Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells

Smad2 is a well-established regulator involved in tissue development and in the pathogenesis of endothelial-to-mesenchymal transition (EndMT) mediated by TGF-β signaling. However, the mechanism underlying the regulation of SMAD2 in human coronary artery endothelial cells (HCAECs), particularly the identity of the responsible E3 ubiquitin ligase and its role during EndMT remain unclear. In this study, we identified Smad ubiquitination regulatory factor 1 (Smurf1) as a negative regulator of Smad2 protein levels in HCAECs and demonstrated that the E3 ligase activity of Smurf1 is essential for this function. Mechanistically, Smurf1 interacts with Smad2, promoting its ubiquitination, and subsequent proteasomal degradation. Specifically, Smurf1 catalyzes K48-linked polyubiquitination of Smad2 at lysine residues K156, K383 and K420. Functionally, Smad2 was found to promote EndMT in HCAECs, an effect that was partially attenuated either by co-expression of Smurf1 or by mutation of Smad2 at lysine 420 (Smad2-K420R), which replaces lysine with arginine. Taken together, our findings identify, for the first time, specific lysine residues on Smad2 targeted by Smurf1 for K48-linked ubiquitination and highlight their crucial regulatory role in modulating EndMT in HCAECs.