1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

Members of the transforming growth factor-β (TGF-β) superfamily bind to two different serine/threonine kinase receptors, i.e. type I and type II receptors. Upon ligand binding, type I receptors specifically activate intracellular Smad proteins. R-Smads are direct substrates of type I receptors; Smads 2 and 3 are specifically activated by activin/nodal and TGF-β type I receptors, whereas Smads 1, 5 and 8 are activated by BMP type I receptors. Nearly 30 proteins have been identified as members of the TGF-β superfamily in mammals, and can be classified based on whether they activate activin/TGF-β-specific R-Smads (AR-Smads) or BMP-specific R-Smads (BR-Smads). R-Smads form complexes with Co-Smads and translocate into the nucleus, where they regulate the transcription of target genes. AR-Smads bind to various proteins, including transcription factors and transcriptional co-activators or co-repressors, whereas BR-Smads interact with other proteins less efficiently than AR-Smads. Id proteins are induced by BR-Smads, and play important roles in exhibiting some biological effects of BMPs. Understanding the mechanisms of TGF-β superfamily signalling is thus important for the development of new ways to treat various clinical diseases in which TGF-β superfamily signalling is involved.

Introduction: the transforming growth factor-β (TGF-β) superfamily

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

Cytokines of the TGF-β superfamily are dimeric proteins with conserved structures, and have pleiotropic functions in vitro and in vivo (Kawabata et al. 1998a). The TGF-β superfamily includes nearly 30 proteins in mammals, e.g. TGF-βs, activins and inhibins, nodal, myostatin, bone morphogenetic proteins (BMPs), growth/differentiation factors (GDFs), and anti-Müllerian hormone (AMH, also called Müllerian inhibiting substance or MIS) (Fig. 1). TGF-β/BMP-like proteins are found in various species, including Xenopus, Caenorhabditis elegans and Drosophila melanogaster.


Figure 1. A phylogenetic tree of the TGF-β superfamily in human (h), mouse (m), Xenopus (X) and Drosophila (D). The amino acid sequences were aligned by ClustalW version 1.82 (Thompson et al. 1994). Phylogenetic analysis was conducted by the maximum-likelihood method with Molphy version 2.3b3 with the star decomposition option (Adachi & Hasegawa 1996). The C-terminal region of TGF-β1 (amino acid residues 293–327, 355–368 and 382–390) and its corresponding sequences were used for comparison. GDF-7, Xnr2 and Xnr4 were excluded because their C-termini were too short to align. Local bootstrap probabilities are shown on the corresponding branches. Ligands that activate AR-Smads or BR-Smads are shown in red or dark blue, respectively. Ligands that may activate AR-Smads or BR-Smads, but whose receptors and downstream signalling pathways have not been fully determined, are shown in orange and light blue, respectively. Activins are dimers of inhibin-β chain. OP, osteogenic protein.

Download figure to PowerPoint

TGF-βs are the prototype of the TGF-β superfamily (Massagué 1998). They act as potent growth inhibitors for most types of cells, and induce the apoptosis of epithelial cells. In addition, TGF-βs stimulate the production of extracellular matrix proteins, inducing fibrosis in various tissues in vivo. Inhibins were originally identified as cytokines that inhibit the secretion of follicle-stimulating hormone (FSH) from the pituitary gland. Inhibins are composed of disulphide-bonded dimers of inhibin-α and -β chains. In contrast, dimers composed of inhibin-β chains (also called activin-β chains) are activins, which stimulate the production of FSH by the pituitary gland. Activins also play important roles in the induction of dorsal mesoderm during early embryogenesis. In addition, they act on epithelial cells and haematopoietic cells and regulate their growth, differentiation and apoptosis. Nodal plays critical roles in the induction of dorsal mesoderm, anterior patterning, and formation of left–right asymmetry. Myostatin, also known as GDF-8, is produced by cells of the skeletal muscle lineage, and inhibits their growth. Although these TGF-β superfamily proteins (Fig. 1; coloured in red and orange) have divergent biological effects in vivo, they transmit similar, although not identical, intracellular signals in target cells.

BMPs were originally identified as cytokines that induce bone and cartilage tissues in vivo (Rosen & Wozney 2002). BMPs were then shown to have diverse effects on various cells, and to induce the ventral mesoderm during early embryogenesis. GDF-5, also known as cartilage-derived morphogenetic protein 1 (CDMP-1), is structurally related to BMPs, and induces cartilage-like tissue in vivo. AMH induces Müllerian duct regression during embryogenesis and inhibits the transcription of gonadal steroidogenic enzymes. The biological effects of AMH appear to differ from those of BMPs and GDF-5; however, the intracellular signals activated by these cytokines (Fig. 1; coloured in dark and light blue) have been shown to be similar.

In this review, we discuss the signalling pathways activated by members of the TGF-β superfamily. Although the biological effects of these cytokines appear to be complicated, two major pathways involving Smad proteins are activated by members of the TGF-β superfamily (Miyazono et al. 2001). Accordingly, the TGF-β superfamily cytokines can be classified into two subfamilies depending on the Smad signalling pathways they activate. Understanding the mechanisms of TGF-β superfamily signalling will aid in the development of new strategies for the treatment of various clinical diseases to which the TGF-β superfamily signalling is tightly linked.

Receptors for TGF-β superfamily proteins

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

Members of the TGF-β superfamily bind to two distinct receptor types, known as type II and type I receptors (Wrana et al. 1994; Heldin et al. 1997). Both type II and type I receptors are required for signal transduction. In addition, some cell surface proteins, including betaglycan (also known as TGF-β type III receptor), endoglin, and the EGF-CFC family proteins, containing a divergent EGF-like motif and a novel cysteine-rich CFC motif, act as co-receptors for certain members of the TGF-β superfamily.

Both type II and type I receptors contain serine/threonine kinase domains in their intracellular portions. The type II receptor kinases are constitutively active; upon ligand binding, hetero-tetrameric complexes composed of two molecules each of type II and type I receptors are formed (Kirsch et al. 2000; Qin et al. 2002). In the tetrameric receptor complexes, type II receptor kinases transphosphorylate the GS domain of type I receptors, which are located between the transmembrane domain and the kinase domain of type I receptors (Fig. 2). Following phosphorylation of the GS domain, type I receptor kinases are activated and phosphorylate intracellular substrates. Thus, type I receptors act as downstream components of type II receptors in the signalling pathways, and determine the specificity of the intracellular signals induced by the TGF-β superfamily cytokines.


Figure 2. Two major signalling pathways of the TGF-β superfamily. Signalling pathways of TGF-β, activin, nodal and myostatin are shown in panel (A). Panel (B) shows the signalling pathways of BMPs and AMH. In panel (C), TGF-β signalling pathways in endothelial cells are shown. Members of the TGF-β superfamily bind to type II and type I receptors. Ligand binding induces the formation of heteromeric complexes, in which type II receptors phosphorylate the GS domain (yellow box) of type I receptors. Type I receptors then activate R-Smads (Smad2/3 in (A) or Smad1/5/8 in (B)), which associate with Co-Smad (Smad4). The R-Smad and Co-Smad complexes move into the nucleus, and regulate the expression of target genes in cooperation with transcription factors, co-activators and co-repressors. Id proteins are induced in response to the Smad1-Smad5 pathway (B). Id proteins act as negative regulators of bHLH transcription factors by antagonizing the activity of tissue-specific bHLH transcription factors and ubiquitous bHLH factors, which activate the transcription of E-box-containing genes. In endothelial cells (C), TGF-β binds to ALK-1 as well as to ALK-5 in the presence of TβR-II; the former activates Smad1 and Smad5, and transmits BMP-like signals.

