SMURF2 monoubiquitinates SMAD3 on lysine residues in the MH2 domain. Lysines K333 and K378 are likely the major sites for monoubiquitination. Lysine K409 is also monoubiquitinated, and possibly lysine K341. Since lysines K333 and K378 are predicted to stabilize the interaction of SMAD3 with SMAD4, monoubiquitination of these lysine residues is expected to disrupt SMAD2/3:SMAD4 heterotrimer. SMURF2-mediated disruption of endogenous Smad2/3:Smad4 heterotrimers was demonstrated in mouse embryonic fibroblasts. SMURF2 also ubiquitinates SMAD2 phosphorylated in the linker region, but loss of Smurf2 has less impact on Smad2 ubiquitination than on Smad3 in vivo.