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

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Abstract
Smad proteins are signal transducers for the members of the transforming growth factor‐β (TGF‐β) superfamily. Here we show that, in the absence TGF‐β stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo‐oligomers upon phosphorylation by the constitutively active TGF‐β 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‐β superfamily members.