Download figure to PowerPoint

Five type II receptors and seven type I receptors are present in mammals. The TGF-β type II receptor (TβR-II) is the specific receptor for TGF-βs. Activin type II and type IIB receptors (ActR-II and ActR-IIB) serve as type II receptors for activins, but are shared with other TGF-β superfamily members, including nodal and BMPs. BMP type II receptor (BMPR-II) and AMH type II receptor (AMHR-II) specifically bind to BMPs and AMH, respectively.

Of the seven type I receptors, the activin receptor-like kinases (ALK)-4, -5, and -7 are structurally similar to each other. ALK-5, also known as TβR-I, serves as a specific receptor for TGF-βs. ALK-4 binds actvins and nodal, whereas ALK-7 binds only nodal (Reissmann et al. 2001). Receptors for myostatin have not yet been determined. ALK-3 and ALK-6 (also termed BMPR-IA and BMPR-IB, respectively) are structurally similar, and function as BMP type I receptors. In addition, ALK-2, which is distantly related to ALK-3 and -6, acts as a type I receptor for certain BMPs, including BMP-6 and -7 (Macías-Silva et al. 1998; Aoki et al. 2001). ALK-1 is structurally very similar to ALK-2; in contrast to ALK-2, however, ALK-1 is expressed in endothelial cells, and serves as a type I receptor for TGF-β (see below; Oh et al. 2000).

Signalling by three types of Smad proteins

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

Smad proteins are major signalling molecules acting downstream of the serine/threonine kinase receptors (Heldin et al. 1997; Moustakas et al. 2001). Smads are classified into three subclasses, i.e. receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads (I-Smads). R-Smads are further divided into two subclasses; Smad2 and Smad3 (referred to as activin/TGF-β activated R-Smads or AR-Smads in this review) are activated by activin, nodal and TGF-β type I receptors, ALK-4, -5 and -7. Smad1, Smad5 and Smad8 (referred to as BMP activated R-Smads; BR-Smads) are activated by BMP type I receptors, ALK-3 and ALK-6. Smads 1 and 5 are also activated by ALK-1 and -2, although they are less similar to ALK-3/6 than the ALK-4/5/7 are. In contrast, only Smad4, which was originally isolated as the product of the tumour suppressor gene DPC4 (deleted in pancreatic carcinoma, locus 4; Hahn et al. 1996), serves as the Co-Smad in mammals. Smad6 and Smad7 function as I-Smads.

Smads are composed of the N-terminal Mad homology (MH1) domain, followed by linker regions and the C-terminal MH2 domains. MH2 domains are conserved in all three subclasses of Smads, whereas MH1 domains are conserved in R-Smads and Co-Smads, but not in I-Smads. The amino acid sequences of linker regions diverge between Smads. In addition, R-Smads have Ser-Ser-X-Ser motifs in their most C-terminal parts, which are phosphorylated by type I receptors.

R-Smads are directly phosphorylated and activated by type I receptor kinases. Without receptor activation, the MH1 and MH2 domains are physically associated with each other, and R-Smads are anchored as dimers to the plasma membrane through SARA and other molecules (Fig. 2, Table 1) (Qin et al. 2002). Following the receptor activation and phosphorylation of R-Smads, the interaction between the MH1 and MH2 domains is disrupted, and R-Smads form hetero-oligomers with the Co-Smad through their MH2 domains. Although the exact structures of the Smad oligomers have not been fully determined, and hetero-trimer and hetero-dimer models have been suggested (Kawabata et al. 1998b; Qin et al. 2001; Wu et al. 2001), Qin et al. (2001) proposed that the R-Smad-Co-Smad complexes are hetero-trimers, containing two R-Smad molecules and one Co-Smad molecule. The R-Smad-Co-Smad complexes translocate into the nucleus and regulate the transcription of target genes.

