The Smad4 Activation Domain _SAD_ Is a Proline-rich_ p300 by fdh56iuoui


									THE JOURNAL   OF   BIOLOGICAL CHEMISTRY                                                           Vol. 275, No. 3, Issue of January 21, pp. 2115–2122, 2000
                                                                                                                                           Printed in U.S.A.

The Smad4 Activation Domain (SAD) Is a Proline-rich,
p300-dependent Transcriptional Activation Domain*
                                                          (Received for publication, August 31, 1999, and in revised form, October 13, 1999)

                   Mark P. de Caestecker‡, Tetsuro Yahata§, David Wang¶, W. Tony Parks, Shixia Huang,
                   Caroline S. Hill , Toshi Shioda§, Anita B. Roberts**, and Robert J. Lechleider‡‡
                   From the Laboratory of Cell Regulation and Carcinogenesis, NCI, National Institutes of Health,
                   Bethesda, Maryland 20892-5055, the §Laboratory of Tumor Biology, Massachusetts General Hospital Cancer Center,
                   Charleston, Massachusetts 02129-2060, and the Developmental Signaling Laboratory, Imperial Cancer Research Fund,
                   P. O. Box 123, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom

  Transforming growth factor- (TGF- ) family mem-                           to the type II receptor results in recruitment and transphos-
bers signal through a unique set of intracellular pro-                      phorylation of type I receptors, which then signal downstream
teins called Smads. Smad4, previously identified as the                     responses (1). Clues as to the mechanisms regulating down-
tumor suppressor DPC4, is functionally distinct among                       stream signaling responses have been provided by the discov-
the Smad family, and is required for the assembly and                       ery of Smad proteins as direct substrates of the TGF- family of
transcriptional activation of diverse, Smad-DNA com-                        receptor kinases, and mediators of signals from the receptors to
plexes. We previously identified a 48-amino acid proline-                   the nucleus.
rich regulatory element within the middle linker do-                           Receptor-activated Smads (R-Smads) interact transiently

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main of this molecule, the Smad4 activation domain                          with specific, ligand-activated type I receptors and are phos-
(SAD), which is essential for mediating these signaling                     phorylated on highly conserved carboxyl-terminal (COOH-ter-
activities. We now characterize the functional activity of
                                                                            minal)-SS(V/M)S motifs. Smad2 and Smad3 are specific medi-
the SAD. Mutants lacking the SAD are still able to form
                                                                            ators of TGF- and activin signaling pathways, while Smad1,
complexes with other Smad family members and associ-
ated transcription factors, but cannot activate tran-                       Smad5, and Smad8 are involved in bone morphogenetic protein
scription in these complexes. Furthermore, the SAD it-                      responses (1). Following receptor activation, these Smad pro-
self is able to activate transcription in heterologous                      teins translocate to the nucleus where they function as tran-
reporter assays, identifying it as a proline-rich tran-                     scriptional regulators (2). Smads have a domain structure con-
scriptional activation domain, and indicating that the                      sisting of highly conserved amino (NH2)- and (COOH)-terminal
SAD is both necessary and sufficient to activate Smad-                      regions, referred to as Mad homology 1 (MH1) and MH2 do-
dependent transcriptional responses. We show that                           mains, respectively, and an intervening middle linker, which is
transcriptional activation by the SAD is p300-depend-                       of variable length and sequence. The MH2 domain contains the
ent, and demonstrate that this activity is associated                       principal receptor serine-threonine kinase phosphoacceptor
with a physical interaction of the SAD with the amino                       sites (1, 3), and determinants of specific Smad-receptor and
terminus of p300. These data identify a novel function of                   Smad-Smad interactions (4), and is essential for transcrip-
the middle linker region of Smad4, and define the role of                   tional activation (2). Effector functions of the MH2 domain are
the SAD as an important locus determining the tran-                         inhibited by the MH1 domain (5), while the MH1 and linker
scriptional activation of the Smad complex.                                 regions of Drosophila MAD and mammalian Smad3 are respon-
                                                                            sible for their DNA binding (2).
                                                                               Smad4 is functionally unique among the Smads, with an
   TGF- 1 is the prototypic member of a large family of struc-              amino acid sequence more closely related to the Drosophila
turally related cytokines including the TGF- s, activins, and               gene product Medea than to Mad (6). In contrast to the R-
bone morphogenetic proteins which regulate cell fate and ex-                Smads, Smad4 is not regulated by phosphorylation, but acts as
tracellular matrix deposition through the transcriptional reg-              a common mediator of TGF- , activin, and bone morphogenetic
ulation of diverse gene targets. These ligands initiate cellular            protein signaling responses (7–9). Following phosphorylation,
signals by associating with two classes of interacting trans-               R-Smads form hetero-oligomeric complexes with Smad4 which
membrane receptor serine-threonine kinases. Ligand binding                  are then translocated to the nucleus (1, 3). Like the R-Smads,
                                                                            the MH2 domain of Smad4 is responsible for interaction with
  * The costs of publication of this article were defrayed in part by the   other Smad proteins, while autoinhibition of MH2 activity and
payment of page charges. This article must therefore be hereby marked       DNA binding are mediated through its MH1 domain (5).
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to            Smad4 is an essential component of transcriptional com-
indicate this fact.
  ‡ Recipient of a Wellcome Trust Advanced Training Fellowship from         plexes mediating the activation of Smad-dependent target
the United Kingdom.                                                         genes. The transcriptional activity of Smad4 has been ascribed
  ¶ HHMI-National Institutes of Health Research Scholar.                    to its capacity to associate with other Smad-transcription factor
  ** To whom correspondence should be addressed. Tel.: 301-496-5391;        complexes on cis-acting elements of responsive promoters (10 –
Fax: 301-496-8395; E-mail:
  ‡‡ Present address: Dept. of Pharmacology, Uniformed Services Uni-        13), and by participating in R-Smad interactions with the
versity of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD         paralogous bridging co-activators CBP and p300 (14 –18). Re-
20814-4799.                                                                 cent data also suggest that Smad4 plays an active role in
    The abbreviations used are: TGF- , transforming growth factor- ;        recruiting other components of the transcriptional complex
R-Smad, receptor-activated Smad; MH1, Mad homology domain 1; GST,
glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis;
                                                                            involved in target gene activation, including the transcrip-
SAD, Smad activation domain; ARF, activin response factor; ARE,             tional co-activator MSG1 (19). Although several studies have
activin response element; CBP, cAMP-binding protein.                        suggested that the C-terminal MH2 domain is essential for

