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INTRODUCTION |
The transforming growth factor
(TGF)1-
superfamily
cytokines act on a wide variety of cells and organs to regulate
development and homeostasis. Members of the Smad proteins play pivotal
roles in the intracellular signal transduction of TGF-
family
proteins. Activated type I TGF-
family receptors phosphorylate
conserved serine residues at the C terminus of so-called
receptor-regulated Smads, Smad1, -5, and -8, for receptors of bone
morphogenetic proteins (BMP), or Smad2 and -3 for receptors of TGF-
and activin, respectively. The phosphorylated Smads are then
translocated into the nucleus with their common partner Smad4, where
they activate target genes in collaboration with other transcriptional
partners (1-3).
Smad-mediated signaling is regulated or modified through a number of
Smad-interacting proteins. It has recently been shown that in the
cytoplasm, a FYVE domain protein, SARA, binds to both Smad2 and TGF-
receptor, and plays an important role in recruiting Smad2 to the
receptor by controlling the subcellular localization of Smad (4).
Microtubules bind to Smad2, -3, and -4, and negatively regulate
signaling, controlling the rate of Smad2 association to receptor and
subsequent phosphorylation (5).
In the nucleus, Smad proteins form complexes with transcriptional
partners depending on target genes. Whereas Smad4 itself has a DNA
binding activity (6-9), a winged-helix DNA-binding protein, FAST1, is
required for Smad2/4 complexes to activate activin-inducible
Mix.2 gene transcription (10). The general co-activator p300
associates with Smad3 and induces transcriptional activity
synergistically (11, 12). In contrast, Ski family oncoproteins (13-15)
and a homeodomain protein TGIF (16) bind to Smad proteins and repress
transcription through recruiting histone deacetylase complex into
TGF-
-activated Smad complexes. AP-1 enhances but oncoprotein
Evi-1 represses the TGF-
-induced transcription through
binding to Smad3 (17, 18). Moreover, Smad3 has been shown to associate
with vitamin D receptor and to act as a co-activator for vitamin D
receptor-mediated transcription, implicating a cross-talk between
vitamin D and TGF-
signaling pathways (19).
It has recently been shown that Smad signaling is also negatively
regulated by a ubiquitin-dependent degradation. The Hect family of E3 ubiquitin-protein ligase Smurf1 interacts with Smad1 and
Smad5, and triggers their ubiquitinization and subsequent degradation
independently of receptor activation (20). In contrast, ubiquitin
conjugating enzymes bind to Smad2 in an activation dependent manner in the nucleus and cause its multi-ubiquitinization and subsequent degradation (21).
Filamin 1 (also called ABP-280) is the major human non-muscle isoform
of a protein family (22). It is a homodimer of 280 kDa containing
N-terminal actin-binding domain and 24 tandem repeats of 96 amino
acids. The last repeat, 24, represents self-association domain of the
molecule. Filamins are multifunctional proteins (23-28). They
efficiently cross-link actin filaments, connect cortical actin filament
networks to cell membrane receptors, and act as a scaffold for
intracellular proteins involved in signal transduction.
Here we report the identification of filamin as a Smad-binding protein.
Filamin-deficient melanoma cells showed impaired TGF-
signaling
activity compared with filamin-supplemented cells, evidenced by a
decreased signal-dependent Smad2 phosphorylation. The
interaction of filamin with Smad family proteins may represent a
potential regulatory mechanism in TGF-
superfamily signaling.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening--
To construct a bait plasmid
(pAS-Smad5) for yeast two-hybrid system, a mouse Smad5 cDNA
fragment, corresponding to amino acids 5-378 and lacking approximately
half of the MH2 C-terminal domain, was inserted in frame into pAS2-1
GAL4 DNA-binding vector (CLONTECH, Palo Alto, CA).