Table 1.  Smad-interacting proteins
NameBinding to Smads*Interacting domainSignal depen dencyDescriptionReferences
AMSH MH2YEScytosolic signalling moleculeItoh et al. 2001
APC (anaphase promoting complex)     MH2YESubiquitin ligaseStroschein et al. 2001
AR (androgen receptor)    ND transcription activator (nuclear receptor)Kang et al. 2001;Chipuk et al. 2002
ARC105    MH2YESactivator-recruited cofactor componentKato et al. 2002
ATF-2      MH1YEStranscription activator (b-Zip)Sano et al. 1999
Axin   MH2DISscaffold protein (Wnt pathway)Furuhashi et al. 2001
BF-1 (brain factor-1)    MH2NOtranscription repressor (winged-helix, c-Qin)Rodriguez et al. 2001
BRCA2      MH1, 2YEStumour suppressor (BRCT domain)Preobrazhenska et al. 2002
Calmodulin    MH1 calcium binding proteinZimmerman et al. 1998; Scherer & Graff 2000
CBF-C (CCAAT- binding factor C subunit)      MH2NOtranscription activator subunitChen et al. 2002b
Dab2   MH2YEScytosolic signalling molecule (PTB domain)Hocevar et al. 2001
Dok-1     MH2YESras-GAP binding proteinYamakawa et al. 2002
E1A   MH2NOviral oncoprotein (adenovirus)Nishihara et al. 1999
E2F4/5       MH2NOtranscription repressorChen et al. 2002a
E7      MH1NOviral oncoprotein (human papilloma virus)Lee et al. 2002b
ER (oestrogen receptor)     MH2YEStranscription activator (nuclear receptor)Matsuda et al. 2001
Evi-1    MH2YESco-repressor (Zn finger protein, recruiting HDAC)Kurokawa et al. 1998
FAST1    MH2YEStranscription activator (winged-helix)Chen et al. 1997
FAST2      NDYEStranscription activator (winged-helix)Labbéet al. 1998; Liu et al. 1999
Filamin   MH1, L cytoskeletal actin binding proteinSasaki et al. 2001
GATA-3    MH1 transcription activator (b-Zip)Blokzijl et al. 2002
Gli3 (C-terminally truncated)    NDDIStranscription repressor (Zn finger, HH target)Liu et al. 1998
GR (glucocorticoid receptor)      MH2 transcription activator (nuclear receptor)Song et al. 1999
HEF1    MH1, 2 cytoplasmic docking protein (Cas family)Liu et al. 2000
HNF4      NDYEStranscription activator (nuclear receptor, orphan)Kardassis et al. 2000
Hoxa-9     NDNOtranscription repressor (homeodomain)Shi et al. 2001
Hoxc-8    MH2YEStranscription repressor (homeodomain)Shi et al. 1999; Bai et al. 2000
Hrs   NDNOintracellular trafficking (FYVE domain protein)Miura et al. 2000
Importin-b1     MH1YESintracellular traffickingXiao et al. 2000; Kurisaki et al. 2001
Jab1      MH2, L co-activator (for AP1)Wan et al. 2002
c-Jun     MH1YEStranscription activator (b-Zip)Zhang et al. 1998; Liberati et al. 1999
JunB   ND transcription activator (b-Zip)Liberati et al. 1999
JunD      ND transcription activator (b-Zip)Liberati et al. 1999
Lef1    MH1, 2YEStranscription activator (HMG domain, Wnt target)Nishita et al. 2000; Labbéet al. 2000
MEF2       ND transcription activatorQuinn et al. 2001
Menin (MEN1 gene product)     MH2?tumour suppressorKaji et al. 2001
Mixer      MH2YEStranscription activator (homeodomain)Germain et al. 2000
Milk       MH2 transcription activator (homeodomain)Germain et al. 2000
MSG1       MH2, LYESco-activatorShioda et al. 1998
Msx-1     ND homeobox proteinYamamoto et al. 2001
c-Myc    MH2YEStranscription activator (bHLH-Zip)Feng et al. 2002
MyoD    MH1? transcription activator (bHLH)Liu et al. 2001
NF-κB      NDYEStranscription activatorLopez-Rovira et al. 2000
OAZ     MH2YESDNA-binding Zn finger proteinHata et al. 2000
p300/CBP   MH2YESco-activator (HAT)Six references§
P/CAF      MH2NOco-activator (HAT)Itoh et al. 2000
PPARγ      ND transcription activator (nuclear receptor)Fu et al. 2001
pX    MH1NOviral oncoprotein (Hepatitis B viras)Lee et al. 2001
Rb (p107)       MH2NOtumour suppressorChen et al. 2002a
ROC1       MH2YESubiquitin ligase component (SCF complex)Fukuchi et al. 2001
Runx1   MH2YEStranscription activator (Runt domain)Hanai et al. 1999; Pardali et al. 2000a
Runx2    NDYEStranscription activator (Runt domain)Hanai et al. 1999
Runx3    MH2YEStranscription activator (Runt domain)Hanai et al. 1999
SARA  MH2DISscaffold protein (FVYE domain protein)Tsukazaki et al. 1998
SIP1 (Smad interacting protein 1)   MH2YEStranscription repressor (Zn finger-homeodomain)Verschueren et al. 1999
c-Ski MH2YESco-repressor (recruiting HDAC)Akiyoshi et al. 1999; Wang et al. 2000
SnoN     MH2 co-repressor (recruiting HDAC)Stroschein et al. 1999
SKIP (Ski-interacting protein)      MH2 +α?co-activatorLeong et al. 2001
SNIP (Smad nuclear interacting protein)   MH2YESp300 inhibitorKim et al. 2000
SMIF  LYESco-activatorBai et al. 2002
Smurf1L(PY)NOubiquitin ligase (HECT type)Zhu et al. 1999; Ebisawa et al. 2001
Smurf2    L(PY)NOubiquitin ligase (HECT type)Lin et al. 2000; Zhang et al. 2001
Sp1  MH1(2), 2(4)YEStranscription activator (Zn finger)Pardali et al. 2000b; Datta et al. 2000; Feng et al. 2000
STRAP  ND TβRI, II-associated proteinDatta & Moses 2000
Swift      NDYESco-activator (BRCT domain)Shimizu et al. 2001
TAK1       ND MAPKKKKimura et al. 2000
Tax     MH2YESviral oncoprotein (HTLV)Lee et al. 2002a
TFE3      MH1YEStranscription activator (bHLH-Zip)Hua et al. 1999
TGIF    NDYESco-repressor (recruiting HDAC)Wotton et al. 1999
Tob   MH2YESanti-proliferative proteinYoshida et al. 2000
TRAP1MH1 +αYESTβRI-associated proteinWurthner et al. 2001
tublin     NDDISmicrotubulesDong et al. 2000
VDR (vitamin D receptor)      MH1 transcription activator (nuclear receptor)Yanagisawa et al. 1999
Vent-2     MH1 transcription activator/repressor (homeodomain)Henningfeld et al. 2002
WBSCR11      MH1YEStranscription activator (bHLH)Ring et al. 2002
YAP65 (Yes-associated protein)      L(PY) +α WW domain proteinFerrigno et al. 2002

In the nucleus, R-Smads and Co-Smad interact with various DNA-binding proteins (Table 1), which bind to promoter regions of target genes together with the Smads. In addition, R-Smads and Co-Smad bind to transcriptional co-activators and co-repressors, which induce the acetylation and de-acetylation of histones, respectively, and play important roles in transcriptional regulation (Miyazono et al. 2000).

I-Smads associate with activated type I receptors and prevent the activation of R-Smads by the receptors. In addition, Smad6 interacts with activated R-Smads and interferes with the formation of a complex with Smad4. Smad7 inhibits both TGF-β activin and BMP signalling, whereas Smad6 preferentially inhibits BMP signalling (Hanyu et al. 2001). The N-terminal domain of Smad7 physically interacts with its MH2 domain, and plays an important role in the inhibition of TGF-β signalling (Hanyu et al. 2001). The HECT type E3 ubiquitin ligases, Smurf1 and Smurf2, support the inhibitory activities of I-Smads on TGF-β superfamily signalling. When Smurfs bind to Smad7, they induce the nuclear export of Smad7, and enhance the degradation of TGF-β receptors, resulting in the inhibition of TGF-β signalling (Kavsak et al. 2000; Ebisawa et al. 2001). Since I-Smads are induced by various signals, including TGF-β and BMPs, the levels of expression of I-Smads modulate the magnitude and duration of TGF-β superfamily signalling.

Signalling by AR-Smads and BR-Smads

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

The MH1 domains of Smads are responsible for their DNA-binding. One of the AR-Smads, Smad3, and Co-Smad, Smad4, directly bind to ‘CAGA’ and related DNA sequences, and activate the transcription of target genes together with DNA-binding proteins (Dennler et al. 1998; Zawel et al. 1998). Smad2 does not directly bind DNA, but by forming complexes with Smad3 and/or Smad4, it is also able to bind to the CAGA sequence. In addition, AR-Smads bind to certain other DNA sequences, e.g. the TGF-β inhibitory element (TIE), which is present in the promoter regions of c-myc (Chen et al. 2001; Yagi et al. 2002). In contrast, BR-Smads do not efficiently bind the CAGA sequence, but bind other sequences, e.g. GC-rich sequences, with low affinities (Ishida et al. 2000; Kusanagi et al. 2001). Smad4 binds the CAGA sequence in vitro, but does not efficiently activate the transcription of reporter genes containing CAGA sequences when complexed with BR-Smads. Exceptionally, however, a promoter-reporter construct containing the CAGAC sequence from the junB gene strongly responds to TGF-β, as well as to BMPs (Jonk et al. 1998). In general, if some ligands activate AR-Smads and transduce activin/TGF-β-like signals, they strongly transactivate the promoter-reporter constructs containing CAGA sequences. If some ligands activate BR-Smads and transmit BMP-like signals, they activate reporters containing GC-rich sequences. Members of the TGF-β superfamily can be classified based on their abilities to activate CAGA-reporters or GC-rich reporters, even if their receptors have not been determined.

