Journal of Cell Science 113, 1101-1109 (2000) 1101 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1056

COMMENTARY Positive and negative regulation of TGF-β signaling

Kohei Miyazono Department of Biochemistry, The Cancer Institute of the Japanese Foundation for Cancer Research (JFCR), and Research for the Future Program, the Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan *Author for correspondence (e-mail: [email protected])

Published on WWW 7 March 2000

SUMMARY

Cytokines of the transforming β (TGF-β) which limit the magnitude of signals and terminate superfamily, including TGF-βs, activins and bone signaling. Negative regulation is also important for morphogenetic (BMPs), bind to specific formation of gradients of , which is crucial in serine/threonine kinase receptors and transmit developmental processes. In addition, other signaling intracellular signals through Smad proteins. Upon ligand pathways regulate TGF-β and BMP signaling through stimulation, Smads move into the nucleus and function as cross-talk. Nearly 20 BMP isoforms have been identified, components of transcription complexes. TGF-β and BMP and their activities are regulated by various extracellular signaling is regulated positively and negatively through antagonists. Regulation of TGF-β signaling might be tightly various mechanisms. Positive regulation amplifies signals linked to tumor progression, since TGF-β is a potent to a level sufficient for biological activity. Negative growth inhibitor in most cell types. regulation occurs at the extracellular, membrane, cytoplasmic and nuclear levels. TGF-β and BMP signaling is often regulated through negative feedback mechanisms, Key words: TGF-β, BMP, activin, antagonist, Smad

