
Endofin in BMP signaling
Journal of Cell Science
Endofin modulates Smad1 phosphorylation and nuclear
translocation
To examine the functional significance of the endofin-Smad1
interaction, we next performed a loss-of-function study, using
small interfering RNA. Knockdown of endofin using specific
siRNA resulted in a reduction in the level of phosphorylation
of Smad1 (Fig. 4A), which corroborated our hypothesis that
endofin acts as a SARA-like protein in BMP signaling and is
required for Smad1 phosphorylation. Notably, endofin appears
to utilize a mechanism similar to that employed by SARA in
which the PP1c-binding motif acts to negatively regulate
phosphorylation of R-Smad.
As phosphorylation of Smad1 enables its translocation into
the nucleus, we next tested whether endofin also regulates the
nuclear translocation of Smad1. Staining of phosphorylated
Smad1 in C2C12 cells expressing endofin confirmed that
endofin enhanced Smad1 phosphorylation and nuclear
translocation upon simulation of BMP2. Overexpression of
SARA, however, did not have a significant effect on Smad1
activation and localization (Fig. 4B).
Endofin regulates expression of BMP downstream
genes in C2C12 cells and mineralization in human
MSCs
To further examine the effect of endofin on BMP signal
transduction, we generated stable cell lines derived from
C2C12 and human MSCs infected with retroviruses that
express endofin, endofin mutants or the control green
fluorescent protein (GFP). The influence of stable endofin
expression on BMP-induced downstream gene transcription
1219
was monitored in these cell lines using a BMP-responsive
reporter construct (9xSBE-luc). As shown in Fig. 5A, wildtype endofin slightly enhanced the expression of the BMPresponsive luciferase gene. The FYVE(C753S) mutant, which
leads to cytosolic mislocalization of endofin and possibly its
associated proteins including Smad1, decreased the reporter
gene transcription. As expected, deletion of the Smad-binding
domain (Endofin⌬SBD) also inhibited BMP-induced gene
expression. By contrast, mutation of the PP1c-binding domain
PBD(F872A) enhanced transcription of the reporter
transcription; a finding that is consistent with our observation
that this mutant increased ALK3 phosphorylation (Fig. 3A).
BMPs were originally purified from bone matrix and have
been shown to regulate bone development and induce
osteoblast differentiation. We therefore examined the effects of
expression of mutant endofin on BMP-stimulated expression
of the osteoblast differentiation marker, alkaline phosphatase
(ALP), as well as osteoblast-mediated mineralization. C2C12
cells with stable overexpression of the PBD(F872A) mutant
showed higher ALP activity than cells infected with wild-type
endofin (Fig. 5B,C). Similarly, human MSCs with stable
overexpression of the PBD(F872A) mutant showed enhanced
mineralization (Fig. 5D).
Regulation of BMP-dependent mesodermal induction by
endofin in Xenopus embryos
BMPs play diverse roles during early Xenopus development,
especially in embryonic dorsoventral patterning. When
overexpressed, BMPs can induce ventral types of mesoderm in
Xenopus ectodermal explants (animal caps). To determine
Fig. 2. Identification of the endofin interaction domain in
Smad1 and PP1c. (A) Schematic representation of wild-type
and mutant endofin. Three mutants were generated to disrupt
these conserved domains: FYVE domain (C753S), Smadbinding domain (deletion of amino acids 814-860) and PP1cbinding domain (F872A). (B) Deletion of Smad-binding
domain in endofin diminishes its interaction with Smad1.
COS1 cells were transfected with Flag-Smad1, HA-tagged
endofin and its mutants. Immunoblots in the two lower
panels show the protein expression levels of the COS1 cell
lysates. The upper panel shows anti-HA immunoprecipitates
probed with anti-Flag antibody. (C) Co-IP of PP1c with
endofin and the PBD(F872A) mutant. HA-tagged endofin
and its mutant with disrupted PP1c-binding domain were cotransfected with PP1c into COS1 cells. The lysate was subject to immunoprecipitation with anti-HA. Immunoblots indicating the protein
expression levels of the lysates of COS1 cells are shown in the two lower panels. The upper panel shows anti-HA immunoprecipitates probed
with anti-Flag antibody.
Journal of Cell Science
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Journal of Cell Science 120 (7)
Fig. 3. The BMP type I receptor is the substrate of PP1.
