Novel dominant negative Smad antagonists to TGFbeta signaling

Cell Signal. 2007 Jul;19(7):1565-74. doi: 10.1016/j.cellsig.2007.02.001. Epub 2007 Feb 15.


We previously identified a critical serine/threonine residue within the linker domain of Smad2/3, phosphorylated by the kinase GRK2 which plays a critical role in regulating Smad signaling. To define the mechanism by which GRK2-mediated phosphorylation modifies Smad2/3 behavior at the molecular level, we generated mutant Smads where the GRK2 phosphorylation site was replaced with an aspartic acid (D) to mimic the properties of a phospho-residue or an alanine (A) as a control. Interestingly, overexpression of either the D or A mutant inhibits TGFbeta signaling, but through two distinct mechanisms. The D mutant is properly localized and released from the plasma membrane upon ligand stimulation, but fails to interact with the type I receptor kinase. The A mutant properly interacts with and is phosphorylated by the type I receptor, translocates to the nucleus and homodimerizes with wild-type R-Smads, but it fails to form a heterocomplex with Smad4. As a result, both mutants act as antagonists of endogenous TGFbeta signaling through divergent mechanisms. The D mutant acts by blocking endogenous R-Smads phosphorylation and the A mutant acts by preventing endogenous R-Smad/Smad4 heterocomplexes. Thus, mutation of the GRK2 phosphorylation site within the Smad generates dominant negative Smads that efficiently inhibit TGFbeta responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Membrane / metabolism
  • Cell Nucleus / metabolism
  • Cricetinae
  • Cricetulus
  • Dimerization
  • Gene Expression
  • Genes, Dominant*
  • Molecular Mimicry
  • Monomeric GTP-Binding Proteins / metabolism
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Phosphorylation
  • Promoter Regions, Genetic / genetics
  • Signal Transduction*
  • Smad Proteins / genetics*
  • Smad Proteins / metabolism*
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism
  • Smad3 Protein / metabolism
  • Smad4 Protein / metabolism
  • Transforming Growth Factor beta / antagonists & inhibitors*
  • beta-Adrenergic Receptor Kinases / metabolism


  • Mutant Proteins
  • Smad Proteins
  • Smad2 Protein
  • Smad3 Protein
  • Smad4 Protein
  • Transforming Growth Factor beta
  • beta-Adrenergic Receptor Kinases
  • Monomeric GTP-Binding Proteins