INTRODUCTION to two different types of serine/threonine kinase : type I and type II (Fig. 1; Heldin et al., 1997; Massague, Transforming growth factor (TGF)-β and related factors are 1998; Zhang and Derynck, 1999). The type II receptor multifunctional that regulate growth, kinases are constitutively active; upon ligand binding, the differentiation, adhesion and apoptosis of various cell types type II receptors activate the type I receptor kinases through (Roberts and Sporn, 1990). More than 30 proteins have been phosphorylation of the juxtamembrane domain (mainly the identified as members of the TGF-β superfamily, which glycine-serine-rich domain or GS domain) of type I receptors. includes TGF-βs, activins and bone morphogenetic proteins The type I receptor kinases then activate intracellular (BMPs). Activins and BMPs play important roles in early substrates, the central signal messengers being Smad proteins embryogenesis (Kingsley, 1994; Hogan, 1996; Harland and (Miyazono et al., 2000). Smads include three subclasses: Gerhart, 1997); activins induce dorsal in receptor-regulated Smads (R-Smads), common-partner , whereas BMPs induce ventral mesoderm. BMPs Smads (Co-Smads) and inhibitory Smads (I-Smads). R- also play critical roles in of various tissues. Smads are anchored to the cell membrane through TGF-β plays an important role in early embryonic membrane-bound proteins, including Smad-anchor for development (Goumans et al., 1999), but might play more receptor activation (SARA; Tsukazaki et al., 1998). R-Smads crucial roles at relatively late stages of development and in directly interact with and become phosphorylated by type I adult tissues. TGF-β acts as a potent growth inhibitor for most receptors. R-Smads then form complexes with Co-Smads and types of cells, including epithelial cells, endothelial cells, migrate into the nucleus, where they regulate transcription of hematopoietic cells and lymphocytes (Roberts and Sporn, target (Fig. 1). In mammals, Smad2 and Smad3 are 1990; Miyazono et al., 1994). In addition, TGF-β functions TGF-β/activin-specific R-Smads, whereas Smad1, Smad5 as a fibrogenic factor and is responsible for tissue sclerosis and, presumably, Smad8 are BMP-specific R-Smads. Smad4 of liver, kidney, , skin and other tissues. is the only Co-Smad in mammals, but two Co-Smads, TGF-β and related factors are produced as dimeric Smad4α and Smad4β, have been identified in Xenopus precursors, in which the C-terminal portions form active (Howell et al., 1999; Masuyama et al., 1999). Smad6 and ligands following proteolytic processing (Miyazono et al., Smad7 act as I-Smads. 1993; Kingsley, 1994). The secreted TGF-β-like factors bind Signaling by TGF-β-like factors is regulated in both positive 1102 K. Miyazono and others and negative fashions, and is tightly controlled temporally and EXTRACELLULAR ANTAGONISTS spatially through multiple mechanisms at the extracellular, membrane, cytoplasmic and nuclear levels. Positive regulation TGF-β is secreted as a latent complex, which must be activated could be critical for amplification of signaling by TGF-β-like to exhibit its biological effects (Saharinen et al., 1999). TGF- factors. Negative regulation plays an important role in βs are synthesized as precursor forms; the N-terminal portions restriction and termination of signaling, and often occurs of the TGF-β precursors are cleaved off, but remain bound to through a negative feedback loop (Fig. 2). Negative regulation the C-terminal active dimers and maintain them in inactive is also crucial in early and forms (Miyazono et al., 1993). In contrast, activins and BMPs morphogenetic processes, limiting the range of signaling by do not form such TGF-β-like latent complexes and therefore TGF-β-like factors and forming a gradient of ligand activity. do not require prior activation to exert biological effects; Signaling by TGF-β-like factors is also regulated through however, their activities are tightly regulated by specific cross-talk with other pathways, including antagonists. Two different types of antagonist have been MAP kinase pathways and JAK/STAT pathways. Perturbation identified: those that directly bind ligands, and those that of the negative regulation of TGF-β signaling might be linked belong to the TGF-β superfamily and interfere with binding of to the pathogenesis of various clinical disorders, especially ligands to specific receptors. progression of tumors. Ligand-binding antagonists Various antagonists that directly bind BMPs have been POSITIVE REGULATION OF TGF-β AND BMP identified. These include , , and its SIGNALING related proteins, and . Cerberus, , caronte, DAN and other