From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606
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ABSTRACT |
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The Smad family of intracellular proteins
mediates signals generated by activin and other transforming growth
factor -related proteins via specific heteromeric complexes of
transmembrane receptor serine kinases (1, 2). xSmad2 has been
implicated as an activin signal mediator that may participate in
transcriptional regulation (3, 4). We have employed an interaction
cloning strategy to identify xSmad2-binding proteins and found that
calmodulin directly associated with Smads. xSmad2, generated either by
in vitro translation or by overexpression in COS cells,
specifically bound to calmodulin-agarose; the association was
calcium-dependent and required xSmad2 N-terminal residues.
In the same assay, xSmad1 and hSmads 2, 3, and 4 also bound to
calmodulin-agarose. Furthermore, a calmodulin antagonist, W13,
increased expression of the activin-inducible transcriptional reporter,
3TP-Lux, whereas overexpression of calmodulin suppressed this reporter.
These observations demonstrate that Smad proteins interact with
calmodulin in a calcium-dependent way through conserved N-terminal amino acids and suggest a role for calmodulin in regulating Smad function.
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INTRODUCTION |
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Activins and structurally related family members including the
transforming growth factors
(TGF)1 and bone
morphogenetic proteins initiate signaling through heteromeric complexes
of transmembrane receptors with intrinsic protein serine/threonine
kinase activity (5). Extensive searches to identify receptor substrates
by two-hybrid analysis have provided little insight into the mechanism
of signal transmission. However, genetic analysis of both
Drosophila melanogaster and Caenorhabditis elegans has led to the identification of a family of intracellular molecules that are required for signal propagation by TGF
family members. Smad (a combination of the gene names from C. elegans, Sma, and Drosophila, Mad) homologs have now
been identified in vertebrates including frogs, mice, and humans (1,
2). Functional analysis in Xenopus laevis indicates that
overexpression of Smad2 mimics the effects of activin, whereas
overexpression of Smad1 recapitulates bone morphogenetic protein
effects (3); this result suggests that different ligands selectively
employ specific Smads. The primary structure of the Smads, which
contain highly conserved N-terminal and C-terminal domains separated by
a divergent linker region, does not provide any clue as to the
biochemical function of this family of molecules (1, 2).
Biochemical analysis of Smad2 indicates that this molecule is rapidly
phosphorylated in response to both activin and TGF on extreme
C-terminal serine residues (6). Bacterially expressed affinity-purified
GST-Smad2 fusion protein can be phosphorylated by TGF
receptor
complexes immunoprecipitated from cultured cells, suggesting that Smad2
may be a direct receptor target (6). Phosphorylation of Smad2 in
vivo correlates with its nuclear accumulation (6); the C-terminal
domain of Smad2 expressed alone is constitutively localized in the
nucleus (7). Furthermore, analysis of both Smad1 and Smad4 has shown
that the conserved C-terminal domain is capable of inducing
transcription when fused to a heterologous DNA-binding domain (8);
based on the sequence conservation of this domain, it is expected that
the other Smads may also demonstrate this function. Activin and TGF
also lead to the specific association of Smad2 and Smad4 as assessed by
co-immunoprecipitation from cultured cells following ligand binding
(9). Smad2 has been identified as a component of an activin-specific
transcriptional complex (4, 10), and there is now evidence that Mad
directly binds DNA via its conserved N-terminal domain (11). These
observations have led to the proposal that activin stimulates Smad2
phosphorylation via ActR complexes, which leads to nuclear accumulation
and complex formation with Smad4 and participation in transcriptional
activation of activin-responsive genes.
To identify proteins that physically and functionally interact with
Smad2, we screened a bacteriophage expression library using
radiolabeled Smad2 as probe. We have identified calmodulin as a
Smad2-interacting protein; we demonstrate that the association is
calcium-dependent and requires amino acids between
Ile77 and Ala208 in the N-terminal half of
Smad2. We also provide evidence that calmodulin can influence gene
expression from an activin and TGF-responsive promoter.