This paper is available on line at                  2115
2116                                              Transcriptional Activity of Smad4
mediating Smad4 transcriptional activation (11, 20), recent                PAGE and Coomassie staining following affinity purification on gluta-
studies show that a Smad4 mutant lacking the whole MH2                     thione beads. Equal amounts of fusion protein were incubated in lysis
                                                                           buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM EGTA, 1 mM
domain retains the capacity to activate transcription (21). Fur-
                                                                           EDTA, 1 mM dithiothreitol, 10% glycerol, 1% Triton X-100 with protease
thermore, we previously showed that the Smad4 MH2 domain                   and phosphatase inhibitors) with lysates from COS cells transiently
is interchangeable with the Smad1 MH2 domain, and that a                   transfected with the Gal4-SAD expression construct. Lysates were im-
48-amino acid segment within the middle linker called the                  munoprecipitated with anti-GST antibody (Santa Cruz Biotechnology)
Smad4 activation domain, or SAD, is required for the activa-               and analyzed by SDS-PAGE and immunblotting with anti-Gal4
tion of Smad4-dependent signaling responses (7). These data                antibody.
                                                                              Indirect Immunofluorescence—NMuMg cells were transiently trans-
provide evidence that elements within the middle linker region
                                                                           fected in chamber slides with the FLAG-tagged Smad4 constructs and
of Smad4 are required for the activation of Smad-dependent                 Myc-Smad2, with or without the activated T RI (T204D), and serum
transcriptional responses, but it is unknown how these regu-               starved overnight 24 h later. Cells were then fixed and permeabilized,
late the function of Smad4.                                                as described previously (24), and FLAG epitopes detected by incubating
   In this study, we show that the SAD is in fact a transcrip-             with the anti-FLAG M2 monoclonal antibody overnight at 4 °C. This
tional activation domain which is both necessary and sufficient            was followed by incubation with a goat anti-mouse fluorescein isothio-
                                                                           cyanate secondary antibody, and mounting in medium containing 4,6-
for the activation of transcription by Smad4. Mutants lacking
                                                                           diamino-2-phenylindole (Vectorshield, Vector Labs). The percentage of
the SAD are still able to form complexes with other Smad                   nuclear localization represents 100 cells counted by a trained observer
family members and associated transcription factors, but can-              (W. T. P.), blinded as to the constructs transfected.
not activate transcription by these complexes. We show that                   Gel Mobility Shift and Supershift Assays—MDA-MB468 and
the SAD has intrinsic, p300-dependent, transcriptional activ-              NMuMg cells were transfected, serum starved overnight, and treated
ity, and determine that this activity is associated with a phys-           with TGF- for 1 h. Cell lysates were prepared in hypertonic buffer
                                                                           containing 400 mM KCl, 0.4% Triton X-100, 10% glycerol, 20 mM
ical interaction between the SAD and the NH2 terminus of
                                                                           HEPES pH 7.5, 10 mM EGTA, 5 mM EDTA, and 1 mM dithiothreitol with
p300. These findings identify a novel function for the middle              protease and phosphatase inhibitors. Following clarification, lysates
linker region of Smad4, and define the role of the SAD as an               were snap frozen and stored at 80 °C. DNA binding assays were