The same cDNA fragment was subcloned in frame into pGBT9 to
generate pGBT9-Smad5. A yeast strain, Y190, was co-transformed with
pAS-Smad5 and human chondrocyte cDNA library constructed in pACT2
(CLONTECH). The transformants were first selected
for HIS3 gene transactivation by virtue of growth in the
absence of Trp, Leu, and His and in the presence of 40 mM 3-amino-1,2,4-triazole. Next, colonies grown on the selection media
were selected for LacZ gene transactivation by
-galactosidase activity in filter assay. pLAM5' encoding GAL4
DNA-binding domain/human lamin C fusion protein was used as a negative
control. Prey plasmids were recovered from
-galactosidase-positive
colonies, and sequence analysis was performed with ABI PRISMTM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
Construction of Expression Vectors--
A C-terminal fragment of
filamin corresponding to amino acids 2163-2647 (ABP-C) was obtained
from the positive clones. Various deletion mutants of filamin were
generated by polymerase chain reaction and subcloned in frame into
pGEX-5X-1 (Amersham Pharmacia Biotech, Uppsala, Sweden) or pcDNA3
(Invitrogen, Carlsbad, CA) with HA epitope at N terminus. cDNAs for
full-length Smad1, -2, -4, -5, and -6 and deletion mutants of Smad5
were generated by polymerase chain reaction and were subcloned into
pcDNA3 with FLAG epitope at the N terminus. All constructs were
fully sequenced.
pEFBOS-filamin-
N, an expression vector for a truncated filamin that
lacks most of the N-terminal actin-binding domain and repeat 1-13, was
generated by excision of SalI/BamHI fragment from pEFBOS-filamin1 (an expression vector for full-length human filamin) and self-ligation with an oligonucleotide spacer for adjusting
reading frame.
Cell Culture--
HEK293 cells were cultured in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) supplemented with
9% fetal bovine serum (FBS). M2, a human melanoma cell line lacking
filamin, and its subline, A7, transfected stably with a full-length
filamin cDNA, were cultured in MEM (Life Technologies, Inc.)
supplemented with 8% newborn calf serum and 2% FBS (23). A7 cells
were cultured in the presence of 0.3 mg/ml G418 (Life Technologies,
Inc.) to maintain filamin expression.
GST Pull-down Assay--
GST and GST fusion proteins were
expressed in DH5
induced by 0.25 mM
isopropyl-1-thio-
-D-galactopyranoside and purified by
affinity chromatography using glutathione-Sepharose beads (Amersham Pharmacia Biotech). [35S]Methionine-labeled Smad5 and its
deletion mutant proteins were generated by in vitro
transcription/translation system (Promega, Madison, WI). Aliquots were
incubated with GST- or GST-ABP-C-conjugated glutathione-Sepharose beads
in binding buffer (50 mM Tris-HCl, pH 7.5, 138 mM KCl, 1 mM EDTA, 0.2% Nonidet P-40, 5%
glycerol, and 5% BSA). 35S-Labeled Smad5-
C (Smad5
lacking MH2 domain) was synthesized as above and incubated with GST or
GST-ABP deletion mutant proteins in the same binding buffer, followed
by precipitation with glutathione-Sepharose beads. Bound proteins were
eluted from beads by boiling in SDS sample buffer, separated by
SDS-PAGE, and visualized by autoradiography.
Immunoprecipitation and Immunoblotting--
HA epitope-tagged
ABP-C expression vector (pHA-ABP-C) and FLAG epitope-tagged expression
vectors for Smad1-6 (pF-Smad1-6) were co-transfected transiently into
HEK293 cells using LipofectAMINE reagent (Life Technologies, Inc.). At
24 h after transfection, cells were lysed with radioimmune
precipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) supplemented with a mixture of proteinase inhibitors (CompleteTM, Roche Molecular Biochemicals, Mannheim, Germany). Precleared cell lysates were subjected to immunoprecipitation with anti-FLAG M2 antibody (Sigma) or anti-HA 3F10 antibody (Roche Biochemicals) following adsorption to protein G-Sepharose beads (Amersham Pharmacia Biotech). In some experiments, agarose-conjugated anti-FLAG antibody and FLAG peptide as a competitor (Sigma) were used. Bound proteins were eluted from beads by boiling in
SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane. Immunoblotting was performed using
anti-FLAG M5 antibody (Sigma) or anti-HA 3F10 antibody, and visualized
by ECL Plus reagents (Amersham Pharmacia Biotech).
Reporter Gene Assay--
Constitutively active (pALK5-TD) and
kinase negative (pALK5-KR) type I TGF-
receptor expression vectors
were kindly provided by Dr. K. Miyazono (University of Tokyo, Tokyo,
Japan). These ALK5 receptor plasmids were transiently co-transfected
with a TGF-
-responsive reporter plasmid, p3TP-lux, using FuGENETM 6 reagent (Roche Molecular Biochemicals). In all transfection
experiments, pCMV-RL, Renilla luciferase expression vector
(Promega), was used as an internal control for normalization of
transfection efficiency. Cells were lysed in Passive Lysis Buffer
(Promega) and assayed for firefly and Renilla luciferase
activities (Lumat LB 9507, EG&G Berthold, Bad Wildbad, Germany). Each
assay was carried out at least in triplicate.
