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Signal transduction by bone morphogenetic protein receptors: functional roles of Smad proteins
Bone Vol. 25, No. 1 July 1999:91–93
Signal Transduction by Bone Morphogenetic Protein Receptors: Functional Roles of Smad Proteins K. MIYAZONO Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for the Promotion of Science, Tokyo, Japan
Smad (Sma and Mad) proteins. Since the type I receptor acts as a downstream component of the type II receptor, specificities of the intracellular signals are determined by the type I receptor. BMPs bind to three different type II receptors, i.e., BMP type II receptor (BMPR-II) and activin type II and type IIB receptors (ActR-II and ActR-IIB). Among type I receptors, BMP type IA (BMPR-IA, originally termed activin receptor-like kinase 3 or ALK-3) and type IB receptors (BMPR-IB/ALK-6) specifically bind BMPs.13 ALK-2 was originally identified as an activin type I receptor (ActR-I), but recent findings have shown that it functions as a type I receptor for certain types of BMPs, e.g., BMP-7/OP-1 (osteogenic protein-1), instead of activins.2,19,31 Smad proteins are signal transducers for the serine/threonine kinase receptors. Here, we discuss the signaling mechanism by Smad proteins, focusing in particular on BMP signaling pathways.
Intracellular signals for bone morphogenetic proteins (BMPs) and other members in the transforming growth factor (TGF)-b superfamily are mediated by Smad proteins. Receptor-regulated Smads (R-Smads) are activated by serine/threonine kinase receptors upon ligand binding. RSmads then form hetero-oligomeric complexes with a common-mediator Smad (co-Smad) and translocate into the nucleus, where they regulate transcription of target genes. Smads 1, 5, and 8 are R-Smads activated by BMP receptors, whereas Smads 2 and 3 are activated by TGF-b and activin receptors. Smad4 is the only co-Smad isolated in mammals, and is shared by BMP and TGF-b/activin signaling pathways. Smads 6 and 7 are anti-Smads, which block signals by preventing the activation of R-Smads by serine/threonine kinase receptors. Anti-Smads are induced by ligand stimulation, suggesting that they constitute a negative feedback loop in the signal transduction pathways of the TGF-b superfamily. (Bone 25:91–93; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Structures and Functions of Smad Proteins Eight Smad proteins (Smads 1 through 8) have thus far been isolated in mammals.9,20 Smads can be classified into three subtypes by structure and function, i.e., receptor-regulated (or pathway-restricted) Smads (R-Smads), common-mediator Smads (co-Smads), and inhibitory Smads (anti-Smads). R-Smads are the prototype of Smad proteins, which can be further classified into those activated by BMP receptors and those activated by TGF-b and activin receptors. Smad1, Smad5, and Smad8 (originally termed MADH6) are R-Smads activated by BMP receptors, whereas Smad2 and Smad3 are R-Smads activated by TGF-b and activin receptors. Smad4 is the only co-Smad identified in mammals. Smad6 and Smad7 are anti-Smads. In the NH2- and COOH-terminal regions, Smads have conserved regions termed Mad homology-1 (MH1) and -2 (MH2) domains, respectively, which are bridged by a linker region of variable length and amino acid sequence. Both MH1 and MH2 domains are observed in R- and co-Smads, but a typical MH1like structure is not found in anti-Smads. In addition, R-Smads, but not other types of Smads, have a Ser-Ser-X-Ser (SSXS) motif in their most COOH-terminal regions. R-Smads serve as direct substrates of type I serine/threonine kinase receptors. They directly interact with and are phosphorylated at their COOH-terminal SSXS motif by type I receptors.18,37 Activation of type I receptor by type II receptor is required for direct interaction between type I receptor and R-Smad; however, the kinase activity of type I receptor is not required for this interaction.18,22 Since R-Smads are rapidly released from type I receptor after phosphorylation, their interaction can be detected in vitro only when type I receptor or