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important locus determining the transcriptional activation of              performed using a radiolabeled ARE probe generated by polymerase
the Smad complex.                                                          chain reaction with [32P]dCTP and [32P]dATP, using two overlapping
                                                                           ARE oligonucleotides (5 -CCGACTAGTATCTGCTGCCCTAAAATGTG-
                   EXPERIMENTAL PROCEDURES                                 TATTCCATGGAAATG-3 and 5 -CCGGCTAGCTAGGGAGAGAAGGG-
   Cell Lines and Expression Constructs—MDA-MB468, NMuMg, and              CAGACATTTCCATGGAATAC-3 ), to generate a 71-base pair probe.
COS-1 cells were maintained in Dulbecco’s modified Eagle’s medium          The radiolabeled probe was added to the cell lysates for 30 min after
with 10% fetal bovine serum and antibiotics. Cells were transfected        equilibrating the freshly thawed lysates to 200 mM KCl with 20 mM
with the indicated constructs using Superfect (Qiagen) or Lipo-            HEPES pH 7.5, 10% glycerol, 10 mM EGTA, and 5 mM EDTA, and
fectAMINE (Life Technologies), according to the manufacturer’s proto-      adding an equal volume of probe mixture containing 20 mM KCl, 11 mM
cols. 3 FLAG-tagged Smad4 mutant and deletion constructs, and 5            MgCl2, 20% glycerol, and 200 g/ml poly(dI-dC). For antibody super-
double Myc-tagged Smad2 were generated by polymerase chain reac-           shift experiments, 200 mM KCl equilibrated cell lysates were incubated
tion using a proofreading polymerase and subcloned into pcDNA3 (In-        for 10 min with anti-Myc 9E10, or anti-FLAG M2 monoclonal antibodies
vitrogen) or the pSG424 (22) expression vectors. All polymerase chain      in the probe mixture, prior to addition of the radiolabeled ARE probe.
reaction-generated products were sequenced using the dideoxynucle-         Protein-DNA complexes were resolved on a 5% nondenaturing poly-
otide method.                                                              acrylamide gel.
   Transcriptional Response Assays—The 3TP-Lux reporter was used to
measure TGF- -induced gene expression, while pG5E1B-Luc, contain-                                        RESULTS
ing the Gal4 upstream activating sequence linked to a luciferase re-          Smad4 SAD Deletion Results in Loss of Function—Smad4
porter, was co-transfected with the indicated Gal4-Smad fusion protein
                                                                           restores TGF- responsive p3TP-Lux reporter gene activation
constructs in heterologous DNA binding assays. For the ARE-reporter
assays, ARE-Luc containing three tandem repeats of the ARE linked to       when co-transfected into Smad4 null MDA-MB468 cells (7).
the luciferase reporter in pGL3 was co-transfected with Myc-FAST1,         Using the same functional assay, we have previously shown
Myc-Smad2, and various Smad4-FLAG constructs. Transfection, TGF-           that deletion of the COOH-terminal portion of the middle
treatment and luciferase assays were performed as described previ-         linker region of Smad4 (amino acids 275–322), the Smad4
ously, co-transfecting pSV- -galactosidase to allow for normalization of   activation domain (SAD), results in loss of function, and that
transfection efficiency (7). The total amount of transfected DNA was
                                                                           the NH2 terminus of Smad4 enhances ligand-dependent re-
standardized by addition of pcDNA3 control vector as required, and all
assays were performed in triplicate, and represented as mean ( S.E.) of    porter gene activation in Smad1/Smad4 chimeras (7). The
three independent transfections.                                           mechanism whereby Smad4( 275–322) interferes with Smad-
   Immunoprecipitation and Western Blots—COS-1 and NMuMg cells             mediated signaling is unknown. To explore this further, we
were transfected with the indicated constructs, with or without, the       used a heterologous transcriptional activation assay to deter-
activated TGF- type I receptor (T204D) point mutant (23). After 24 h       mine the ability of this Smad4 mutant to restore transcrip-
cells were switched to 0.2% serum overnight, and lysed in 0.5 ml of
                                                                           tional activity of a Gal4-Smad2 fusion protein, which has pre-
Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, and 50 mM
Tris, pH 8.0) in the presence of phosphatase and protease inhibitors.      viously been shown to be dependent on Smad4 expression (11).
Lysates were either directly separated by sodium dodecyl sulfate-poly-     Co-transfection of Smad4 with Gal4-Smad2 restored ligand-de-
acrylamide gel electrophoresis and transferred onto Immobilon-P mem-       pendent transcriptional activation of the Gal4 reporter gene in
branes (Millipore), and/or first immunoprecipitated for 1 h using          Smad4-null MDA-MB 468 cells, while Smad4( 275–322) only
epitope-specific 9E10 anti-Myc (Zymed Laboratories Inc.) monoclonal        weakly restored transcriptional activity of the fusion protein,
antibodies. For the p300 interaction assays, cells were lysed in Nonidet
                                                                           despite comparable levels of Gal4-Smad2 fusion protein expres-
P-40 lysis buffer containing 100 mM NaCl, and endogenous p300 im-
munoprecipitated with rabbit anti-p300 (N15, Santa Cruz). Western          sion (Fig. 1A).
blots were performed using mouse monoclonal anti-Myc, anti-FLAG M2            To determine if the differences in ability to activate tran-
(Kodak), anti-HA (Roche Molecular Biochemicals), rabbit anti-Gal4          scription were dependent on Smad4 directly, we performed the
DNA-binding domain (Santa Cruz), or rabbit anti-p300 (C-20, Santa          same experiments using Gal4-Smad4 fusion constructs. Trans-
Cruz), as indicated, detected using the appropriate horseradish perox-     fection of Gal4 fusion proteins containing full-length Smad4, or
idase-conjugated secondary antibody, and visualized by chemilumines-
                                                                           a truncated Smad4 encompassing amino acids 266 –552,
cence (Pierce).
   For GST affinity assays, glutathione S-transferase (GST) fusion pro-    showed a ligand dependent activation of the Gal4 reporter,
teins for the N (amino acids 1–596), M (744 –1571), or C (1572–2370)       with a larger absolute activation by the 266 –552 construct,
regions of p300 were expressed in bacteria and quantitated by SDS-         consistent with relief of autoinhibition by the MH1 domain (5,
                                                  Transcriptional Activity of Smad4                                                       2117