Immunofluorescence Microscopic Examination--
M2 and A7 cells
were plated on chamber slides and cultured in MEM with 0.5% FBS for
24 h. Cells were treated with 10 ng/ml human recombinant TGF-
1
(Roche Molecular Biochemicals) for 90 min, fixed with 4%
paraformaldehyde, and then permeabilized with methanol. Smad2 proteins
were detected by indirect immunofluorescence technique using anti-Smad2
monoclonal antibody (Transduction Laboratory, Lexington, KY) and
FITC-conjugated anti-mouse IgG (Sigma). Cells were counterstained with
4,6-diamidino-2-phenylindole to visualize nuclei. Microscopic
examination was performed (Axiovert S100, Carl Zeiss, Jena, Germany)
and visualized with an image analysis system (Spot Software version
2.2, Diagnostic Instruments, Inc., Sterling Heights, MI).
In Vivo Phosphorylation--
M2 and A7 cells were co-transfected
with pF-Smad2 and pALK5-TD or pALK5-KR in serum-free medium containing
0.1% BSA. At 24 h after transfection, cells were lysed with
radioimmune precipitation buffer containing 50 mM NaF, 1 mM Na3VO4 and the proteinase
inhibitor mixture (CompleteTM). The cell lysates were
immunoprecipitated with anti-FLAG antibody, and the precipitates were
subjected to immunoblotting as described above. The phosphorylated
proteins were detected with anti-phosphoserine antibody
(Zymed Laboratories Inc., San Francisco, CA).
M2 and A7 cells were cultured in phosphate-free MEM with 0.5% FBS for
24 h, and then labeled with [32P]orthophosphate for
3 h following treatment with human recombinant TGF-
1 (10 ng/ml)
for 20 min. After TGF-
treatment, cells were harvested in lysis
buffer (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.5% Triton X-100) supplemented with 50 mM NaF, 1 mM Na3VO4,
and the proteinase inhibitor mixture (CompleteTM). Precleared cell
lysates were subjected to immunoprecipitation with anti-Smad2 antibody
(Santa Cruz Biotechnology, Santa Cruz, CA) following protein
G-Sepharose bead adsorption. Precipitated and eluted proteins were
separated on SDS-PAGE and visualized by autoradiography.
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RESULTS |
Identification of Filamin as a Smad5-interacting
Protein--
Three million transformants of yeast were screened by
interaction trap, and 110
-galactosidase-positive colonies were
obtained using Smad5 as a bait. The prey plasmids were recovered from
47 positive clones and sequenced, resulting in isolation of 4 independent clones encoding the C-terminal portion of filamin. In
addition to filamin, clones encoding ABPL, a homologue of filamin (29), were isolated. At least two clones encoding Smad4 were also detected (data not shown). These positive clones were re-introduced into yeast
with pGBT9-Smad5 to confirm specific interaction. Only pGBT9-Smad5, but
not pAS2-1 or pLAM5'showed transactivation of HIS3 and
lacZ with these prey plasmids encoding filamin, ABPL, and
Smad4 (data not shown).
Sequence analysis of the positive clones revealed that all of the
clones encoding filamin started at repeat 20 (Fig.
1), it is reasonable to assume that this
region contains a Smad-binding site. The shared domain, encoded by
clone 22, was herein termed ABP-C.

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Fig. 1.
Identification of cDNA clones encoding
filamin (ABP-280) in yeast two-hybrid screening. Schematic diagram
of filamin structure (upper) and four filamin-encoding
cDNA clones obtained in yeast two-hybrid screening
(lower). The first amino acid residue of filamin in each
clone is indicated as one-letter symbol. The C-terminal
portion of filamin encoded by clone 22 was termed ABP-C.