Key Words: Bone morphogenetic protein; Transforming growth factor-b; Serine/threonine kinase receptor; Smad; Signal transduction. Introduction Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b (TGF-b) superfamily, which includes TGF-bs, activins, inhibins, and Mu¨llerian inhibiting substance.13,26 BMPs play multiple roles in regulation of growth, differentiation, and apoptosis of various cell types. They exhibit important in vivo functions during embryonal development and tissue morphogenesis, including bone and cartilage formation. Homologues of BMPs have been identified in invertebrates, e.g., Decapentaplegic (Dpp) in Drosophila. Members of the TGF-b superfamily exert their biological effects via binding to two types of serine/threonine kinase receptors (type I and type II), both of which are required for signaling activity. The type II receptor transphosphorylates type I receptor, leading to the activation of intracellular substrates, including Address for correspondence and reprints: Kohei Miyazono, Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 1708455, Japan. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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R-Smads are functionally inactive. After phosphorylation by serine/threonine kinase receptors, R-Smads interact with coSmad to form hetero-oligomeric complexes,17,37 which then translocate into the nucleus and regulate the transcription of various target genes. Our recent finding,14 together with the finding that the MH2 domain of Smad4 forms a trimer in solution,27 suggested that the hetero-oligomers may be heterotrimers, composed of two and one molecules, or one and two molecules of R-Smads and Smad4, respectively. Although phosphorylated R-Smads can form oligomers and translocate into the nucleus even in the absence of co-Smad, co-Smad stabilizes the structures of the Smad oligomers and is thus required for efficient transcriptional activity of the Smad complexes. R-Smads activated by BMP receptors appear to be essential and probably sufficient for the differentiation of osteoprogenitor cells into osteoblasts induced by BMPs.24,34 BMPs bind to three different type I receptors, i.e., ALK-3/BMPR-IA, ALK-6/ BMPR-IB, and ALK-2/ActR-I, which in turn activate Smads 1, 5, and 8.19,30 Smads 1 and 5 have been shown to induce the differentiation of C2C12 cells even in the absence of stimulation by ligands or BMP receptors.24,34 R-Smads are observed throughout the cell in the absence of ligand stimulation and they cannot efficiently induce differentiation of C2C12 cells; however, R-Smads translocate into the nucleus upon receptor activation, and induce cellular differentiation. Nuclear translocation is thus one of the most critical events in the function of R-Smads. Activities of Smads in the Nucleus In the nucleus, Smad proteins exert transcriptional activity through direct binding to DNA as well as through association with other DNA-binding proteins. The MH1 domain is responsible for the DNA-binding of Smad proteins. Drosophila Mad, which is structurally similar to Smads 1, 5, and 8, was the first to be shown to bind to DNA.15 Subsequently, Smad3 and Smad4 have been shown to bind to specific DNA sequences through the MH1 domains.4,35,36 Direct DNA-binding has been demonstrated for Smad3 and Smad4, but not for Smad2. Smads 2 and 3 are structurally and functionally highly similar to each other, but our recent data revealed that a unique 30-amino acid region encoded by exon 3 of the Smad2 gene and located in the middle of the MH1 domain interferes with the binding of Smad2 to DNA.33 This finding was further supported by analysis of the three-dimensional structure of the MH1 domain of Smad3.28 Smad2 is functionally less potent than Smad3 with regard to transactivation of p3TP-lux promoter. Smad2 that lacks exon 3 is able to bind to DNA, and thus has transcriptional activity as potent as that of Smad3. R-Smads activated by TGF-b receptors specifically bind to the DNA sequence “AGAC” or its complementary “GTCT” sequence. Drosophila Mad binds to the “GCCGNCGC” sequence;15 however, the consensus sequence for the binding of Smads 1, 5, and 8 remains to be determined. Smads interact with specific DNA sequences via other DNAbinding proteins. Xenopus FAST-1 and its mammalian homologues associate with Smad2, and play important roles in the transduction of certain activin signals.3,16,39 Other transcription factors, including c-Jun/c-Fos,38 have also been shown to interact with Smad3. Thus far, no proteins that specifically interact with R-Smads activated by BMP receptors have been reported in mammals. Smads 2 and 3 interact with transcriptional coactivators, p300/CBP, upon ligand stimulation; this interaction may be crucial for the efficient transcriptional activation of target genes.5,12,23,25 The MH2 domain plays important roles in various functions of Smads, including receptor interaction, oligomer formation, and transcriptional activation. Interaction of R-Smads with