TGF-βs bind to ALK-5, and activins and nodal bind to ALK-4 and/or -7; these ligands thus activate AR-Smads (Fig. 2A). Although the receptors for myostatin have not been determined, it activates CAGA-reporters (Zimmers et al. 2002), and may thus transduce signals similar to actvins and TGF-βs through AR-Smads. In contrast, most BMPs, including BMP-2, BMP-4, BMP-6, BMP-7 and GDF-5, activate BR-Smads (Aoki et al. 2001). AMH binds to ALK-2 and ALK-6, thus activating BR-Smads (Fig. 2B) (Gouédard et al. 2000; Visser et al. 2001; Clarke et al. 2001). BMP-3 was originally isolated as a member of the BMP family. BMP-3, however, does not have the ability to induce bone formation in vivo, but transmits activin/TGF-β-like signals through ActR-II and ALK-4 (Daluiski et al. 2001).

Certain members of the TGF-β superfamily, including some GDFs, have been isolated by virtue of their amino acid sequence similarities with other members of the TGF-β superfamily. Receptors and downstream signals have not been determined for most of these ligands (Fig. 1); it will be important to determine whether they activate AR-Smads or BR-Smads to perform their activities. Although the inhibin-α chain is a member of the TGF-β superfamily, it is not known whether inhibins display their effects by antagonizing activins, or by binding to specific signalling receptors (Bernard et al. 2002). Inhibins bind to betaglycan; however, it is not known whether they bind to serine/threonine kinase receptors.

Although both TGF-βs and activins activate Smad2 and Smad3, their biological activities are in most cases not identical. Analyses by gene targeting in mice have suggested that Smad2 plays important roles in embryogenesis, whereas Smad3 is of critical importance in cell growth and migration after birth (Nomura & Li 1998; Zhu et al. 1998; Yang et al. 1999; Datto et al. 1999). Thus, the effects of Smad2 may be more closely linked to activins, and those of Smad3 to TGF-βs. It is not currently known whether ALK-4 and ALK-5 differentially activate Smad2 and Smad3 in vivo. It will also be important to determine whether Smad-independent pathways are involved in the signalling pathways of TGF-β and other factors.

In classifying TGF-β superfamily cytokines based on their intracellular signals, an important exception is observed in TGF-β signalling in endothelial cells (Fig. 2C). TGF-β binds to ALK-5 in almost all TGF-β responsive cells, and transmits typical TGF-β-like signals. In addition, however, TGF-β binds to ALK-1 in endothelial cells and possibly in certain other types of cells (Oh et al. 2000). ALK-1 is structurally similar to ALK-2 and activates BR-Smads. TGF-β is thus able to transmit intracellular signals through AR-Smads and BR-Smads in endothelial cells. The balance of activation of ALK-5/AR-Smads and ALK-1/BR-Smads determines the activation status of endothelial cells in the development and homeostasis of vascular tissues (Goumans et al. 2002).

Targets of AR-Smads and BR-Smads

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

AR-Smads interact with various DNA-binding proteins (Table 1), which play important roles in exhibiting multiple biological activities of TGF-βs and activins. In contrast, only a few DNA-binding partners of BR-Smads have been identified, and some of them are shared with AR-Smads. Similarly, the transcriptional co-activators p300 and CBP, and the co-repressors c-Ski and SnoN, interact strongly with AR-Smads, but only weakly with BR-Smads. Why do BR-Smads interact with only a limited number of proteins compared to AR-Smads? As shown in Fig. 3, the surface of the trimer composed of the MH2 domain of Smad3 is rich in charged amino acid residues. The N-terminal upper side is rich in acidic amino acids, and the lower side is rich in basic amino acids. In contrast, the surface of the Smad1 MH2 trimer is much less charged than that of the Smad3 MH2 trimer. Although the Smad1/3 MH2 domain may interact with other proteins through hydrophobic amino acid residues in protein–protein interaction surfaces, this finding suggests that Smad3 MH2 is able to interact with other proteins more efficiently than Smad1 MH2.


Figure 3. Three-dimensional structure models of the homo-trimers of Smad1 and Smad3. The N-terminal upper side (upper panels) and bottom side (lower panels) of the homotrimer of MH2 domains are shown. Molecular surfaces are represented by the Grasp program (Nicholls et al. 1991). Left panels show the electrostatic mapping in molecular surface of Smad1 homotrimer X-ray model, and right panels represent the electrostatic mapping in molecular surface of Smad3 homotrimer theoretical model. Regions coloured in blue show positive charges, while those coloured in red show negative charges.

Download figure to PowerPoint

Through interactions with various DNA-binding partners, AR-Smads transactivate various target genes, including plasminogen activator inhibitor-1 (PAI-1), type I collagen, junB, Smad7 and Mix.2. For inhibition of cell growth by TGF-β, AR-Smads induce the transcription of cyclin-dependent kinase (CDK) inhibitors p21 and p15 (Fig. 2A). In addition, Smad3 binds directly to the promoter region of c-myc through TIE, and represses the transcription of c-myc. In contrast, only a few target genes for BMPs have been identified, including Id (inhibitor of differentiation or inhibitor of DNA-binding) 1 through 3, Smad6, Vent-2, and Tlx-2 (Fig. 2B). Id proteins, however, appear to play important roles in the exhibition of the multiple biological activities of BMPs (Miyazono & Miyazawa 2002).

Id proteins act as negative regulators of cell differentiation and positive regulators of cell proliferation (Norton et al. 1998; Yokota & Mori 2002). Four Id proteins, Id1 through Id4, have been identified in mammals. They have overlapping profiles of expression, and elicit similar, but not identical, biological activities. Id proteins have a helix-loop-helix (HLH) dimerization domain, which binds to ubiquitously expressed transcription factors containing the basic HLH (bHLH) domain (Fig. 2B). Ubiquitously expressed bHLH transcription factors interact with tissue-specific bHLH transcription factors, and these complexes activate the transcription of genes containing an E-box in their promoter regions. In contrast to bHLH transcription factors, Id proteins lack the basic region responsible for binding to DNA. Id proteins thus sequester ubiquitous bHLH transcription factors and inhibit their transcriptional activities. Tissue-specific bHLH transcription factors include MyoD in muscle, and neurogenin and NueroD in neurones. Inhibition of the transcriptional activities of MyoD and neurogenin/NeuroD by Id proteins results in the inhibition of myogenesis and neurogenesis, respectively. These findings suggest that BMPs may elicit some of their biological activities through Id proteins (Nakashima et al. 2001; Goumans et al. 2002). In addition, Id2 interacts with the hypo-phosphorylated forms of Rb family proteins and inhibits their effects. Id2 may therefore play a role in the positive regulation of cell growth. BMP has been shown to both stimulate and inhibit the growth of cells, depending on cell type and culture conditions. Whether Id proteins play a role in the growth stimulation induced by BMPs is an important question to be answered in the future.

Conclusion: relation to clinical diseases

  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

Members of the TGF-β superfamily exhibit various biological activities, and perturbations of their signalling are linked to certain clinical disorders. For example, TGF-β induces fibrosis in various tissues, including lung fibrosis, glomerulonephritis and liver fibrosis (Blobe et al. 2000). Inhibition of TGF-β signalling may therefore result in the prevention of fibrosis in these disorders. Recently, inhibitors of ALK-4, -5, and -7 kinases have been generated (Laping et al. 2002; Inman et al. 2002). SB-431542 inhibits ALK-4/5/7 signals, but not ALK-3/6 signals; it thus inhibits TGF-β signals without affecting BMP signals. However, these type I receptor kinase inhibitors should be used with care, since they may inhibit not only TGF-β, but also activins, nodal and myostatin. Understanding the signalling pathways of the TGF-β superfamily is therefore important for the clinical application of various agents which regulate the signalling activities of serine/threonine kinase receptors and Smads.