structurally related proteins are collectively Positive regulation of TGF-β and BMP signaling, especially termed the DAN family (Hsu et al., 1998). Proteins of the DAN the induction of ligands and their signaling components, often family have a conserved cystine-knot motif, which is also occurs through the action of TGF-β-like factors themselves. found in other growth factors, including TGF-β-like factors For example, three mammalian isoforms of TGF-β (TGF-β1, (Pearce et al., 1999; Rodriguez Esteban et al., 1999). However, TGF-β2, and TGF-β3) are auto- and cross-induced by different other BMP antagonists lack sequence similarity with each TGF-β isoforms (Bascom et al., 1989; Kim et al., 1990; other. O’Reilly et al., 1992). Nodal and its related proteins, which Why are there so many antagonists of BMPs? One important play an important role in early embryogenesis and act through reason may be that these antagonists have distinct expression activin receptors and Smad2 (Nomura and Li, 1998), are also profiles and regulate different biological responses in vivo. induced by signaling (Meno et al., 1999). In certain types Noggin and chordin are secreted by Spemann’s organizer, and of cell, TGF-β receptors might be induced by ligand induce neural tissue from and dorsalize ventral stimulation (Bloom et al., 1996). mesoderm (Piccolo et al., 1996; Zimmerman et al., 1996). Transcription factors that function as targets of TGF-β- Cerberus plays an essential role in formation of head-like like factors are also induced by ligand stimulation. TGF-β structure. A cerberus-like , caronte, plays a critical role induces production of a , Runx3 (formerly in the establishment of left-right asymmetry (Rodriguez termed PEBP2αC/Cbfa3/AML2), in B lymphocytes (Shi and Esteban et al., 1999; Yokouchi et al., 1999). Limb development Stavnezer, 1998). Newly synthesized Runx3 in turn forms a is controlled by various BMP antagonists, including noggin, complex with Smad3 activated by TGF-β, and cooperatively chordin, follistatin and gremlin, which have distinct roles in induces IgA class switching in B lymphocytes (Hanai et al., limb morphogenesis (McMahon et al., 1998; Capdevila et al., 1999). c-Jun is induced by TGF-β (Wong et al., 1999) and 1999; Merino et al., 1999). Noggin is also involved in hair- regulates the transcription of target genes in concert with follicle induction (Botchkarev et al., 1999). Smads (Zhang et al., 1998; Liberati et al., 1999). Another important reason may be that these antagonists have Smad signaling is also positively modulated through cross- different affinities for various BMP isoforms (some of them are talk with other signaling pathways. Smads might be activated termed growth/differentiation factors or GDFs) as well as other by tyrosine kinase receptor signals under certain conditions (de factors. Both noggin and chordin directly and specifically bind Caestecker et al., 1998). TGF-β activates non-Smad pathways, BMPs with high affinity, and abolish the activity of BMPs. including the three distinct MAP kinase pathways: the Erk, c- Noggin binds to BMP-2, BMP-4 and GDF-6 with high affinity, Jun N-terminal kinase (JNK) and p38 MAP kinase pathways but to BMP-7 with low affinity (Zimmerman et al., 1996; (Hartsough and Mulder, 1995; Atfi et al., 1997; Adachi- Chang and Hemmati-Brivanlou, 1999). Follistatin was Yamada et al., 1999; Hocevar et al., 1999; Zhou et al., 1999). originally identified as an antagonist of activins, but it has also JNK and p38 MAP kinase phosphorylate c-Jun and ATF-2 been shown to bind BMPs (Yamashita et al., 1995; Iemura et (also called CRE-BP-1), respectively. c-Jun is a component of al., 1998). Caronte binds to BMP-4, BMP-7 and nodal, but not the AP-1 transcription factor, whereas ATF-2 acts as a to activin A (Rodriguez Esteban et al., 1999; Yokouchi et al., homodimer as well as as a heterodimer with c-Jun. Smad3 1999). Cerberus binds to multiple growth factors, including physically interacts with phosphorylated c-Jun and ATF-2, BMPs, nodal-like proteins and a non-TGF-β-superfamily although phosphorylation of c-Jun and ATF-2 appears not to protein, Wnt, but not to TGF-β1 (Hsu et al., 1998; Piccolo et be required for interaction with Smad3. Smad3 and Smad4 al., 1999). Interestingly, the binding of cerberus to these growth then act in concert with c-Jun and ATF-2 in transcriptional factors occurs through independent binding sites in the activation of target genes (Zhang et al., 1998; Liberati et al., cerberus molecule (Piccolo et al., 1999). In contrast, various 1999; Sano et al., 1999). BMP antagonists might interact with similar domains in the Regulation of TGF-β signaling 1103

