Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP)

J Biol Chem. 2011 May 6;286(18):15883-94. doi: 10.1074/jbc.M110.201814. Epub 2011 Mar 16.


The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • HEK293 Cells
  • HSC70 Heat-Shock Proteins / chemistry
  • HSC70 Heat-Shock Proteins / genetics
  • HSC70 Heat-Shock Proteins / metabolism
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Signal Transduction / physiology
  • Smad1 Protein / chemistry
  • Smad1 Protein / genetics
  • Smad1 Protein / metabolism*
  • Smad5 Protein / chemistry
  • Smad5 Protein / genetics
  • Smad5 Protein / metabolism*
  • Transforming Growth Factor beta / chemistry
  • Transforming Growth Factor beta / genetics
  • Transforming Growth Factor beta / metabolism
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination / physiology


  • HSC70 Heat-Shock Proteins
  • HSPA8 protein, human
  • Peptides
  • SMAD1 protein, human
  • SMAD5 protein, human
  • Smad1 Protein
  • Smad5 Protein
  • Transforming Growth Factor beta
  • STUB1 protein, human
  • Ubiquitin-Protein Ligases

Associated data

  • PDB/3Q47
  • PDB/3Q49
  • PDB/3Q4A