  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References

We thank Ms Hisako Hirano for preparing the list of references. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


  1. Top of page
  2. Abstract
  3. Introduction: the transforming growth factor-β (TGF-β) superfamily
  4. Receptors for TGF-β superfamily proteins
  5. Signalling by three types of Smad proteins
  6. Signalling by AR-Smads and BR-Smads
  7. Targets of AR-Smads and BR-Smads
  8. Conclusion: relation to clinical diseases
  9. Acknowledgements
  10. References
  • Adachi, J. & Hasegawa, M. (1996) MOLPHY, version 2.3: Programs for molecular phylogenetics based on maximum likelihood. Computer Science Monographs 28. Institute of Statistical Mathematics, Tokyo.
  • Akiyoshi, S., Inoue, H., Hanai, J.-i., et al. (1999) c-Ski acts as a transcriptional co-repressor in transforming growth factor-β signaling through interaction with Smads. J. Biol. Chem. 274, 3526935277.
  • Aoki, H., Fujii, M., Imamura, T., et al. (2001) Synergistic effects of different bone morphogenetic protein type I receptors on alkaline phosphatase induction. J. Cell Sci. 114, 14831489.
  • Bai, R.-Y., Koester, C., Ouyang, T., et al. (2002) SMIF, a Smad4-interacting protein that functions as a co-activator in TGFβ signaling. Nature Cell Biol. 4, 181191.
  • Bai, S., Shi, X., Yang, X. & Cao, X. (2000) Smad6 as a transcriptional corepressor. J. Biol. Chem. 275, 82678270.
  • Bernard, D.J., Chapman, S.C. & Woodruff, T.K. (2002) Inhibin binding protein (InhBP/p120), betaglycan, and the continuing search for the inhibin receptor. Mol. Endocrinol. 16, 207212.
  • Blobe, G.C., Schiemann, W.P. & Lodish, H.F. (2000) Role of transforming growth factor-β in human disease. N. Engl. J. Med. 342, 13501358.
  • Blokzijl, A., Ten Dijke, P. & Ibañez, C.F. (2002) Physical and functional interaction between GATA-3 and Smad3 allows TGF-β regulation of GATA target genes. Curr. Biol. 12, 3545.
  • Chen, C.-R., Kang, Y. & Massagué, J. (2001) Defective repression of c-myc in breast cancer cells: a loss at the core of the transforming growth factor growth-β arrest program. Proc. Natl. Acad. Sci. USA 98, 992999.
  • Chen, C.-R., Kang, Y., Siegel, P.M. & Massagué, J. (2002a) E2F4/5 and p107 as Smad cofactors linking the TGFβ receptor to c-myc repression. Cell 110, 1932.
  • Chen, F., Ogawa, K., Liu, X., Stringfield, T.M. & Chen, Y. (2002b) Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit. Biochem. J. 364, 571577.
  • Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G. & Whitman, M. (1997) Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389, 8589.
  • Chipuk, J.E., Cornelius, S.C., Pultz, N.J., et al. (2002) The androgen receptor represses transforming growth factor–β signaling through interaction with Smad3. J. Biol. Chem. 277, 12401248.
  • Clarke, T.R., Hoshiya, Y., Yi, S.E., Liu, X., Lyons, K.M. & Donahoe, P.K. (2001) Müllerian inhibiting substance signaling uses a bone morphogenetic protein (BMP)-like pathway mediated by ALK2 and induces Smad6 expression. Mol. Endocrinol. 15, 946959.
  • Daluiski, A., Engstrand, T., Bahamonde, M.E., et al. (2001) Bone morphogenetic protein-3 is a negative regulator of bone density. Nature Genet. 27, 8488.
  • Datta, P.K., Blake, M.C. & Moses, H.L. (2000) Regulation of plasminogen activator inhibitor-1 expression by transforming growth factor-β induced physical and functional interactions between Smads and Sp1. J. Biol. Chem. 275, 4001440019.
  • Datta, P.K. & Moses, H.L. (2000) STRAP and Smad7 synergize in the inhibition of transforming growth factor-β signaling. Mol. Cell. Biol. 20, 31573167.
  • Datto, M.B., Frederick, J.P., Pan, L., Borton, A.J., Zhuang, Y. & Wang, X.-F. (1999) Targeted disruption of smad3 reveals an essential role in transforming growth factor β-mediated signal transduction. Mol. Cell. Biol. 19, 24952504.
  • Dennler, S., Itoh, S., Vivien, D., Ten Dijke, P., Huet, S. & Gauthier, J.M. (1998) Direct binding of Smad3 and Smad4 to critical TGFβ-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 17, 30913100.
  • Dong, C., Li, Z., Alvarez, R. Jr, Feng, X.-H. & Goldschmidt-Clermont, P.J. (2000) Microtubule binding to Smads may regulate TGFβ activity. Mol. Cell 5, 2734.
  • Ebisawa, T., Fukuchi, M., Murakami, G., et al. (2001) Smurf1 interacts with transforming growth factor-β type I receptor through Smad7 and induces receptor degradation. J. Biol. Chem. 276, 1247712480.
  • Feng, X.-H., Liang, Y.-Y., Liang, M., Zhai, W. & Lin, X. (2002) Direct interaction of c-Myc with Smad2 and Smad3 to inhibit TGF-β-mediated induction of the CDK inhibitor p15Ink4B. Mol. Cell 9, 133143.
  • Feng, X.-H., Lin, X. & Derynck, R. (2000) Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15Ink4B transcription in response to TGF-β. EMBO J. 19, 51785193.
  • Feng, X.-H., Zhang, Y., Wu, R.Y. & Derynck, R. (1998) The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smad3 in TGF-β-induced transcriptional activation. Genes Dev. 12, 21532163.
  • Ferrigno, O., Lallemand, F., Verrecchia, F., et al. (2002) Yes-associated protein (YAP65) interacts with Smad7 and potentiates its inhibitory activity against TGF-β/Smad signaling. Oncogene 21, 48794884.
  • Fu, M., Zhang, J., Zhu, X., et al. (2001) Peroxisome proliferator-activated receptor γ inhibits transforming growth factor β-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3. J. Biol. Chem. 276, 4588845894.
  • Fukuchi, M., Imamura, T., Chiba, T., et al. (2001) Ligand-dependent degradation of Smad3 by a ubiquitin ligase complex of ROC1 and associated proteins. Mol. Biol. Cell 12, 14311443.
  • Furuhashi, M., Yagi, K., Yamamoto, H., et al. (2001) Axin facilitates Smad3 activation in the transforming growth factor β signaling pathway. Mol. Cell. Biol. 21, 51325141.
  • Germain, S., Howell, M., Esslemont, G.M. & Hill, C.S. (2000) Homeodomain and winged-helix transcription factors recruit activated Smads to distinct promoter elements via a common Smad interaction motif. Genes Dev. 14, 435451.
