Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

SMIF, a Smad4-interacting protein that functions as a co-activator in TGFβ signalling

Nature Cell Biology volume 4, pages 181–190 (2002)Cite this article

Abstract

Proteins of the transforming growth factor β(TGFβ) superfamily regulate diverse cellular responses, including cell growth and differentiation. After TGFβ stimulation, receptor-associated Smads are phosphorylated and form a complex with the common mediator Smad4. Here, we report the cloning of SMIF, a ubiquitously expressed, Smad4-interacting transcriptional co-activator. SMIF forms a TGFβ/bone morphogenetic protein 4 (BMP4)-inducible complex with Smad4, but not with others Smads, and translocates to the nucleus in a TGFβ/BMP4-inducible and Smad4-dependent manner. SMIF possesses strong intrinsic TGFβ-inducible transcriptional activity, which is dependent on Smad4 in mammalian cells and requires p300/CBP. A point mutation in Smad4 abolished binding to SMIF and impaired its activity in transcriptional assays. Overexpression of wild-type SMIF enhanced expression of TGFβ/BMP regulated genes, whereas a dominant-negative SMIF mutant suppressed expression. Furthermore, dominant-negative SMIF is able to block TGFβ-induced growth inhibition. In a knockdown approach with morpholino-antisense oligonucleotides targeting zebrafish SMIF, severe but distinct phenotypic defects were observed in zebrafish embryos. Thus, we propose that SMIF is a crucial activator of TGFβ signalling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: SMIF encodes a ubiquitously expressed 70K protein.
Figure 2: Interaction of SMIF and Smad4.
Figure 3: SMIF translocates to the nucleus in Mv1Lu cells after stimulation with TGFβ or BMP4.
Figure 4: The nuclear translocation of SMIF depends on Smad4.
Figure 5: Transcriptional activity of SMIF depends on Smad4 and p300/CBP.
Figure 6: SMIF is functionally involved in the TGFβ/BMP signalling pathway.
Figure 7: Morpholino antisense knockdown of smif results in severe phenotypic defects in zebrafish embryos.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Attisano, L. & Wrana, J. L. Smads as transcriptional co-modulators. Curr. Opin. Cell Biol. 12, 235–243 (2000).

    Article  CAS  PubMed  Google Scholar 

  2. Massague, J., Blain, S. W. & Lo, R. S. TGFβ signalling in growth control, cancer, and heritable disorders. Cell 103, 295–309 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Heldin, C. H., Miyazono, K. & ten Dijke, P. TGF-β signalling from cell membrane to nucleus through SMAD proteins. Nature 390, 465–471 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Massague, J. & Wotton, D. Transcriptional control by the TGF-β/Smad signalling system. EMBO J. 19, 1745–1754 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Massague, J. & Chen, Y. G. Controlling TGF-β signalling. Genes Dev. 14, 627–644 (2000).

    CAS  PubMed  Google Scholar 

  6. Wrana, J. L. & Attisano, L. The Smad pathway. Cytokine Growth Factor Rev. 11, 5–13 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Chen, X., Rubock, M. J. & Whitman, M. A transcriptional partner for MAD proteins in TGF-β signalling. Nature 383, 691–696. (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Verschueren, K. et al. 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, 20489–20498 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Wotton, D., Lo, R. S., Lee, S. & Massague, J. A Smad transcriptional corepressor. Cell 97, 29–39 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Shioda, T. et al. Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator. Proc. Natl Acad. Sci. USA 95, 9785–9790 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hata, A. et al. OAZ uses distinct DNA- and protein-binding zinc fingers in separate BMP-Smad and Olf signalling pathways. Cell 100, 229–240 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Feng, X. H., Zhang, Y., Wu, R. Y. & Derynck, R. The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are co-activators for smad3 in TGF-β-induced transcriptional activation. Genes Dev. 12, 2153–2163 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Shimizu, K., Bourillot, P.-Y., Nielsen, S. J., Zorn, A. M. & Gurdon, J. B. Swift is a novel BRCT domain co-activator of Smad2 in Transforming Growth Factor β signalling. Mol. Cell Biol. 21, 3901–3912 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pouponnot, C., Jayaraman, L. & Massague, J. Physical and functional interaction of SMADs and p300/CBP. J. Biol. Chem. 273, 22865–22868 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Itoh, S., Ericsson, J., Nishikawa, J., Heldin, C. H. & ten Dijke, P. The transcriptional co-activator P/CAF potentiates TGF-β/Smad signalling. Nucleic Acids Res. 28: 4291–4298 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Stroschein, S. L., Wang, W., Zhou, S., Zhou, Q. & Luo, K. Negative feedback regulation of TGF-β signalling by the SnoN oncoprotein. Science 286, 771–774 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Luo, K. et al. The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signalling. Genes Dev. 13, 2196–2206 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lange, D. et al. Expression of TGF-b related Smad proteins in human epithelial skin tumors. Int. J. Oncol. 14, 1049–1056 (1999).