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   FIG. 1. The Smad4 SAD is essential for maximal transcrip-
                                                                              FIG. 2. Smad4( 275–322) associates with Smad2 and is trans-
tional activation of Smad2 and Smad4. A, the Smad4 SAD domain
                                                                           located to the nucleus. A, Smad4( 275–322) associates with Smad2.
is essential for maximal transcriptional activation of Smad2. B, the
                                                                           COS-1 cells were transfected with the indicated constructs and lysates
SAD domain is essential for transcriptional activation of Smad4. In
                                                                           analyzed by immunoprecipitation and Western blotting, as indicated.
these heterologous DNA binding assays, MDA-MB468 cells (A) or
                                                                           T RI* indicates transfection with the activating point mutant of the
NMuMg cells (B) were transfected with equal amounts of the indicated
                                                                           TGF- type 1 receptor (T204D). B, Smad4( 275–322) and wild-type
Gal4-Smad fusion proteins with or without FLAG-tagged Smad4 con-
                                                                           Smad4 translocate to the nucleus in the presence of Smad2. NMuMg
structs, and the pSV- -galactosidase and pG5E1B luciferase reporters.
                                                                           cells were transiently transfected with the indicated FLAG-tagged ex-
Following transfection, cells were serum starved overnight and treated
                                                                           pression constructs, Myc-tagged Smad2, with or without T RI*, and
for 20 h with 10 ng/ml TGF- 1. Cell lysate luciferase activity was
                                                                           processed for anti-FLAG immunofluorescence. The percentage of cells
detected, and activities corrected on the basis of -galactosidase activ-
                                                                           with nuclear staining is indicated. Quantification is depicted as mean
ity. Results are expressed as mean ( S.E.) of triplicate assays. Protein
                                                                           ( S.E.) from five separate experiments, counted by an independent,
expression was determined in parallel experiments following transient
                                                                           blinded observer.
transfection of the indicated constructs in COS-1 cells.

                                                                           Is Distinct from Its Stabilizing Effects on Protein-DNA Com-
20). Compatible with the above findings, deleting the SAD from
                                                                           plexes—To determine how deletion of the SAD disrupts the
these constructs caused a complete loss of signal, despite com-
                                                                           nuclear functions of Smad4, we reconstituted a defined tran-
parable expression levels (Fig. 1B).
                                                                           scriptional response from Xenopus in mammalian cells. Activin
   Smad4 SAD Deletion Does Not Affect the Known Cytoplasmic
                                                                           and TGF- signaling induce the formation of an activin re-
Functions of Smad4 —Interactions between Smad4 and
                                                                           sponse factor (ARF) that contains Smad2, Smad4, and FAST1,
R-Smad proteins are particularly sensitive to deletion and mu-
                                                                           and binds to the activin response element (ARE) on the Xeno-
tation within the COOH-terminal domain of Smad4 (25–27). As
                                                                           pus Mix.2 promotor (10, 11, 29). Smad4 is a critical component
the Smad4( 275–322) deletion is in close apposition to the
                                                                           of this complex, enabling transcriptional activation of the ARE-
MH2 domain (amino acids 323–552), we felt that a possible
                                                                           containing reporter construct (10, 11). Previous studies have
explanation for its lack of function was through disruption of
                                                                           shown that wild type Smad4 forms ternary complexes with
the MH2 tertiary structure. We therefore tested the known
                                                                           FAST1 in the presence of Smad2 (10, 11). We reproduced these
cytoplasmic functions of the MH2 domain of Smad4, including
                                                                           findings with wild type Smad4, and also showed that the
hetero-oligomerization with R-Smads and nuclear transloca-
                                                                           Smad4( 275–322) mutant co-immunoprecipitates with FAST1
tion of Smad-containing complexes. Smad4( 275–322) formed
                                                                           in the presence of Smad2.2 We sought to determine if loss of the
heteromeric complexes with Smad2 (Lane 4, Fig. 2A), and, as
                                                                           SAD would alter the DNA binding ability of the Smad-FAST1
described for the wild type molecule (11, 28), underwent ligand-
                                                                           complex. We performed gel shift experiments using lysates
dependent nuclear translocation in the presence of overex-
                                                                           from Smad4-null MDA-MB468 cells transfected with various
pressed Smad2 (Fig. 2B). This differentiates it from COOH-
                                                                           components of the ARF and a 32P-labeled ARE probe. No TGF-
terminal point mutants of Smad4, G508S and D351H, which
                                                                             -inducible ARE binding complexes were detected in cells
disrupt the normal COOH-terminal structure of Smad4 and
                                                                           transfected with vector alone, FAST1, or Smad2 and FAST1.
fail to form heteromeric complexes with activated Smad2 (25).
                                                                           However, co-transfection with either wild type Smad4 or the
Furthermore, Smad4G508S and Smad4D351H did not undergo
                                                                           Smad4( 275–322) SAD deletion mutant yielded TGF- -induc-
nuclear translocation following receptor activation (Fig. 2B).
                                                                           ible gel-shifted complexes (Lanes 9 and 11, Fig. 3A). To confirm
This suggests that the COOH-terminal structure of Smad4 is
                                                                           that wild type Smad4, Smad4( 275–322), Smad2 and FAST1
preserved in Smad4( 275–322), and indicates that the princi-
                                                                           all participated in these ARE-binding complexes, we used an-
pal effect of the SAD deletion is to interfere with the nuclear
activities of Smad4.
   Transcriptional Activation by Smad4 Requires the SAD and                      R. Lechleider, unpublished data.
2118                                          Transcriptional Activity of Smad4