  • Gouédard, L., Chen, Y.-G., Thevenet, L., et al. (2000) Engagement of bone morphogenetic protein type IB receptor and Smad1 signaling by anti-Müllerian hormone and its type II receptor. J. Biol. Chem. 275, 2797327978.
  • Goumans, M.-J., Valdimarsdottir, G., Itoh, S., Rosendahl, A., Sideras, P. & Ten Dijke, P. (2002) Balancing the activation state of the endothelium via two distinct TGF-β type I receptors. EMBO J. 21, 17431753.
  • Hahn, S.A., Schutte, M., Hoque, A.T.M.S., et al. (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21. 1. Science 271, 350353.
  • Hanai, J.-i., Chen, L.F., Kanno, T., et al. (1999) Interaction and functional cooperation of PEBP2/CBF with Smads: Synergistic induction of the immunoglobulin germline Cα promoter. J. Biol. Chem. 274, 3157731582.
  • Hanyu, A., Ishidou, Y., Ebisawa, T., Shimanuki, T., Imamura, T. & Miyazono, K. (2001) The N-domain of Smad7 is essential for specific inhibition of transforming growth factor-β signaling. J. Cell Biol. 155, 10171028.
  • Hata, A., Seoane, J., Lagna, G., Montalvo, E., Hemmati-Brivanlou, A. & Massagué, J. (2000) OAZ uses distinct DNA- and protein-binding Zinc fingers in separate BMP-Smad and Olf signaling pathways. Cell 100, 229240.
  • Heldin, C.-H., Miyazono, K. & Ten Dijke, P. (1997) TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465471.
  • Henningfeld, K.A., Friedle, H., Rastegar, S. & Knöchel, W. (2002) Autoregulation of Xvent-2B; direct interaction and functional cooperation of Xvent-2 and Smad1. J. Biol. Chem. 277, 20972103.
  • Hocevar, B.A., Smine, A., Xu, X.-X. & Howe, P.H. (2001) The adaptor molecule Disabled-2 links the transforming growth factor-β receptors to the Smad pathway. EMBO J. 20, 27892801.
  • Hua, X., Miller, Z.A., Wu, G., Shi, Y. & Lodish, H.F. (1999) Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor µE3, and Smad proteins. Proc. Natl. Acad. Sci. USA 96, 1313013135.
  • Inman, G.J., Nicolas, F.J., Callahan, J.F., et al. (2002) SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 6574.
  • Ishida, W., Hamamoto, K., Kusanagi, K., et al. (2000) Smad6 is a Smad1/5-induced Smad inhibitor: Characterization of bone morphogenetic protein-responsive element in the mouse Smad6 promoter. J. Biol. Chem. 275, 60756079.
  • Itoh, F., Asao, H., Sugamura, K., Heldin, C.-H., Ten Dijke, P. & Itoh, S. (2001) Promoting bone morphogenetic protein signaling through negative regulation of inhibitory Smads. EMBO J. 20, 41324142.
  • Itoh, S., Ericsson, J., Nishikawa, J., Heldin, C.-H. & Ten Dijke, P. (2000) The transcriptional co-activator P/CAF potentiates TGF-β/Smad signaling. Nucl. Acids Res. 28, 42914298.
  • Janknecht, R., Wells, N.J. & Hunter, T. (1998) TGF-β-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev. 12, 21142119.
  • Jonk, L.J., Itoh, S., Heldin, C.-H., Ten Dijke, P. & Kruijer, W. (1998) Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-β, activin, and bone morphogenetic protein-inducible enhancer. J. Biol. Chem. 273, 2114521152.
  • Kaji, H., Canaff, L., Lebrun, J.-J., Goltzman, D. & Hendy, G.N. (2001) Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling. Proc. Natl. Acad. Sci. USA 98, 38373842.
  • Kang, H.-Y., Lin, H.-K., Hu, Y.-C., Yeh, S. & Chang, C. (2001) From transforming growth factor-β signaling to androgen action: Identification of Smad3 as an androgen receptor coregulator in prostate cancer cells. Proc. Natl. Acad. Sci. USA 98, 30183023.
  • Kardassis, D., Pardali, K. & Zannis, V.I. (2000) Smad proteins transactivate the human ApoC III promoter by interacting physically and functionally with hepatocyte nuclear factor 4. J. Biol. Chem. 275, 4140541414.
  • Wurthner, Y., Habas, R., Katsuyama, Y., Näär, A.M. & He, X. (2002) A component of the ARC/Mediator complex required for TGFβ/Nodal signaling. Nature 418, 641646.
  • Kavsak, P., Rasmussen, R.K., Causing, C.G., et al. (2000) Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGFβ receptor for degradation. Mol. Cell 6, 13651375.
  • Kawabata, M., Imamura, T. & Miyazono, K. (1998a) Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev. 9, 4961.
  • Kawabata, M., Inoue, H., Hanyu, A., Imamura, T. & Miyazono, K. (1998b) Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors. EMBO J. 17, 40564065.
  • Kim, R.H., Wang, D., Tsang, M., et al. (2000) A novel Smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-β signal transduction. Genes Dev. 14, 16051616.
  • Kimura, N., Matsuo, R., Shibuya, H., Nakashima, K. & Taga, T. (2000) BMP2-induced apoptosis is mediated by activation of the TAK1-p38 kinase pathway that is negatively regulated by Smad6. J. Biol. Chem. 275, 1764717652.
  • Kirsch, T., Sebald, W. & Dreyer, M.K. (2000) Crystal structure of the BMP-2-BRIA ectodomain complex. Nature Struct. Biol. 7, 492496.
  • Kurisaki, A., Kose, S., Yoneda, Y., Heldin, C.-H. & Moustakas, A. (2001) Transforming growth factor-β induces nuclear import of Smad3 in an importin-β1 and Ran-dependent manner. Mol. Biol. Cell 12, 10791091.
  • Kurokawa, M., Mitani, K., Irie, K., et al. (1998) The oncoprotein Evi-1 represses TGF-β signalling by inhibiting Smad3. Nature 394, 9296.
  • Kusanagi, K., Kawabata, M., Mishima, H.K. & Miyazono, K. (2001) α-Helix 2 in the amino-terminal Mad homology 1 domain is responsible for specific DNA-binding of Smad3. J. Biol. Chem. 276, 2815528163.
  • Labbé, E., Letamendia, A. & Attisano, L. (2000) Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-β and Wnt pathways. Proc. Natl. Acad. Sci. USA 97, 83588363.
  • Labbé, E., Silvestri, C., Hoodless, P.A., Wrana, J.L. & Attisano, L. (1998) Smad2 and Smad3 positively and negatively regulate TGFβ-dependent transcription through the forkhead DNA-binding protein FAST2. Mol. Cell 2, 109120.
  • Laping, N.J., Grygielko, E., Mathur, A., et al. (2002) Inhibition of transforming growth factor (TGF) β-1-induced extracellular matrix with a novel inhibitor of the TGF-β type I receptor kinase activity: SB-431542. Mol. Pharmacol. 62, 5864.
  • Lee, D.K., Kim, B.-C., Brady, J.N., Jeang, K.-T. & Kim, S.J. (2002a) Human T-cell lymphotropic virus 1-Tax inhibits TGF-β signaling by blocking the association of Smad proteins with Smad binding element. J. Biol. Chem. 277, 3376633775.
