1Program in Membrane Biology and 2Renal Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Charlestown 02129; and 3Molecular Cardiology Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111
Submitted 6 February 2003 ; accepted in final form 2 June 2003
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ABSTRACT |
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signal transduction; mitogen-activated protein kinase; phosphopeptide mapping; extracellular signal-regulated kinase phosphorylation site; luciferase reporter
Smad4 is the product of the tumor suppressor gene deleted in pancreatic carcinoma-4 (DPC-4) (10). Its MH1 domain can directly interact with DNA (26) but is also thought to bind to the phosphorylated C-tail of activated R-Smads (11). The MH2 domain is important for transcriptional activation (3, 5, 27) and for direct protein interactions with the MH1 domain of R-Smads (23). Smad4 lacks the COOH-terminal SSV/MS motif found in R-Smads and neither binds nor is phosphorylated by activated type I receptors (15, 17). However, Smad4 has been shown to be constitutively phosphorylated (17), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 have never been elucidated.
A domain in the COOH-terminal part of the linker region between amino acid
residues 275 and 322, named the Smad activation domain (SAD), was demonstrated
to be important for full transcriptional activity of Smad4
(3). How the SAD regulates
Smad4 transcriptional activity is unknown but may involve binding to
transcriptional coactivators
(5). It was suggested that
Smad4 shuttles continuously between the nucleus and the cytoplasm as a
consequence of a functional nuclear localization signal (NLS) located in the
MH1 domain and a nuclear export signal (NES) located in the
NH2-terminal part of the linker region
(18). Activation by TGF-
causes Smad4 to accumulate in the nucleus, but little is known about the
mechanism of this nuclear accumulation.
An interplay between TGF-/activin/bone morphogenetic protein (BMP)
signaling and Ras-activating mitogens was demonstrated by the identification
of functional ERK phosphorylation sites in the linker regions of R-Smads that
are distinct from the receptor-mediated phosphorylation sites at the COOH
terminus. ERK-mediated phosphorylation of R-Smads could potentially inhibit
the nuclear accumulation of these proteins and block TGF-
/BMP-induced
signaling (13,
14). In contrast, others
demonstrated that phosphorylation of R-Smads by mitogen-activated protein
(MAP) kinases leads to enhanced nuclear accumulation of R-Smads
(4,
7).
This report describes the identification and characterization of a
consensus ERK phosphorylation site in the linker region of the Co-Smad Smad4
at threonine 276 (Thr276). Phosphorylation of Thr276 is
shown to be important for TGF--induced nuclear accumulation and, as a
consequence, transcriptional activity of Smad4.
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EXPERIMENTAL PROCEDURES |
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Immunoprecipitation, SDS-PAGE, and Western blot analysis. Cells
were lysed in lysis buffer [50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 1x protease inhibitor cocktail (Sigma)]. Protein
extracts were immunoprecipitated with M2 anti-Flag antibody resin (Sigma) or
analyzed directly. SDS-PAGE was performed with precast NuPAGE Novex 4-12%
Bis-Tris gels (Invitrogen). Samples were electroblotted to polyvinylidene
difluoride (PVDF) membranes (Bio-Rad, Hercules, CA), and membranes were probed
with anti-human Smad4 rabbit polyclonal IgG (1:400; Santa Cruz, Santa Cruz,
CA), monoclonal M2 anti-Flag antibody (1:5,000; Sigma), anti-Smad4 (1:500;
Santa Cruz), anti-P-ERK1/2 (1:1,000; Cell Signaling Technology, Beverly, MA),
anti-ERK1/2 (1:1,000; Cell Signaling Technology), or monoclonal
anti--actin antibody (1:5,000; Abcam, Cambridge, UK) followed by
anti-rabbit or anti-mouse horseradish peroxidase (HRP) antibody (1:10,000;
Santa Cruz). Antibody binding was detected with chemiluminescence reagent (NEN
Life Sciences Products, Boston, MA) and exposure to X-Omat film (Kodak,
Rochester, NY).