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FAST-1 as well as with p300/CBP is also mediated through the MH2 domain of R-Smads. The MH2 domain appears to be important for the interaction of Smads with most other proteins, although Smad3 interacts with c-Jun through the MH1 domain.38 R-Smads are activated by BMP receptors in osteoblastic cells as well as in cells of many other types. Smads 1 and 5 can efficiently induce the differentiation of C2C12 cells into osteoblast-like cells when they translocate into the nucleus.24,34 However, most cells types expressing BMP receptors and their downstream Smad proteins do not differentiate into osteoblast-like cells. It is possible that DNA-binding proteins interacting with Smads are at least in part responsible for the determination of cellular fate after BMP stimulation. Anti-Smads Act as Negative Regulators in Signaling Pathways Smad6 and Smad7 are distantly related members of the Smad family and are classified as anti-Smads. Anti-Smads exert their inhibitory effects by binding to type I receptors and competing with R-Smads activated by the receptors.8,10,21 In contrast to R-Smads, however, anti-Smads are not immediately released from the receptors. It was also shown that Smad6 binds to activated Smad1, and thus competes with Smad4 for heteromer formation with Smad1.7 Both Smad6 and Smad7 inhibit BMP signaling, while Smad7 is more potent in inhibiting TGF-b/ activin signals than Smad6.11 Similar to anti-Smads, certain R-Smad mutants stably bind type I receptors. A Smad3 mutant, Smad3D507E, binds to the TGF-b type I receptor and inhibits the phosphorylation of Smad2 and Smad3.6 In contrast, Smad1 phosphorylation is not inhibited by this Smad3 mutant, suggesting that R-Smad mutants function dominant-negatively and pathway-specifically in signaling pathways. Similar findings were reported using a Smad5 mutant (Smad5G419S).24 Expression of anti-Smads is induced by ligand stimulation. mRNAs for Smad6 and Smad7 are induced by BMPs and TGF-b.1,29 However, these inductions may occur by different mechanisms. Smad6 expression is induced 3 h after ligand stimulation and continues for more than 48 h; in contrast, Smad7 induction is rapid but only transient. A Drosophila homologue of Smad6/7, Daughters against Dpp (Dad), is also induced by Dpp signal.32 These findings indicate that anti-Smads are induced by signaling Smads, and regulate their activity by a negative feedback loop. Conclusion More than a dozen BMP-like molecules have thus far been identified in mammals. However, only three type II receptors and three type I receptors have been shown to bind BMPs. Although some of these receptors, e.g., ALK-3/6 vs. ALK-2, are structurally different, the specific signals induced by these receptors are not fully understood. BMPs activate Smad1, Smad5, and Smad8. The specificities of the signals induced by these BMP-activated R-Smads have not been determined. Since BMPs transduce different responses in different cell types, it may be important to understand the differences in biological activity between distinct receptor molecules and Smad proteins. It will also be important to identify proteins that cooperate with Smads in signal transduction. Modulation of the activities of Smads may be useful in establishing new approaches to the treatment of various clinical disorders.
