Home LiteratureArticle Details
PMID: 9670020 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

The EMBO journal ·Vol. 17 ·No. 14 ·1998-07-15 ·Pages 4056-65

Kawabata M, Inoue H, Hanyu A, Imamura T, Miyazono K

Abstract

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.

MeSH Terms
Activin Receptors, Type I Animals Binding, Competitive COS Cells Carcinoma Chromatography, Gel/methods Colonic Neoplasms Cross-Linking Reagents DNA/metabolism DNA-Binding Proteins/chemistry,genetics,metabolism Molecular Weight Phosphorylation Protein Conformation Protein Serine-Threonine Kinases/genetics,physiology Receptor, Transforming Growth Factor-beta Type I Receptors, Transforming Growth Factor beta/genetics,physiology Signal Transduction/physiology Smad2 Protein Smad3 Protein Succinimides Trans-Activators/chemistry,genetics,metabolism Transfection Tumor Cells, Cultured
Chemicals
Cross-Linking Reagents DNA-Binding Proteins Receptors, Transforming Growth Factor beta Smad2 Protein Smad3 Protein Succinimides Trans-Activators DNA Protein Serine-Threonine Kinases Activin Receptors, Type I Receptor, Transforming Growth Factor-beta Type I disuccinimidyl suberate
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Kawabata M
Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research (JFCR), and Research for the Future Program, Japan Society for the Promotion of Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan.
Inoue H
Hanyu A
Imamura T
Miyazono K
References (50)
50 references, click to expand
  1. Xenopus Mad proteins transduce distinct subsets of signals for the TGF beta superfamily.
    Cell. 1996 May 17;85(4):479-87 PMID: 8653784
  2. Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium.
    Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9314-9 PMID: 9256479
  3. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways.
    Nature. 1996 Oct 31;383(6603):832-6 PMID: 8893010
  4. A structural basis for mutational inactivation of the tumour suppressor Smad4.
    Nature. 1997 Jul 3;388(6637):87-93 PMID: 9214508
  5. Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling.
    J Biol Chem. 1997 Oct 31;272(44):28107-15 PMID: 9346966
  6. Smad6 inhibits signalling by the TGF-beta superfamily.
    Nature. 1997 Oct 9;389(6651):622-6 PMID: 9335505
  7. GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex.
    EMBO J. 1995 May 15;14(10):2199-208 PMID: 7774578
  8. Homomeric interactions between type II transforming growth factor-beta receptors.
    J Biol Chem. 1994 Sep 9;269(36):22868-74 PMID: 7521335
  9. TGF-beta signalling from cell membrane to nucleus through SMAD proteins.
    Nature. 1997 Dec 4;390(6659):465-71 PMID: 9393997
  10. Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells.
    Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10669-74 PMID: 9380693
  11. 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):127-40 PMID: 9076683
  12. Interaction of Drosophila inhibitors of apoptosis with thick veins, a type I serine/threonine kinase receptor for decapentaplegic.
    J Biol Chem. 1998 Apr 17;273(16):9353-6 PMID: 9545255
  13. Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor.
    Genes Dev. 1998 Jan 15;12(2):186-97 PMID: 9436979
  14. Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster.
    Genetics. 1995 Mar;139(3):1347-58 PMID: 7768443
  15. A transcriptional partner for MAD proteins in TGF-beta signalling.
    Nature. 1996 Oct 24;383(6602):691-6 PMID: 8878477
  16. Cloning and characterization of a novel member of the human Mad gene family (MADH6).
    Genomics. 1997 Jun 15;42(3):446-51 PMID: 9205116
  17. Receptor-associated Mad homologues synergize as effectors of the TGF-beta response.
    Nature. 1996 Sep 12;383(6596):168-72 PMID: 8774881
  18. 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. 1992 Mar;12(3):1387-95 PMID: 1347643
  19. MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling.
    Cell. 1996 Dec 27;87(7):1215-24 PMID: 8980228
  20. Smad8 mediates the signaling of the ALK-2 [corrected] receptor serine kinase.
    Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12938-43 PMID: 9371779
  21. Smad4 and FAST-1 in the assembly of activin-responsive factor.
    Nature. 1997 Sep 4;389(6646):85-9 PMID: 9288972
  22. Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein.
    Mol Cell Biol. 1997 Dec;17(12):7019-28 PMID: 9372933
  23. TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.
    EMBO J. 1997 Sep 1;16(17):5353-62 PMID: 9311995
  24. Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.
    J Cell Biol. 1998 Feb 23;140(4):767-77 PMID: 9472030
  25. Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4.
    Nature. 1997 Jul 3;388(6637):82-7 PMID: 9214507
  26. Cellular interpretation of multiple TGF-beta signals: intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated by Smads.
    Development. 1997 Nov;124(22):4467-80 PMID: 9409665
  27. The types II and III transforming growth factor-beta receptors form homo-oligomers.
    J Cell Biol. 1994 Jul;126(1):139-54 PMID: 8027173
  28. Formation of hetero-oligomeric complexes of type I and type II receptors for transforming growth factor-beta.
    J Biol Chem. 1994 Aug 5;269(31):20172-8 PMID: 8051105
  29. Modifications of vectors pEF-BOS, pcDNA1 and pcDNA3 result in improved convenience and expression.
    Biotechniques. 1996 Dec;21(6):1013-5 PMID: 8969825
  30. A kinase subdomain of transforming growth factor-beta (TGF-beta) type I receptor determines the TGF-beta intracellular signaling specificity.
    EMBO J. 1997 Jul 1;16(13):3912-23 PMID: 9233801
  31. Functional analysis of the transforming growth factor beta responsive elements in the WAF1/Cip1/p21 promoter.
    J Biol Chem. 1995 Dec 1;270(48):28623-8 PMID: 7499379
  32. TGF-beta signalling through the Smad pathway.
    Trends Cell Biol. 1997 May;7(5):187-92 PMID: 17708943
  33. The MAD-related protein Smad7 associates with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling.
    Cell. 1997 Jun 27;89(7):1165-73 PMID: 9215638
  34. A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway.
    Genes Dev. 1996 Aug 1;10(15):1880-9 PMID: 8756346
  35. 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):790-4 PMID: 8570636
  36. Human Smad3 and Smad4 are sequence-specific transcription activators.
    Mol Cell. 1998 Mar;1(4):611-7 PMID: 9660945
  37. TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling.
    J Biol Chem. 1997 Oct 31;272(44):27678-85 PMID: 9346908
  38. Dual role of the Smad4/DPC4 tumor suppressor in TGFbeta-inducible transcriptional complexes.
    Genes Dev. 1997 Dec 1;11(23):3157-67 PMID: 9389648
  39. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.
    Nature. 1997 Oct 9;389(6651):631-5 PMID: 9335507
  40. Heteromeric and homomeric interactions correlate with signaling activity and functional cooperativity of Smad3 and Smad4/DPC4.
    Mol Cell Biol. 1997 May;17(5):2521-8 PMID: 9111321
  41. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.
    Science. 1996 Jan 19;271(5247):350-3 PMID: 8553070
  42. MADR1, a MAD-related protein that functions in BMP2 signaling pathways.
    Cell. 1996 May 17;85(4):489-500 PMID: 8653785
  43. Mad-related genes in the human.
    Nat Genet. 1996 Jul;13(3):347-9 PMID: 8673135
  44. A human Mad protein acting as a BMP-regulated transcriptional activator.
    Nature. 1996 Jun 13;381(6583):620-3 PMID: 8637600
  45. Mechanism of activation of the TGF-beta receptor.
    Nature. 1994 Aug 4;370(6488):341-7 PMID: 8047140
  46. Type I receptors specify growth-inhibitory and transcriptional responses to transforming growth factor beta and activin.
    Mol Cell Biol. 1994 Jun;14(6):3810-21 PMID: 8196624
  47. Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic.
    Nature. 1997 Jul 17;388(6639):304-8 PMID: 9230443
  48. Complementation between kinase-defective and activation-defective TGF-beta receptors reveals a novel form of receptor cooperativity essential for signaling.
    EMBO J. 1996 Jan 15;15(2):276-89 PMID: 8617203
  49. Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex.
    Mol Cell Biol. 1995 Mar;15(3):1573-81 PMID: 7862150
  50. TGF-beta receptor signaling.
    Biochim Biophys Acta. 1997 Oct 24;1333(2):F105-50 PMID: 9395284
Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/17.14.4056
Published
1998-07-15
Pages
4056-65
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1170738
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/17.14.4056 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2