In vitro phosphorylation. The cDNAs encoding Smad4 wild-type and
Smad4-T276A were inserted into a pGEX 4T vector (Amersham, Piscataway, NJ) and
transformed into BL21 Escherichia coli cells for glutathione
S-transferase (GST)-fusion protein expression induced by adding 0.2
mM isopropyl--D-thiogalactopyranoside. Purification of the
proteins was performed with GST beads according to the manufacturer's protocol
(Novagen, Madison, WI). One microgram of each protein was incubated with 100 U
of recombinant ERK2 (New England Biolabs, Beverly, MA), MAP kinase buffer, 100
µM ATP, and 5 µCi of [
-32P]ATP (NEN Life Sciences
Products) for 30 min at 30°C. The samples were analyzed by SDS-PAGE,
electroblotting, and autoradiography.
Luciferase assay. Cells were transiently transfected with the
(CAGA)12Luc reporter construct
(6) and with a pRL-TK vector
(Promega). Cells were then serum starved and treated with 400 pM TGF-1
(16 h). Experiments were performed in triplicate wells. Cells were lysed, and
luciferase activity was determined with the Dual Reporter Assay (Promega).
Relative light units were calculated as ratios of firefly (reporter) and
Renilla (transfection control) luciferase values.
Orthophosphate labeling, phosphoamino acid analysis, and tryptic
phosphopeptide mapping. Subconfluent cells were washed with
phosphate-free medium and labeled with 500 µCi/ml
[32P]orthophosphate (NEN Life Science Products) for 4 h in
phosphate-free medium containing 1% bovine serum albumin (Sigma) in the
presence or absence of TGF-1. Cells were lysed with (in mM) 50 Tris
· HCl pH 7.4, 150 NaCl, 1 EDTA, 1
-glycerophosphate (Sigma), and
1 phenylmethylsulfonyl fluoride (Sigma) with 1% Triton X-100 and subjected to
immunoprecipitation with M2 anti-Flag-antibody (Sigma). Samples separated by
SDS-PAGE were electroblotted onto PVDF membrane and subjected to
autoradiography. The Smad4 bands were excised and prepared for phosphoamino
acid analysis as described previously
(2). Samples were
electrophoresed in one dimension on TLC plates in pH 3.5 buffer. Unlabeled
standards were visualized with ninhydrin spray (Sigma), and the plates were
subjected to autoradiography. For tryptic phosphopeptide mapping, Smad4 bands
were excised directly from the gel, prepared as described previously
(2), and electrophoresed on TLC
plates in pH 1.9 buffer. The TLC plates were subsequently subjected to
chromatography for 6 h (2).
Radioactivity was detected with phosphor-imager screens.
Immunofluorescence. Cells grown on coverslips were serum starved
(8 h) before treatment with TGF-1 for 1 h, fixed in 4% paraformaldehyde,
incubated with M5 anti-Flag monoclonal antibody (dilution 1:300; Sigma)
followed by incubation with anti-mouse CY-3 (dilution 1:800; Jackson
Immunoresearch, West Grove, PA), and mounted with mounting medium (Kirkegaard
and Perry Laboratories, Gaithersburg, MD). Epifluorescence analysis was
performed on an Eclipse E8000 microscope (Nikon). For quantification of
nuclear accumulation, the number of nuclei in which the fluorescence was
brighter than in the cytoplasm was counted on photographs by an independent
observer. Cells were counter-stained with 4',6-diamidino-2-phenylindole
(DAPI) to identify nuclei. At least 70 nuclei were counted for each
condition.
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RESULTS |
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Smad4 wild type and Smad4-T276A are both phosphoproteins. We
subsequently investigated whether there is a difference in phosphorylation in
vivo between Smad4 wild type and Smad4 in which Thr276 was mutated
to Ala (Smad4-T276A). Wild-type LLC-PK1 (porcine kidney epithelial
cells) and LLC-PK1 stably transfected with mammalian expression
vectors with cDNA constructs coding for Flag-Smad4 wild type and
Flag-Smad4-T276A were metabolically labeled with
[32P]orthophosphate in the presence or absence of TGF-1.
Whole cell lysates were subsequently subjected to immunoprecipitation with an
antibody against Flag. Flag-Smad4 wild type was already phosphorylated in the
absence of TGF-
, and that remained unchanged after stimulation with
TGF-
(Fig. 2A).
Flag-Smad4-T276A was also phosphorylated, irrespective of the presence of
TGF-
, and no decrease was observed in gross phosphorylation levels
compared with Smad4 wild type (Fig.