(A) C2C12 cells transfected with different combinations of
genes were labeled with [32P]orthophosphate in the
presence or absence of BMP2 as indicated. ALK3-HA was
immunoprecipitated from lysates of treated cells and
separated by 8.5% SDS-PAGE. Gels were dried and
exposed to Biomax Mr film (Eastman Kodak). PBD
(F872A) is a mutant with the disrupted PP1c-binding
domain. Phosphatase inhibitor-1 is an inhibitor for PP1.
(B) BMP type I receptors interact with PP1 regulatory
subunit GADD34. COS1 cells transfected with HA-tagged
type I receptors (ALK3 and ALK6) and Flag-tagged
GADD34. Lysates were subjected to coimmunoprecipitation with anti-Flag and the presence of
ALK3 and ALK6 were probed with antibody against HA. (C) Unlike in the TGF␤ signaling pathway, GADD34 interacts with the BMP type I
receptor without the bridging of inhibitory Smads. The GADD34-ALK3/6 complex was further confirmed by a sequential
immunoprecipitation. COS1 cells were first co-transfected with ALK3/6-HA with or without Flag-Smad6/7 and GADD34. The lysates were
subjected to immunoprecipitation with HA antibody, and the resultant precipitates were eluted from protein-G-Sepharose beads by HA peptide
competition and then subjected to a second immunoprecipitation with anti-Flag antibody. The final precipitates were immunoblotted with
antibodies against all these components.
whether endofin can modulate BMP signaling in vivo, we
examined the effect of endofin overexpression on BMPdependent endogenous mesodermal marker expression. In this
experiment, we injected BMP4 RNA with or without RNAs
encoding endofin or its mutants in animal poles of two-cell
stage embryos and dissected animal caps from injected
embryos at blastula stages 8.5-9. The caps were incubated to
gastrula stages before total RNA was extracted for use in a
reverse transcription (RT)-PCR assay of marker gene
expression. As shown in Fig. 6, BMP2 induced the expression
of the early mesodermal markers Xbra, Xhox3 and Xwnt8.
Expression of wild-type endofin did not affect the level of
induction of these genes, possibly because of the presence of
a high level of endogenous Xenopus endofin-like molecule.
Expression of the FYVE(C735S) mutant led to a dramatic
inhibition of Xbra, Xhox3 and Xwnt8, whereas expression of
the endofin ⌬SBD mutant resulted in a modest reduction in the
marker gene expression (Fig. 6). By contrast, expression of the
PBD(F872A) mutant enhanced the expression of Xbra, Xhox3
and Xwnt8: a result further supporting the notion that
recruitment of PP1c by endofin plays an important role in
negative feedback regulation of BMP signaling in vivo.
Discussion
The FYVE domain protein, SARA, was identified as a key
molecule that anchors Smad2/3 and promotes receptor
activation of the TGF␤ signaling pathway (Tsukazaki et al.,
1998; Bennett and Alphey, 2002; Wu et al., 2000). The
similarities among members of the TGF␤ superfamily
prompted us to search for evidence of a SARA-like molecule
associated with BMP signaling. GenBank search for sequences
with homology to functional regions of SARA identified a
FYVE domain protein, KIAA0305, which had a C-terminal
region that was closely related to the C-terminus of SARA.
KIAA0305, also known as endofin (Seet and Hong, 2001), was
first sequenced by investigators at the Kazusa Institute in
Japan, but its function remained unknown. The conserved
domain structures between SARA and endofin suggest that
endofin may function like SARA as a membrane anchor
protein for R-Smads; however, the Smad-binding domain is
highly divergent between the two proteins. Accordingly, it has
been shown that although endofin localizes to early
endosomes, it does not bind to Smad2 nor influence TGF␤
signals (Seet and Hong, 2001). We therefore investigate the
possibility that endofin acts like SARA but binds different RSmads. Here, we report that using co-immunoprecipitation,
protein-fragment
complementation
assay
and
immunolocalization approaches, we observed consistently that
endofin preferentially binds to Smad1, not Smad2, suggesting
that endofin may act as a Smad anchor for receptor activation
in BMP signaling. This idea is consistent with our finding that
endofin, but not SARA, facilitated the BMP signalling, and
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Journal of Cell Science
Endofin in BMP signaling
Fig. 4. Endofin modulates Smad1 phosphorylation and nuclear translocation. (A) Silencing of endofin expression by RNA interference
inhibited Smad1 phosphorylation. C2C12 cells were transfected with Flag-tagged Smad1 and either vector, construct for GFP or endofin
siRNA. On the third day following 3-hour BMP2 treatment, Flag-tagged Smad1 was precipitated with anti-flag and its phosphorylation level
was detected with anti-phospho-Smad1. (B) Endofin, but not SARA, regulates BMP-specific phosphorylation and intracellular translocation of
Smad1. C2C12 cells were transfected with either HA-SARA or HA-Endofin, then stimulated with BMP (100 ng/ml) for 3 hours. After fixation
with paraformaldehyde (PFA), cells were immunostained with either anti-HA (green) or anti-phospho-Smad1 (red). Nuclei were visualized
using Hoechst 33342. Representative images are shown. Bar, 20 ␮m.
