TGF-β signalling is regulated by post-translational modifications of Smad 
proteins to translate quantitative difference in ligand concentration into 
proportional transcriptional output. Previous studies in cell culture systems 
suggested that Smad ubiquitination regulatory factors (Smurfs) act in this 
regulation by targeting Smads for proteasomal degradation, but whether this 
mechanism operates under physiological conditions is not clear. Here, we 
generated mice harbouring a target-disrupted Smurf2 allele. Using primary mouse 
embryonic fibroblasts and dermal fibroblasts, we show that TGF-β-mediated, 
Smad-dependent transcriptional responses are elevated in the absence of Smurf2. 
Instead of promoting poly-ubiquitination and degradation, we show that Smurf2 
actually induces multiple mono-ubiquitination of Smad3 in vivo. Phosphorylation 
of T179, immediately upstream of the Smad3 PY motif, enhances Smurf2 and Smad3 
interaction and Smad3 ubiquitination. We have mapped Smurf2-induced Smad3 
ubiquitination sites to lysine residues at the MH2 domain, and demonstrate that 
Smad3 ubiquitination inhibits the formation of Smad3 complexes. Thus, our data 
support a model in which Smurf2 negatively regulates TGF-β signalling by 
attenuating the activity of Smad3 rather than promoting its degradation.