2A). These results indicate that there are also other
residues in Smad4 that are targets for phosphorylation. Phosphoamino acid
analysis revealed phosphorylation predominantly on serine residues and on
threonine residues to a lesser extent. No phosphorylation was observed on
tyrosine (Fig. 2B).
Smad4-T276A still was phosphorylated on threonine residues but to a lesser
extent than Smad4 wild type, indicating that threonine residues other than
Thr276 are phosphorylated. Tryptic phosphopeptide mapping revealed
multiple phosphopeptides, most of these migrating to the cathode. Comparison
of the maps of Smad4 wild type and Smad4-T276A with the map of in vitro
phosphorylated GST-Smad4 wild type revealed a significant spot that is present
in the phosphopeptide maps of in vitro and in vivo phosphorylated Smad4 wild
type but absent in the map of Smad4-T276A
(Fig. 2C). This spot
most likely represents phosphorylation at Thr276.
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Thr276 is important for TGF--induced nuclear
localization of Smad4. To determine the functional significance of Smad4
phosphorylation at amino acid residue Thr276 in cells, the
subcellular distribution of Flag-Smad4 in LLC-PK1 cells stably
transfected with cDNA constructs coding for Flag-Smad4 wild type and for
Flag-Smad4-T276A was followed with indirect immunofluorescence with an
anti-Flag antibody. In the absence of exogenous TGF-
, Flag-Smad4
wild-type protein and Flag-Smad4-T276A mutant protein were present throughout
the cell (Fig. 3A). On
TGF-
addition, Flag-Smad4 wild-type protein accumulated in the nucleus,
but, in contrast, the Flag-Smad4-T276A protein was predominantly cytoplasmic
in distribution (Fig.
3A). Quantification of the immunofluorescence data showed
that in cells expressing Smad4 wild type, a marked increase is observed in the
percentage of cells in which Flag-Smad4 is predominantly in the nucleus after
stimulation with TGF-
, whereas this percentage remains similar to
control values in cells expressing Smad4-T276A
(Fig. 3B). Even after
6 h of stimulation with TGF-
, Flag-Smad4-T276A localization was
predominantly cytoplasmic (data not shown), indicating that the diminished
TGF-
-induced nuclear accumulation is not simply due to slower kinetics.
These results demonstrate that Thr276 is an important residue for
efficient nuclear accumulation of Smad4 in response to TGF-
and suggest
that the phosphorylation of Thr276 can shift the nuclear import vs.
export ratio of Smad4.
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Thr276 is required for full transcriptional activity of
Smad4. We next used the stably transfected LLC-PK1 cells to
examine the ability to induce transcription of a (CAGA)12-Luc
reporter, which is specific for Smad3/Smad4 signaling by TGF-/activin
(6). Because LLC-PK1
cells express endogenous Smad4, a TGF-
response was observed in
untransfected cells after stimulation with TGF-
1 for 16 h
(Fig. 4A; compare
bars 1 and 2). In stable LLC-PK1 cells expressing
Flag-Smad4 wild type, luciferase activation was enhanced compared with
untransfected cells after stimulation with TGF-
(Fig. 4A; compare
bars 4 and 2). However, in the cell lines expressing the
Flag-Smad4-T276A mutant protein, the TGF-
-induced activation of
luciferase was not enhanced and was similar to that in control cells
(Fig. 4A; compare
bars 6 and 2). As a control for protein expression in the
stable cells, whole cell extracts were immunoprecipitated with anti-Flag
antibody followed by immunoblotting with an anti-Smad4 antibody
(Fig. 4B). The results
demonstrate that equal amounts of protein are made by the different Smad4
variants in their respective stable cell lines and indicate that the
Flag-Smad4-T276A mutant protein is not grossly mis-folded or misaggregated
compared with the Flag-Smad4 wild-type protein. Analysis of total cell lysates
revealed that the endogenous levels of Smad4 in LLC-PK1 cells are
relatively low and that the increase of Smad4 expression in the stable cell
lines was approximately sevenfold (Fig.