   FIG. 3. Smad4( 275–322) partici-
pates in Smad transcriptional com-
plexes, but only weakly activates
transcription. A, Smad4( 275–322)
forms a gel-shifted complex in Smad4 null
cells. MDA-MB468 cells were transfected
with the indicated constructs, serum
starved overnight, treated with or with-
out 10 g/ml TGF- for 1 h, and the ex-
tracts incubated with the 71-base pair
ARE probe. Lane 1 contains the ARE
probe only (P). B, Smad4( 275–322) is a
component of the ARF in NMuMg cells.
NMuMg cells were transiently trans-
fected with Myc-tagged Smad2 and
FAST1, and FLAG-tagged Smad4 con-
structs. After treatment with or without
10 g/ml TGF- for 1 h, cell extracts were
then incubated with the ARE probe, and
gel shifted complexes super-shifted with

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either anti-Myc (M) or anti-FLAG (F) an-
tibodies, as indicated. C, Smad4, and not
Smad4( 275–322), can fully activate the
ARE in Smad4 null cells. Smad4 null
MDA-MB468 cells were transfected with
the ARE-Luc reporter and the indicated
constructs, and luciferase activity meas-
ured after TGF- treatment for 20 h. D,
wild type but not mutant Smad4 aug-
ments the ARE response in NMuMg cells.
NMuMg cells were transfected with the
indicated constructs, and luciferase activ-
ity measured after TGF- treatment. All
results are expressed as mean ( S.E.) of
triplicate assays, corrected for -galacto-
sidase activity. E, expression of the vari-
ous constructs. NMuMg cell lysates were
transfected and probed as indicated.

tibodies directed against the epitopes expressed on these con-        -responsive NMuMg cells (Fig. 3D). The Smad4( 275–322)
structs to supershift the complexes. TGF- -responsive NMuMg         mutant only weakly enhanced ARE-luciferase activity in
cells were used as these gave a reproducibly high level of          Smad4 null cells when compared with the Smad4-independent
protein expression following transient transfection (Fig. 3E). In   activation response (Fig. 3C). Furthermore, in NMuMg cells,
these cells, ligand-dependent gel-shifted complexes are seen in     both basal and ligand-dependent activation of the ARE lucifer-
the absence of exogenous Smad4 (Lane 3, Fig. 3B), presumably        ase reporter were reduced in the presence of Smad4( 275–322)
as a result of binding to complexes containing endogenous           (Fig. 3D), indicating that Smad4( 275–322) has dominant neg-
Smad4. Incubation with Myc antibody reduced the mobility of         ative effects on TGF- signaling. In these experiments, various
the ARE-binding complex (Lanes 4, 8, and 12), confirming the        defined components of this transcriptional complex were ex-
presence of Myc-Smad2 and/or Myc-FAST1 in all of the com-           pressed at comparable levels (Fig. 3E). Taken together, these
plexes, while the FLAG antibody shifted a component of the          data indicate that maximal activation of Smad-dependent tran-
complex either with transfected wild type Smad4, or the             scriptional responses by Smad4 requires the SAD, and that this
Smad4( 275–322) deletion mutant (Lanes 9 and 13). The               activity is distinct from its ability to participate in these tran-
FLAG antibody did not lead to any supershift in lysates from        scriptional complexes.
cells transfected with FAST1 and Smad2 alone (Lane 5), con-            The SAD Is a Proline-rich Transcriptional Activation Do-
firming the specificity of these findings. These data show that     main That Binds p300 —Although we have shown that Smad4
the Smad4( 275–322) deletion mutant participates in ligand-         mutants lacking the SAD only weakly activate transcription of
dependent DNA binding complexes.                                    Smad-dependent transcriptional complexes, we were unable to
   The ability of wild type Smad4 to participate in these tran-     identify any defects in the other known biochemical functions
scriptional complexes correlates with its ability to enhance        of Smad4. Analysis of the SAD amino acid sequence (Fig. 4A)
ligand-dependent transcriptional activation of the ARE-lucif-       shows that it is rich in proline residues, much like the tran-
erase reporter both in Smad4 null cells (Fig. 3C), and in TGF-      scriptional activation domains of other well characterized tran-
                                             Transcriptional Activity of Smad4                                               2119