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EXPERIMENTAL PROCEDURES |
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Materials-- Purified activin A was generously provided by Dr. Yuzuru Eto and by the National Institutes of Health Hormone Distribution Program, NIDDK, NHPP, NICHHD, and the USDA (lot 15365-36(1)). 4,5-Dihydro-2-[6-hydroxy-2-benzothiazolyl]-4-thiazolecarboxylic acid (D-Luciferin), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13), N-(4-aminobutyl)-2-naphthalenesulfonamide (W12), thrombin, and calmodulin-agarose were obtained from Sigma.
DNA Constructs-- For expression in E. coli, xSmad2 cDNAs were subcloned into the vector pGEX-2T, which was modified to include a consensus protein kinase A phosphorylation site. For expression in mammalian cells, xSmad cDNAs provided by Jon Graff were subcloned into the vector pcDNA1 (InVitrogen) or into pcDNA1 modified to contain three iterations of the HA epitope tag at the C terminus of the protein (12); CaM cDNA (CaM A, locus MUSCAM, accession number M27844) was also subcloned into pcDNA1; a genomic CaM clone was provided by Tony Means. ActR DNAs and p3TP-Lux were as described (12). xSmad2 mutants and truncations were generated by polymerase chain reaction; all subclones were fully sequenced. Expression vectors for hSmads 2, 3, and 4 tagged with the FLAG epitope at the C terminus were generously provided by Dr. Rik Derynk.
Screening of EXlox Library--
A
EXlox cDNA library
from a 16-day mouse embryo was screened essentially as described (13).
Phage were plated to yield 40,000 plaques/150-mm plate and incubated at
37 °C for 12 h before being overlaid with nitrocellulose
circles impregnated with 1 mM
isopropyl-
-D-thiogalactoside. Following an additional
12 h at 37 °C, filters were washed and probed as described
(13). The xSmad2 probe was expressed in and purified from
Escherichia coli as a GST fusion protein by standard methods
(14) and cleaved from GST by incubation with thrombin before labeling
as described (13). The labeled probe was then separated from
unincorporated 32P on Sephadex G-50 Fine, Quick Spin
Columns (Boehringer Mannheim) before incubation with the filters.
Positive clones were plaque purified, and phage were converted to
plasmids prior to double-stranded sequencing by standard methods
(Sequenase version 2, U. S. Biochemical Corp./Amersham Corp.).
In Vitro Binding Assay-- Smads, generated by coupled in vitro transcription/translation using rabbit reticulocyte lysate (Promega), were mixed with 50 µl of either calcium or EGTA buffer-washed calmodulin-agarose in 500 µl of either calcium or EGTA buffer. The mixture was incubated at 4 °C for 1 h with gentle rocking before being washed five times in the corresponding buffer. Bound protein was eluted by boiling in SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis under reducing conditions, and visualized by fluorography. Buffer consisted of 50 mM HEPES, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 0.1 mM dithiothreitol, 0.5% Triton X-100, and either 0.2 mM CaCl2 or 100 mM EGTA, with a mixture of protease inhibitors.
Luciferase Assay--
The L17 cell line was cultured and
transfected as described (12). Cells were transiently transfected with
0.5 µg of the p3TP-Lux reporter plasmid and combinations of 0.5 µg
of calmodulin and 0.5 µg of ALK DNAs as indicated. Cells were
co-transfected with a plasmid expressing -galactosidase. Following
transfection, cells recovered for 30 h and were then stimulated
with 2 nM activin A, or 100 pM TGF
in 0.25 ml of minimum essential medium containing 0.2% fetal bovine serum
(v/v) with or without increasing amounts of W12 or W13 as indicated to
induce transcription of 3TP-Lux. Luciferase activity was measured in 40 µl of a 200-µl lysate and normalized to
-galactosidase activity
measured in 20 µl of lysate as described (12).