    CAS  PubMed  Google Scholar 

  19. Liu, F., Pouponnot, C. & Massague, J. Dual role of the Smad4/DPC4 tumor suppressor in TGFβ-inducible transcriptional complexes. Genes Dev. 11, 3157–3167 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhou, S. et al. Targeted deletion of Smad4 shows it is required for transforming growth factor β and activin signalling in colorectal cancer cells. Proc. Natl Acad. Sci. USA 95, 2412–2416 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Adams, M. D. et al. The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 (2000).

    Article  PubMed  Google Scholar 

  22. Kozak, M. The scanning model for translation: an update. J. Cell. Biol. 108, 229–241 (1989).

    Article  CAS  PubMed  Google Scholar 

  23. de Caestecker, M. P. et al. The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain. J. Biol. Chem. 275, 2115–2122 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Goldman, P. S., Tran, V. K. & Goodman, R. H. The multifunctional role of the co-activator CBP in transcriptional regulation. Recent Prog. Horm. Res. 52, 103–119 (1997).

    CAS  PubMed  Google Scholar 

  25. Shikama, N., Lyon, J. & La Thangue, N. B. The p300/CBP family: integrating signals with transcription factors and chromatin. Trends Cell Biol. 7, 230–236 (1997).

    Article  CAS  Google Scholar 

  26. Liu, F. et al. A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381, 620–623 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Labbe, E., Silvestri, C., Hoodless, P. A., Wrana, J. L. & Attisano, L. Smad2 and Smad3 positively and negatively regulate TGF b-dependent transcription through the forkhead DNA-binding protein FAST2. Mol. Cell 2, 109–120 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Pertovaara, L. et al. Enhanced jun gene expression is an early genomic response to transforming growth factor β stimulation. Mol. Cell Biol. 9, 1255–1262 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jonk, L. J., Itoh, S., Heldin, C. H., ten Dijke, P. & Kruijer, W. 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, 21145–21152 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Marazzi, G., Wang, Y. & Sassoon, D. Msx2 is a transcriptional regulator in the BMP4-mediated programmed cell death pathway. Dev. Biol. 186, 127–138 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Phippard, D. J. et al. Regulation of Msx-1, Msx-2, Bmp-2 and Bmp-4 during foetal and postnatal mammary gland development. Development 122, 2729–2737 (1996).

    CAS  PubMed  Google Scholar 

  32. Nasevicius, A. & Ekker, S. C. Effective targeted gene 'knockdown' in zebrafish. Nature Genet. 26, 216–220 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Lele, Z., Bakkers, J. & Hammerschmidt, M. Morpholino phenocopies of the swirl, snailhouse, somitabun, minifin, silberblick, and pipetail mutations. Genesis 30, 190–194 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Solnica-Krezel, L. Pattern formation in zebrafish – fruitful liaisons between embryology and genetics. Curr. Top. Dev. Biol. 41, 1–35 (1999).

    CAS  PubMed  Google Scholar 

  35. Schier, A. F. & Shen, M. M. Nodal signalling in vertebrate development. Nature 403, 385–389 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Davidson, A. J. & Zon, L. I. Turning mesoderm into blood: the formation of hematopoietic stem cells during embryogenesis. Curr. Top. Dev. Biol. 50, 45–60 (2000).

    Article  CAS  PubMed  Google Scholar 

  37. de Caestecker, M. P. et al. Characterization of functional domains within Smad4/DPC4. J. Biol. Chem. 272, 13690–13696 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Shen, X. et al. TGF-b-induced phosphorylation of Smad3 regulates its interaction with co-activator p300/CREB-binding protein. Mol. Biol. Cell 9, 3309–3319 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Nishihara, A. et al. Role of p300, a transcriptional co-activator, in signalling of TGF-β. Genes Cells 3, 613–623 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Yahata, T. et al. The MSG1 non-DNA-binding transactivator binds to the p300/CBP co-activators, enhancing their functional link to the Smad transcription factors. J. Biol. Chem. 275, 8825–8834 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Shioda, T., Fenner, M. H. & Isselbacher, K. J. Msg1, a novel melanocyte-specific gene, encodes a nuclear protein and is associated with pigmentation. Proc. Natl Acad. Sci. USA 93, 12298–12303 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dunwoodie, S. L., Rodriguez, T. A. & Beddington, R. S. Msg1 and Mrg1, founding members of a gene family, show distinct patterns of gene expression during mouse embryogenesis. Mech. Dev. 72, 27–40 (1998).

