Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.
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.
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.
M
M
Kawabata
H
H
Inoue
A
A
Hanyu
T
T
Imamura
K
K
Miyazono
D030201:Activin Receptors, Type I; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002277:Carcinoma; D002850:Chromatography, Gel / Q000379:methods; D003110:Colonic Neoplasms; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein; D051900:Smad3 Protein; D013388:Succinimides; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; D051900:Smad3 Protein; D013388:Succinimides; D015534:Trans-Activators; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I; C019358:disuccinimidyl suberate