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Smad5 Binds to Filamin in Vitro--
To examine whether Smad5
protein directly binds to filamin, GST pull-down assay was performed
using GST-fused ABP-C protein and 35S-labeled Smad5. As
shown in Fig. 2A,
35S-labeled Smad5 was precipitated with GST-ABP-C, but not
with GST, indicating that the binding of Smad5 is specific to ABP-C. The interacting site in Smad5 was then determined by GST pull-down assay with Smad5 deletion mutants. Smad5-
C lacking the MH2 domain bound to GST-ABP-C stronger than full-length Smad5 and Smad5-
N. Smad5-MH1, Smad5-l
N, and Smad5-l bound only weakly to GST-ABP-C and
Smad5-MH2 did not (Fig. 2A). The results of binding analysis with Smad5 deletion mutants are summarized in Fig. 2C. Based
on these assays, it is suggested that a filamin-binding site is located within the MH1 domain and the linker portion of Smad5, which is consistent with the result of yeast two-hybrid screening.

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Fig. 2.
In vitro binding of Smad5 to
ABP-C. A, in vitro binding of Smad5 deletion
mutants with ABP-C. FLAG tagged-Smad5 and its deletion mutant proteins
were synthesized in vitro with 35S labeling and
incubated with glutathione-Sepharose beads conjugated with GST
(G) or GST-ABP-C (A) in binding buffer as
described under "Experimental Procedures." Precipitated proteins
were separated by SDS-PAGE (left two
panels). 10% of 35S-labeled proteins were
separated without pull-down to evaluate protein synthesis
(right two panels). B,
in vitro binding of ABP deletion mutants with Smad5- C.
FLAG tagged-Smad5- C was synthesized in vitro with
35S labeling and incubated with GST or GST-ABP deletion
mutant proteins, followed by precipitation with glutathione-Sepharose
beads. Precipitated proteins were separated by SDS-PAGE
(left panel). 5% of 35S-labeled
F-Smad5- C was separated without pull-down to evaluate protein
synthesis (far right lane). C, schematic diagram
of the structure of Smad5 deletion mutants and summary of GST pull-down
assay. D, schematic diagram of the structure of filamin
deletion mutants and summary of GST pull-down and yeast two-hybrid
assays.
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A Smad5-binding site in filamin was also determined using deletion
mutants of ABP-C. In GST pull-down assay, GST-ABP-
3, which lacks the
24th repeat of filamin, bound to Smad5-
C efficiently or even
stronger than GST-ABP-C (Fig. 2B), indicating that the repeat is not essential for binding to Smad5. Almost identical results
were obtained by yeast two-hybrid system (data not shown). The results
of binding analysis with ABP-C deletion mutants are summarized in Fig.
2D.
Smad5 and Other Smad Proteins Bind to Filamin in Vivo--
We next
examined the association of Smad5 protein to filamin in
vivo. HA-tagged ABP-C (HA-ABP-C) and FLAG-tagged Smad5 (F-Smad5) were transiently overexpressed in HEK293 cells (Fig.
3C). As shown in Fig.
3A, HA-ABP-C was co-immunoprecipitated with F-Smad5 from lysates of HEK293 transfectants (lane 3).
Inversely, F-Smad5 was co-immunoprecipitated with HA-ABP-C (Fig.
3B, lane 4), suggesting that Smad5
forms complexes with ABP-C in mammalian cells.

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Fig. 3.
Interaction of Smad5 (A and
B) and other Smad proteins (D) with
ABP-C in vivo. HEK293 cells were transiently
transfected with the indicated plasmids. Cell lysates were
immunoprecipitated (IP) with anti-FLAG antibody
(A) or with anti-HA antibody (B and
D). FLAG peptide was used as a specific competitor in
A. The immunoprecipitates were resolved by SDS-PAGE, and
precipitated proteins were detected by immunoblotting with anti-HA
antibody (A) or anti-FLAG antibody (B and
D), respectively. Aliquots of cellular extracts were
immunoblotted without immunoprecipitation to evaluate protein
expression (C and the lower panel of
D).
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We further examined whether filamin binds to other members of Smad
proteins in vivo. As expected, Smad1, which bears ~90% amino acid identity to Smad5, was co-precipitated with HA-ABP-C. In
addition, Smad2, Smad4, and Smad6 were also shown to be co-precipitated with HA-ABP-C in vivo (Fig. 3D).
Defective TGF-
Signaling in Filamin-deficient Cells--
In
order to determine whether or not the interaction of Smad family
proteins with filamin affects TGF-
superfamily signaling, a
filamin-deficient human melanoma cell line, M2, and its subline, A7,
stably transfected with full-length human filamin cDNA (23) were
analyzed using GAL4-Smad5 reporter system. The extracts from A7 cells
co-transfected with pBIND-Smad5 (GAL4-Smad5 fusion construct) and
pG5-luc (GAL4-dependent luciferase reporter construct)
showed ~2.5-fold increase in the luciferase activity, compared with
those transfected with pBIND control vector and pG5-luc. Whereas M2 cells did not show such increased luciferase activity (data not shown).