  • Lee, D.K., Kim, B.-C., Kim, I.Y., Cho, E.-A., Satterwhite, D.J. & Kim, S.-J. (2002b) The human papilloma virus E7 oncoprotein inhibits TGF-β signaling by blocking of the Smad complex to its target sequence. J. Biol. Chem. 277, 3855738564.
  • Lee, D.K., Park, S.H., Yi, Y., et al. (2001) The hepatitis B virus encoded oncoprotein pX amplifies TGF-β family signaling through direct interaction with Smad4: potential mechanism of hepatitis B virus-induced liver fibrosis. Genes Dev. 15, 455466.
  • Leong, G.M., Subramaniam, N., Figueroa, J., et al. (2001) Ski-interacting protein interacts with Smad proteins to augment transforming growth factor-β-dependent transcription. J. Biol. Chem. 276, 1824318248.
  • Liberati, N.T., Datto, M.B., Frederick, J.P., et al. (1999) Smads bind directly to the Jun family of AP-1 transcription factors. Proc. Natl. Acad. Sci. USA 96, 48444849.
  • Lin, X., Liang, M. & Feng, X.-H. (2000) Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in TGF-β signaling. J. Biol. Chem. 275, 3681836822.
  • Liu, D., Black, B.L. & Derynck, R. (2001) TGF-β inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3. Genes Dev. 15, 29502966.
  • Liu, B., Dou, C.L., Prabhu, L. & Lai, E. (1999) FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor β signals. Mol. Cell. Biol. 19, 424430.
  • Liu, X., Elia, A.E.H., Law, S.F., Golemis, E.A., Farley, J. & Wang, T. (2000) A novel ability of Smad3 to regulate proteasomal degradation of a Cas family member HEF1. EMBO J. 19, 67596769.
  • Liu, F., Massagué, J. & Ruiz i Altaba, A. (1998) Carboxy-terminally truncated Gli3 proteins associate with Smads. Nature Genet. 20, 325326.
  • Lopez-Rovira, T., Chalaux, E., Rosa, J.L., Bartrons, R. & Ventura, F. (2000) Interaction and functional cooperation of NFκB with Smads. J. Biol. Chem. 275, 2893728946.
  • Macías-Silva, M., Hoodless, P.A., Tang, S.J., Buchwald, M. & Wrana, J.L. (1998) Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J. Biol. Chem. 273, 2562825636.
  • Massagué, J. (1998) TGF-β signal transduction. Annu. Rev. Biochem. 67, 753791.
  • Matsuda, T., Yamamoto, T., Muraguchi, A. & Saatcioglu, F. (2001) Cross-talk between transforming growth factor-β and estrogen receptor signaling through Smad3. J. Biol. Chem. 276, 4290842914.
  • Miura, S., Takeshita, T., Asao, H., et al. (2000) Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol. Cell. Biol. 20, 93469355.
  • Miyazono, K., Kusanagi, K. & Inoue, H. (2001) Divergence and convergence of TGF-β/BMP signaling. J. Cell. Physiol. 187, 265276.
  • Miyazono, K. & Miyazawa, K. (2002) Id: a target of BMP signaling. Sci. STKE, in press.
  • Miyazono, K., Ten Dijke, P. & Heldin, C.-H. (2000) TGF-β signaling by Smad proteins. Adv. Immunol. 75, 115157.
  • Moustakas, A., Souchelnytskyi, S. & Heldin, C.-H. (2001) Smad regulation in TGF-β signal transduction. J. Cell Sci. 114, 43594369.
  • Nakashima, K., Takizawa, T., Ochiai, W., et al. (2001) BMP2-mediated alteration in the developmental pathway of fetal mouse brain cells from neurogenesis to astrocytogenesis. Proc. Natl. Acad. Sci. USA 98, 58685873.
  • Nicholls, A., Sharp, K.A. & Honig, B. (1991) Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. General 11, 282296.
  • Nishihara, A., Hanai, J.-i., Imamura, T., Miyazono, K. & Kawabata, M. (1999) E1A inhibits transforming growth factor-β signaling through binding to Smad proteins. J. Biol. Chem. 274, 2871628723.
  • Nishihara, A., Hanai, J.-i., Okamoto, N., et al. (1998) Role of p300, a transcriptional coactivator, in signalling of TGF-β. Genes Cells 3, 613623.
  • Nishita, H., Hashimoto, M.K., Ogata, S., et al. (2000) Interaction between Wnt and TGF-β signalling pathways during formation of Spemann's organizer. Nature 403, 781785.
  • Nomura, M. & Li, E. (1998) Smad2 role in mesoderm formation, left-right patterning and craniofacial development. Nature 393, 786790.
  • Norton, J.D., Deed, R.W., Craggs, G. & Sabilitzky, F. (1998) Id helix-loop-helix proteins in cell growth and differentiation. Trends Cell Biol. 8, 5865.
  • Oh, S.P., Seki, T., Goss, K.A., et al. (2000) Activin receptor-like kinase 1 modulates transforming growth factor-β1 signaling in the regulation of angiogenesis. Proc. Natl. Acad. Sci. USA 97, 26262631.
  • Pardali, K., Kurisaki, A., Moren, A., Ten Dijke, P., Kardassis, D. & Moustakas, A. (2000b) Role of Smad proteins and transcription factor Sp1 in p21Waf-1/Cip-1 regulation by transforming growth factor-β. J. Biol. Chem. 275, 2924429256.
  • Pardali, E., Xie, X.Q., Tsapogas, P., et al. (2000a) Smad and AML proteins synergistically confer transforming growth factor β1 responsiveness to human germ-line IgA genes. J. Biol. Chem. 275, 35523560.
  • Pouponnot, C., Jayaraman, L. & Massagué, J. (1998) Physical and functional interaction of SMADs and p300/CBP. J. Biol. Chem. 273, 2286522868.
  • Preobrazhenska, O., Yakymovych, M., Kanamoto, T., et al. (2002) BRCA2 and Smad3 synergize in regulation of gene transcription. Oncogene 21, 56605664.
  • Qin, B.Y., Chacko, B.M., Lam, S.S., De Caestecker, M.P., Correia, J.J. & Lin, K. (2001) Structural basis of Smad1 activation by receptor kinase phosphorylation. Mol. Cell 6, 13031312.
  • Qin, B.Y., Lam, S.S., Correia, J.J. & Lin, K. (2002) Smad3 allostery links TGF-β receptor kinase activation to transcriptional control. Genes Dev. 16, 19501963.
  • Quinn, Z.A., Yang, C.-C., Wrana, J.L. & McDermott, J.C. (2001) Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins. Nucl. Acids Res. 29, 732742.
  • Reissmann, E., Jörnvall, H., Blokzijl, A., et al. (2001) The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev. 15, 20102022.
  • Ring, C., Ogata, S., Meek, L., et al. (2002) The role of a Williams–Beuren syndrome-associated helix-loop-helix domain-containing transcription factor in activin/nodal signaling. Genes Dev. 15, 455466.
  • Rodriguez, C., Huang, L.J.-S., Son, J.K., McKee, A., Xiao, Z. & Lodish, H.F. (2001) Functional cloning of the proto-oncogene brain factor-1 (BF-1) as a Smad-binding antagonist of transforming growth factor-β signaling. J. Biol. Chem. 276, 3022430230.