knockdown of endofin with siRNA reduced the level of Smad1
phosphorylation.
The specificity of the Smad anchor for receptor activation
proteins may be determined by critical residues in the Smadbinding domains, which define preferential interactions with
TGF␤- or BMP-specific R-Smads (Wu et al., 2000). The
crystal structure of the Smad2 MH2 domain bound by the
Smad-binding domain of SARA has revealed that the MH2
domain interacts with an extended proline-rich motif
consisting of a proline-rich coil, an ␣ helix and a ␤ strand in
the Smad-binding domain of SARA (Wu et al., 2000). The
sequence of this region differs in endofin and SARA, which
may explain why the two proteins bind distinct R-Smads.
Furthermore, it has been shown that an asparagine residue
(N381) in Smad2 makes extensive contacts with the Smadbinding domain of SARA (Wu et al., 2000). Notably, in Smad1
this residue is a serine. Substitution of N381 in Smad2 with
serine interferes with the binding of SARA to Smad2 and leads
to a failure of SARA to properly localize the mutant Smad2
for activation. We also found that substitution of the serine at
position 379 in Smad1 with asparagine interfered with the
binding of endofin to Smad1 (our unpublished data). This
would suggest that a serine at this position is critical for contact
of Smad1 with endofin.
The SARA-like proteins appear to be capable of both
enhancing and dampening the level of phosphorylation of
certain phospho-proteins. Based on our results, it seems that
when ligand binding initiates the signaling events, the endofin
recruits the Smad effectors thereby enabling their
phosphorylation, subsequent nuclear translocation and further
regulation of downstream gene expression (Tsukazaki et al.,
1998; Bennett and Alphey, 2002; Shi et al., 2004). To balance
the stimulated signaling, PP1c is recruited by endofin for
dephosphorylation of Smad1 and this enables the signaling
Journal of Cell Science
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Journal of Cell Science 120 (7)
Fig. 5. Endofin regulates expression of BMP downstream genes and mineralization in human MSCs. (A) Transcriptional response assay. C2C12
cells stably expressing GFP (control), or endofin or its mutants were transfected with BMP signaling reporter construct 9XSBE-luc. Transfected
cells were incubated in the presence or absence of BMP2 (200 ng/ml). Luciferase activity was normalized and plotted as the mean ± s.d. of
triplicates from a representative experiment. *P<0.05 compared with the second group. (B) Alkaline phosphatase activity assay. C2C12 cells
stably expressing GFP (control) or endofin or its mutant PBD(F872A) were cultured in 24-well plates treated with or without BMP2 and
harvested at day 5 for alkaline phosphatase activity assay. Relative alkaline phosphatase activity was normalized and plotted as the mean ± s.d.
of triplicates from a representative experiment. *P<0.05 compared with second group. (C) Alkaline phosphatase staining. C2C12 were cultured
and treated as in alkaline phosphatase activity assay and harvested at day 3. After fixation with 4% paraformaldehyde, cells were subjected to
alkaline phosphatase staining. Bar, 20 ␮m. (D) Overexpression of mutated endofin with disrupted PP1c-binding domain enhanced
mineralization activity of human MSCs (von Kossa Assay). Human MSCs were cultured in Dulbecco’s Modified Eagle’s Medium: low glucose,
1⫻ penicillin-streptomycin, 10% fetal bovine serum (BioWhittaker, MSC serum), 10 mM ␤-glycerol phosphate, 50 ␮M Ascorbic acid 2phosphate (AsAP) and 200 ng/ml BMP2. Mineralization assay was performed at day 24. After silver nitrate was added, a calcium deposit was
visible as a black structure or focal dot. i, GFP; ii, GFP+BMP2; iii, Endofin+BMP2; iv, Endofin-PBD(F872A)+BMP2. Bar, 30 ␮m.