4B). Equal protein expression was confirmed by
immunoblotting of whole cell lysates with an anti-
-actin antibody
(Fig. 4B). We next
analyzed the transcriptional activity of Flag-Smad4 wild type and
Flag-Smad4-T276A in cells that have a homozygous deletion of the Smad4 gene
and thus lack endogenous Smad4 expression, the human breast tumor cell line
MDA-MB468 (22,
25). Transient transfection of
Flag-Smad4 wild type restored TGF-
-dependent reporter gene activation in
these cells as measured by (CAGA)12-Luc induction
(Fig. 4C). In
comparison, transient transfection of Flag-Smad4-T276A in these cells was only
partially able to restore TGF-
-specific transcriptional response
(Fig. 4C). The levels
of Smad4 overexpression in these cells were analyzed by immunoblotting with an
anti-Flag antibody, and the results demonstrate that the decrease in
luciferase activity in Smad4-T276A-expressing cells was not due to lower
levels of expression (Fig.
4D). These results demonstrate that Thr276 is
required for full transcriptional activity of Smad4 and suggest that
phosphorylation of Smad4 at Thr276 enhances its activity.
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To further investigate the effect of phosphorylation by ERK on Smad4
cellular distribution, LLC-PK1 cells stably expressing Flag-Smad4
wild-type and mutant proteins were pretreated with the MEK inhibitor U0126
(8) for 1 h before stimulation
with TGF-1. Analysis of lysates from LLC-PK1 cells confirmed
that cells treated for 1 h with U0126 exhibit a dramatic reduction in levels
of ERK phosphorylation (Fig.
5B). Treatment for 1 h with TGF-
resulted in a
minor increase in levels of phosphorylated ERK
(Fig. 5B). In cells
expressing Smad4 wild type, Smad4 accumulated in the nucleus after exposure to
TGF-
. However, pretreatment of these cells with U0126 inhibited the
TGF-
-induced nuclear accumulation of Smad4
(Fig. 5A). We have
used concentrations of U0126 ranging from 10 to 70 µM and observed similar
results with all concentrations, although the results were more pronounced at
the higher concentrations (data not shown). Pretreatment with U0126 did not
affect the distribution of Smad4-T276A, which was predominantly cytoplasmic
even in the presence of TGF-
(Fig.
5A).
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It was previously demonstrated that Smad4 nuclear export requires the CRM1
protein and that inhibition of CRM1-mediated nuclear export with LMB results
in accumulation of Smad4 in the nucleus even in the absence of TGF-
(18). Treatment of cells
expressing Flag-Smad4 wild type with LMB for 1 h resulted in nuclear
localization of Smad4 (Fig.
5A). In cells expressing Flag-Smad4-T276A, treatment with
LMB led to strong nuclear accumulation of Flag-Smad4-T276A
(Fig. 5A), suggesting
that Smad4-T276A transports to the nucleus and that the diminished
TGF-
-induced nuclear accumulation of Smad4-T276A is not due to an
absolute inability of Smad4-T276A to enter the nucleus but rather to a
decreased nuclear import-to-export ratio. When Smad4 wild type or Smad4-T276A
was forced to accumulate in the nucleus with LMB, the transcriptional activity
in the absence of TGF-
was enhanced approximately twofold
(Fig. 5C). When
LLC-PK1 cells were simultaneously treated with LMB and TGF-
1, the fold
increase in transcriptional activity of Smad4-T276A reached levels similar to
the transcriptional activity of Smad4 wild type with TGF-
1
(Fig. 5D), indicating
that the reduced transcriptional activity observed with the Smad4-T276A mutant
protein results from a reduced nuclear accumulation.
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DISCUSSION |
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The consensus ERK site in Smad4 is a viable phosphorylation site in vitro (Fig. 1B) and seems to be conserved in most mammals but is absent in porcine Smad4. Importantly, the LLC-PK1 cells used in this study are porcine epithelial cells, whose endogenous Smad4 was confirmed by RT-PCR to contain a proline and not a threonine at the analogous site (data not shown). Therefore, the endogenous porcine Smad4 will not result in background Thr276 phosphorylation in the metabolic labeling experiments. The significance of the substitution of threonine by proline at the analogous site is unknown. Phosphoamino acid analysis of Flag-tagged mouse Smad4 expressed in LLC-PK1 cells showed phosphorylation on serine and threonine but not on tyrosine residues (Fig. 2B), although our results do not preclude tyrosine phosphorylation of Smad4 under other physiological conditions.
Smad4 subcellular distribution is thought to involve NLS and NES domains
that would cause Smad4 to shuttle continuously between the cytoplasm and the
nucleus (18). Our data suggest
that phosphorylation of Thr276 can regulate Smad4 subcellular
distribution, because Smad4-T276A mutants, which cannot be phosphorylated at
position 276, do not accumulate efficiently in the nucleus in response to
TGF-1 (Fig. 3A)
and pretreatment of LLC-PK1 cells expressing mouse Smad4 wild type
with U0126 inhibited the TGF-
-induced nuclear accumulation
(Fig. 5A). It is
unlikely that phosphorylation of Smad4 at Thr276 is sufficient to
cause nuclear accumulation in the absence of TGF-
, because stimulation
of ERK activity with EGF alone did not result in enhanced nuclear localization
of Smad4 (data not shown).
Interestingly, residue Thr276 is in the COOH-terminal part of
the linker region previously identified as important for full Smad4
activation. The significance of amino acids 275-322 in the linker region for
TGF--induced transcriptional activity was recognized by examining the
function of deletion mutants or naturally occurring splice variants of Smad4
in this region (3,
5,
18,
25). These Smad4 deletion
mutants were transcriptionally less efficient compared with Smad4 wild type,
although, importantly, signaling activity was not completely abolished
(3). We demonstrate a
consistent finding because the signaling activity of Smad4-T276A is decreased
compared with Smad4 wild type but not completely absent
(Fig. 3). The decreased
activity that we observed is most likely not due to impaired
heterooligomerization with the R-Smads, because Smad4 mutants that lacked
regions containing Thr276, or in fact the entire linker region,
were still able to physically interact with R-Smads
(5,
18). In addition, when LMB,
which inhibits Smad4 nuclear export
(18) and therefore forces
Smad4 to accumulate in the nucleus, was added to cells, the TGF-
-induced
transcriptional activity was similar for Smad4 wild type and Smad4-T276A
(Fig. 5D). Therefore,
we hypothesize that phosphorylation of Thr276 only affects the
nuclear import vs. export ratio of Smad4, but once Smad4 is in the nucleus
Smad4 wild type and Smad4-T276A have equal transcriptional activity. In
contrast to previous studies, we observe a lack of nuclear accumulation of
Smad-T276A, whereas others have reported no effect on nuclear accumulation of
Smad4 mutants lacking the SAD. This could be explained either by the
importance of residues in the SAD other than Thr276 or by a
difference in assay conditions, because we examined nuclear accumulation
within 1 h of addition of TGF-
, whereas others used a constitutively
active type I receptor and examined nuclear accumulation after 24 h
(5). Several studies have
pointed to a significant cross talk between the TGF-
and the MAP kinase
pathways, most of them demonstrating activation of MAP kinase pathways by
TGF-
(16). Conversely,
the MAP kinase pathways have been shown to regulate TGF-
signaling.
Phosphorylation of R-Smads by MAP kinases was shown either to enhance
(13,
14) or inhibit
(7) TGF-
-induced nuclear
accumulation of the R-Smads. Our results now also incorporate regulation of
Smad4 activity in the MAP kinase pathway, enhancing the complexity of the
interactions between the TGF-
and MAP kinase pathways. These effects are
likely to be cell type dependent. Previously, it was reported that in RIE
cells, stimulation of the ERK pathway by high levels of oncogenic Ras
repressed TGF-
signaling as a result of degradation of Smad4 through the
ubiquitin-proteasome pathway
(21). These results are
opposite to our findings, possibly because of differences in cell context.
In conclusion, we propose that Smad4 nuclear localization can be regulated
by MAP kinase phosphorylation of residue Thr276 to promote nuclear
accumulation of Smad4 and therefore to enhance Smad signaling. This regulation
of Smad activity could occur during important physiological and
pathophysiological processes such as organogenesis and wound healing, in which
TGF- and the MAP kinase signaling pathways are known to function in a
cooperative manner. In vivo examples of such activities would include the
synergistic or enhanced effects of TGF-
/BMP and EGF/FGF signaling during
induction of chondrogenesis during otic capsule formation
(9), paraxial mesoderm
myogenesis (24), cardiogenic
induction of non-precardiac mesoderm
(1), and tubule formation in
metanephric mesenchyme
(19).
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address of B. A. J. Roelen: Dept. of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584 CL Utrecht, The Netherlands.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* B. A. J. Roelen and O. S. Cohen contributed equally to this work.
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