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RESULTS |
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Cloning of xSmad2-binding Proteins--
To identify proteins that
functionally interact with xSmad2, we screened a bacteriophage
expression library of 16-day mouse embryo cDNA using radiolabeled
xSmad2 as probe. Screening of 1.5 × 106 total plaques
resulted in the isolation of seven cDNAs encoding xSmad2-binding
proteins. Partial cDNA sequencing indicated that all seven were
CaM; five different cDNAs representing three CaM genes were
recovered (Fig. 1). The longest cDNAs
included the entire CaM coding region plus 57 5 nucleotides that
encode 19 additional amino acids. Two cDNAs encoded CaM lacking
only the initial methionine, whereas one cDNA encoded only the 122 C-terminal amino acids of CaM (of 148 total), indicating that this
region is sufficient for interaction with xSmad2.
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In Vitro Association of Smads with Calmodulin-- To determine whether xSmad2 associates with CaM in vitro, we tested xSmad2 for binding to CaM-agarose affinity resin. xSmad2, tagged with three iterations of the HA epitope at its C terminus, was transcribed and translated in vitro with [35S]methionine and then mixed with CaM-agarose in the presence of calcium or EGTA. xSmad2 was retained on CaM-agarose in the presence of calcium but not in its absence. xSmad2 was also retained on CaM-agarose in the presence of calcium when a peptide corresponding to the HA epitope was included in the binding reaction but not when a known CaM-binding peptide was present (Fig. 2). Similar results were obtained using xSmad2 generated by overexpression in COS cells (data not shown). The slight difference in mobility of in vitro transcribed/translated Smads following incubation in the binding mixture compared with input (Figs. 2 and 3) was not apparent when Smads were generated by overexpression in COS cells; furthermore, this difference was not dependent on Ca2+ or CaM, nor was it the result of phosphorylation.
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Mapping the CaM-binding Domain of xSmad2-- To determine which region of xSmad2 is responsible for binding to CaM, we generated deletion mutants encoding either the conserved N-terminal domain with the linker region (amino acids 1-263) or the conserved C-terminal domain (amino acids 261-467) of the molecule and tested these proteins for association with CaM as described above. In the presence of calcium, both wild-type xSmad2 and xSmad2-N were retained on CaM-agarose, whereas xSmad2-C was not (Fig. 3). This observation is consistent with the earlier observation that the purified radiolabeled C-terminal domain of xSmad2 did not bind phage-encoded CaM on nitrocellulose filters.
To further localize the CaM-binding domain of xSmad2, we used additional deletion mutants that lack increasing amounts of the N terminus; xSmad2 mutants lacking the initial 26 (xSmad2CaM Function in Activin Signaling-- Because CaM physically associates with Smads and may therefore regulate Smad function, we tested whether CaM could modulate expression of an activin-responsive reporter gene. L17 mink lung epithelial cells stably transfected with the type I activin receptor, ALK4, were treated with a calmodulin antagonist, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13) or its 10-fold less active chemical analog, N-(4-aminobutyl)-2-naphthalenesulfonamide (W12) as a negative control. Activin responsiveness was evaluated by measuring expression of an activin-inducible reporter gene, 3TP-Lux. In the absence of calmodulin inhibitor, a 4-fold activin-dependent increase in luciferase expression from 3TP-Lux was observed. Similarly, at all doses of W12, activin treatment resulted in a 4-5-fold increase in luciferase expression with no changes in overall levels of luciferase expression relative to cells not treated with inhibitor. In contrast, treatment with W13 resulted in a dose-dependent proportional increase in overall luciferase expression both in the presence and the absence of activin (Fig. 4A).
