Differential ubiquitination of Smad1 mediated by CHIP: implications in the regulation of the bone morphogenetic protein signaling pathway.
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 alpha-NH(2) group of the N terminus and the epsilon-NH(2) 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.
Li
Ren-Feng
RF
Department of Biological Sciences and Biotechnology, State-Key Lab of Biomembranes and Membrane Biotechnology, Tsinghua University, Beijing 100084, China.
Shang
Yu
Y
Liu
Di
D
Ren
Ze-Song
ZS
Chang
Zhijie
Z
Sui
Sen-Fang
SF
D015153:Blotting, Western; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D015398:Signal Transduction; D051898:Smad1 Protein / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
D019485:Bone Morphogenetic Proteins; D051898:Smad1 Protein; D025801:Ubiquitin; C467728:STUB1 protein, human; D044767:Ubiquitin-Protein Ligases