    Article  CAS  PubMed  Google Scholar 

  43. Bai, R. Y. et al. The SH2-containing adapter protein GRB10 interacts with BCR-ABL. Oncogene 17, 941–948 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Boyd, F. T. & Massague, J. Transforming growth factor-b inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J. Biol. Chem. 264, 2272–2278 (1989).

    CAS  PubMed  Google Scholar 

  45. Laiho, M., Weis, M. B. & Massague, J. Concomitant loss of transforming growth factor (TGF)-β receptor types I and II in TGF-β-resistant cell mutants implicates both receptor types in signal transduction. J. Biol. Chem. 265, 18518–18524 (1990).

    CAS  PubMed  Google Scholar 

  46. Goyette, M. C. et al. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol. Cell. Biol. 12, 1387–1395 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bai, R. Y., Dieter, P., Peschel, C., Morris, S. W. & Duyster, J. Nucleophosmin-anaplastic lymphoma kinase of large-cell anaplastic lymphoma is a constitutively active tyrosine kinase that utilizes phospholipase C-γ to mediate its mitogenicity. Mol. Cell. Biol. 18, 6951–6961 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Hoodless, P. A. et al. MADR1, a MAD-related protein that functions in BMP2 signalling pathways. Cell 85, 489–500 (1996).

    Article  CAS  PubMed  Google Scholar 

  49. Hammerschmidt, M. et al. Dino and mercedes, two genes regulating dorsal development in the zebrafish embryo. Development 123, 95–102 (1996).

    CAS  PubMed  Google Scholar 

  50. Molitor, J. A., Walker, W. H., Doerre, S., Ballard, D. W. & Greene, W. C. NF-kappa B: a family of inducible and differentially expressed enhancer- binding proteins in human T cells. Proc. Natl Acad. Sci. USA 87, 10028–10032 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank B. Vogelstein and K.W. Kinzler for the HCT116 and HCT116-SMAD4−/− cell lines and for the FAST1 cDNA, J. Wrana and L. Attisano for the ARE-lux construct, H.F. Lodish for TGFβ-RII cDNA, C.H. Heldin for TGFβ-RI and BMP-RI (ALK4) constructs and the Mv1lu-R42 cell line, M. Kawabata for BMP-RII and Smad6 cDNAs, R. Derynck for Smad3 cDNA, X.F. Wang for mSmad5 cDNA, B. Hill for Msx2 mRNA fragment, F. Oswald and R. Schmid for E1A constructs, E.J. Stanbridge for the SW480.7 cell line and J. Massague for Smad1, Smad2 and 3TP-lux constructs. We also thank C. Miething, N. von Bubnoff and J. Metzger for helpful technical advice, J. Sänger for technical help and K. Götze for critical reading of the manuscript. K.N. Astrahantseff is acknowledged for sharing unpublished data. This work was supported by a grant to JD from the German José-Carreras Stiftung.

Author information

Authors and Affiliations

  1. Department of Internal Medicine III, Laboratory of Leukemogenesis, Technical University of Munich, Ismaningerstrasse 22, Munich, 81675, Germany

    Ren-Yuan Bai, Tao Ouyang, Christian Peschel & Justus Duyster

  2. Hans-Spemann-Laboratory, Max-Plank Institute for Immunobiology, Stuebeweg 51, Freiburg, D79108

    Christina Koester & Matthias Hammerschmidt

  3. Department of Internal Medicine, Ruhr-University, In der Schornau 23, Bochum, D44892, Germany

    Stephan A. Hahn

Authors
  1. Ren-Yuan Bai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christina Koester
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tao Ouyang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephan A. Hahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Matthias Hammerschmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christian Peschel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Justus Duyster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Justus Duyster.

Supplementary information

Supplementary figures

Figure S1 SMIF immunoblot (PDF 481 kb)

Figure S2 Experiments similar to Figure 5d and e were performed in Mv1Lu-R42 cells, which express endogenous Smad4.

Figure S3 HCT Smad4-/- cells were transfected with TGFβ receptor II (TRII) and constructs as decribed in Figure 5f, with or without Smad4.

Figure S4 SMIF-Δ(124-390) competes with wildtype F-SMIF for the binding to Smad4.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bai, RY., Koester, C., Ouyang, T. et al. SMIF, a Smad4-interacting protein that functions as a co-activator in TGFβ signalling. Nat Cell Biol 4, 181–190 (2002). https://doi.org/10.1038/ncb753

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb753

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing