Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway |
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Authors: | Li Ren-Feng Shang Yu Liu Di Ren Ze-Song Chang Zhijie Sui Sen-Fang |
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Affiliation: | 1 Department of Biological Sciences and Biotechnology, State-Key Lab of Biomembranes and Membrane Biotechnology, Tsinghua University, Beijing 100084, China 2 School of Medicine, Institute of Biomedicine, Tsinghua University, Beijing 100084, China |
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Abstract: | Smad1, a downstream regulator of the bone morphogenetic protein (BMP) receptors, is tightly regulated by the ubiquitin-proteasomal degradation system. To dissect the mechanisms that underlie the regulation of Smad1, it is important to investigate the specific ubiquitination site(s) in Smad1. Here we report that the α-NH2 group of the N terminus and the ε-NH2 groups of internal lysine residues 116, 118 and 269 (K116, K118 and K269) of Smad1 are ubiquitin acceptor sites mediated by the carboxyl terminus of Hsc70-interacting protein (CHIP). The in vitro degradation assay indicates that ubiquitination at the N terminus partially contributes to the degradation of Smad1. Furthermore, we demonstrate that the ubiquitination level of pseudo-phosphorylated Smad1 by CHIP is stronger than that of wild-type Smad1 and can be strongly inhibited by a phosphorylated tail of Smad1, PIS(pS)V(pS). Third, our results indicate that Hsp70 facilitates CHIP-mediated poly-ubiquitination of Smad1 whereas it attenuates CHIP-meditated mono-ubiquitination of Smad1. Finally, consistent with the in vitro observation, we show that CHIP preferentially mediates the degradation of phospho-Smad1/5 in vivo. Taken together, these results provide us a hint that CHIP might preferentially regulate phosphorylated Smad1 and thus the BMP signaling. |
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Keywords: | TGF-β, transforming growth factor-β BMP, bone morphogenetic protein CHIP, carboxyl terminus of Hsc70-interacting protein TPR, tetratricopeptide repeat R-Smads, receptor-regulated Smads Smurf1, Smad ubiquitination regulatory factor-1 WT, wild-type Ubiquitin(K0), all lysine residues of ubiquitin were mutated to arginine Ub, ubiquitin GST, glutathione S-transferase E1, ubiquitin-activating enzyme E2, ubiquitin-conjugating enzyme E3, ubiquitin ligase |
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