Abstract
Smad4 mediates signaling by the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Smad signaling is negatively regulated by inhibitory (I) Smads and ubiquitin-mediated processes. Known mechanisms of proteasomal degradation of Smads depend on the direct interaction of specific E3 ligases with Smads. Alternatively, I-Smads elicit degradation of the TGF-beta receptor by recruiting the WW and HECT domain E3 ligases, Smurfs, WWP1, or NEDD4-2. We describe an equivalent mechanism of degradation of Smad4 by the above E3 ligases, via formation of ternary complexes between Smad4 and Smurfs, mediated by R-Smads (Smad2) or I-Smads (Smad6/7), acting as adaptors. Smurfs, which otherwise cannot directly bind to Smad4, mediated poly-ubiquitination of Smad4 in the presence of Smad6 or Smad7. Smad4 co-localized with Smad7 and Smurf1 primarily in the cytoplasm and in peripheral cell protrusions. Smad2 or Smad7 mutants defective in Smad4 interaction failed to induce Smurf1-mediated down-regulation of Smad4. A Smad4 mutant defective in Smad2 or Smad7 interaction could not be effectively down-regulated by Smurf1. We propose that Smad4 is targeted for degradation by multiple ubiquitin ligases that can simultaneously act on R-Smads and signaling receptors. Such mechanisms of down-regulation of TGF-beta signaling may be critical for proper physiological response to this pathway.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenoviridae / metabolism
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Cell Line
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Cytoplasm / metabolism
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / metabolism
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Down-Regulation
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Endosomal Sorting Complexes Required for Transport
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Genetic Vectors
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins / metabolism
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Humans
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Microscopy, Fluorescence
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Mutation
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Nedd4 Ubiquitin Protein Ligases
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Plasmids / metabolism
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Proteasome Endopeptidase Complex / metabolism
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Protein Binding
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Protein Structure, Tertiary
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Receptors, Transforming Growth Factor beta / metabolism
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Signal Transduction*
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Smad2 Protein
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Smad4 Protein
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Smad7 Protein
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Trans-Activators / chemistry*
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Trans-Activators / metabolism
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Transfection
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Transforming Growth Factor beta / metabolism
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Ubiquitin / chemistry
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Ubiquitin / metabolism
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Ubiquitin-Protein Ligases / chemistry*
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Ubiquitin-Protein Ligases / metabolism
Substances
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DNA-Binding Proteins
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Endosomal Sorting Complexes Required for Transport
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Receptors, Transforming Growth Factor beta
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SMAD2 protein, human
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SMAD4 protein, human
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SMAD7 protein, human
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Smad2 Protein
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Smad4 Protein
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Smad7 Protein
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Trans-Activators
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Transforming Growth Factor beta
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Ubiquitin
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Green Fluorescent Proteins
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Nedd4 Ubiquitin Protein Ligases
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Nedd4 protein, human
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Nedd4L protein, human
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SMURF1 protein, human
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SMURF2 protein, human
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WWP1 protein, human
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Ubiquitin-Protein Ligases
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Glutathione Transferase
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Proteasome Endopeptidase Complex