   FIG. 4. The SAD is a proline-rich,
p300-dependent transcriptional acti-
vation domain. A, amino acid sequence
of the SAD. The Smad4 amino acid se-
quence from 275–322 is represented using
standard single letter abbreviations. Pro-
lines are bolded and underlined. B, Gal4-
SAD fusion proteins can activate tran-
scription. Full-length Gal4-Smad4 (WT),
Gal4-Smad4 (266 –552), or Gal4-SAD fu-
sion constructs were transfected into
NMuMg cells, along with the reporter
plasmid and luciferase activity deter-
mined before and after TGF- treatment
for 20 h. C, Gal4-SAD transcriptional ac-
tivity is dependent on p300/CBP. Gal4-
SAD was co-transfected with E1A wild
type or p300/CBP binding mutant ( 2–36)
constructs, and Gal4-dependent lucifer-
ase was activity determined. The effect of

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EIA transfection on Gal4-SAD expression
was determined in parallel experiments
in which COS-1 cells were transfected
with Gal4-SAD along with the indicated
EIA constructs, and the cell lysates im-
munoblotted with anti-Gal4 antibodies.
D, p300 partially restores E1A-dependent
transcriptional repression. Gal4-SAD was
co-transfected with the indicated expres-
sion constructs and Gal4-dependent lucif-
erase activity determined. All results are
expressed as the mean ( S.E.) of tripli-
cate assays corrected for -galactosidase

scriptional activators including AP-2 and CTF/NF-1 (30, 31). To    hibition of Gal4-SAD (Fig. 4D). This indicates that transcrip-
test our hypothesis that the SAD acts as an intrinsic transcrip-   tional activation by the SAD is functionally dependent on p300.
tional activation domain, we performed heterologous activation        These studies suggest that there is a functional co-operativ-
assays using a Gal4-SAD fusion protein. These experiments          ity between CBP/p300 and the SAD, but do not define the
demonstrate that the SAD is a strong, ligand-independent           nature of this interaction. To determine if the p300-dependent
transcriptional activator, with levels of activation comparable    activation of Gal4-SAD is mediated by a direct interaction with
to or higher than the most active Gal4-Smad4 fusion construct      p300, we performed co-immunoprecipitation assays. Initially
(Fig. 4B).                                                         we looked for interactions between endogenous p300 and
   Recent studies suggest that the transcriptional activity of     epitope-tagged Smad4 constructs. We used Smad4 266 –552 as
Smad proteins is dependent on their interaction with the           the backbone for these studies as this was expressed at high
paralogous bridging co-activators CBP and p300, linking the        levels following transfection, has previously been shown to
Smad-DNA binding complex to the basal transcriptional ma-          interact with p300 and Smad2 (5, 18), and is transcriptionally
chinery (14 –18, 32). In order to determine if SAD transcrip-      active (Fig. 1B). In these studies, the SAD deletion mutant was
tional activity was dependent on interaction with these co-        still able to bind to endogenous p300 in a ligand-dependent
activators, we co-transfected adenoviral E1A, an inhibitor of      manner (Fig. 5A, Lanes 7 and 9). Furthermore, binding of both
CBP/p300 activity (33), along with Gal4-SAD, in the heterolo-      the wild type and the SAD deletion mutant was enhanced by
gous activation assay. E1A overexpression markedly reduced         overexpression of Smad2 (Lanes 5 and 9), indicating that the
the level of Gal4-SAD transcriptional activity, while a mutant     principal interaction of Smad4 with p300 may be indirect,
form of E1A lacking the CBP/p300-binding site ( 2–36) (34)         mediated by stronger interactions between the R-Smad,
had no effect on this response (Fig. 4C). The levels of Gal4-SAD   Smad2, and p300, and the R-Smad with Smad4.
protein expression were unaffected by E1A transfection. To            As our functional data provided evidence supporting the role
confirm that this effect was dependent on p300 activity, we        of the SAD as a CBP/p300-dependent transcriptional activation
co-transfected increasing amounts of p300 along with E1A in        domain, we went on to determine whether the SAD itself could
the Gal4-SAD reporter assay. p300 partially relieved E1A in-       independently interact with p300. For this, GST-p300 frag-
2120                                         Transcriptional Activity of Smad4

   FIG. 5. Physical interactions be-
tween Smad4 and p300. A, Smad2-de-
pendent binding of Smad 4 to endogenous
p300. COS-1 cells were transfected with
the indicated constructs and the lysates
analyzed by immunoprecipitation and
Western blotting, as indicated. T R-I* in-
dicates transfection with the activating
point mutant of the TGF- type 1 receptor
(T204D). B and C, the SAD interacts with
the NH2-terminal region of p300. Equal
amounts of bacterially produced GST fu-

                                                                                                                                     Downloaded from by guest, on July 26, 2011
sion fragments of p300 containing the
CH1 (GST-N), CH2 (GST-M), or CH3
(GST-C) regions (B), were incubated with
cell lysates from COS cells transiently
transfected with Gal4-SAD, and precipi-
tated by an anti-GST antibody (C). Co-
precipitated Gal4-SAD was detected fol-
lowing SDS-PAGE by Western blotting
using an anti-GAL4 antibody (top panel).
Coomassie Blue staining of p300 fusion
proteins demonstrates equal expression
(bottom panel).