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References 1. Afrakhte, M., More´n, A., Jossan, S., et al. Induction of inhibitory Smad6 and Smad7 mRNA by TGF-b family members. Biochem Biophys Res Commun 249:505–511; 1998. 2. Armes, N. A. and Smith, J. C. The ALK-2 and ALK-4 activin receptors transduce distinct mesoderm-inducing signals during early Xenopus development but do not co-operate to establish thresholds. Development 124:3797– 3804; 1997. 3. Chen, X., Rubock, M. J., and Whitman, M. A transcriptional partner for MAD proteins in TGF-b signalling. Nature 383:691– 696; 1996. 4. Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. Direct binding of Smad3 and Smad4 to critical TGFb-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. Eur Mol Biol Organ J 17:3091–3100; 1998. 5. Feng, X. H., Zhang, Y., Wu, R. Y., and Derynck, R. The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smad3 in TGF-b-induced transcriptional activation. Genes Dev 12:2153–2163; 1998. 6. Goto, D., Yagi, K., Inoue, H., et al. A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF-b signals. FEBS Lett 430:201–204; 1998. 7. Hata, A., Lagna, G., Massague´, J., and Hemmati-Brivanlou, A. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes Dev 12:186 –197; 1998. 8. Hayashi, H., Abdollah, S., Qiu, Y., et al. The MAD-related protein Smad7 associates with the TGFb receptor and functions as an antagonist of TGFb signaling. Cell 89:1165–1173; 1997. 9. Heldin, C.-H., Miyazono, K., and ten Dijke, P. TGF-b signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465– 471; 1997. 10. Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J.-i., Kawabata, M., and Miyazono, K. Smad6 inhibits signalling by the TGF-b superfamily. Nature 389:622– 626; 1997. 11. Itoh, S., Landstro¨m, M., Hermansson, A., et al. Transforming growth factor-b1 induces nuclear export of inhibitory Smad7. J Biol Chem 273:29195–21201; 1998. 12. Janknecht, R., Wells, N. J., and Hunter, T. TGF-b-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev 12:2114 –2119; 1998. 13. Kawabata, M., Imamura, T., and Miyazono, K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev 9:49 – 61; 1998. 14. Kawabata, M., Inoue, H., Hanyu, A., Imamura, T., and Miyazono, K. Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors. Eur Mol Biol Organ J 17:4056 – 4065; 1998. 15. Kim, J., Johnson, K., Chen, H. J., Carroll, S., and Laughon, A. Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic. Nature 388:304 –308; 1997. 16. Labbe, E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. Smad2 and Smad3 positively and negatively regulate TGFb-dependent transcription through the forkhead DNA-binding protein FAST2. Mol Cell 2:109 –120; 1998. 17. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague´, J. Partnership between DPC4 and SMAD proteins in TGF-b signalling pathways. Nature 383:832– 836; 1996. 18. Macı´as-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. MADR2 is a substrate of the TGFb receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87:1215–1224; 1996. 19. Macı´as-Silva, M., Hoodless, P. A., Tang, S. J., Buchwald, M., and Wrana, J. L. Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2. J Biol Chem 273:25628 –25636; 1998.
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20. Massague´, J. TGF-b signal transduction. Annu Rev Biochem 67:753–791; 1998. 21. Nakao, A., Afrakhte, M., More´n, A., et al. Identification of Smad7, a TGFbinducible antagonist of TGF-b signalling. Nature 389:631– 635; 1997. 22. Nakao, A., Imamura, T., Souchelnytskyi, S., et al. TGF-b receptor-mediated signalling through Smad2, Smad3 and Smad4. Eur Mol Biol Organ J 16:5353– 5362; 1997. 23. Nishihara, A., Hanai, J.-i., Okamoto, N., Yanagisawa, J., Kato, S., Miyazono, K., and Kawabata, M. Role of p300, a transcriptional coactivator, in signaling of TGF-b. Genes Cells 3:611– 621; 1998. 24. Nishimura, R., Kato, Y., Chen, D., Harris, S. E., Mundy, G. R., and Yoneda, T. Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem 273:1872–1879; 1998. 25. Pouponnot, C., Jayaraman, L., and Massague´, J. Physical and functional interaction of SMADs and p300/CBP. J Biol Chem 273:22865–22868; 1998. 26. Reddi, A. H. Bone and cartilage differentiation. Curr Opin Genet Develop 4:737–744; 1994. 27. Shi, Y., Hata, A., Lo, R. S., Massague´, J., and Pavletich, N. P. A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 388:87–93; 1997. 28. Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague´, J., and Pavletich, N. P. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-b signaling. Cell 94:585–594; 1998. 29. Takase, M., Imamura, T., Sampath, T. K., et al. Induction of Smad6 mRNA by bone morphogenetic proteins. Biochem Biophys Res Commun 244:6 –29; 1998. 30. Tamaki, K., Souchelnytskyi, S., Itoh, S., et al. Intracellular signaling of osteogenic protein-1 through Smad5 activation. J Cell Physiol 177:355–363; 1998. 31. ten Dijke, P., Yamashita, H., Sampath, T. K., et al. Identification of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J Biol Chem 269:16985–16988; 1994. 32. Tsuneizumi, K., Nakayama, T., Kamoshida, Y., Kornberg, T. B., Christian, J. L., and Tabata, T. Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389:627– 631; 1998. 33. Yagi, K., Goto, D., Hamamoto, T., Takenoshita, S., Kato, M., and Miyazono, K. Alternatively-spliced variant of Smad2 lacking exon 3: Comparison with wild-type Smad2 and Smad3. J Biol Chem 274:703–709; 1999. 34. Yamamoto, N., Akiyama, S., Katagiri, T., Namiki, M., Kurokawa, T., and Suda, T. Smad1 and Smad5 act downstream of intracellular signalings of BMP-2 that inhibits myogenic differentiation and induces osteoblast differentiation in C2C12 myoblasts. Biochem Biophys Res Commun 238:574 –580; 1997. 35. Yingling, J. M., Datto, M. B., Wong, C., Frederick, J. P., Liberati, N. T., and Wang, X.-F. Tumor suppressor Smad4 is a transforming growth factor b-inducible DNA binding protein. Mol Cell Biol 17:7019 –7028; 1997. 36. Zawel, L., Dai, J. L., Buckhaults, P., et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1:611– 617; 1998. 37. Zhang, Y., Feng, X.-H., Wu, R.-Y., and Derynck, R. Receptor-associated Mad homologues synergize as effectors of the TGF-b response. Nature 383:168 – 172; 1996. 38. Zhang, Y., Feng, X. H., and Derynck, R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-b-induced transcription. Nature 394:909 –913; 1998. 39. Zhou, S., Zawel, L., Lengauer, C., Kinzler, K. W., and Vogelstein, B. Characterization of human FAST-1, a TGFb and activin signal transducer. Mol Cell 2:121–127; 1998.
Signal Transduction by Bone Morphogenetic Protein Receptors: Functional Roles of Smad Proteins K. MIYAZONO Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for the Promotion of Science, Tokyo, Japan
Smad (Sma and Mad) proteins. Since the type I receptor acts as a downstream component of the type II receptor, specificities of the intracellular signals are determined by the type I receptor. BMPs bind to three different type II receptors, i.e., BMP type II receptor (BMPR-II) and activin type II and type IIB receptors (ActR-II and ActR-IIB). Among type I receptors, BMP type IA (BMPR-IA, originally termed activin receptor-like kinase 3 or ALK-3) and type IB receptors (BMPR-IB/ALK-6) specifically bind BMPs.13 ALK-2 was originally identified as an activin type I receptor (ActR-I), but recent findings have shown that it functions as a type I receptor for certain types of BMPs, e.g., BMP-7/OP-1 (osteogenic protein-1), instead of activins.2,19,31 Smad proteins are signal transducers for the serine/threonine kinase receptors. Here, we discuss the signaling mechanism by Smad proteins, focusing in particular on BMP signaling pathways.
Intracellular signals for bone morphogenetic proteins (BMPs) and other members in the transforming growth factor (TGF)-b superfamily are mediated by Smad proteins. Receptor-regulated Smads (R-Smads) are activated by serine/threonine kinase receptors upon ligand binding. RSmads then form hetero-oligomeric complexes with a common-mediator Smad (co-Smad) and translocate into the nucleus, where they regulate transcription of target genes. Smads 1, 5, and 8 are R-Smads activated by BMP receptors, whereas Smads 2 and 3 are activated by TGF-b and activin receptors. Smad4 is the only co-Smad isolated in mammals, and is shared by BMP and TGF-b/activin signaling pathways. Smads 6 and 7 are anti-Smads, which block signals by preventing the activation of R-Smads by serine/threonine kinase receptors. Anti-Smads are induced by ligand stimulation, suggesting that they constitute a negative feedback loop in the signal transduction pathways of the TGF-b superfamily. (Bone 25:91–93; 1999) © 1999 by Elsevier Science Inc. All rights reserved.