Although this may suggest that BMP signaling activity was reduced in
filamin-defective cells, both M2 and A7 cells failed to enhance
ligand-dependent luciferase activity with co-transfection of the constitutive active type I BMP receptor or with BMP4 treatment (data not shown). We therefore switched to TGF-
-responsive p3TP-lux reporter system, because filamin was also shown to be associated with
TGF-
/activin-specific Smad2 (Fig. 3D). As shown in Fig. 4A, A7 showed ~8-fold
induction of p3TP-lux reporter activity when co-transfected with
constitutive active type I TGF-
receptor plasmid, whereas M2
exhibited impaired response (~3-fold). A7 cells also showed ~6-fold
induction of p3TP-lux reporter activity in response to TGF-
1 (data
not shown).

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Fig. 4.
Defective transcriptional response to
TGF- (A and
B) and nuclear accumulation of Smad2
(C) in filamin-deficient M2 cells. A,
filamin-deficient M2 cells and filamin-supplemented A7 cells were
co-transfected with p3TP-lux reporter construct and pALK5-TD
(constitutive active type I TGF- receptor) or pALK5-KR (kinase
negative type I TGF- receptor). Twenty-four hours after
transfection, luciferase activities in cell lysates were determined.
B, M2 cells were transfected with p3TP-lux reporter
construct and increasing amounts of pEFBOS-filamin1 (full-length
filamin) or pEFBOS-filamin- N (N-terminal truncated filamin) in the
presence or absence of pALK5-TD. 24 h after transfection,
luciferase activities in cell lysates were determined. The activity is
shown as -fold induction by pALK5-TD. C, M2 and A7 cells
were treated with TGF- 1 (10 ng/ml) for 90 min prior to fixation.
Immunostaining was performed with anti-Smad2 monoclonal antibody and
fluorescence-conjugated secondary antibody. Cells were counterstained
with 4,6-diamidino-2-phenylindole (DAPI) to visualize
nuclei. Note that TGF- -induced nuclear accumulation of Smad2 is
observed only in A7 cells.
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To rule out the possibility that the difference in the responsiveness
to TGF-
was due to the clonal difference between M2 and A7 cells, an
expression vector for full-length human filamin, pEFBOS-filamin1, was
transfected transiently into filamin-deficient M2 cells, and the
reporter gene assay was performed using p3TP-lux. M2 cells restored the
response to the constitutive active receptor, dependently on the
amounts of transfected pEFBOS-filamin1 (Fig. 4B). M2 cells
also restored the response following transfection of N-terminal
truncated filamin, suggesting that the actin-binding domain is
dispensable for TGF-
signaling.
TGF-
-induced nuclear translocation of Smad2 was also impaired in M2
cells. As shown in Fig. 4C, nuclear accumulation of
endogenous Smad2 in response to TGF-
1 was observed in A7 cells,
whereas the nuclear accumulation was not seen in M2 cells following
TGF-
1 treatment. The expression level of Smad2 protein per cell was comparable between A7 and M2 cells in immunoblotting analysis (data not
shown). To examine whether the distribution of filamin may change upon
TGF-
treatment, we observed filamin by anti-filamin antibody and
fluorescein-labeled secondary antibody in A7 cells with or without
TGF-
treatment. Filamin was distributed throughout the cytoplasm,
occasionally concentrated at filopodia and lamellipodia, with no change
in distribution upon TGF-
1 treatment (data not shown).
Impaired Smad2 Phosphorylation in Filamin-deficient Cells--
The
result of impaired nuclear translocation in M2 cells suggests that
Smad2-mediated signaling is impaired at cytoplasmic level in
filamin-deficient M2 cells. We therefore compared Smad2 phosphorylation
in M2 and A7 cells after stimulation with constitutive active TGF-
receptor. As shown in Fig. 5A,
F-Smad2 was serine-phosphorylated in A7 cells following co-transfection
with pALK5-TD, whereas phosphorylation of F-Smad2 was impaired in M2
cells.

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Fig. 5.
