title: Molecular mechanism of the negative regulation of Smad1/5 protein by carboxyl terminus of Hsc70-interacting protein (CHIP).
abstract: 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.
authors: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China.
authors: Le
authors: L
authors: Wang
authors: Yi-Tong
authors: YT
authors: Liu
authors: Rui
authors: R
authors: Hao
authors: Lei
authors: L
authors: Chen
authors: Zhijie
authors: Z
authors: Chang
authors: Hong-Rui
authors: HR
authors: Wang
authors: Zhi-Xin
authors: ZX
authors: Wang
authors: Jia-Wei
authors: JW
authors: Wu
mesh_terms: D020816:Amino Acid Motifs; D057809:HEK293 Cells; D050883:HSC70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D051898:Smad1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051902:Smad5 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D050883:HSC70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D010455:Peptides; C497399:SMAD1 protein, human; C497797:SMAD5 protein, human; D051898:Smad1 Protein; D051902:Smad5 Protein; D016212:Transforming Growth Factor beta; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases