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. 1997 Sep 1;16(17):5353–5362. doi: 10.1093/emboj/16.17.5353

TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.

A Nakao 1, T Imamura 1, S Souchelnytskyi 1, M Kawabata 1, A Ishisaki 1, E Oeda 1, K Tamaki 1, J Hanai 1, C H Heldin 1, K Miyazono 1, P ten Dijke 1
PMCID: PMC1170167  PMID: 9311995

Abstract

Smad family members are newly identified essential intracellular signalling components of the transforming growth factor-beta (TGF-beta) superfamily. Smad2 and Smad3 are structurally highly similar and mediate TGF-beta signals. Smad4 is distantly related to Smads 2 and 3, and forms a heteromeric complex with Smad2 after TGF-beta or activin stimulation. Here we show that Smad2 and Smad3 interacted with the kinase-deficient TGF-beta type I receptor (TbetaR)-I after it was phosphorylated by TbetaR-II kinase. TGF-beta1 induced phosphorylation of Smad2 and Smad3 in Mv1Lu mink lung epithelial cells. Smad4 was found to be constitutively phosphorylated in Mv1Lu cells, the phosphorylation level remaining unchanged upon TGF-beta1 stimulation. Similar results were obtained using HSC4 cells, which are also growth-inhibited by TGF-beta. Smads 2 and 3 interacted with Smad4 after TbetaR activation in transfected COS cells. In addition, we observed TbetaR-activation-dependent interaction between Smad2 and Smad3. Smads 2, 3 and 4 accumulated in the nucleus upon TGF-beta1 treatment in Mv1Lu cells, and showed a synergistic effect in a transcriptional reporter assay using the TGF-beta-inducible plasminogen activator inhibitor-1 promoter. Dominant-negative Smad3 inhibited the transcriptional synergistic response by Smad2 and Smad4. These data suggest that TGF-beta induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-beta signal transduction.

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Selected References

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  1. Baker J. C., Harland R. M. A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway. Genes Dev. 1996 Aug 1;10(15):1880–1889. doi: 10.1101/gad.10.15.1880. [DOI] [PubMed] [Google Scholar]
  2. Chen X., Rubock M. J., Whitman M. A transcriptional partner for MAD proteins in TGF-beta signalling. Nature. 1996 Oct 24;383(6602):691–696. doi: 10.1038/383691a0. [DOI] [PubMed] [Google Scholar]
  3. Derynck R., Gelbart W. M., Harland R. M., Heldin C. H., Kern S. E., Massagué J., Melton D. A., Mlodzik M., Padgett R. W., Roberts A. B. Nomenclature: vertebrate mediators of TGFbeta family signals. Cell. 1996 Oct 18;87(2):173–173. doi: 10.1016/s0092-8674(00)81335-5. [DOI] [PubMed] [Google Scholar]
  4. Eppert K., Scherer S. W., Ozcelik H., Pirone R., Hoodless P., Kim H., Tsui L. C., Bapat B., Gallinger S., Andrulis I. L. MADR2 maps to 18q21 and encodes a TGFbeta-regulated MAD-related protein that is functionally mutated in colorectal carcinoma. Cell. 1996 Aug 23;86(4):543–552. doi: 10.1016/s0092-8674(00)80128-2. [DOI] [PubMed] [Google Scholar]
  5. Franzén P., ten Dijke P., Ichijo H., Yamashita H., Schulz P., Heldin C. H., Miyazono K. Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor. Cell. 1993 Nov 19;75(4):681–692. doi: 10.1016/0092-8674(93)90489-d. [DOI] [PubMed] [Google Scholar]
  6. Frolik C. A., Wakefield L. M., Smith D. M., Sporn M. B. Characterization of a membrane receptor for transforming growth factor-beta in normal rat kidney fibroblasts. J Biol Chem. 1984 Sep 10;259(17):10995–11000. [PubMed] [Google Scholar]
  7. Graff J. M., Bansal A., Melton D. A. Xenopus Mad proteins transduce distinct subsets of signals for the TGF beta superfamily. Cell. 1996 May 17;85(4):479–487. doi: 10.1016/s0092-8674(00)81249-0. [DOI] [PubMed] [Google Scholar]
  8. Hahn S. A., Schutte M., Hoque A. T., Moskaluk C. A., da Costa L. T., Rozenblum E., Weinstein C. L., Fischer A., Yeo C. J., Hruban R. H. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science. 1996 Jan 19;271(5247):350–353. doi: 10.1126/science.271.5247.350. [DOI] [PubMed] [Google Scholar]
  9. Hoodless P. A., Haerry T., Abdollah S., Stapleton M., O'Connor M. B., Attisano L., Wrana J. L. MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell. 1996 May 17;85(4):489–500. doi: 10.1016/s0092-8674(00)81250-7. [DOI] [PubMed] [Google Scholar]
  10. Ichijo H., Momose F., Miyazono K. Biological effects and binding properties of transforming growth factor-beta on human oral squamous cell carcinoma cells. Exp Cell Res. 1990 Apr;187(2):263–269. doi: 10.1016/0014-4827(90)90090-w. [DOI] [PubMed] [Google Scholar]
  11. Jameson B. A., Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci. 1988 Mar;4(1):181–186. doi: 10.1093/bioinformatics/4.1.181. [DOI] [PubMed] [Google Scholar]
  12. Lagna G., Hata A., Hemmati-Brivanlou A., Massagué J. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature. 1996 Oct 31;383(6603):832–836. doi: 10.1038/383832a0. [DOI] [PubMed] [Google Scholar]
  13. Lechleider R. J., de Caestecker M. P., Dehejia A., Polymeropoulos M. H., Roberts A. B. Serine phosphorylation, chromosomal localization, and transforming growth factor-beta signal transduction by human bsp-1. J Biol Chem. 1996 Jul 26;271(30):17617–17620. doi: 10.1074/jbc.271.30.17617. [DOI] [PubMed] [Google Scholar]
  14. Liu F., Hata A., Baker J. C., Doody J., Cárcamo J., Harland R. M., Massagué J. A human Mad protein acting as a BMP-regulated transcriptional activator. Nature. 1996 Jun 13;381(6583):620–623. doi: 10.1038/381620a0. [DOI] [PubMed] [Google Scholar]
  15. Macías-Silva M., Abdollah S., Hoodless P. A., Pirone R., Attisano L., Wrana J. L. MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell. 1996 Dec 27;87(7):1215–1224. doi: 10.1016/s0092-8674(00)81817-6. [DOI] [PubMed] [Google Scholar]
  16. Massagué J. TGFbeta signaling: receptors, transducers, and Mad proteins. Cell. 1996 Jun 28;85(7):947–950. doi: 10.1016/s0092-8674(00)81296-9. [DOI] [PubMed] [Google Scholar]
  17. Massagué J., Weis-Garcia F. Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals. Cancer Surv. 1996;27:41–64. [PubMed] [Google Scholar]
  18. Meersseman G., Verschueren K., Nelles L., Blumenstock C., Kraft H., Wuytens G., Remacle J., Kozak C. A., Tylzanowski P., Niehrs C. The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation. Mech Dev. 1997 Jan;61(1-2):127–140. doi: 10.1016/s0925-4773(96)00629-6. [DOI] [PubMed] [Google Scholar]
  19. Nakao A., Röijer E., Imamura T., Souchelnytskyi S., Stenman G., Heldin C. H., ten Dijke P. Identification of Smad2, a human Mad-related protein in the transforming growth factor beta signaling pathway. J Biol Chem. 1997 Jan 31;272(5):2896–2900. doi: 10.1074/jbc.272.5.2896. [DOI] [PubMed] [Google Scholar]
  20. Riggins G. J., Thiagalingam S., Rozenblum E., Weinstein C. L., Kern S. E., Hamilton S. R., Willson J. K., Markowitz S. D., Kinzler K. W., Vogelstein B. Mad-related genes in the human. Nat Genet. 1996 Jul;13(3):347–349. doi: 10.1038/ng0796-347. [DOI] [PubMed] [Google Scholar]
  21. Savage C., Das P., Finelli A. L., Townsend S. R., Sun C. Y., Baird S. E., Padgett R. W. Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):790–794. doi: 10.1073/pnas.93.2.790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sekelsky J. J., Newfeld S. J., Raftery L. A., Chartoff E. H., Gelbart W. M. Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster. Genetics. 1995 Mar;139(3):1347–1358. doi: 10.1093/genetics/139.3.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thomsen G. H. Xenopus mothers against decapentaplegic is an embryonic ventralizing agent that acts downstream of the BMP-2/4 receptor. Development. 1996 Aug;122(8):2359–2366. doi: 10.1242/dev.122.8.2359. [DOI] [PubMed] [Google Scholar]
  24. Wrana J. L., Attisano L., Wieser R., Ventura F., Massagué J. Mechanism of activation of the TGF-beta receptor. Nature. 1994 Aug 4;370(6488):341–347. doi: 10.1038/370341a0. [DOI] [PubMed] [Google Scholar]
  25. Yamaguchi K., Shirakabe K., Shibuya H., Irie K., Oishi I., Ueno N., Taniguchi T., Nishida E., Matsumoto K. Identification of a member of the MAPKKK family as a potential mediator of TGF-beta signal transduction. Science. 1995 Dec 22;270(5244):2008–2011. doi: 10.1126/science.270.5244.2008. [DOI] [PubMed] [Google Scholar]
  26. Yamashita H., ten Dijke P., Huylebroeck D., Sampath T. K., Andries M., Smith J. C., Heldin C. H., Miyazono K. Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J Cell Biol. 1995 Jul;130(1):217–226. doi: 10.1083/jcb.130.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Yingling J. M., Das P., Savage C., Zhang M., Padgett R. W., Wang X. F. Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8940–8944. doi: 10.1073/pnas.93.17.8940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang Y., Feng X., We R., Derynck R. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response. Nature. 1996 Sep 12;383(6596):168–172. doi: 10.1038/383168a0. [DOI] [PubMed] [Google Scholar]
  29. de Winter J. P., Roelen B. A., ten Dijke P., van der Burg B., van den Eijnden-van Raaij A. J. DPC4 (SMAD4) mediates transforming growth factor-beta1 (TGF-beta1) induced growth inhibition and transcriptional response in breast tumour cells. Oncogene. 1997 Apr 24;14(16):1891–1899. doi: 10.1038/sj.onc.1201017. [DOI] [PubMed] [Google Scholar]
  30. ten Dijke P., Miyazono K., Heldin C. H. Signaling via hetero-oligomeric complexes of type I and type II serine/threonine kinase receptors. Curr Opin Cell Biol. 1996 Apr;8(2):139–145. doi: 10.1016/s0955-0674(96)80058-5. [DOI] [PubMed] [Google Scholar]
  31. ten Dijke P., Yamashita H., Ichijo H., Franzén P., Laiho M., Miyazono K., Heldin C. H. Characterization of type I receptors for transforming growth factor-beta and activin. Science. 1994 Apr 1;264(5155):101–104. doi: 10.1126/science.8140412. [DOI] [PubMed] [Google Scholar]

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