Fig. 6. Regulation of BMP-dependent mesodermal induction
by endofin in Xenopus embryos. Capped RNAs encoding
endofin (wild type and mutants) and BMP4 were synthesized
in vitro and injected alone or in combination into both animal
poles of two-cell-stage embryos. The ectodermal explants
(animal caps) from injected embryos were dissected at
blastula stages (stage 8.5-9) and cultured until gastrula stages
(stage 11) before total RNA was extracted. RT-PCR was
performed using the primers for different mesodermal marker
genes. EF1-␣ serves as a loading control. Although mutations
in the FYVE and SBD domains reduced expression of BMPresponsive genes, the mutation in the PBD domain enhanced
BMP-induced marker gene expression.
Journal of Cell Science
Endofin in BMP signaling
back to a basal unstimulated state. Endofin preferentially binds
unphosphorylated R-Smads. Therefore, once most R-Smads
are phosphorylated and dislodged from endofin, PP1c will
readily bind to endofin (Shi et al., 2004). The availability of RSmads and phosphorylated R-Smads or the ratio of the two
determines whether endofin facilitates BMP signaling
positively or negatively. Thus, the immediate question is the
identity of the substrate of the PP1c once it is recruited to
endofin. Smad1 is not likely to be the substrate because it
dissociates from endofin once phosphorylated (Bennett and
Alphey, 2002; Shi et al., 2004). However, as shown previously,
GADD34, a PP1 regulatory subunit (He et al., 1996; Novoa et
al., 2001), interacts with Smad7 to direct the PP1 holoenzyme
to the type I receptor of TGF␤, ALK5 for its dephosphorylation
(Shi et al., 2004). Here we demonstrated a Smad7-independent
interaction between GADD34 and the type I receptor of BMP.
Endofin, acting as a Smad anchor for receptor activation,
delivers PP1c to GADD34 to form a holoenzyme that
dephosphorylates BMP type I receptor. This dual function also
explains why overexpression of wild-type endofin and SARA
has only a modest affect on phosphorylation of Smads and
osteoblast differentiation (Fig. 4B, Figs 5 and 6) (Bennett and
Alphey, 2002; Shi et al., 2004). These dual regulatory
mechanisms ensure the proper level of signaling activity.
However, once the dephosphorylation activity is blunted by
mutation, the positive regulatory effect of the Smad anchor for
receptor activation becomes evident immediately (Figs 5 and
6). Endofin and its mutants affected the expression of the
osteoblast differentiation marker alkaline phosphatase,
osteoblast-mediated mineralization as well as transcription of
endogenous BMP-responsive genes in vivo in Xenopus upon
BMP stimulation (Figs 5 and 6), which suggests that endofin
utilizes membrane anchoring (FYVE domain) and PP1c
dephosphorylation (PBD domain) to modulate the levels of
BMP signaling during in vitro osteoblast differentiation and in
vivo Xenopus development.
TGF␤ signaling is modulated mainly through regulation of
the phosphorylation status of key components of the signaling
pathway (Massague, 1998; Derynck, 1994). Continuous
receptor activity is required to maintain localization of active
Smads in the nucleus and for TGF␤-induced transcription
(Inman et al., 2002). This implies that depletion of active
Smads in the nucleus is a prerequisite for dampening of the
signaling. It has been reported that both Smads and receptors
can be degraded in the cytoplasm through proteosome
degradation (Datto and Wang, 2005; Kuratomi et al., 2005; Di
Guglielmo et al., 2003). However, accumulating evidence
suggests that R-Smads are recycled and that R-Smads are
shuttled continuously between the nucleus and cytoplasm (Xu
et al., 2002; Inman et al., 2002). After phosphorylation by a
type I receptor and translocation into the nucleus, the R-Smads
reappear in the cytoplasm unphosphorylated by a
dephosphorylation mechanism. Recently, such a phosphatase
was finally identified (Knockaert et al., 2006; Lin et al., 2006).
In addition to the direct dephosphorylation mechanism of RSmads, we identified a BMP-specific inhibitory mechanism
by which endofin recruits PP1 complex to dephosphorylate
type I receptors. As a result, like SARA, endofin indirectly
regulates the R-Smads phosphorylation level positively and
negatively in controlling R-Smads recycling, and maintains
balanced BMP signaling. Thus, upon BMP stimulation and
1223
Smad1 phosphorylation, endofin releases the phosphorylated
Smad1 for its translocation to the nucleus. At the same time,
there is a signal-dependent increase in binding of PP1c to
endofin for negative feedback inhibition of the BMP signals.