Defective Smad2 phosphorylation in
filamin-deficient M2 cells. A, M2 and A7 cells were
co-transfected with pF-Smad2 and pALK5-TD (TD), pALK5-KR
(KR), or pcDNA3 (C). 24 h after
transfection, cells were lysed, immunoprecipitated (IP) with
anti-FLAG antibody, followed by immunoblotting with anti-phosphoserine
antibody (upper panel) or anti-FLAG antibody
(lower panel). B, M2 and A7 cells were
labeled with [32P]orthophosphate for 3 h and treated
with TGF- 1 (10 ng/ml) for 20 min. Cell lysates were
immunoprecipitated with anti-Smad2 antibody and resolved by SDS-PAGE
(upper panel). Lysates of unlabeled cells treated
with TGF- 1 in the same way were immunoprecipitated and immunoblotted
with anti-Smad2 antibody to evaluate the amounts of immunoprecipitated
Smad2 protein (lower panel). C, M2 and
A7 cells were co-transfected with pF-Smad2 and pALK5-TD, lysed,
immunoprecipitated, and immunoblotted in the same way as A.
M2 cells were also co-transfected with pEFBOS-filamin1 (F),
pEFBOS-filamin- N (F N), or pcDNA3
(C). D, schematic representation of a potential
function of filamin in Smad phosphorylation. Filamin may serve as an
anchor protein to recruit Smad proteins to control its localization
near the cell surface receptors (a). Alternatively, filamin
may prevent Smad proteins to undergo inactive conformation induced by
self-association, and thereby help phosphorylation or subsequent
hetero-oligomerization (b).
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Next, we investigated phosphorylation of endogenous Smad2 in response
to TGF-
1 treatment. The anti-Smad2 immunoprecipitates from
[32P]orthophosphate-labeled cells were examined by
SDS-PAGE to detect phosphorylation of Smad2. As shown in Fig.
5B, A7 cells increased Smad2 phosphorylation in response to
TGF-
1 within 20 min, whereas M2 cells did not.
To investigate whether M2 cells restore Smad2 phosphorylation, the
full-length and a truncated filamin lacking N-terminal actin-binding
domain were transiently transfected to M2 cells, and the Smad2
phosphorylation were assessed as described above. In agreement with the
results of reporter gene assay (Fig. 4B), both the
full-length and the N-terminal truncated filamin augmented Smad2 serine
phosphorylation in M2 cells upon stimulation with constitutive active
TGF-
receptor (Fig. 5C).
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DISCUSSION |
Subcellular localization, phosphorylation, nuclear translocation,
transcriptional activity, and turnover of Smad proteins are thought to
be regulated by Smad-interacting molecules. In the present study we
have identified filamin, a cytoskeletal protein, as a Smad-interacting
protein, and demonstrated the involvement of filamin in Smad2-mediated
TGF-
signaling in the cytoplasm.
Interaction of Smad family proteins with filamin suggested the
involvement of filamin in TGF-
superfamily signaling.
Filamin-deficient M2 cells showed impaired TGF-
signaling compared
with filamin-supplemented A7 cells (Fig. 4, A and
C) and the defect in M2 cells was shown to be due to
decreased Smad2 phosphorylation in response to TGF-
stimulation
(Fig. 5, A and B). The reduced TGF-
signaling
activity was restored following supplement of filamin by transfection
(Figs. 4B and 5C). The N-terminal truncated
filamin also rescued TGF-
responsiveness in M2 cells, indicating
that the binding of filamin to actin with subsequent actin network
reorganization and stabilization is dispensable for the function of
filamin at least in TGF-
signaling. Since phosphorylation of Smad is
recognized as a trigger for Smad-mediated signaling, interaction with
filamin may be essential for effective Smad phosphorylation in TGF-
superfamily signaling pathway. However, it is also possible that
nuclear translocation of Smads is impaired in M2 cells, through
distinct mechanism from phosphorylation of Smad protein.
How does filamin work in the phosphorylation of Smad? There are several
possible explanations for the potential function of filamin in Smad
pathway (Fig. 5D). First, filamin may serve as an anchor
protein, like SARA, to control localization of Smad proteins near the
cell surface receptors. Supporting this idea is the observation that
filamin is concentrated in cortical regions of the cytoplasm tethered
by certain cell surface molecules (25, 27, 30). Second,
filamin-associated Smad proteins may keep their conformation suitable
for receptor-mediated phosphorylation. MH1 domains of Smad2 and Smad4
can interact with their corresponding MH2 domains to form inactive
conformation (31), and association with filamin may interrupt this
self-association to promote phosphorylation or subsequent
hetero-oligomerization. There is a possibility that filamin protects
Smad proteins from ubiquitin-dependent degradation by
masking a ubiquitin ligase-binding site of Smads. However, the
expression level of Smad2 in both M2 and A7 cells did not change
following treatment with a proteasome inhibitor, MG-132 (data not
shown), which suggest that absence of filamin did not cause an
increased turnover of Smad2 protein in M2 cells.
