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.