Endofin-regulated dephosphorylation occurs in a similar
fashion to that of SARA, but with a distinct mechanism:
inhibitory Smad7 is not involved, since type I receptors of
BMPs directly interact with GADD34 in the absence of
inhibitory Smads (Fig. 3). Furthermore, unlike direct
dephosphorylation of R-Smads by their phosphatase
(Knockaert et al., 2006; Lin et al., 2006), SARA- and/or
endofin-mediated dephosphorylation is at the receptor level,
upstream of R-Smads. It is unclear whether endofin
interaction with Smad1 and/or PP1c is also modulated by
other proteins, and whether this constitutes another level of
control for BMP signals in a cell-type-specific manner.
Materials and Methods
Antibodies and reagents
For endogenous co-immunoprecipitation, with the assistance of Cytomol Corp.
(Mountain View, CA) we developed a rabbit anti-endofin polyclonal antibody raised
against a peptide (amino acids 41-59, CSVSSELASSQRTSLLPKD) in the Nterminus of human endofin. All other antibodies were obtained from commercial
sources: monoclonal anti-Flag M2 and anti-␤-actin (Sigma-Aldrich), anti-HA
(Babco), anti-phospho-Smad1 (Ser463/465) rabbit polyclonal IgG (Upstate), antiphospho-Smad2 (Ser465/467) rabbit polyclonal IgG (Biosource), mouse
monoclonal anti-Smad1 and monoclonal anti-PP1 (Santa Cruz). Texas-Redconjugated and FITC-conjugated (donkey anti-mouse, donkey anti-rabbit)
secondary antibodies were from Jackson Immunoresearch Laboratories (West
Grove, PA).
cDNA constructs and retroviral vectors
The human cDNA of endofin (KIAA0305) was kindly provided by the Kazusa DNA
Research Institute and cloned into the pcDNA3 vector. For FYP fragment fusion
expression vectors, Smad1 and endofin were subcloned respectively at the 3⬘ and
5⬘ end of the N-terminal fragment FYP1 (a.a. 1-158) and C-terminal fragment FYP2
(a.a. 159-239) of YFP (Clontech, Palo Alto, CA) into the pcDNA3.1 vector. The
mutations in the FYVE domain and PP1c-binding domain were generated by sitedirected mutagenesis using the Quik-Change® Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA). The deletion mutant endofin⌬SBD was generated by
PCR. We used the murine stem cell virus (MSCV) vector, pMSCVneo (Clontech),
to generate cell lines expressing endofin or its mutants. Endofin and its mutants
were subcloned into pMSCVneo, and the viruses were prepared by transfection of
these retroviral constructs into a packaging cell line, 293GPG, using Lipofectamine.
Human mesenchymal stromal cells (MSCs) were infected with these viruses, and
stable expression of endofin or its mutants was achieved through neomycin
resistance gene selection. pMSCVneo-EGFP was used as a transfection and
infection control.
Immunoprecipitation and immunoblotting
Cells transfected by Lipofectamine (Gibco-BRL) were lysed with
radioimmunoprecipitation buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1%
Nonidet-P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease
inhibitors (10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 1.0 mM PMSF) and phosphatase
inhibitors (10 mM sodium orthovanadate, 50 nM inhibitor-1 and 50 mM sodium ␤glycerophosphate). Lysates were immunoprecipitated by incubation with the
appropriate antibodies, followed by adsorption to protein-G-Sepharose.
Immunoprecipitates were separated by SDS-PAGE, blotted onto a PVDF membrane
(Bio-Rad Laboratories), and visualized by enhanced chemiluminescence (ECL Kit;
Amersham Biosciences).
Small interfering RNA (siRNA) constructs and transfection
To generate the construct for silencing endogenous endofin expression, a 21nucleotide oligo (oligo 1) corresponding to nucleotides 390-410 of the mouse
endofin coding region was first inserted into ApaI-HindIII-digested pBS/U6 (Sui et
al., 2002). The inverted motif that contains the six-nucleotide spacer and five Ts
(oligo 2) was then subcloned into the EcoRI-HindIII sites of the intermediate
plasmid to generate BS–U6–si-Endofin for endofin silencing. The sequence of oligo
1 is 5⬘-GGTAA CTTAG TGCAT GCCAC A-3⬘ (forward) and 5⬘-AGCTT GTGGC
ATGCA CTAAG TTACC-3⬘ (reverse). The sequence of Oligo 2 is 5⬘-AGCTT
GTGGC ATGCA CTAAG TTACC CTTTT TG-3⬘ (forward) and 5⬘-AATTC
AAAAA GGGTA ACTTA GTGCA TGCCA CA-3⬘ (reverse). For the control
construct for silencing of green fluorescence protein (GFP) expression, pBS-U6-
1224
Journal of Cell Science 120 (7)
siGFP, a 22-nucleotide oligo (oligo 1) corresponding to nucleotides 106-127 of the
GFP coding region was used as described previously (Sui et al., 2002). A BLAST
search of the NCBI database ensured specific targeting of the cognate mRNA.