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DISCUSSION |
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Smad proteins are responsible for mediating signals initiated by
specific receptor serine kinases (RSKs) in response to activin and
other TGF-related ligands. The central role of this family of
molecules in RSK signaling is highlighted by the observation that cells
compromised for either Smad2 or Smad4 activity are resistant to RSK
activation. Because 1) Smads accumulate in the nucleus following ligand
treatment, 2) Smad2 has been identified as a component of
activin-dependent DNA-binding complexes, 3) Mad binds DNA
directly, and 4) Smads1 and 4 can support transcriptional activation
when fused to a heterologous DNA-binding domain, Smads are thought to
act as transcriptional regulators. We have shown that Smads are capable
of interacting with CaM; the interaction is dependent on calcium and
requires at least residues between Ile77 and
Ala208 in the N-terminal portion of Smad2. This region
contains a putative (residues Leu123 to Trp132)
1-5-10 basic amphiphilic
-helix, which is a known CaM target motif
(15), and may be responsible for mediating this interaction. We have
also shown that CaM can modulate gene expression from an activin- and
TGF
-responsive promoter. These results demonstrate that CaM can
function as a negative regulator of activin signaling, possibly through
physical association with Smad proteins.
CaM is the primary intracellular receptor for calcium ions and mediates the effects of signals that cause intracellular calcium flux (16). Calcium-saturated CaM binds to and regulates a wide variety of well characterized enzymes, in addition to structural proteins of the cytoskeleton and transcription factors (16). Ca2+/CaM can stimulate transcription factors such as c-Fos, c-Jun, cAMP response element-binding protein, and serum response factor indirectly via activation of CaM-dependent kinases (17) and can inhibit specific members of the basic helix-loop-helix (bHLH) family of transcription factors by direct protein-protein interaction (18-20). The mechanism of transcriptional inhibition of the CaM-sensitive class of bHLH proteins involves direct binding to the bHLH domains, which interferes with dimerization and prevents DNA binding (20-22). A recent report demonstrates that Mad is capable of direct sequence-specific DNA binding via an N-terminal domain requiring residues between 120 and 159 (11). This region corresponds to that of Smad2 which is required for CaM binding, and suggests that CaM may also inhibit Smad2 by interfering with DNA binding. Alternatively, CaM may interfere with assembly of the transcriptional complex formed in response to activin, which includes Smad2 and a winged helix DNA-binding protein, FAST-1 (4, 10). Although CaM is found throughout the cell, it is also possible that CaM regulates the subcellular distribution of Smads.
The potential role of calcium as a mediator of activin signaling has been investigated, but no consensus has been reached (5). We measured intracellular calcium in L17 cells stably transfected with ALK4 and did not observe any change when they were assayed under conditions identical to those that yielded induction of luciferase expression from 3TP-Lux (data not shown). The precise mechanism by which CaM negatively regulates gene expression from activin-responsive promoters remains to be elucidated; however, the identification of a calcium-dependent physical association between Smads and CaM suggests complex regulation of Smad function. Biochemical analysis of the role of CaM in regulating Smad2 activity will provide additional insight into the process of activin signal transduction.
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ACKNOWLEDGEMENTS |
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We thank Sue Willis, Kunliang Guan, Jack Dixon, Jon Graff, and Tony Means for stimulating discussions and for comments on the manuscript. We also thank Ben Margolis for reagents and advice in carrying out the screen and Owen Wittekindt for technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants RO1 GM-50416 (to L. S. M.) and T32 GM-08353 (to C. M. Z.) and by funds from the Searle Scholars Program/The Chicago Community Trust (to L. S. M.).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.
To whom correspondence should be addressed: Dept. of Biological
Chemistry, University of Michigan, 1301 Catherine Rd., Ann Arbor, MI
48109-0606. Tel.: 313-936-3907; Fax: 313-763-4581; E-mail: lmathews{at}umich.edu.
1 The abbreviations used are: TGF, transforming growth factor(s); CaM, calmodulin; GST, glutathione S-transferase; HA, hemaglutinin; RSK, receptor serine kinase; bHLH, basic helix-loop-helix; ALK, activin-like kinase.
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REFERENCES |
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