Molecular Mechanism Of The Negative Regulation Of Smad1/5Protein By CHIP

The transforming growth factor-β (TGF-β) superfamily of signaling pathwaysexists in species ranging from flies and worms to mammals, regulating cellularprocesses including cell proliferation, differentiation, apoptosis and specification ofdevelopmental fate, with its dysfunction associated with many types of human cancer.The Smad family proteins are key components for the intracellular signaling cascade.The TGF-β superfamily of ligands signals along two intracellular pathways,Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bonemorphogenetic protein (BMP) pathway. Carboxyl terminus of Hsc70-interacting protein(CHIP) serves as an E3ubiquitin ligase to mediate the degradation of Smad proteinsand many other signaling proteins. However, the molecular mechanism forCHIP-mediated down-regulation of TGF-β signaling remains unclear.The thesis work presented here employed a combined research strategy ofstructural biology and biochemistry at protein and cell culture levels to investigate themolecular mechanisms of CHIP recognizing and regulating Smad family members.Based on successful expression and purification of various forms of recombinant CHIPand Smad proteins, our research work shows that the extreme C-terminal sequence ofSmad1plays an indispensable role in its direct association with the TPR domain ofCHIP. Interestingly, Smad1undergoes CHIP-mediated polyubiquitination in theabsence of molecular chaperones, and phosphorylation of the C-terminal SXS motif ofSmad1enhances the interaction and ubiquitination. We also found that CHIPpreferentially binds to Smad1/5and specifically disrupts the core signaling complex ofSmad1/5and Smad4. We determined the crystal structures of CHIP-TPR in complexwith the phosphorylated/pseudophosphorylated Smad1peptides and with anHsp70/Hsc70C-terminal peptide. Structural analyses and subsequent biochemicalstudies revealed that the distinct CHIP-binding affinities of Smad1/5or Smad2/3resultfrom the nonconservative hydrophobic residues at R-Smad C-termini. Unexpectedly,the C-terminal peptides from Smad1and molecular chaperones including Hsp70/Hsc70and Hsp90bind in the same groove of CHIP-TPR, and these heat shock proteinscompete with Smad1/5for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination.Taken together, we conclude that CHIP inhibits the signaling activities of Smad1/5by recruiting Smad1/5from the functional R-/Co-Smad complex and further promotingthe ubiquitination/degradation of Smad1/5in a chaperone-independent manner.