Structures and Functions of Smad Proteins Eight Smad proteins (Smads 1 through 8) have thus far been isolated in mammals.9,20 Smads can be classified into three subtypes by structure and function, i.e., receptor-regulated (or pathway-restricted) Smads (R-Smads), common-mediator Smads (co-Smads), and inhibitory Smads (anti-Smads). R-Smads are the prototype of Smad proteins, which can be further classified into those activated by BMP receptors and those activated by TGF-b and activin receptors. Smad1, Smad5, and Smad8 (originally termed MADH6) are R-Smads activated by BMP receptors, whereas Smad2 and Smad3 are R-Smads activated by TGF-b and activin receptors. Smad4 is the only co-Smad identified in mammals. Smad6 and Smad7 are anti-Smads. In the NH2- and COOH-terminal regions, Smads have conserved regions termed Mad homology-1 (MH1) and -2 (MH2) domains, respectively, which are bridged by a linker region of variable length and amino acid sequence. Both MH1 and MH2 domains are observed in R- and co-Smads, but a typical MH1like structure is not found in anti-Smads. In addition, R-Smads, but not other types of Smads, have a Ser-Ser-X-Ser (SSXS) motif in their most COOH-terminal regions. R-Smads serve as direct substrates of type I serine/threonine kinase receptors. They directly interact with and are phosphorylated at their COOH-terminal SSXS motif by type I receptors.18,37 Activation of type I receptor by type II receptor is required for direct interaction between type I receptor and R-Smad; however, the kinase activity of type I receptor is not required for this interaction.18,22 Since R-Smads are rapidly released from type I receptor after phosphorylation, their interaction can be detected in vitro only when type I receptor or
Key Words: Bone morphogenetic protein; Transforming growth factor-b; Serine/threonine kinase receptor; Smad; Signal transduction. Introduction Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-b (TGF-b) superfamily, which includes TGF-bs, activins, inhibins, and Mu¨llerian inhibiting substance.13,26 BMPs play multiple roles in regulation of growth, differentiation, and apoptosis of various cell types. They exhibit important in vivo functions during embryonal development and tissue morphogenesis, including bone and cartilage formation. Homologues of BMPs have been identified in invertebrates, e.g., Decapentaplegic (Dpp) in Drosophila. Members of the TGF-b superfamily exert their biological effects via binding to two types of serine/threonine kinase receptors (type I and type II), both of which are required for signaling activity. The type II receptor transphosphorylates type I receptor, leading to the activation of intracellular substrates, including Address for correspondence and reprints: Kohei Miyazono, Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, and Research for the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 1708455, Japan. E-mail: [email protected] © 1999 by Elsevier Science Inc. All rights reserved.
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R-Smads are functionally inactive. After phosphorylation by serine/threonine kinase receptors, R-Smads interact with coSmad to form hetero-oligomeric complexes,17,37 which then translocate into the nucleus and regulate the transcription of various target genes. Our recent finding,14 together with the finding that the MH2 domain of Smad4 forms a trimer in solution,27 suggested that the hetero-oligomers may be heterotrimers, composed of two and one molecules, or one and two molecules of R-Smads and Smad4, respectively. Although phosphorylated R-Smads can form oligomers and translocate into the nucleus even in the absence of co-Smad, co-Smad stabilizes the structures of the Smad oligomers and is thus required for efficient transcriptional activity of the Smad complexes. R-Smads activated by BMP receptors appear to be essential and probably sufficient for the differentiation of osteoprogenitor cells into osteoblasts induced by BMPs.24,34 BMPs bind to three different type I receptors, i.e., ALK-3/BMPR-IA, ALK-6/ BMPR-IB, and ALK-2/ActR-I, which in turn activate Smads 1, 5, and 8.19,30 Smads 1 and 5 have been shown to induce the differentiation of C2C12 cells even in the absence of stimulation by ligands or BMP receptors.24,34 R-Smads are observed throughout the cell in the absence of ligand stimulation and they cannot efficiently induce differentiation of C2C12 cells; however, R-Smads translocate into the nucleus upon receptor activation, and induce cellular differentiation. Nuclear translocation is thus one of the most critical events in the function of R-Smads. Activities of Smads in the Nucleus In the nucleus, Smad proteins exert transcriptional activity through direct binding to DNA as well as through association with other DNA-binding proteins. The MH1 domain is responsible for the DNA-binding of Smad proteins. Drosophila Mad, which is structurally similar to Smads 1, 5, and 8, was the first to be shown to bind to DNA.15 Subsequently, Smad3 and Smad4 have been shown to bind to specific DNA sequences through the MH1 domains.4,35,36 Direct DNA-binding has been