BMP molecules, given that noggin competes with cerberus, type II receptors were unable to recruit type I receptors, which gremlin and DAN for binding to BMP-2 (Hsu et al., 1998). are essential for signaling (Xu et al., 1995; Lebrun and Vale, A third reason may be that these antagonists diffuse in 1997). However, certain bioactivities of inhibins (e.g. tissues at different rates. Chordin is ~120 kDa, which is much inhibition of FSH release) might not be elicited by the larger than other BMP antagonists, and might thus diffuse less antagonistic effects against activins (Lebrun and Vale, 1997); efficiently in tissues (Piccolo et al., 1996). Formation of in fact, specific inhibin-receptor-like proteins have been gradients is critical for pattern formation in early isolated in gonadal tumors obtained from mice lacking the development (Shimizu and Gurdon, 1999): BMPs and activins inhibin α subunit (Draper et al., 1998), which suggests that differentially activate target genes, depending on their inhibin is not a simple pseudoligand for activin receptors but concentrations. A gradient of BMP-4 activity is produced by instead exerts effects through specific cell surface receptors in the interaction between BMP-4 and noggin (or chordin; Holley certain types of cell. et al., 1996). High concentrations of BMP-4 induce blood formation in Xenopus embryos, whereas complete loss of BMP activity caused by chordin results in neurogenesis (Fig. 2B). REGULATION OF RECEPTOR FUNCTION Importantly, low concentrations of BMP-4, which are partially antagonized by chordin, lead to formation of muscle. Signaling is regulated at the cell membrane and in the These BMP antagonists can also exert effects though a cytoplasm through mechanisms common to both the TGF- negative feedback loop (Fig. 2A); expression of noggin is β/activin and BMP signaling pathways. BAMBI is a induced by BMP-2, BMP-4 and BMP-6 in osteoprogenitor pseudoreceptor for serine/threonine kinase receptors found in cells, and noggin in turn abolishes the bioactivity of BMPs Xenopus embryos (Onichtchouk et al., 1999) and exhibits a (Gazzero et al., 1998). Interestingly, TGF-β1 also transiently high degree of sequence similarity to the human nma induces the expression of noggin (Gazzero et al., 1998), which product (Degen et al., 1996). nma is downregulated in suggests that BMP signaling is cross-regulated by TGF-β1 metastatic melanoma cell lines (Degen et al., 1996). BAMBI through noggin. is structurally similar to type I serine/threonine kinase Pseudoligand-type antagonists Most of the BMP antagonists act by binding ligands directly, Precursor whereas certain activin antagonists bind to receptors, Form preventing ligand binding. Mammalian -1, lefty-2 and zebrafish antivin form a subfamily of the TGF-β superfamily (Meno et al., 1998, 1999; Thisse and Thisse, 1999). Lefty/antivin plays an essential role in the formation of left- right asymmetry. Lefty and antivin might function as Active monomers, since these molecules lack both the long α helix Ligand loop (α3) required for dimerization and the cysteine residue involved in intermolecular disulfide bridging. Lefty/antivin is an inhibitor of nodal and its related proteins. Because the R-II R-I biological effects of lefty/antivin are overridden by overexpression of the extracellular domains of activin type II P receptors, lefty/antivin blocks nodal signaling through Cytoplasm P P competition for binding to type II receptors, and thus functions P as a pseudoligand (Meno et al., 1999; Thisse and Thisse 1999). BMPs also bind to activin type II receptors (Yamashita et al., R-Smad 1995), but it is currently unknown whether lefty/antivin blocks BMP signaling. Nodal upregulates the expression of nodal Co-Smad itself, but at the same time induces the expression of I-Smad lefty/antivin and regulates its own bioactivity by a negative PP feedback mechanism (Meno et al., 1999; Saijoh et al., 2000). PP Ubiquitin-dependent An important question remaining to be answered is whether degradation the inhibin α chain acts as a pseudoligand, or whether inhibin exerts activity through binding to specific receptors. Activins Nucleus are disulfide-linked dimers composed of β chains; in mammals, Coactivator or four β chain isoforms (βA, βB, βC and βE) have been PP PP Corepressor identified. Inhibins are heterodimers composed of α and β chains. Both α and β chains are members of the TGF-β DNA binding superfamily, but the α chain is distantly related to other protein members of this superfamily. Inhibin might antagonize activins in certain types of cell. In human hepatoma cells and Fig. 1. Signaling by TGF-β through serine/threonine kinase receptors erythroleukemia cells, inhibin was shown to bind to activin and Smad proteins. I-Smads inhibit signaling by R-Smad-Co-Smad type II receptors through β subunits and to compete with complexes. R-I and R-II represent type I and type II receptors, activins for receptor binding. Upon binding of inhibin, activin respectively. 1104 K. Miyazono and others receptors but lacks an intracellular domain. The expression form a negative feedback loop in the signaling pathways profile of BAMBI is similar to that of BMP-4 in Xenopus activated by the TGF-β superfamily (Fig. 2A). Moreover, embryos, and BAMBI requires BMP signaling for expression. Smad7 is an inhibitor of both TGF-β/activins and BMPs; BAMBI stably interacts with various type I serine/threonine therefore, Smad7 induced by TGF-β cross-regulates the kinase receptors, as well as type II receptors, and abolishes activity of BMPs. Smad6 and Smad7 are highly expressed in signaling by BMPs, activins and TGF-βs. BAMBI might thus pancreatic cancers (Kleeff et al., 1999a,b), which results in be induced by BMPs and auto-regulate BMP signaling; in resistance of cells to the growth inhibitory effects of TGF-β, addition, it might cross-regulate signaling by other members of and possibly tumor progression. the TGF-β superfamily. The FK506-binding immunophilin, FKBP12, interacts with Regulation of Smad signaling by the Erk MAP kinase type I receptors for TGF-β family proteins in yeast two-hybrid pathway screens (Wang et al., 1994). FKBP12 is an abundant protein, The Erk MAP kinase pathway is activated by growth and binds to a Leu-Pro sequence in the GS domain of type I factors, including epidermal growth factor (EGF) and receptors. Loss of binding of FKBP12 to type I receptors leads hepatocyte growth factor (HGF). Erk phosphorylates serine or to spontaneous receptor activation in the absence of ligand threonine residues in the PX(S/T)P or (S/T)P motif in the linker stimulation (Wang et al., 1996; Chen et al., 1997). FKBP12 regions of Smads. Although R-Smads phosphorylated by Erk might therefore safeguard TGF-β-like-factor signaling through can form complexes with Smad4, they do not migrate into the protection against ligand-independent activation of type I nucleus, and thereby Erk inhibits the signaling by TGF-β and receptors by type II receptors. However, analysis by gene BMPs (Kretzschmar et al., 1997, 1999). Ras is involved in targeting failed to demonstrate a role of FKBP12 in signaling the activation of the Erk MAP kinase pathway by tyrosine by TGF-β-superfamily members (Shou et al., 1998). Further kinase receptors in peptide growth factor signaling. Cells studies may be needed to elucidate the functional importance transformed by oncogenic activated Ras become resistant to of FKBP12 in the TGF-β signal transduction. the growth inhibitory effect of TGF-β; this might be due to the inhibition of nuclear translocation of TGF-β-specific R-Smads (Kretzschmar et al., 1999). NEGATIVE REGULATION IN THE CYTOPLASM