  • Rosen, V. & Wozney, J.M. (2002) Bone morphogenetic proteins. In: Principles of Bone Biology (eds J.P.Bilezikian, L.G.Raisz, G.A.Rodan, et al.), 2nd edn, pp. 919928. San Diego: Academic Press.
  • Sano, Y., Harada, J., Tashiro, S., Gotoh-Mandeville, R., Maekawa, T. & Ishii, S. (1999) ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-β signaling. J. Biol. Chem. 274, 89498957.
  • Sasaki, A., Masuda, Y., Ohta, Y., Ikeda, K. & Watanabe, K. (2001) Filamin associates with Smads and regulates transforming growth factor-β signaling. J. Biol. Chem. 274, 1787117877.
  • Scherer, A. & Graff, J.M. (2000) Calmodulin differentially modulates Smad1 and Smad2 signaling. J. Biol. Chem. 275, 4143041438.
  • Shen, X., Hu, P.P., Liberati, N.T., Datto, M.B., Frederick, J.P. & Wang, X.-F. (1998) TGF-β-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein. Mol. Biol. Cell 9, 33093319.
  • Shi, X., Bai, S., Li, L. & Cao, X. (2001) Hoxa-9 represses TGF-β-induced osteopontin gene transcription. J. Biol. Chem. 276, 850855.
  • Shi, X., Yang, X., Chen, D., Chang, D. & Cao, X. (1999) Smad1 interacts with homeobox DNA-binding proteins in bone morphogenetic protein signaling. J. Biol. Chem. 274, 1371113717.
  • Shimizu, K., Bourillot, P.-Y., Nielsen, S.J., Zorn, A. & Gurdon, J.B. (2001) Swift is a novel BRCT domain coactivator of Smad2 in transforming growth factor β signaling. Mol. Cell. Biol. 21, 39013912.
  • Shioda, T., Lechleider, R.J., Dunwoodie, S.L., et al. (1998) Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator. Proc. Natl. Acad. Sci. USA 95, 97859790.
  • Song, C.Z., Tian, X. & Gelehrter, T.D. (1999) Glucocorticoid receptor inhibits transforming growth factor-β signaling by directly targeting the transcriptional activation function of Smad3. Proc. Natl. Acad. Sci. USA 96, 1177611781.
  • Stroschein, S.L., Wang, W., Zhou, S., Zhou, Q. & Luo, K. (1999) Negative feedback regulation of TGF-β signaling by the SnoN oncoprotein. Science 286, 771774.
  • Stroschein, S.L., Bonni, S., Wrana, J.L. & Luo, K. (2001) Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev. 15, 28822836.
  • Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 46734680.
  • Topper, J.N., DiChiara, M.R., Brown, J.D., et al. (1998) CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor β transcriptional responses in endothelial cells. Proc. Natl. Acad. Sci. USA 95, 95069511.
  • Tsukazaki, T., Chiang, T.A., Davison, A.F., Attisano, L. & Wrana, J.L. (1998) SARA, a FYVE domain protein that recruits Smad2 to the TGFβ receptor. Cell 11, 779791.
  • Verschueren, K., Remacle, J.E., Collart, C., et al. (1999) SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5′-CACCT sequences in candidate target genes. J. Biol. Chem. 274, 2048920498.
  • Visser, J.A., Olaso, R., Verhoef-Post, M., Kramer, P., Themmen, A.P.N. & Ingraham, H.A. (2001) The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling. Mol. Endocrinol. 15, 936945.
  • Wan, M., Cao, X., Wu, Y., et al. (2002) Jab1 antagonizes TGF-β signaling by inducing Smad4 degradation. EMBO Report 3, 171176.
  • Wang, W., Mariani, F.V., Harland, R.M. & Luo, K. (2000) Ski represses bone morphogenetic protein signaling in Xenopus and mammalian cells. Proc. Natl. Acad. Sci. USA 97, 1439314399.
  • Wotton, D., Lo, R.S., Lee, S. & Massagué, J. (1999) A Smad transcriptional corepressor. Cell 97, 2939.
  • Wrana, J.L., Attisano, L., Wieser, R., Ventura, F. & Massagué, J. (1994) Mechanism of activation of the TGF-β receptor. Nature 370, 341347.
  • Wu, J.W., Hu, M., Chai, J., et al. (2001) Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-β signaling. Mol. Cell 8, 12771289.
  • Wurthner, J.U., Frank, D.B., Felici, A., et al. (2001) Transforming growth factor-β receptor-associated protein 1 is a Smad4 chaperone. J. Biol. Chem. 276, 1949519502.
  • Xiao, Z., Liu, X. & Lodish, H.F. (2000) Importin β mediates nuclear translocation of Smad 3. J. Biol. Chem. 275, 2342523428.
  • Yagi, K., Furuhashi, M., Aoki, H., et al. (2002) c-myc is a downstream target of Smad pathway. J. Biol. Chem. 277, 854861.
  • Yamakawa, N., Tsuchida, K. & Sugino, H. (2002) The ras GAP-binding protein, Dok-1, mediates activin signaling via serine/threonine kinase receptors. EMBO J. 21, 16841694.
  • Yamamoto, T.S., Takagi, C., Hyodo, A.C. & Ueno, N. (2001) Suppression of head formation by Xmsx-1 through the inhibition of intracellular nodal signaling. Development 128, 27692779.
  • Yanagisawa, J., Yanagi, Y., Masuhiro, Y., et al. (1999) Convergence of transforming growth factor-β and vitamin D signaling pathways on SMAD transcriptional coactivators. Science. 283, 13171321.
  • Yang, X., Letterio, J.J., Lechleider, R.J., et al. (1999) Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-β. EMBO J. 18, 12801291.
  • Yokota, Y. & Mori, S. (2002) Role of Id family proteins in growth control. J. Cell. Physiol. 190, 2128.
  • Yoshida, Y., Tanaka, S., Umemori, H., et al. (2000) Negative regulation of BMP/Smad signaling by Tob in osteoblasts. Cell 103, 10851097.
  • Zawel, L., Le Dai, J., Buckhaults, P., et al. (1998) Human Smad3 and Smad4 are sequence-specific transcription activators. Mol. Cell 1, 611617.
  • Zhang, Y., Chang, C., Gehling, D.J., Hemmati-Brivanlou, A. & Derynck, R. (2001) Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc. Natl. Acad. Sci. USA 98, 974979.
  • Zhang, Y., Feng, X.-H. & Derynck, R. (1998) Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-β-induced transcription. Nature 394, 909913.
  • Zhu, H., Kavsak, P., Abdollah, S., Wrana, J.L. & Thomsen, G.H. (1999) A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature 400, 687693.
  • Zhu, Y., Richardson, J.A., Parada, L.F. & Graff, J.M. (1998) Smad3 mutant mice develop metastatic colorectal cancer. Cell 94, 703714.
  • Zimmerman, C.M., Kariapper, M.S. & Mathews, L.S. (1998) Smad proteins physically interact with calmodulin. J. Biol. Chem. 273, 677680.
  • Zimmers, T.A., Davies, M.V., Koniaris, L.G., et al. (2002) Induction of cachexia in mice by systemically administered myostatin. Science 296, 14861488.