ments purified by affinity chromatography (Fig. 5B), were in-     the Gal4-Smad4 266 –552 construct in these studies as this has
cubated with lysates from COS cells transiently transfected       particularly strong ligand-dependent transcriptional activation
with Gal4-SAD or the Gal4 vector alone, and immunoprecipi-        when compared with the wild type Smad4 fusion protein (Fig.
tated using anti-GST antibodies (Fig. 5C). Immunoblotting of      1B). Overexpression of an NH2-terminal p300 construct (1–
the immunoprecipitates revealed an association of Gal4-SAD        1736), which contains the SAD-binding site but lacks the glu-
with the CH1-containing NH2-terminal region of p300 (Fig. 5C,     tamine-rich transactivation domain of p300, strongly repressed
middle panel, Lane GST-N). Gal4-SAD showed a weak inter-          both basal and ligand-dependent activation of Gal4 Smad4
action with the middle (GST-M) region and did not interact        266 –552. In contrast, the COOH-terminal p300 construct
with the COOH-terminal (GST-C) region of p300, and there          (1737–2414), which lacks the SAD-binding site but has the
was no interaction between the Gal4 protein alone and the         capacity to bind to R-Smads, only partially inhibited this tran-
NH2-terminal GST-p300 fusion protein. This SAD-interaction        scriptional response (Fig. 6A). This is in keeping with the
domain is distinct from the COOH-terminal p300-binding site       observation that p300 (1737–2414) only partially blocks TGF-
for the MH2 domains of the R-Smads (14, 15, 17, 18, 32), and is     -dependent activation of the p3TP-Lux reporter, and probably
consistent with a weak interaction observed between Smad4         reflects inhibition of R-Smad binding to endogenous p300 (32).
and the NH2 terminus of CBP in a mammalian two-hybrid             As Smad4 266 –552 contains the SAD(275–322), we sought to
assay (14).                                                       determine whether p300 (1–1736) might be interfering with the
   In order to define the functional significance of this Smad4   transcriptional activation of Gal4-Smad4 266 –552 by inhibit-
SAD-p300 interaction, we sought to determine whether over-        ing p300-dependent transcriptional activation of the SAD.
expression of the SAD-binding region of p300 could interfere      Transfection of increasing amounts of p300 (1–1736) inhibits
with transcriptional activation by the SAD. Initially, we used    the transcriptional activity of Gal4-SAD, while the COOH-
                                                 Transcriptional Activity of Smad4                                                    2121
                                                                          binding assays in Smad4 null cells (11). This suggests that
                                                                          Smad4 has an additional function as a transcriptional
                                                                             We first identified the Smad4 activation domain, or SAD,
                                                                          from a deletional analysis of the Smad4 middle linker (7).
                                                                          Deletion of the SAD strongly reduces the ability of Smad4 to
                                                                          activate transcription of a variety of target genes both in ho-
                                                                          mologous and heterologous reporter gene assays. SAD deletion
                                                                          mutants do not affect R-Smad-Smad4 hetero-oligomerization,
                                                                          nor the ability of Smad4 to participate in ligand-dependent
                                                                          DNA binding of Smad-containing transcriptional complexes.
                                                                          This contrasts with mutations and deletions within the COOH-
                                                                          terminal MH2 domain of Smad4, which interfere with tran-
                                                                          scriptional responses by disrupting hetero-oligomerization of
                                                                          Smad4 with R-Smads (26, 35, 36). Thus, while Smad4 requires
                                                                          its COOH terminus to interact with R-Smads and form ligand-
                                                                          dependent transcriptional complexes, this activity is distinct
                                                                          from the transactivating activity of Smad4 which requires the
                                                                             The only other inactivating deletion in the middle linker
                                                                          region which has been studied ( 223–301) is a splice variant of
                                                                          Smad4 found in MDA-MB231, a breast cancer cell line (37).
                                                                          Unlike the SAD deletion, which can inhibit TGF- -dependent

                                                                                                                                               Downloaded from by guest, on July 26, 2011
                                                                          responsive reporter gene activity,3 this mutant lacks dominant
                                                                          negative activity, suggesting that the deletion of additional
                                                                          sequences upstream of the SAD may interfere with other func-
                                                                          tions of Smad4, for example, its association with other R-
                                                                          Smads in the cytoplasm. Partial mapping of Smad4 transacti-
                                                                          vator domains using heterologous DNA binding assays
                                                                          confirms that Gal4-Smad4(266 –552) strongly activates tran-
   FIG. 6. Functional interactions between Smad4 and p300. A,             scription of the luciferase reporter in a ligand-dependent man-
the NH2 terminus of p300 inhibits ligand-dependent transcriptional        ner, while deletion of the SAD in this construct completely
activation of Smad4. NMuMg cells were transfected with Gal4-Smad4         abolishes this activity. Our experiments in Smad4 null cells
266 –552 or Gal4-Smad4 266 –552 ( 275–322) fusion constructs, 1.5 g
                                                                          also show that the transactivating activity of Gal4-Smad2 is
of the indicated p300 constructs (1–1736) and (1737–2414), the appro-
priate reporter plasmids, and luciferase activity determined before and   absolutely dependent on the presence of exogenous Smad4, and
after TGF- treatment for 20 h. Results are expressed as mean ( S.E.)      that this response is markedly reduced following deletion of the
of triplicate assays, corrected for -galactosidase activity. B, the NH2   SAD. These data confirm that Smad4 is involved in mediating
terminus of p300 inhibits transcriptional activity of the SAD. NMuMg      Smad-dependent transcriptional responses, and support our
cells were transfected with Gal4-SAD, reporter constructs, and increas-
ing amounts (0.75 and 1.5 g) of the indicated p300 constructs. Lucif-     conclusion that this activity is dependent on the SAD.
erase activity was determined 40 h following transfection.                   We now demonstrate that the SAD is an intrinsic transcrip-
                                                                          tional activation domain, rich in proline residues, and that it is
terminal, R-Smad binding p300 construct (1737–2414), had no               not only necessary, but also sufficient to activate maximal
effect on this response (Fig. 6B). This suggests that p300 (1–            Smad-dependent transcriptional responses. Similar proline-
1736) is competing with endogenous p300 for binding to the                rich domains have been described in a number of other tran-
SAD, and therefore interfering with transcriptional activation            scriptional activators such as AP-2 and CTF/NF1 (30), suggest-
by the Gal4-SAD fusion protein. Taken together, these data                ing a common mechanism of action. A feature of these
indicate that the interaction between the Smad4 SAD and the               activation domains is that they interact with diverse compo-
NH2 terminus of p300 is functionally significant, and points to           nents within the general transcriptional machinery, recruiting
a novel role of the middle linker in regulating the transcrip-            multicomponent complexes of proteins into juxtaposition with
tional activity of Smad4.                                                 the transcription factor-DNA complex. In this context, the crys-
                                                                          tal structure of a transcriptionally active Smad4 fragment
                                                                          (273–552) has recently been solved (38), providing key insights
   Many components of the transcriptional complexes mediat-               into the structural basis for the transcriptional responses me-
ing Smad-dependent activation of target genes have recently               diated by the SAD. This structure contrasts with the previously
been identified. These include Smad4, R-Smads, shared com-                published structure of an inactive Smad4 fragment (319 –543)
ponents of the basal transcriptional machinery, and the CBP/              (25) as the additional residues stabilize a previously disordered
p300 histone acetyltransferase bridging co-activators (14 –17,            structure within the MH2 domain of Smad4, forming a glu-
32). Studies in Smad4 null cells indicate that the presence of            tamine-rich extension from the trimeric core. Interestingly,
Smad4 is essential for many of these transcriptional responses            this glutamine-rich extension is reinforced by the SAD, which
(7–9). In certain contexts, Smad4 may be required for stabili-            is stabilized by flanking sequences that interact with the struc-
zation of the Smad-DNA transcriptional complex (11, 13), while            tural core of Smad4. The proline-rich, hydrophobic surface of
in others this function may result from a co-operative interac-           the SAD is located on the same surface as the solvent accessible
tion between Smad4 and R-Smads (12, 14). However, while the               glutamine-rich extension of the MH2 domain at the periphery
NH2-terminal MH1 domain of Smad4 is required to stabilize                 of the trimeric disc, suggesting that this energetically unfavor-
these transcriptional complexes, for example, the TGF- induc-
ible FAST1/Smad2 ARF, Smad4 mutants lacking the NH2 ter-
minus can still transactivate Gal4-Smad2 in heterologous DNA                    M. P. de Caestecker, unpublished data.
2122                                        Transcriptional Activity of Smad4
able hydrophobic surface could be stabilized by interaction with   meric R-Smad/Smad4 interaction enables interaction of the
a transcriptional co-factor. This model provides a structural      SAD with the NH2 terminus of CBP/p300. This interaction is
basis for the unique functional role we have ascribed to the       essential for maximal activation by the Smad complex, result-
SAD.                                                               ing in activation of the basal transcriptional machinery, re-
   Residual ligand-dependent reporter gene activation of the       cruitment of other members of the complex, and/or alterations
SAD deletion constructs suggests that other domains of Smad4       in histone acetyltransferase activity.
may also contribute to the maximal activation of Smad tran-
scriptional complexes. Artificial nuclear localization of Smad4       Acknowledgments—We thank Kai Lin for sharing unpublished data
                                                                   on the crystal structure of Smad4, D. M Livingston and S. R. Grossman
using an estrogen receptor fusion protein indicates that loss of                                      ´
                                                                   for p300 constructs, J. Massague for the p3TP-Lux reporter, M.
the COOH-terminal 37 amino acids prevents Smad4-depend-            Montminy and C. R. Goding for E1A constructs, M. Ptashne for
ent transcriptional activation (27). This result resembles our     pSG424J, M. Whitman for the Xenopus Myc-tagged FAST1 and ARE-
findings with the SAD deletion mutant, and suggests that           Luc constructs, and J. Wrana and L. Attisano for the p147-Gal4 re-
                                                                   porter, pG5E1B, and activated TGF- type I receptor T204D. C. Eng
there may be an additional transcriptional activation domain       and J. Ryan provided expert technical assistance.
in the COOH terminus of Smad4. This would explain our
observation that there is residual, ligand-dependent activation                                       REFERENCES
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