Inhibition by I-Smads TRANSCRIPTIONAL CO-REPRESSORS REGULATE I-Smads belong to the Smad protein family and function as TGF-β SIGNALING antagonists of R-Smad and Co-Smad signaling. Smads have conserved N- and C-terminal domains termed Mad homology After translocation into the nucleus, Smads regulate (MH) 1 and MH2 domains, respectively, which are linked by transcription of target genes by binding to their consensus nonconserved linker regions. I-Smads have MH2 domains, but DNA sequences, interacting with other transcription factors their N-terminal sequences are highly divergent from the MH1 and recruiting transcriptional coactivators or co-repressors domains of other Smads. In a similar way to R-Smads, I-Smads (Fig. 1; Derynck et al., 1998; Miyazono et al., 2000). For interact with type I receptors activated by type II receptors; example, Smads activated by TGF-β directly bind to the Smad- whereas R-Smads rapidly dissociate from activated type I binding elements (SBE) in the promoter of the gene that receptors, I-Smads stably interact with them (Fig. 1; Hayashi encodes plasminogen activator inhibitor 1 (PAI-1; Dennler et et al., 1997; Imamura et al., 1997; Nakao et al., 1997; Inoue et al., 1998; Song et al., 1998; Stroschein et al., 1999a). Smads al., 1998). I-Smads have also been reported to compete with also physically interact with a transcription factor, TFE3, that Co-Smad for formation of complexes with R-Smads (Hata et binds to an E-box sequence located adjacent to SBE in the PAI- al., 1998). 1 promoter (Hua et al., 1998, 1999). TFE3 thus functionally Expression of I-Smads is induced by various extracellular synergizes with Smads in activation of the PAI-1 promoter. stimuli, including peptide growth factors and mechanical stress Smads also recruit the transcriptional coactivator p300/CBP (Topper et al., 1997; Afrakhte et al., 1998). Cells treated with (Feng et al., 1998; Janknecht et al., 1998; Nishihara et al., interferon (IFN)-γ become resistant to the effects of TGF-β 1998; Pouponnot et al., 1998; Shen et al., 1998; Topper et al., (Ulloa et al., 1999). IFN-γ activates the JAK/STAT pathway; 1998), which has histone acetyltransferase activity (Fig. 3). STAT1 activated by IFN-γ induces the expression of Smad7, Through acetylation of core histones and possibly other which in turn blocks TGF-β signaling. TGF-β signaling is thus proteins, p300/CBP loosens the structure of and regulated through cross-talk with the IFN-γ/STAT pathway via increases accessibility of transcriptional complexes to the basal production of Smad7 (Fig. 2C). transcriptional machinery (Travers, 1999). Importantly, I-Smad expression is upregulated by TGF- Several transcriptional co-repressors interact with Smads. β/activins and BMPs (Tsuneizumi et al., 1997; Afrakhte et al., Interestingly, these co-repressors preferentially modulate 1998; Ishisaki et al., 1998; Takase et al., 1998). Smad7 is a signaling by TGF-β, but not that by BMPs. A homeodomain potent inhibitor of signaling by both TGF-β/activins and protein of the TALE class, TGIF, is the first transcriptional co- BMPs, and expression of Smad7 is induced by direct effects repressor shown to interact with Smads (Wotton et al., 1999). of Smad3 and Smad4 on the Smad7 promoter (Nagarajan et al., c-Ski and its related protein SnoN (Ski-related novel gene) are 1999). Smad6 preferentially inhibits BMP signaling rather than also Smad-binding transcriptional co-repressors; c-Ski binds to TGF-β signaling, and Smad6 expression is induced by direct Smad2, Smad3 and Smad4, but not to BMP-activated Smads effects of BMP-activated Smads on the Smad6 promoter (Akiyoshi et al., 1999; Sun et al., 1999a; Luo et al., 1999). ski (Ishida et al., 2000). R-Smads/Co-Smads and I-Smads thus was discovered as an oncogene in the avian Sloan Kettering Regulation of TGF-β signaling 1105