It was recently reported that cytoskeletal microtubules bind to Smad
proteins, and negatively regulate the signaling (5). Our study strongly
suggests that filamin, which is localized in the juxtamembrane region
where receptors and their signaling mediators interact, work positively
on Smad phosphorylation. Although it remains a possibility that
cytoplasmic filamin far from cell surface membrane may negatively
regulate Smad signaling like microtubules, filamin is essential for
maximal function of Smad-mediated signaling, evidenced by the results
from the experiments using filamin-deficient cells. It is suggested
that the mode of the regulation in Smad signaling is different between
these cytoskeletal elements, filamins and tubulins, depending on their
cytoplasmic organization and localization.
A variety of proteins have been reported to interact with filamin,
including cell surface protein integrin
chains (24, 30, 32),
glycoprotein Ib
(33), immunoglobulin G Fc receptor I (34), tissue
factor (25), presenilin-1 (35), processing enzyme furin (28),
cytoplasmic MAPK protein SEK1 (36), the small GTPase RalA (27), and
tumor necrosis factor receptor-associated factor 2 (37). Among these
molecules, integrin
chains, presenilin-1, and SEK1 bind to a
portion of filamin similar to the Smad-binding site identified in
current study, namely from repeat 20 to repeat 23 or 24.
Accumulating evidence suggests that MAPK cascade closely correlates
with TGF-
superfamily signaling. The extracellular signal-regulated kinase subfamily of MAPK phosphorylates specific sites in the linker
region of Smads, thereby inhibiting (38, 39), or activating (40)
nuclear translocation. MAPK cascade is also included by TGF-
signaling itself. It was reported that c-Jun N-terminal kinase is also
activated in a Smad-independent manner (41, 42). p38 is activated by
TGF-
superfamily stimulation in rat pheochromocytoma cells (43),
human gingival fibroblasts (44), and developing Drosophila
wing (45), possibly via TAK1-MKK6/3-p38 cascade (46). c-Jun N-terminal
kinase-activated AP-1 complex and p38-activated ATF-2 work
synergistically with Smad complex in TGF-
- induced gene
transcription (47, 48). Interestingly, SEK1, which binds to the
C-terminal region of filamin, is activated by TAK1 (49). Therefore, it
is possible that filamin mediate cross-talk between Smad and MAPK pathways.
On the other hand, cross-talk between integrins and TGF-
signaling
system has also been reported. Expression of TGF-
receptors is
controlled by ligand-activated integrins in breast cancer (50) and
osteoblastic (51, 52) cells. In bleomycin-induced pulmonary fibrosis,
integrin
v
6 binds to latency-associated
peptide and induces TGF-
activation to induce pulmonary inflammation
(53). MAPK is activated by integrin stimulation in fibroblasts (54) and
osteoblasts (52) through focal adhesion protein-tyrosine kinase
activation. Since MAPK phosphorylates linker portion of Smads and
regulates their nuclear translocation, it is possible that activated
integrin modulates Smad-mediated signaling via MAPK pathway.
It has been recently shown that periventricular heterotopia (PH), a
human X-linked dominant disorder, is caused by a mutation of the
filamin1 gene (55). Most patients of PH suffer from seizures because a subset of neuron cells fail to migrate into developing cerebral cortex and persist as nodules of neurons lining the
ventricular surface. PH patients also show shortened
digits, syndactyly, and clinodactyly. These anomalies have also been
observed in impairment of TGF-
superfamily protein (56, 57), and
defective Smad-mediated signaling due to mutations in filamin may
underline the pathogenesis of PH.
In conclusion, we have identified a cytoskeletal protein, filamin, as a
Smad-associating protein, and demonstrated this interaction plays an
important role in receptor-mediated phosphorylation of Smad. In view of
its association with various mediators of intracellular signaling,
filamin may provide a scaffold of cross-talk between TGF-
superfamily and other signal transduction pathways.