C2C12 myoblasts were transfected using Lipofectamine (Invitrogen).
Detection of in vivo phosphorylation
Thirty-six hours after transfection with different combinations of genes, cells were
washed twice with phosphate-free DMEM containing 2% dialyzed fetal calf serum,
incubated in the same medium for 4 hours and then treated with rhBMP2 (SigmaAldrich) for an additional 2 hours at 37°C. The cells were washed again with the
same medium and incubated with complete DMEM/2% FBS for a further 2 hours.
The cells were then washed with ice-cold PBS and lysed with
radioimmunoprecipitation assay buffer. Flag-Smad1 and HA-ALK3 was
immunoprecipitated with anti-Flag and anti-HA, respectively, as described above.
The resultant precipitates were separated by 8.5% SDS-PAGE, blotted onto PVDF
membranes and visualized using the ECLPlus western blotting detection system
(Amersham Biosciences). For metabolic labeling, the cells were labeled with 1
mCi/ml [32P]orthophosphate (PerkinElmer) for an additional 2 hours at 37°C in the
absence or presence of BMP2 (200 ␮g/ml), then subjected to the procedures
described above. Finally, the gels were dried and exposed to Biomax Mr or MS film
(Eastman Kodak Co.). For confirmation of equal loading, the dried gels were
rehydrated with transfer buffer after autoradiographic analysis, reblotted onto a
PVDF membrane, and the transfected HA-ALK3 visualized using the ECLPlus
western blotting detection system (Amersham Biosciences).
Journal of Cell Science
Subcellular localization
Immunolocalization was performed as described previously (Shi et al., 2004).
Briefly, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with
0.1% Triton X-100, and the lysates incubated with primary antibody incubation,
followed by incubation with chromophore-conjugated secondary antibody. Digital
pictures were taken using an Olympus, IX TRINOC camera fitted to an Olympus,
IX70 Inverted Research Microscope (Olympus) with objective lenses of Hoffman
Modulation Contrast®, HMC 10 LWD PL FL, 0.3NA ⬁/1, (OPTICS Inc.) at room
temperature, and processed using MagnaFire® SP imaging software (Optronics). A
Zeiss TCs SP2 system was used for confocal imaging.
Alkaline phosphatase (ALP) and von Kossa assays
Cells were cultured in osteogenic induction medium (100 nM ascorbic acid, 10 mM
glycerophosphate) for osteogenic differentiation. ALP activity and staining were
determined using the ALP activity assay kit (APF-1KT) and ALP staining kit (86c1KT) from Sigma according to the manuals provided with the kits. For von Kossa
staining, human MSCS cells were cultured in osteoinduction medium with or
without BMP2 (200 ng/ml). Medium was changed every third or fourth day and the
mineralization assay was performed at day 24.
Xenopus animal cap assay
Capped RNAs encoding endofin (wild-type and mutants) and BMP4 were
synthesized in vitro using the mMessage Machine kit (Ambion). The RNAs were
then injected alone or in combination into both animal poles of two-cell stage
embryos. The ectodermal explants (animal caps) from injected embryos were
dissected at blastula stages (stage 8.5-9) and incubated until they reached the gastrula
stage (stage 11) before total RNA was extracted. Reverse transcription-PCR was
performed using the primers for different marker genes as described previously
(Chang et al., 1997; Chang and Hemmati-Brivanlou, 1999). First-strand cDNAs were
synthesized from 1 ␮g total RNAs with oligo(dT) priming using Multiscript Reverse
Transcriptase (Promega) at 37°C for 1 hour. 1.5 ␮Ci [␣-32P]dATP was added to each
PCR sample, and the PCR products were resolved by electrophoresis on a 5%
polyacrylamide gel. As a negative control, PCR was performed with whole embryo
RNA that had not been reverse-transcribed to check for DNA contamination.
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