(A) Negative feedback (C) Cross-talk

PP PP

(B) Gradient formation

Cytokine Antagonist

Fig. 2. Negative regulation of TGF-β signaling. (A) Negative regulation can occur through a negative feedback mechanism. (B) A gradient of morphogens is produced by negative regulation by specific antagonists. (C) Cross-talk with other signaling pathways may regulate TGF-β signaling. retroviruses, and its overexpression leads to transformation of Since SnoN is structurally related to c-Ski and recruits avian fibroblasts (Colmenares and Stavnezer, 1989). TGIF and HDAC1 (Nomura et al., 1999), it might repress the c-Ski compete with p300/CBP for interaction with TGF-β- transcription of TGF-β-responsive genes through a mechanism specific R-Smads, and they repress the transcription of target similar to that of c-Ski. However, SnoN is a more effective genes induced by TGF-β (Fig. 3). Both TGIF and c-Ski recruit histone deacetylases (HDACs) to Smad complexes, which leads to the transcriptional repression of target genes (Akiyoshi (A) Coactivator et al., 1999; Wotton et al., 1999). Depending on their levels of p300/CBP expression, TGIF and c-Ski could limit the magnitude of TGF- β signaling. Expression of c-Ski is modulated under various Smad Acetylation conditions; for example, it is induced during differentiation of complex HAT erythroleukemia cells into megakaryocytic cells (Namciu et al., 1994). When the expression of c-Ski is upregulated during oncogenesis (Nomura et al., 1989), cells become resistant to DNA-binding On the growth inhibitory effect of TGF-β and, therefore, protein transformed (Luo et al., 1999; Sun et al., 1999a).