demonstrated for Smad3 and Smad4, but not for Smad2. Smads 2 and 3 are structurally and functionally highly similar to each other, but our recent data revealed that a unique 30-amino acid region encoded by exon 3 of the Smad2 gene and located in the middle of the MH1 domain interferes with the binding of Smad2 to DNA.33 This finding was further supported by analysis of the three-dimensional structure of the MH1 domain of Smad3.28 Smad2 is functionally less potent than Smad3 with regard to transactivation of p3TP-lux promoter. Smad2 that lacks exon 3 is able to bind to DNA, and thus has transcriptional activity as potent as that of Smad3. R-Smads activated by TGF-b receptors specifically bind to the DNA sequence “AGAC” or its complementary “GTCT” sequence. Drosophila Mad binds to the “GCCGNCGC” sequence;15 however, the consensus sequence for the binding of Smads 1, 5, and 8 remains to be determined. Smads interact with specific DNA sequences via other DNAbinding proteins. Xenopus FAST-1 and its mammalian homologues associate with Smad2, and play important roles in the transduction of certain activin signals.3,16,39 Other transcription factors, including c-Jun/c-Fos,38 have also been shown to interact with Smad3. Thus far, no proteins that specifically interact with R-Smads activated by BMP receptors have been reported in mammals. Smads 2 and 3 interact with transcriptional coactivators, p300/CBP, upon ligand stimulation; this interaction may be crucial for the efficient transcriptional activation of target genes.5,12,23,25 The MH2 domain plays important roles in various functions of Smads, including receptor interaction, oligomer formation, and transcriptional activation. Interaction of R-Smads with
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FAST-1 as well as with p300/CBP is also mediated through the MH2 domain of R-Smads. The MH2 domain appears to be important for the interaction of Smads with most other proteins, although Smad3 interacts with c-Jun through the MH1 domain.38 R-Smads are activated by BMP receptors in osteoblastic cells as well as in cells of many other types. Smads 1 and 5 can efficiently induce the differentiation of C2C12 cells into osteoblast-like cells when they translocate into the nucleus.24,34 However, most cells types expressing BMP receptors and their downstream Smad proteins do not differentiate into osteoblast-like cells. It is possible that DNA-binding proteins interacting with Smads are at least in part responsible for the determination of cellular fate after BMP stimulation. Anti-Smads Act as Negative Regulators in Signaling Pathways Smad6 and Smad7 are distantly related members of the Smad family and are classified as anti-Smads. Anti-Smads exert their inhibitory effects by binding to type I receptors and competing with R-Smads activated by the receptors.8,10,21 In contrast to R-Smads, however, anti-Smads are not immediately released from the receptors. It was also shown that Smad6 binds to activated Smad1, and thus competes with Smad4 for heteromer formation with Smad1.7 Both Smad6 and Smad7 inhibit BMP signaling, while Smad7 is more potent in inhibiting TGF-b/ activin signals than Smad6.11 Similar to anti-Smads, certain R-Smad mutants stably bind type I receptors. A Smad3 mutant, Smad3D507E, binds to the TGF-b type I receptor and inhibits the phosphorylation of Smad2 and Smad3.6 In contrast, Smad1 phosphorylation is not inhibited by this Smad3 mutant, suggesting that R-Smad mutants function dominant-negatively and pathway-specifically in signaling pathways. Similar findings were reported using a Smad5 mutant (Smad5G419S).24 Expression of anti-Smads is induced by ligand stimulation. mRNAs for Smad6 and Smad7 are induced by BMPs and TGF-b.1,29 However, these inductions may occur by different mechanisms. Smad6 expression is induced 3 h after ligand stimulation and continues for more than 48 h; in contrast, Smad7 induction is rapid but only transient. A Drosophila homologue of Smad6/7, Daughters against Dpp (Dad), is also induced by Dpp signal.32 These findings indicate that anti-Smads are induced by signaling Smads, and regulate their activity by a negative feedback loop. Conclusion More than a dozen BMP-like molecules have thus far been identified in mammals. However, only three type II receptors and three type I receptors have been shown to bind BMPs. Although some of these receptors, e.g., ALK-3/6 vs. ALK-2, are structurally different, the specific signals induced by these receptors are not fully understood. BMPs activate Smad1, Smad5, and Smad8. The specificities of the signals induced by these BMP-activated R-Smads have not been determined. Since BMPs transduce different responses in different cell types, it may be important to understand the differences in biological activity between distinct receptor molecules and Smad proteins. It will also be important to identify proteins that cooperate with Smads in signal transduction. Modulation of the activities of Smads may be useful in establishing new approaches to the treatment of various clinical disorders.
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