Wojtowicz S, Lee S, Chan E, Ng E, Campbell CI, Di Guglielmo GM

TGF?-dependent signal transduction is facilitated by Smad anchor for receptor activation (SARA) and inhibited by the inhibitory-Smad, Smad7, which recruits the E3 ubiquitin ligase, Smurf2, to catalyze the degradation of TGF? receptors. Since the signalling and degradation pathways target active receptor complexes, we assessed if SARA and Smurf2/Smad7 interact and ...

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if Smad7/Smurf2 would affect SARA steady state levels. We observed that the Smurf2/Smad7 complex induces a decrease of SARA steady state levels in a process that is dependent on the HECT ubiquitin E3 ligase activity of Smurf2 but is independent of SARA associating with TGF? receptors or Smad2. We observed that Smurf2/Smad7-dependent reduction of SARA levels is dependent on proteasome activity, as the pharmacological inhibition of the proteasome using MG132 blocked degradation of SARA. When we assessed the functional outcome of reducing endogenous SARA levels via siRNA-mediated silencing, we observed that siRNA directed at SARA decreased both TGF?-dependent Smad2 membrane recruitment and phosphorylation, as assessed by subcellular fractionation and western blotting. Furthermore, siRNA targeting SARA decreased TGF?-dependent epithelial to mesenchymal transition, as measured by cellular E- and N-Cadherin protein levels, and the reorganization of actin from cortical actin to stress fiber formation. These data describe a previously undescribed mechanism where the robustness of the TGF? signalling is regulated by interplay between SARA and Smurf2/Smad7 complexes.

Date: Dec. 01, 2019

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