Fig. 3. Transcriptional regulation by recruitment of transcriptional (B) Co-repressor coactivators or corepressors. (A) Transcriptional coactivators HDAC TGIF/Ski/SnoN (p300/CBP) induce acetylation of core histones through their histone acetyltransferase (HAT) domains and activate transcription. (B) Transcriptional co-repressors, including TGIF, c-Ski and SnoN, Deacetylation recruit histone deacetylases (HDACs) directly or indirectly via bridging molecules, including N-CoR and mSin3A (Nomura et al., 1999). They induce deacetylation of core histones and repress transcription. Off 1106 K. Miyazono and others repressor of transcription induced by Smad2 than of that CONCLUSION induced by Smad3 (Stroschein et al., 1999b), and regulates TGF-β activity through a complex mechanism. In the absence The activities of TGF-β, activins and BMPs are regulated of ligand stimulation, SnoN represses the spontaneous through multiple mechanisms. Regulation by direct binding of activation of TGF-β-responsive genes. Upon TGF-β ligands by extracellular antagonists appears to be very stimulation and nuclear accumulation of Smad3, SnoN is important for the regulation of BMP activities. There are nearly rapidly degraded by cellular (Stroschein et al., 20 BMP isoforms in mammals (Kawabata et al., 1998; 1999b; Sun et al., 1999b). c-Ski is also degraded upon TGF-β Kawabata and Miyazono, 2000); these isoforms have different stimulation, although the half-life of c-Ski is longer than that profiles of expression, different affinities for receptors and of SnoN (Sun et al., 1999b). SnoN might thus transiently therefore unique biological activities in vivo. It is thus disappear from cells after activation of Smad3 by TGF-β and reasonable to propose that a wide variety of extracellular thereby make TGF-β activity more overt. After a time lag, antagonists that have different specificities regulate the actions however, the expression of SnoN is induced by TGF-β of BMPs during early development and morphogenetic signaling, and SnoN in turn terminates TGF-β signaling processes. Activins and nodal-like proteins exert effects through negative feedback regulation. through activin receptors, and they are also regulated by extracellular antagonists. In addition to proteins that directly bind ligands, several activin/nodal antagonists are -DEPENDENT DEGRADATION OF pseudoligand-type molecules that compete with ligands for SMAD PROTEINS receptor binding. Regulation of TGF-β and BMP signaling at cell membrane Smads are degraded by the ubiquitin-proteasome pathway in and cytoplasmic levels is less specific to each signaling both ligand-dependent and ligand-independent fashions. pathway. BAMBI and FKBP12 modulate the effects of both Ubiquitination of proteins occurs by E1 ubiquitin-activating TGF-β/activin and BMP signaling. Smad6 activity is specific , E2 ubiquitin-conjugating enzymes and E3 to BMP signaling but Smad7 blocks both TGF-β/activin and ubiquitin-ligases. Although ubiquitination can occur in the BMP signaling. Moreover, regulation by Erk occurs in both the absence of E3 ligases on certain proteins, E3 ligases are TGF-β and BMP signaling pathways. Although a Hect family important for specific recognition of target proteins and, E3 ligase has been identified only for BMP-specific Smads, ultimately, degradation by the 26S proteasomes (Laney and other E3 ligases that act on TGF-β-specific R-Smads or other Hochstrasser, 1999). Smurf1 is an E3 ubiquitin-ligase of the Smads probably exist. Negative modulation in the nucleus by Hect family that specifically interacts with Smad1 and Smad5 transcriptional corepressors appears to be unique to TGF-β specific for BMP signaling pathways (Zhu et al., 1999). signaling; thus far, negative regulation of BMP signaling in the Smurf1 has two copies of the WW motif, which is responsible nucleus has not been demonstrated. Both TGIF and c-Ski for binding to a PY motif (PPXY sequence) in the linker specifically bind to Smad2 and Smad3, but not efficiently to regions of Smad1 and Smad5. Smurf1 degrades Smad1 and BMP-specific R-Smads. Perturbation of TGF-β signaling is Smad5 ligand-independently, limiting the intracellular one of the most crucial events in tumor progression. Mutations amounts of Smad1 and Smad5. Since the intracellular pool of of the TGF-β type II receptor and Smad4 have been identified Smad4 is limited in cells, and since Smad4 is a component in various cancers and frequently occur in certain colorectal shared by the TGF-β/activin and BMP pathways (Candia et cancers and pancreatic cancers (Markowitz et al., 1995; Hahn al., 1997), these two pathways might compete with each other et al., 1996; Miyaki et al., 1999). Current findings have shown for formation of complexes with Smad4 in signal that overexpression or constitutive activation of negative transduction. Ectopic expression of Smurf1 in Xenopus regulators of TGF-β signaling (i.e. I-Smads, Ras and c-Ski) embryos results in the repression of the ability of cells to might also lead to resistance to the growth-inhibitory effect of respond to BMPs and, at the same time, enhances their TGF-β and transformation of cells. It will be interesting to responsiveness to activins (Zhu et al., 1999). Smurf1 might determine how these regulatory molecules are expressed, thus influence the balance of BMP and activin signaling in activated and orchestrated in the TGF-β and BMP signaling Xenopus embryos. Whether Smurf1-like molecules for the pathways under various physiological and pathological TGF-β-specific R-Smads or other Smads exist remains to be conditions. determined. The half-life of Smad2 activated by TGF-β is shorter than I thank the colleagues in my laboratory for valuable discussion. that of non-activated Smad2. Since treatment by proteasome inhibitors prolongs the half-life of activated Smad2, degradation of activated Smad2 might occur through the ubiquitin-proteasome pathway (Lo and Massague, 1999; Fig. REFERENCES 1). Phosphorylation of Smad2 is not required for the Adachi-Yamada, T., Nakamura, M., Irie, K., Tomoyasu, Y., Sano, Y., Mori, degradation of Smad2; instead, nuclear localization of Smad2 E., Goto, S., Ueno, N., Nishida, Y. and Matsumoto, K. 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